WO2025207474A1 - Methods and compositions relating to angioinvasive lung adenocarcinoma - Google Patents

Methods and compositions relating to angioinvasive lung adenocarcinoma

Info

Publication number
WO2025207474A1
WO2025207474A1 PCT/US2025/021084 US2025021084W WO2025207474A1 WO 2025207474 A1 WO2025207474 A1 WO 2025207474A1 US 2025021084 W US2025021084 W US 2025021084W WO 2025207474 A1 WO2025207474 A1 WO 2025207474A1
Authority
WO
WIPO (PCT)
Prior art keywords
therapy
aspects
subject
administered
lobectomy
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/021084
Other languages
French (fr)
Inventor
Marc LENBURG
Jennifer Beane
Dylan STEINER
Eric BURKS
Jiarui ZHANG
Sarah Mazzilli
Kimberly RIEGER-CHRIST
Travis Sullivan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Boston University
Boston Medical Center Corp
Lahey Hospital and Medical Center
Original Assignee
Boston University
Boston Medical Center Corp
Lahey Hospital and Medical Center
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Boston University, Boston Medical Center Corp, Lahey Hospital and Medical Center filed Critical Boston University
Publication of WO2025207474A1 publication Critical patent/WO2025207474A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5752—Immunoassay; Biospecific binding assay; Materials therefor for cancer of the lungs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the technology described herein relates to the treatment and prognosis of lung cancer, e.g., particularly stage I lung adenocarcinoma.
  • Described herein are methods relating to gene expression biomarkers of vascular invasion that can accurately identify subjects with vascular invasion. This provides the ability to predict angioinvasive LU AD from small biopsy specimens, allowing for more tailored treatment prior to and after surgery.
  • a method comprising, determining the expression of at least one gene selected from the group consisting of: SHROOM4; NBEAL1; MUC16; NOTCH3; H19; COL1A1; and GARS1 in a subject with lung cancer.
  • a method of treating or prognosing lung cancer comprising: administering adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and/or lymphadenectomy to a subject having decreased expression of at least one gene selected from the group consisting of: SHROOM4 and NBEAL1; and/or increased expression of at least one gene selected from the group consisting of: MUC16; N0TCH3; H19; C0L1A1; and GARS1.
  • the method described herein further comprises administering active surveillance, wedge resection of the lung, or a segmentectomy of the lung to a subject not having decreased expression of at least one gene selected from the group consisting of:
  • SHR00M4 and NBEAL1 and/or not having increased expression of at least one gene selected from the group consisting of: MUC16; NOTCH3; H19; COL1A1; and GARS1.
  • the at least one gene is SHR00M4 or NBEAL1. In some embodiments of any of the aspects, the at least one gene is SHR00M4. In some embodiments of any of the aspects, the at least one gene is NBEAL1. In some embodiments of any of the aspects, the at least one gene is MUC16; NOTCH3; H19; or COL1A1. In some embodiments of any of the aspects, the at least one gene is SHROOM4; NBEAL1; MUC16; NOTCH3; H19; and COL1A1.
  • BRCA1; HLTF; and FANCA at least one gene selected from the group consisting of:
  • PRKDC TPI1; PKM; GAPDH; AHNAK2; YWHAZ; ATP 13 A3; GARS1; CP;
  • PRKDC TPI1; PKM; GAPDH; AHNAK2; YWHAZ; ATP 13 A3; GARS1; CP;
  • ANKHD1; NBEAL1; MPRIP; SHR00M4; and TET2 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and/or lymphadenectomy.
  • the increased expression and/or decreased expression is relative to an average for subjects with lung cancer. In some embodiments of any of the aspects, the increased expression and/or decreased expression is relative to an average for subjects with lung adenocarcinoma. In some embodiments of any of the aspects, the increased expression and/or decreased expression is relative to an average for subjects with lung cancerthat is not vascular invasive. [0013] In some embodiments of any of the aspects, the subject having increased expression of the at least one gene is determined to have increased expression of the at least one gene. In some embodiments of any of the aspects, the subject having increased expression of the at least one gene is determined to have increased expression of the at least one gene in a sample of tumor cells. In some embodiments of any of the aspects, the level of expression is the level of mRNA.
  • the subject having increased expression of the at least one gene is not administered a lobectomy. In some embodiments of any of the aspects, the subject having increased expression of the at least one gene is not administered a sublobar resection. In some embodiments of any of the aspects, the subject having increased expression of the at least one gene is not administered a lobectomy or sublobar resection.
  • the lung cancer is non-small cell lung cancer (NSCLC). In some embodiments of any of the aspects, the lung cancer is lung adenocarcinoma. In some embodiments of any of the aspects, the lung cancer comprises vascular invasion. In some embodiments of any of the aspects, the lung cancer is vascular invasive lung adenocarcinoma. In some embodiments of any of the aspects, the lung cancer is stage I lung adenocarcinoma.
  • NSCLC non-small cell lung cancer
  • Figs. 1A-1D Distinct gene expression changes associated with VI in LUAD.
  • Fig. 1A Overview of cohorts, sequencing technologies, and analyses utilized in the study. BMC, Boston Medical Center; LHMC, Lahey Hospital and Medical Center.
  • VI vascular invasion
  • NST no special type
  • LMP low malignant potential
  • VPI visceral pleural invasion
  • LI lymphatic invasion.
  • Figs. 1C-1D Top 10 Biological enrichment terms of genes within each gene co-expression cluster.
  • Figs. 2A-2D Spatial transcriptomics of early-stage LUAD reveals association of VI gene clusters with specific LUAD histopathology features.
  • Fig. 2B Association between expression of Vi-associated clusters and pathology across all 15 samples. Only results with FDR ⁇ 0.01 are shown. FDR values were derived from a linear-mixed model predicting cluster expression with sample as random effect.
  • Fig. 2C The reason for a linear-mixed model predicting cluster expression with sample as random effect.
  • Fig. 2D Spot-wise scoring of VP and VI + stRNA- seq samples for expression of VI gene expression clusters in spots annotated with high-grade LUAD patterns (solid, micropapillary, and cribriform). P values were derived from a linear-mixed model predicting cluster expression with sample as a random effect.
  • Figs. 3A-3C The VI signature is composed of both tumor-specific and tumormicroenvironment changes reflective of angioinvasion.
  • Fig. 3A Cell type proportions by VI status (with the VI focus excluded) in the stRNA-seq data, revealed by spot deconvolution.
  • Fig. 3B Peribronchial fibroblast and macrophage proportions by VI status in the bulk RNA-seq discovery cohort.
  • Fig. 3C Mean spatially weighted correlation of VI gene clusters and stage I LUAD cell type signatures from the Salcher et al. lung cancer atlas across all stRNA-seq samples.
  • Figs. 4A-4I A VI predictor derived from the signature validates in an independent stage I LUAD cohort.
  • Fig. 4A Cross-validation approach for feature and model selection.
  • Fig. 4C Feature importance for the final VI predictor.
  • Fig. 4F Predictor association with RFS in the full validation cohort.
  • Fig. 4G Predictor association with RFS in the Vi-negative tumors. KM p values were calculated using the log-rank test.
  • Fig. 4H Predictor performance in the validation cohort by histologic pattern (n varies). Error bar shows bootstrapped 95% CI.
  • Fig. 41 Predictor performance when classifying other LUAD invasion types in the validation cohort.
  • Figs. 5A-5E The VI predictor is robust to intra-tumor heterogeneity.
  • Fig. 5A Examples of binning VI + tumors into distal VI + (spots > 1mm outside invaded foci boundary) and proximal VI + (spots ⁇ 1mm from and including the invaded foci). Any VI + tumors that did not contain VI foci in the capture area were considered as proximal VI + .
  • Fig. 5B Spot-wise expression of the VI predictor (predictor up gene enrichment minus predictor down gene enrichment) between distal VI + and VI" tumors. P values are derived from a type II Anova of a linear mixed model predicting gene expression with sample as a random effect.
  • Fig. 5A Examples of binning VI + tumors into distal VI + (spots > 1mm outside invaded foci boundary) and proximal VI + (spots ⁇ 1mm from and including the invaded foci). Any VI + tumors that did not contain
  • Fig. 5D The enrichment of the 48 VI predictor genes among all filtered genes ranked by correlation between two randomly selected regions of the same tumor. P value calculated by gene set enrichment analysis (GSEA).
  • Fig. 5E Differences in scores between unmatched regions (inter-tumor heterogeneity) and matched regions (intra-tumor heterogeneity).
  • Figs. 6A-6K VI is the LU AD invasion type most associated with recurrence.
  • Figs. 6B-6E Association of VI, STAS, VPI, and LI pathology, respectively, with RFS.
  • Fig. 6F Co-occurrence of invasion types in the stage I LUAD clinical cohort.
  • Fig. 6G Association of VI with RFS when controlling for common clinical variables, collection site (LHMC - Lahey Hospital & Medical Center, BMC - Boston Medical Center) and the other invasion types.
  • Fig. 6H Association between invasion types and LUAD growth patterns.
  • Fig. 6K Sub-distribution HR at the indicated sites of recurrence.
  • Figs. 7A-7B The four VI gene expression clusters individually predict VI even in the absence of LMP tumors.
  • Fig. 7A ROC curves for predicting VI+ vs. VI- tumors using the mean z- score of each gene expression cluster.
  • Fig. 7B ROC curves for predicting VI vs. NST tumors using the mean z-score of each gene expression cluster. P values are reported for the Wilcoxon test where the null hypothesis is that the AUROC is equal to 0.5.
  • Figs. 8A-8C VI but not LI is associated with expression of tissue remodeling genes and larger tumor invasive size.
  • Fig. 8B Heatmap of 15 genes associated with LI contrasts (FDR ⁇ 0.01). Genes are annotated if they belong to one of the clusters defined in Fig IB.
  • Fig. 8C Gene-set enrichment analysis (GSEA) results of the 4 VI gene clusters against a ranked list of genes ordered by association with LI.
  • GSEA Gene-set enrichment analysis
  • Figs. 9A-9E Bulk RNA-seq and stRNA-seq data show strong concordance in gene expression.
  • Fig. 9A The association of spot-wise pathology annotations with tumor-level VI status in stRNA-seq data. FDR values were calculated by chi-square test.
  • Fig. 9B Features per spot and
  • Fig. 9C UMI counts per spot by sample in the stRNA-seq data. Sample 6 failed QC and was excluded from downstream analysis.
  • Fig. 9D Correlation of mean gene expression between pseudo bulked stRNA-seq data and tumor-matched bulk RNA-seq data. P value shown for Spearman rank coefficient.
  • Fig. 10A-10C Cell type enrichment in stRNA-seq data.
  • Fig. 10A Representative images showing the association of cell type enrichment signatures with pathology.
  • Fig. 10B Proportions of plasma cells from deconvolution of the bulk RNA-seq discovery cohort, stratified by pathologist annotated plasma cell grade.
  • Fig. 10C Correlation of cell type markers (using the average expression of the top 50 differentially expressed marker genes) in the stage I LU AD samples from the Salcher lung cancer atlas.
  • Figs. 11A-1 IE VI predictor development and validation.
  • Fig. 11A Cross-validation performance for the four AutoML models.
  • IASLC International Association for the Study of Lung Cancer
  • WHO World Health Organization
  • Figs. 11C Association of pathology features with VI predictor scores in VI- tumors within the validation cohort. A univariate linear model was used to calculate FDR values for each pathology feature, with predictor scores as the dependent variable.
  • Figs. 1 ID-1 IE Association of mean predictor scores per tumor with LVI+ (Fig. 1 ID) and ctDNA+ tumors (Fig. HE).
  • Figs. 12A-12E depict VI predictor genes that are most correlated with overall VI predictor score. Individual VI predictor genes ranked by their correlation with the overall cluster mean expression in the validation cohort for clusters 1 (Fig. 12A), 2 (Fig. 12B), 3 (Fig. 12C), and. 4 (Fig. 12D). (Fig. 12E) shows the location of genes (MKI67, COL1A1, PRKDC, CHD2) that have strong correlation with mean cluster expression in the validation cohort, are high ranking in the VI model, and have ISH probes readily available.
  • genes MKI67, COL1A1, PRKDC, CHD2
  • Figs. 13A-13B demonstrate that a minimal VI gene predictor built from 4 genes performs well in predicting VI and is robust to ITH.
  • Fig. 13A A VI gene predictor built from the 4 genes with the highest GLM standardized coefficients per cluster in the validation cohort.
  • Fig. 13B A VI gene predictor built from the 4 genes(l from each cluster) that had strong correlation with mean cluster expression in the validation cohort, are high ranking in the VI model, and have ISH probes readily available. Performance is shown for predicting VI in the discovery and validation cohorts, and also robustness to intra-tumor heterogeneity in the TRACERx cohort.
  • Figs. 14A-14B demonstrate that VI predictor score is associated with lymph node positivity in TRACERx stage I and stage II LU AD.
  • Fig. 14A An unsupervised heatmap of the VI predictor genes’ expression in the TRACERx stage I and stage II LU AD cohort.
  • Fig. 14B Mean VI predictor score across regions of the same tumors is significantly associated with lymph node status.
  • Figs. 15A-15B demonstrate that the VI gene cluster 2 may be important in LN metastasis.
  • Fig. 15A Genes associated with presence of positive lymph nodes in TRACERx stage II LU AD. 671 genes up with LN positivity and 755 genes down at FDR ⁇ 0.05 were derived using a negative binomial model of ⁇ 1+Lymph Node+Stage+LVI.
  • Fig. 15B When examining the enrichment of the VI gene clusters against the ranked list of these genes associated with LN positivity in TRACERx, VI gene cluster 2 was significantly enriched, despite not being enriched in genes associated with LI.
  • Fig. 16 demonstrates that VI gene cluster 2 and genes from VI gene cluster 2 are associated with myofibroblasts in scRNAseq LU AD data.
  • Figs. 17A-17D demonstrate that COL1A1, a key gene from VI gene cluster 2 that we demonstrated is significantly associated with predicted myofibroblast proportion within the stRNAseq data, shows a similar pattern within a VI+ tumor sample to THY-1 protein, a known marker of myofibroblasts/cancer associated fibroblasts.
  • FIG. 17A Expression pattern of COL1A1 in Visium stRNAseq data.
  • Fig. 17B Expression pattern of COL1A1 RNA by ISH RNAscope labeling.
  • Fig. 17C Expression pattern of COL1A1 protein by IHC.
  • Fig. 17D Expression pattern of THY-1 protein by IHC.
  • FIG. 18 demonstrates that spatial transcriptomics of early-stage LU AD reveals an association of VI gene clusters with specific LU AD histopathology features.
  • Figure depicts spotwise scoring of VI- and VI+ stRNA-seq samples for expression of VI gene expression clusters in spots annotated with high-grade LU AD patterns (solid, micropapillary, and cribriform). P values are derived form a type II Anova of a linear mixed model predicting gene expression with sample as a random effect and high-grade pattern as a fixed effect.
  • Fig. 19 demonstrates that a VI predictor derived from the signature validates in an independent stage I LU AD cohort.
  • Figure depicts the predictor association with outcome across Stage I LU AD patient cohorts (discovery (RFS), validation (RFS), TRACERx (RFS), TCGA (OS), Uppsala (OS)). P values were each calculated using univariate cox proportional hazards regression.
  • Figs. 20A-20B demonstrate that the predictor can identify patients with high risk as measured by 5-year RFS, with improvement over TNM staging.
  • the inventors have identified characteristics of lung cancer which are indicative of vascular invasion. Because these characteristics are associated with a pathological feature of the cancer and not a specific prognosis or therapeutic response, the characteristics are more robust and broadly applicable as they are not influenced by patient comorbidities or variability in local treatment centers. This permits clinicians to reliably identify the patients whose tumors are aggressive by nature of vascular invasion. These patients are in need of aggressive treatment, while patients without vascular invasion can be treated with less aggressive approaches which carry less risk of side effects, morbidity, and mortality.
  • the relevant characteristics are therefore used to identify or predict angioinvasive lung adenocarcinoma.
  • This biomarker can predict vascular invasion based on pre-surgical lung nodule biopsy and impact decision making about the extent of surgical resection, neo-adjuvant, or other ablative therapies. It can also be used on resected tumor tissue to detect occult VI or guide the use of adjuvant therapy. It is contemplated herein that the biomarker can also be applied to pre-surgical needle biopsy tissues from lung nodules, therefore being able to predict whether a tumor has vascular invasion prior to resection. Further variations can include measuring the biomarker in circulating tumor cells or from circulating tumor DNA or by staining of tumor material for biomarker components.
  • the methods described herein relate to determining tumor pathology (specifically, a novel grading approach to tumor pathology) rather than patient outcome, which distinguishes this method from existing molecular biomarkers that predict recurrence or overall survival in NSCLC, which may be influenced by patient comorbidities, treatment response and standard of care.
  • the methods described herein determine gene expression in pre-surgical biopsies. Such determinations in pre-surgical biopsies permit decision making prior to curative therapy, as opposed to existing technologies that predict aggressiveness in an adjuvant setting.
  • a method comprising, determining the expression of at least one gene selected from the group consisting of: SHR00M4; NBEAL1; MUC16; NOTCH3; H19; COL1A1; and GARS1 in a subject with lung cancer.
  • the at least one gene comprises SHR00M4. In some embodiments of any of the aspects, the at least one gene comprises NBEAL1. In some embodiments of any of the aspects, the at least one gene comprises SHR00M4 and NBEAL1. In some embodiments of any of the aspects, the at least one gene comprises SHR00M4; NBEAL1; MUC16; NOTCH3; H19; COL1A1; and GARS1. In some embodiments of any of the aspects, the at least one gene comprises SHR00M4; NBEAL1; MUC16; NOTCH3; H19; and COL1A1.
  • the at least one gene comprises SHR00M4; NBEAL1; and MUC16. In some embodiments of any of the aspects, the at least one gene comprises SHROOM4; NBEAL1; MUC16; and NOTCH3. In some embodiments of any of the aspects, the at least one gene comprises MUC16. [0041] In some embodiments of any of the aspects, the at least one gene is SHROOM4. In some embodiments of any of the aspects, the at least one gene is NBEAL1. In some embodiments of any of the aspects, the at least one gene is SHROOM4 and NBEAL1.
  • the at least one gene is SHROOM4; NBEAL1; MUC16; NOTCH3; H19; COL1A1; and GARS1. In some embodiments of any of the aspects, the at least one gene is SHR00M4; NBEAL1; MUC16; N0TCH3; H19; and COL1A1. In some embodiments of any of the aspects, the at least one gene is SHR00M4; NBEAL1; and MUC16. In some embodiments of any of the aspects, the at least one gene is SHR00M4; NBEAL1; MUC16; and N0TCH3. In some embodiments of any of the aspects, the at least one gene is MUC16.
  • a method comprising, determining the expression of: at least one gene selected from the group consisting of: MKI67; ASPM; ATAD2; TOP2A; FANCI; PLOD2; NCAPG2; CENPF; KPNA2; BRCA1; HLTF; and FANCA; at least one gene selected from the group consisting of: COL1A2; COL3A1; COL1A1; and COL6A3; at least one gene selected from the group consisting of: PRKDC; TPI1; PKM; GAPDH;
  • a method comprising, determining the expression of: at least one gene selected from the group consisting of: MKI67; ASPM; ATAD2; TOP2A; FANCI; PLOD2; NCAPG2; CENPF; KPNA2; BRCA1; HLTF; and FANCA; at least one gene selected from the group consisting of: COL1A2; COL3A1; COL1A1; and COL6A3; at least one gene selected from the group consisting of: PRKDC; TPI1; PKM; GAPDH;
  • a method comprising, determining the expression of: at least one gene selected from the group consisting of: COL1A2; COL3A1; COL1A1; and COL6A3; at least one gene selected from the group consisting of: PRKDC; TPI1; PKM; GAPDH; AHNAK2; YWHAZ; ATP13A3; GARS1; CP; MUC16; TUBA1C; H19; PPFIA1; NOTCH3; ACLY; GCLC; CAD; and CCT6A; or at least one gene selected from the group consisting of: SPTBN1; SFTPB; LRRK2; AKAP13;
  • a method comprising determining the expression of: at least one gene selected from the group consisting of: COL1A2; COL3A1; COL1A1; and COL6A3; at least one gene selected from the group consisting of: PRKDC; TPI1; PKM; GAPDH;
  • AHNAK2 AHNAK2; YWHAZ; ATP13A3; GARS1; CP; MUC16; TUBA1C; H19; PPFIA1; NOTCH3;
  • a method comprising determining the expression of: at least one gene selected from the group consisting of: MKI67; ASPM; ATAD2; TOP2A;
  • FANCI FANCI
  • PLOD2 at least one gene selected from the group consisting of: COL1A2; COL3A1; COL1A1; and COL6A3; at least one gene selected from the group consisting of: PRKDC; TPI1; PKM; GAPDH;
  • AHNAK2 AHNAK2
  • YWHAZ AHNAK2
  • a method comprising, determining the expression of: at least one gene selected from the group consisting of: MKI67; ASPM; ATAD2; and TOP2A; at least one gene selected from the group consisting of: COL1A2; COL3A1; COL1A1; and COL6A3; at least one gene selected from the group consisting of: PRKDC; TPI1; PKM; and GAPDH; or at least one gene selected from the group consisting of: SPTBN1; SFTPB; LRRK2; and AKAP13.
  • a method comprising determining the expression of: at least one gene selected from the group consisting of: MKI67; and ASPM; at least one gene selected from the group consisting of: COL1A2; and COL3A1; at least one gene selected from the group consisting of: PRKDC; and TPI1; or at least one gene selected from the group consisting of: SPTBN1; and SFTPB.
  • a method comprising determining the expression of: at least one gene selected from the group consisting of: MKI67; and ASPM; at least one gene selected from the group consisting of: COL1A2; and COL3A1; at least one gene selected from the group consisting of: PRKDC; and TPI1; or at least one gene selected from the group consisting of: SHROOM4; and NBEAL1.
  • a method comprising determining the expression of: at least one gene selected from the group consisting of: MKI67; ASPM; ATAD2; TOP2A;
  • a method comprising determining the expression of: at least one gene selected from the group consisting of: COL1A2; COL3A1; COL1A1; and COL6A3; at least one gene selected from the group consisting of: PRKDC; TPI1; PKM; GAPDH;
  • a method comprising determining the expression of: at least one gene selected from the group consisting of: COL1A2; COL3A1; COL1A1; and COL6A3; at least one gene selected from the group consisting of: PRKDC; TPI1; PKM; and GAPDH; and at least one gene selected from the group consisting of: SPTBN1; SFTPB; LRRK2; and AKAPI3.
  • a method comprising determining the expression of: at least one gene selected from the group consisting of: COL1A2; and COL3A1; at least one gene selected from the group consisting of: PRKDC; and TPI1; and at least one gene selected from the group consisting of: SPTBN1; and SFTPB.
  • a method comprising determining the expression of: at least one gene selected from the group consisting of: COL1A2; and COL3A1; at least one gene selected from the group consisting of: PRKDC; and TPI1; and at least one gene selected from the group consisting of: SHROOM4 and NBEAL1.
  • PRKDC TPI1; PKM; GAPDH; AHNAK2; and YWHAZ;
  • PRKDC TPI1; PKM; GAPDH; AHNAK2; and YWHAZ;
  • sequences for the genes described herein are known in the art, e.g., the human sequences for the genes described herein are available in the NCBI database, e.g., associated with the EntrezID provided in the tables herein, or the ENSEMBL database.
  • the NCBI database provides the sequences of homologs and orthologs for numerous species.
  • a gene, mRNA, or protein has the sequence provided in the NCBI database for that ID number as of March 24, 2025. Where reference is made to database entries, e.g., NCBI database entries, reference is made to the information and sequences available as of March 24, 2025 under the indicated ID and/or Accession Number.
  • a gene expression product has a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or greater sequence identity to a sequence provided in the NCBI database for that ID number. In some embodiments of any of the aspects, a gene expression product has a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or greater sequence identity to a sequence provided in the NCBI database forthat ID number and retaining the same activity as the reference sequence.
  • a gene expression product has a sequence with at least at least 95% sequence identity to a sequence provided in the NCBI database for that ID number and retaining the same activity as the reference sequence. In some embodiments of any of the aspects, a gene expression product has a sequence with at least 95% sequence identity to a sequence provided in the NCBI database for that ID number and retaining the same activity as the reference sequence.
  • a SHROOM4 expression product is a polynucleotide having the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, a SHROOM4 expression product is a polynucleotide having a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, a SHROOM4 expression product is a polynucleotide having a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, a SHROOM4 expression product is a polynucleotide having a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 1.
  • a SHROOM4 expression product is a polynucleotide having a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, a SHROOM4 expression product is a polynucleotide having a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 1.
  • a SHR00M4 expression product is a polypeptide having the sequence of SEQ ID NO: 2. In some embodiments of any of the aspects, a SHR00M4 expression product is a polypeptide having a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 2. In some embodiments of any of the aspects, a SHROOM4 expression product is a polypeptide having a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 2. In some embodiments of any of the aspects, a SHR00M4 expression product is a polypeptide having a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 2.
  • a SHROOM4 expression product is a polypeptide having a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 2. In some embodiments of any of the aspects, a SHROOM4 expression product is a polypeptide having a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 2.
  • a NBEAL1 expression product is a polynucleotide having the sequence of SEQ ID NO: 3 or 4. In some embodiments of any of the aspects, a NBEAL1 expression product is a polynucleotide having a sequence with at least 80% sequence identity to the sequence of SEQ ID NO:3 or 4. In some embodiments of any of the aspects, a NBEAL1 expression product is a polynucleotide having a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 3 or 4.
  • a MUC16 expression product is a polynucleotide having a sequence with at least 90% sequence identity to the sequence of one of SEQ ID NOs: 7-10. In some embodiments of any of the aspects, a MUC16 expression product is a polynucleotide having a sequence with at least 95% sequence identity to the sequence of one of SEQ ID NOs: 7-10. In some embodiments of any of the aspects, a MUC16 expression product is a polynucleotide having a sequence with at least 98% sequence identity to the sequence of one of SEQ ID NOs: 7-10.
  • a H19 expression product is a polynucleotide having the sequence of one of SEQ ID NOs: 17-23. In some embodiments of any of the aspects, a H19 expression product is a polynucleotide having a sequence with at least 80% sequence identity to the sequence of one of SEQ ID NOs: 17-23. In some embodiments of any of the aspects, a H19 expression product is a polynucleotide having a sequence with at least 85% sequence identity to the sequence of one of SEQ ID NOs: 17-23.
  • a COL1A1 expression product is a polypeptide having a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 25. In some embodiments of any of the aspects, a COL1A1 expression product is a polypeptide having a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 25.
  • a level which is less than a reference level can be a level which is less by at least about 10%, at least about 20%, at least about 50%, at least about 60%, at least about 80%, at least about 90%, or less relative to the reference level. In some embodiments of any of the aspects, a level which is less than a reference level can be a level which is statistically significantly less than the reference level.
  • the reference can be a level of the target molecule in a population of subjects who do not have or are not diagnosed as having, and/or do not exhibit signs or symptoms of lung cancer. In some embodiments of any of the aspects, the reference can be a level of the target molecule in a population of subjects who do not have or are not diagnosed as having, and/or do not exhibit signs or symptoms of lung adenocarcinoma. In some embodiments of any of the aspects, the reference can be a level of the target molecule in a population of subjects who do not have or are not diagnosed as having, and/or do not exhibit signs or symptoms of stage I lung adenocarcinoma.
  • the reference can be a level of the target molecule in a population of subjects who do not have or are not diagnosed as having, and/or do not exhibit signs or symptoms of vascular invasive lung adenocarcinoma. In some embodiments of any of the aspects, the reference can also be a level of expression of the target molecule in a control sample, a pooled sample of control individuals or a numeric value or range of values based on the same.
  • the reference can be the level of a target molecule in a sample obtained from the same subject at an earlier point in time, e.g., the methods described herein can be used to determine if a subject’s sensitivity or response to a given therapy is changing over time.
  • an increase or decrease in expression is an increase or decrease relative to an average level for subjects with lung cancer. In some embodiments of any of the aspects, an increase or decrease in expression is an increase or decrease relative to an average level for subjects with non-small cell lung cancer (NSCLC). In some embodiments of any of the aspects, an increase or decrease in expression is an increase or decrease relative to an average level for subjects with lung adenocarcinoma. In some embodiments of any of the aspects, an increase or decrease in expression is an increase or decrease relative to an average level for subjects with stage I lung adenocarcinoma.
  • NSCLC non-small cell lung cancer
  • an increase or decrease in expression is an increase or decrease relative to an average level for subjects with lung cancer which does not exhibit vascular invasion. In some embodiments of any of the aspects, an increase or decrease in expression is an increase or decrease relative to an average level for subjects with non-small cell lung cancer (NSCLC) which does not exhibit vascular invasion. In some embodiments of any of the aspects, an increase or decrease in expression is an increase or decrease relative to an average level for subjects with lung adenocarcinoma which does not exhibit vascular invasion. In some embodiments of any of the aspects, an increase or decrease in expression is an increase or decrease relative to an average level for subjects with stage I lung adenocarcinoma which does not exhibit vascular invasion.
  • NSCLC non-small cell lung cancer
  • the level of expression products of no more than 200 other genes is determined. In some embodiments of any of the aspects, the level of expression products of no more than 100 other genes is determined. In some embodiments of any of the aspects, the level of expression products of no more than 20 other genes is determined. In some embodiments of any of the aspects, the level of expression products of no more than 10 other genes is determined.
  • the expression level of a given gene can be normalized relative to the expression level of one or more reference genes or reference proteins.
  • the reference level can be the level in a sample of similar cell type, sample type, sample processing, and/or obtained from a subject of similar age, sex and other demographic parameters as the sample/subject for which the level of the at least one gene is to be determined.
  • the test sample and control reference sample are of the same type, that is, obtained from the same biological source, and comprising the same composition, e.g. the same number and type of cells.
  • sample or “test sample” as used herein denotes a sample taken or isolated from a biological organism, e.g., cells or expression product samples from a subject.
  • the present invention encompasses several examples of a biological sample.
  • the biological sample is cells, or tissue, or peripheral blood, or bodily fluid.
  • Exemplary biological samples include, but are not limited to, a biopsy, a tumor sample, biofluid sample; blood; serum; plasma; urine; sperm; mucus; tissue biopsy; organ biopsy; synovial fluid; bile fluid; cerebrospinal fluid; mucosal secretion; effusion; sweat; saliva; and/or tissue sample etc.
  • the term also includes a mixture of the above-mentioned samples.
  • test sample also includes untreated or pretreated (or pre-processed) biological samples.
  • a test sample can comprise cells from a subject.
  • the expression is determined in a sample, or is the expression in a sample comprising material selected from the group consisting of: tumor cells, cancer cells from the airway, circulating tumor cells, biopsied lung tissue, a lung tissue section, circulating tumor DNA (ctDNA), bronchial brushing sample, nasal epithelial sample, bronchial biopsy, bronchial epithelium sample, airway epithelium sample, nasal brushing sample, and bronchoalveolar lavage sample.
  • ctDNA tumor DNA
  • the expression is determined in a sample, or is the expression in a sample comprising material selected from the group consisting of: tumor cells, cancer cells from the airway, circulating tumor cells, biopsied lung tissue, a lung tissue section, and circulating tumor DNA (ctDNA). In some embodiments of any of the aspects, the expression is determined in a sample, or is the expression in a sample comprising biopsied lung tissue.
  • the sample is tumor cells, cancer cells from the airway, circulating tumor cells, biopsied lung tissue, a lung tissue section, circulating tumor DNA (ctDNA), bronchial brushing sample, nasal epithelial sample, bronchial biopsy, bronchial epithelium sample, airway epithelium sample, nasal brushing sample, or bronchoalveolar lavage sample.
  • the sample is: tumor cells, cancer cells from the airway, circulating tumor cells, biopsied lung tissue, a lung tissue section, or circulating tumor DNA (ctDNA).
  • the sample is biopsied lung tissue.
  • the test sample can be obtained by removing a sample from a subject but can also be accomplished by using a previously isolated sample (e.g. isolated at a prior timepoint and isolated by the same or another person).
  • the test sample can be an untreated test sample.
  • untreated test sample refers to a test sample that has not had any prior sample pre-treatment except for dilution and/or suspension in a solution.
  • Exemplary methods for treating a test sample include, but are not limited to, centrifugation, fdtration, sonication, homogenization, heating, freezing and thawing, and combinations thereof.
  • the test sample can be a frozen test sample, e.g., a frozen tissue. The frozen sample can be thawed before employing methods, assays and systems described herein.
  • a frozen sample can be centrifuged before being subjected to methods, assays and systems described herein.
  • the test sample is a clarified test sample, for example, by centrifugation and collection of a supernatant comprising the clarified test sample.
  • a test sample can be a pre-processed test sample, for example, supernatant or filtrate resulting from a treatment selected from the group consisting of centrifugation, filtration, thawing, purification, and any combinations thereof.
  • the test sample can be treated with a chemical and/or biological reagent.
  • Chemical and/or biological reagents can be employed to protect and/or maintain the stability of the sample, including biomolecules (e.g., nucleic acid and protein) therein, during processing.
  • One exemplary reagent is a protease inhibitor, which is generally used to protect or maintain the stability of protein during processing.
  • the skilled artisan is well aware of methods and processes appropriate for pre-processing of biological samples required for determination of the level of an expression product as described herein.
  • the methods, assays, and systems described herein can further comprise a step of obtaining or having obtained a test sample from a subject.
  • the subject can be a human subject.
  • the subject can be a subject in need of treatment for (e.g. having or diagnosed as having) lung cancer or a subject at risk of or at increased risk of developing lung cancer as described elsewhere herein.
  • the sample obtained from a subject can be a biopsy sample. In some embodiments of any of the aspects, the sample obtained from a subject can be a blood or serum sample.
  • the lung cancer is non-small cell lung cancer (NSCLC). In some embodiments of any of the aspects, the lung cancer is stage I and/or stage II non- small cell lung cancer (NSCLC). In some embodiments of any of the aspects, the lung cancer is stage I non-small cell lung cancer (NSCLC). In some embodiments of any of the aspects, the lung cancer is stage II non-small cell lung cancer (NSCLC).
  • the lung cancer is stage II lung adenocarcinoma.
  • One of skill in the art is aware of lung cancer staging and how to perform it.
  • An exemplary staging system can be found in Asamura, H. (Ed). (2016) The IASLC Staging Handbook in Thoracic Oncology (2nd Edition) includes 8th Edition Tumor, Node, and Metastasis Classification (TNM) information for Lung Cancer, Plural Mesothelioma, Thymic Malignancies, and Carcinoma of the Oesophagus and of Oesophagogastric Junction. International Association for the Study of Lung Cancer; which is incorporated by reference herein in its entirety.
  • the lung cancer comprises vascular invasion.
  • the subject is administered adjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, and ablation therapy.
  • the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy.
  • the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, ablation therapy, radiation therapy, and a lobectomy.
  • the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is ablation therapy, radiation therapy, and lymphadenectomy.
  • the subject is administered ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, and radiation therapy.
  • the subject is administered adjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered, neoadjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered ablation therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered radiation therapy and a lobectomy. In some embodiments of any of the aspects, the subject is administered radiation therapy and lymphadenectomy.
  • the subject is administered a lobectomy and ablation therapy. In some embodiments of any of the aspects, the subject is administered lymphadenectomy and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lobectomy, and ablation therapy.
  • the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy.
  • the subject is administered neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy.
  • the subject is administered adjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy.
  • the subject is administered ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy.
  • the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, and radiation therapy.
  • the subject is administered adjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered, neoadjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered ablation therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered radiation therapy and a lobectomy. In some embodiments of any of the aspects, the subject is administered radiation therapy and lymphadenectomy.
  • the subject is administered a lobectomy and ablation therapy. In some embodiments of any of the aspects, the subject is administered lymphadenectomy and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lobectomy, and ablation therapy.
  • the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy.
  • the subject is administered neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy.
  • the subject is administered adjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy.
  • the subject is administered ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy.
  • the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, and radiation therapy.
  • the subject is radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and lymphadenectomy.
  • the subject is administered adjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered, neoadjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered ablation therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered radiation therapy and a lobectomy. In some embodiments of any of the aspects, the subject is administered radiation therapy and lymphadenectomy.
  • a method of treating lung cancer comprising administering adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and/or lymphadenectomy to a subject having decreased expression of at least one gene selected from the group consisting of: SHROOM4 and NBEAL1, and increased expression of at least one gene selected from the group consisting of: MUC16, NOTCH3, H19, and COL1A1.
  • the subject is administered adjuvant therapy and ablation therapy.
  • the subject is administered neoadjuvant therapy and ablation therapy.
  • the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy.
  • the subject is administered neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy.
  • the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, and radiation therapy.
  • the subject is radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and lymphadenectomy.
  • the subject is administered adjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered, neoadjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered ablation therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered radiation therapy and a lobectomy. In some embodiments of any of the aspects, the subject is administered radiation therapy and lymphadenectomy.
  • a method of treating lung cancer comprising administering adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and/or lymphadenectomy to a subject having increased expression of at least one gene selected from the group consisting of: MUC16, NOTCH3, H19, and COL1A1.
  • the subject is administered adjuvant therapy and ablation therapy.
  • the subject is administered neoadjuvant therapy and ablation therapy.
  • the subject is administered a lobectomy and ablation therapy.
  • the subject is administered lymphadenectomy and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy.
  • the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy.
  • the subject is administered adjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy.
  • the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy.
  • the subject is administered neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy.
  • the subject is ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and a lobectomy.
  • a method of treating lung cancer comprising administering adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and/or lymphadenectomy to a subject having increased expression of at least one gene selected from the group consisting of: MUC16, NOTCH3, H19, COL1A1, and GARS1.
  • the subject is administered adjuvant therapy and ablation therapy.
  • the subject is administered neoadjuvant therapy and ablation therapy.
  • the subject is administered a lobectomy and ablation therapy.
  • the subject is administered adjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy.
  • the subject is administered neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy.
  • the subject is administered adjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is radiation therapy, a lobectomy, and lymphadenectomy.
  • the subject is ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and a lobectomy.
  • the subject is administered adjuvant therapy, neoadjuvant therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered, neoadjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered ablation therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered radiation therapy and a lobectomy. In some embodiments of any of the aspects, the subject is administered radiation therapy and lymphadenectomy.
  • a method of treating lung cancer comprising administering adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and/or lymphadenectomy to a subject having increased expression of MUC16.
  • the subject is administered adjuvant therapy and ablation therapy.
  • the subject is administered neoadjuvant therapy and ablation therapy.
  • the subject is administered a lobectomy and ablation therapy.
  • the subject is administered lymphadenectomy and ablation therapy.
  • the subject is administered adjuvant therapy, neoadjuvant therapy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy.
  • the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy.
  • the subject is administered neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy.
  • the subject is administered adjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is radiation therapy, a lobectomy, and lymphadenectomy.
  • the subject is ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and a lobectomy.
  • the subject is administered adjuvant therapy, neoadjuvant therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered, neoadjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered ablation therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered radiation therapy and a lobectomy. In some embodiments of any of the aspects, the subject is administered radiation therapy and lymphadenectomy.
  • a method of treating lung cancer comprising administering adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and/or lymphadenectomy to a subject having decreased expression of at least one gene selected from the group consisting of: SHROOM4 and NBEAL1.
  • the subject is administered adjuvant therapy and ablation therapy.
  • the subject is administered neoadjuvant therapy and ablation therapy.
  • the subject is administered a lobectomy and ablation therapy.
  • the subject is administered lymphadenectomy and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy.
  • the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy.
  • the subject is administered adjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy.
  • the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy.
  • the subject is administered neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy.
  • the subject is administered adjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is radiation therapy, a lobectomy, and lymphadenectomy.
  • a method of treating lung cancer comprising administering adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and/or lymphadenectomy to a subject having decreased expression of SHROOM4 and NBEAL1.
  • the subject is administered adjuvant therapy and ablation therapy.
  • the subject is administered neoadjuvant therapy and ablation therapy.
  • the subject is administered a lobectomy and ablation therapy.
  • the subject is administered lymphadenectomy and ablation therapy.
  • the subject is administered adjuvant therapy, neoadjuvant therapy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy.
  • the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy.
  • the subject is administered adjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy.
  • the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy.
  • the subject is administered neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy.
  • the subject is administered adjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is radiation therapy, a lobectomy, and lymphadenectomy.
  • the subject is ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and a lobectomy.
  • the subject is administered adjuvant therapy, neoadjuvant therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered, neoadjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered ablation therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered radiation therapy and a lobectomy. In some embodiments of any of the aspects, the subject is administered radiation therapy and lymphadenectomy.
  • a method of treating lung cancer comprising administering adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and/or lymphadenectomy to a subject having decreased expression of SHROOM4.
  • the subject is administered adjuvant therapy and ablation therapy.
  • the subject is administered neoadjuvant therapy and ablation therapy.
  • the subject is administered a lobectomy and ablation therapy.
  • the subject is administered lymphadenectomy and ablation therapy.
  • the subject is administered adjuvant therapy, neoadjuvant therapy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy.
  • the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy.
  • the subject is administered adjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy.
  • the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy.
  • the subject is administered adjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is radiation therapy, a lobectomy, and lymphadenectomy.
  • the subject is ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and a lobectomy.
  • the subject is administered adjuvant therapy, neoadjuvant therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered, neoadjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered ablation therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered radiation therapy and a lobectomy. In some embodiments of any of the aspects, the subject is administered radiation therapy and lymphadenectomy.
  • a method of treating lung cancer comprising administering adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and/or lymphadenectomy to a subject having decreased expression of NBEAL1.
  • the subject is administered adjuvant therapy and ablation therapy.
  • the subject is administered neoadjuvant therapy and ablation therapy.
  • the subject is administered a lobectomy and ablation therapy.
  • the subject is administered lymphadenectomy and ablation therapy.
  • the subject is administered adjuvant therapy, neoadjuvant therapy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy.
  • the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy.
  • the subject is administered adjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy.
  • the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy.
  • the subject is administered neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy.
  • the subject is ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and a lobectomy.
  • the subject is administered adjuvant therapy, neoadjuvant therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered, neoadjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered ablation therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered radiation therapy and a lobectomy. In some embodiments of any of the aspects, the subject is administered radiation therapy and lymphadenectomy.
  • General chemotherapeutic agents include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5- deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin (Actinomycin
  • immunotherapy refers to any chemical or biological agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth by promoting, preserving, or increasing the activity of immune cells.
  • Immunotherapies include immune checkpoint inhibitors, T-cell transfer therapy (e.g., CAR-T therapies), antibody therapies, treatment vaccines, and immune system modulators.
  • Non-limiting examples of immune checkpoint inhibitors can include :MGA271 (B7-H3: MacroGenics); ipilimumab (CTLA- 4; Bristol Meyers Squibb); pembrolizumab (PD-1; Merck); nivolumab (PD-1; Bristol Meyers Squibb) ; atezolizumab (PD-L1; Genentech); galiximab (B7.1; Biogen); IMP321 (LAG3: Immuntep); BMS- 986016 (LAG3; Bristol Meyers Squibb); SMB-663513 (CD137; Bristol-Meyers Squibb); PF-05082566 (CD137; Pfizer); IPH2101 (KIR; Innate Pharma); KW-0761 (CCR4; Kyowa Kirin); CDX-1127 (CD27; CellDex); MEDI-6769 (0x40; Medl
  • lymphadenectomy refers to the dissection and removal of at least one lymph node.
  • a regional or selective lymphadenectomy removes a sample of the lymph node which is closest to the tumor.
  • a radical, complete, or total lymphadenectomy removes all lymph nodes at a given location.
  • the lymphadenectomy is a regional lymphadenectomy.
  • the lymphadenectomy is a radical lymphadenectomy .
  • the method further comprises administering active surveillance, wedge resection of the lung, or a segmentectomy of the lung to a subject not having decreased expression of at least one gene selected from the group consisting of: SHR00M4 and NBEAL1; and/or not having increased expression of at least one gene selected from the group consisting of: MUC16, NOTCH3, H19, COL1A1, and GARS1.
  • the subject is administered active surveillance.
  • the subject is administered a wedge resection of the lung.
  • the subject is administered a segmentectomy of the lung.
  • the method further comprises administering active surveillance, wedge resection of the lung, or a segmentectomy of the lung to a subject not having decreased expression of at least one gene selected from the group consisting of: SHR00M4 and NBEAL1; and not having increased expression of at least one gene selected from the group consisting of: MUC16, NOTCH3, H19, COL1A1, and GARS1.
  • the subject is administered active surveillance.
  • the subject is administered a wedge resection of the lung.
  • the subject is administered a segmentectomy of the lung.
  • the subject is administered a wedge resection of the lung and is not administered an adjuvant therapy, a neoadjuvant therapy, an ablation therapy, radiation therapy, a lobectomy, and/or a lymphadenectomy.
  • the subject is administered a segmentectomy of the lung and is not administered an adjuvant therapy, a neoadjuvant therapy, an ablation therapy, radiation therapy, a lobectomy, and/or a lymphadenectomy.
  • the method further comprises administering active surveillance, wedge resection of the lung, or a segmentectomy of the lung to a subject not having increased expression of at least one gene selected from the group consisting of: MUC16, NOTCH3, H19, COL1A1, and GARS1.
  • the subject is administered active surveillance.
  • the subject is administered a wedge resection of the lung.
  • the subject is administered a segmentectomy of the lung.
  • the subject is administered a wedge resection of the lung and is not administered an adjuvant or neoadjuvant.
  • the subject is administered a segmentectomy of the lung and is not administered an adjuvant or neoadjuvant. In some embodiments of any of the aspects, the subject is administered a wedge resection of the lung and is not administered an adjuvant therapy, a neoadjuvant therapy, an ablation therapy, radiation therapy, a lobectomy, and/or a lymphadenectomy. In some embodiments of any of the aspects, the subject is administered a segmentectomy of the lung and is not administered an adjuvant therapy, a neoadjuvant therapy, an ablation therapy, radiation therapy, a lobectomy, and/or a lymphadenectomy.
  • the method further comprises administering active surveillance, wedge resection of the lung, or a segmentectomy of the lung to a subject not having decreased expression of SHR00M4 and NBEAL1.
  • the subject is administered active surveillance.
  • the subject is administered a wedge resection of the lung.
  • the subject is administered a segmentectomy of the lung.
  • the subject is administered a wedge resection of the lung and is not administered an adjuvant or neoadjuvant.
  • the subject is administered a segmentectomy of the lung and is not administered an adjuvant or neoadjuvant. In some embodiments of any of the aspects, the subject is administered a wedge resection of the lung and is not administered an adjuvant therapy, a neoadjuvant therapy, an ablation therapy, radiation therapy, a lobectomy, and/or a lymphadenectomy. In some embodiments of any of the aspects, the subject is administered a segmentectomy of the lung and is not administered an adjuvant therapy, a neoadjuvant therapy, an ablation therapy, radiation therapy, a lobectomy, and/or a lymphadenectomy.
  • the method further comprises administering active surveillance, wedge resection of the lung, or a segmentectomy of the lung to a subject not having decreased expression of SHR00M4.
  • the subject is administered active surveillance.
  • the subject is administered a wedge resection of the lung.
  • the subject is administered a segmentectomy of the lung.
  • the subject is administered a wedge resection of the lung and is not administered an adjuvant or neoadjuvant.
  • active surveillance refers to repeated exams, scans, and/or screens without administration of a drug or surgical intervention.
  • active surveillance comprises administration of a CT scan at least twice at intervals of 1-3 months.
  • active surveillance comprises administration of a CT scan at least twice at intervals of 1-3 months, during which time the subject is not administered a drug or surgical intervention.
  • active surveillance comprises administration of a CT scan at least twice at intervals of 1-18 months.
  • active surveillance comprises administration of a CT scan at least twice at intervals of 1-18 months, during which time the subject is not administered a drug or surgical intervention.
  • active surveillance comprises administration of a CT scan at least twice at intervals of 3-6 months.
  • active surveillance comprises administration of a CT scan at least twice at intervals of 3-6 months, during which time the subject is not administered a drug or surgical intervention. In some embodiments, active surveillance comprises administration of a CT scan at least twice at intervals of 1-12 months. In some embodiments, active surveillance comprises administration of a CT scan at least twice at intervals of 1-12 months, during which time the subject is not administered a drug or surgical intervention. In some embodiments, active surveillance comprises administration of a CT scan at least twice at intervals of 3-12 months. In some embodiments, active surveillance comprises administration of a CT scan at least twice at intervals of 3-12 months, during which time the subject is not administered a drug or surgical intervention.
  • active surveillance comprises administration of a CT scan at least twice at intervals of 3-18 months. In some embodiments, active surveillance comprises administration of a CT scan at least twice at intervals of 3-18 months, during which time the subject is not administered a drug or surgical intervention. In some embodiments, active surveillance comprises administration of a CT scan at least twice at intervals of 6-18 months. In some embodiments, active surveillance comprises administration of a CT scan at least twice at intervals of 6- 18 months, during which time the subject is not administered a drug or surgical intervention.
  • edge resection of the lung refers to removing a small section of the lung.
  • the method comprises a first step of determining the level of at least one gene as described above herein in a sample obtained from a subject. In some embodiments of any of the aspects, the method comprises administering the treatment to a subject previously determined to have the indicated level of the at least one gene.
  • the step of determining the expression level comprises i) obtaining or having obtained a sample from the subject and ii) performing or having performed an assay on the sample obtained from the subject to determine/measure the expression level in the subject. In some embodiments of any of the aspects, the step of determining the expression level comprises performing or having performed an assay on a sample obtained from the subject to determine/measure the expression level in the subject. In some embodiments of any of the aspects, the step of determining the expression level comprises comprise ordering or requesting an assay on a sample obtained from the subject to determine/measure the expression level in the subject.
  • the step of determining the expression level can comprise receiving the results of an assay on a sample obtained from the subject to determine/measure the expression level in the subject. In some embodiments of any of the aspects, the step of determining if the expression level can comprise receiving a report, results, or other means of identifying the subject as a subject with certain expression level(s).
  • a method of treating lung cancer in a subject in need thereof comprising: a) determining the expression level of one or more genes as described herein; and b) instructing or directing that the subject be administered the treatment described herein.
  • the step of instructing or directing that the subject be administered a particular treatment can comprise providing a report of the assay results.
  • the step of instructing or directing that the subject be administered a particular treatment can comprise providing a report of the assay results and/or treatment recommendations in view of the assay results.
  • described herein is a method of treating or prognosing lung cancer, the method comprising determining the level of expression: a) at least one gene selected from the group consisting of:
  • ANKHD1; NBEAL1; MPRIP; SHROOM4; and TET2 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
  • PRKDC TPI1; PKM; GAPDH; AHNAK2; YWHAZ; ATP 13 A3; GARS1; CP;
  • ANKHD1; NBEAL1; MPRIP; SHR00M4; and TET2 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
  • MUC16 MUC16; NOTCH3; H19; COL1A1; and GARS1; and/or c. decreased expression of at least one gene selected from the group consisting of:
  • NBEAL1 and SHROOM4 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
  • MUC16; NOTCH3; H19; COL1A1; NBEAL1; and SHROOM4 wherein: b. increased expression of at least one gene selected from the group consisting of: MUC16; NOTCH3; H19; and COL1A1; and/or c. decreased expression of at least one gene selected from the group consisting of: NBEAL1 and SHROOM4 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
  • MUC16; NOTCH3; H19; and COL1A1 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
  • a method of treating or prognosing lung cancer comprising determining the level of expression of MUC16, wherein increased expression of MUC16 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
  • a method of treating or prognosing lung cancer comprising determining the level of expression of MUC16, wherein increased expression of MUC16 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
  • NBEAL1 and SHR00M4 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
  • a method of treating or prognosing lung cancer comprising determining the level of expression of MUC16, NBEAL1, and SHR00M4, wherein: a. increased expression MUC 16; and b. decreased expression of at least one gene selected from the group consisting of: NBEAL1 and SHR00M4 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
  • a method of treating or prognosing lung cancer comprising determining the level of expression of NBEAL1 and SHR00M4 wherein decreased expression of at least one gene selected from the group consisting of: NBEAL1 and SHR00M4 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
  • a method of treating or prognosing lung cancer comprising determining the level of expression of NBEAL1 and SHR00M4, wherein decreased expression of at least one gene selected from the group consisting of: NBEAL1 and SHR00M4 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
  • a method of treating or prognosing lung cancer comprising determining the level of expression of NBEAL1 wherein decreased expression of NBEAL1 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
  • a method of treating or prognosing lung cancer comprising determining the level of expression of NBEAL1, wherein decreased expression of NBEAL1 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
  • a method of treating or prognosing lung cancer comprising determining the level of expression of SHR00M4 wherein decreased expression of SHR00M4 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
  • a method of treating or prognosing lung cancer comprising determining the level of expression of SHR00M4, wherein decreased expression of SHR00M4 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
  • PRKDC at least one gene selected from the group consisting of:
  • PRKDC TPI1; PKM; GAPDH; AHNAK2; and YWHAZ; and/or h) decreased expression of at least one gene selected from the group consisting of:
  • PRKDC PRKDC
  • TPI1 PKM
  • GAPDH GAPDH
  • AHNAK2 GAPDH
  • YWHAZ YWHAZ
  • PRKDC TPI1; PKM; GAPDH; AHNAK2; and YWHAZ; and/or h) decreased expression of at least one gene selected from the group consisting of:
  • PRKDC PRKDC; TPI1; PKM; and GAPDH; or d) at least one gene selected from the group consisting of: SPTBN1; SFTPB; LRRK2; and AKAP13; wherein: e) increased expression of at least one gene selected from the group consisting of:
  • PRKDC PRKDC
  • TPI1 PKM
  • GAPDH GAPDH
  • h decreased expression of at least one gene selected from the group consisting of:
  • SPTBN1; SFTPB; LRRK2; and AKAP13 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
  • PRKDC PRKDC
  • TPI1 PKM
  • GAPDH GAPDH
  • PRKDC PRKDC
  • TPI1 PKM
  • GAPDH GAPDH
  • h decreased expression of at least one gene selected from the group consisting of:
  • SPTBN1; SFTPB; LRRK2; and AKAP13 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
  • PRKDC PRKDC
  • TPIl TPIl
  • COL1A2 COL3A1; g) increased expression of at least one gene selected from the group consisting of:
  • PRKDC PRKDC
  • TPI1 TPI1
  • h decreased expression of at least one gene selected from the group consisting of:
  • SPTBN1 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
  • PRKDC PRKDC
  • TPI1 at least one gene selected from the group consisting of:
  • COL1A2 COL3A1; g) increased expression of at least one gene selected from the group consisting of:
  • PRKDC PRKDC
  • TPI1 TPI1
  • h decreased expression of at least one gene selected from the group consisting of:
  • SPTBN1 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
  • a method of treating or prognosing lung cancer comprising determining the level of expression of: a) MKI67; and ASPM; b) COL1A2; and COL3A1; c) PRKDC; and TPI1; and d) SPTBN1; and SFTPB; wherein: e) increased expression of MKI67; and ASPM; f) increased expression of COL1A2; and COL3A1; g) increased expression of PRKDC; and TPI 1 ; and/or h) decreased expression of SPTBN 1 ; and SFTPB; indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
  • BRCA1; HLTF; and FANCA at least one gene selected from the group consisting of:
  • PRKDC TPI1; PKM; GAPDH; AHNAK2; YWHAZ; ATP 13 A3; GARS1; CP;
  • ANKHD1; NBEAL1; MPRIP; SHR00M4; and TET2 indicates increased likelihood of vascular invasion in a subject with lung cancer.
  • NBEAL 1 and SHR00M4 indicates increased likelihood of vascular invasion in a subject with lung cancer.
  • NBEAL1 and SHR00M4 indicates increased likelihood of vascular invasion in a subject with lung cancer.
  • NBEAL 1 and SHR00M4 indicates increased likelihood of vascular invasion in a subject with lung cancer.
  • a method of determining the likelihood of vascular invasion in a subject with lung cancer comprising determining the level of expression of NBEAL wherein decreased expression ofNBEALl indicates increased likelihood of vascular invasion in a subject with lung cancer.
  • a method of determining the likelihood of vascular invasion in a subject with lung cancer comprising determining the level of expression of SHR00M4 wherein decreased expression of SHR00M4 indicates increased likelihood of vascular invasion in a subject with lung cancer.
  • a method of determining the likelihood of vascular invasion in a subject with lung cancer comprising determining the level of expression: a) at least one gene selected from the group consisting of:
  • PRKDC at least one gene selected from the group consisting of:
  • PRKDC TPI1; PKM; GAPDH; AHNAK2; and YWHAZ; and/or h) decreased expression of at least one gene selected from the group consisting of:
  • PRKDC PRKDC
  • TPI1 PKM
  • GAPDH GAPDH
  • AHNAK2 GAPDH
  • YWHAZ YWHAZ
  • PRKDC TPI1; PKM; GAPDH; AHNAK2; and YWHAZ; and/or h) decreased expression of at least one gene selected from the group consisting of:
  • PRKDC PRKDC
  • TPI1 PKM
  • GAPDH GAPDH
  • PRKDC PRKDC
  • TPI1 PKM
  • GAPDH GAPDH
  • h decreased expression of at least one gene selected from the group consisting of:
  • SPTBN1; SFTPB; LRRK2; and AKAP13 indicates increased likelihood of vascular invasion.
  • PRKDC PRKDC
  • TPI1 PKM
  • GAPDH GAPDH
  • PRKDC PRKDC
  • TPI1 PKM
  • GAPDH GAPDH
  • h decreased expression of at least one gene selected from the group consisting of:
  • SPTBN1; SFTPB; LRRK2; and AKAP13 indicates increased likelihood of vascular invasion.
  • PRKDC PRKDC
  • TPI1 TPI1
  • COL1A2 COL3A1; g) increased expression of at least one gene selected from the group consisting of:
  • PRKDC PRKDC
  • TPI1 TPI1
  • h decreased expression of at least one gene selected from the group consisting of:
  • SPTBN1; and SFTPB indicates increased likelihood of vascular invasion.
  • PRKDC PRKDC
  • TPI1 at least one gene selected from the group consisting of:
  • COL1A2 COL3A1; g) increased expression of at least one gene selected from the group consisting of:
  • PRKDC PRKDC
  • TPI1 TPI1
  • h decreased expression of at least one gene selected from the group consisting of:
  • SPTBN1; and SFTPB indicates increased likelihood of vascular invasion.
  • a method of determining the likelihood of vascular invasion in a subject with lung cancer comprising determining the level of expression of: a) MKI67; and ASPM; b) COL1A2; and COL3A1; c) PRKDC; and TPI1; and d) SPTBN1; and SFTPB; wherein: e) increased expression of MKI67; and ASPM; f) increased expression of COL1A2; and COL3A1; g) increased expression of PRKDC; and TPI 1 ; and/or h) decreased expression of SPTBN 1 ; and SFTPB; indicates increased likelihood of vascular invasion.
  • measurement of the level of a target and/or detection of the level or presence of a target can comprise a transformation.
  • transforming or “transformation” refers to changing an object or a substance, e.g., biological sample, nucleic acid or protein, into another substance.
  • the transformation can be physical, biological or chemical.
  • Exemplary physical transformation includes, but is not limited to, pre-treatment of a biological sample, e.g., from whole blood to blood serum by differential centrifugation.
  • a biological/chemical transformation can involve the action of at least one enzyme and/or a chemical reagent in a reaction.
  • a DNA sample can be digested into fragments by one or more restriction enzymes, or an exogenous molecule can be attached to a fragmented DNA sample with a ligase.
  • a DNA sample can undergo enzymatic replication, e.g., by polymerase chain reaction (PCR).
  • Transformation, measurement, and/or detection of a target molecule can comprise contacting a sample obtained from a subject with a reagent (e.g. a detection reagent) which is specific for the target, e.g., a target-specific reagent.
  • a reagent e.g. a detection reagent
  • the target-specific reagent is detectably labeled.
  • the target-specific reagent is capable of generating a detectable signal.
  • the target-specific reagent generates a detectable signal when the target molecule is present.
  • the PCR procedure describes a method of gene amplification which is comprised of (i) sequence-specific hybridization of primers to specific genes or sequences within a nucleic acid sample or library, (ii) subsequent amplification involving multiple rounds of annealing, elongation, and denaturation using a thermostable DNA polymerase, and (iii) screening the PCR products for a band of the correct size.
  • the primers used are oligonucleotides of sufficient length and appropriate sequence to provide initiation of polymerization, i.e. each primer is specifically designed to be complementary to a strand of the genomic locus to be amplified.
  • the level of an mRNA can be measured by a quantitative sequencing technology, e.g. a quantitative next-generation sequence technology.
  • Methods of sequencing a nucleic acid sequence are well known in the art. Briefly, a sample obtained from a subject can be contacted with one or more primers which specifically hybridize to a single-strand nucleic acid sequence flanking the target gene sequence and a complementary strand is synthesized.
  • an adaptor double or single-stranded
  • the sequence can be determined, e.g.
  • exemplary methods of sequencing include, but are not limited to, Sanger sequencing, dideoxy chain termination, high-throughput sequencing, next generation sequencing, 454 sequencing, SOLiD sequencing, polony sequencing, Illumina sequencing, Ion Torrent sequencing, sequencing by hybridization, nanopore sequencing, Helioscope sequencing, single molecule real time sequencing, RNAP sequencing, long read sequencing, and the like. Methods and protocols for performing these sequencing methods are known in the art, see, e.g. “Next Generation Genome Sequencing” Ed.
  • nucleic acid sequences of the genes described herein have been assigned NCBI accession numbers for different species such as human, mouse and rat. Accordingly, a skilled artisan can design an appropriate primer based on the known sequence for determining the mRNA level of the respective gene.
  • Nucleic acid and ribonucleic acid (RNA) molecules can be isolated from a particular biological sample using any of a number of procedures, which are well-known in the art, the particular isolation procedure chosen being appropriate for the particular biological sample.
  • freeze-thaw and alkaline lysis procedures can be useful for obtaining nucleic acid molecules from solid materials
  • heat and alkaline lysis procedures can be useful for obtaining nucleic acid molecules from urine
  • proteinase K extraction can be used to obtain nucleic acid from blood (Roiff, A et al. PCR: Clinical Diagnostics and Research, Springer (1994)).
  • Methods to measure gene expression products are known to a skilled artisan. Such methods to measure gene expression products, e.g., protein level, include ELISA (enzyme linked immunosorbent assay), western blot, immunoprecipitation, and immunofluorescence using detection reagents such as an antibody or protein binding agents.
  • a peptide can be detected in a subject by introducing into a subject a labeled anti -peptide antibody and other types of detection agent.
  • the antibody can be labeled with a detectable marker whose presence and location in the subject is detected by standard imaging techniques.
  • antibodies for the various targets described herein are commercially available and can be used for the purposes of the invention to measure protein expression levels.
  • amino acid sequences for the targets described herein are known and publicly available at the NCBI website, one of skill in the art can raise their own antibodies against these polypeptides of interest for the purpose of the methods described herein.
  • the amino acid sequences of the polypeptides described herein have been assigned NCBI accession numbers for different species such as human, mouse and rat.
  • immunohistochemistry (“IHC”) and immunocytochemistry (“ICC”) techniques can be used.
  • IHC is the application of immunochemistry to tissue sections
  • ICC is the application of immunochemistry to cells or tissue imprints after they have undergone specific cytological preparations such as, for example, liquid-based preparations.
  • Immunochemistry is a family of techniques based on the use of an antibody, wherein the antibodies are used to specifically target molecules inside or on the surface of cells.
  • the antibody typically contains a marker that will undergo a biochemical reaction, and thereby experience a change of color, upon encountering the targeted molecules.
  • signal amplification can be integrated into the particular protocol, wherein a secondary antibody, that includes the marker stain or marker signal, follows the application of a primary specific antibody.
  • the assay can be a Western blot analysis.
  • proteins can be separated by two-dimensional gel electrophoresis systems. Two- dimensional gel electrophoresis is well known in the art and typically involves iso-electric focusing along a first dimension followed by SDS-PAGE electrophoresis along a second dimension. These methods also require a considerable amount of cellular material.
  • the analysis of 2D SDS-PAGE gels can be performed by determining the intensity of protein spots on the gel or can be performed using immune detection.
  • protein samples are analyzed by mass spectroscopy.
  • Immunological tests can be used with the methods and assays described herein and include, for example, competitive and non-competitive assay systems using techniques such as Western blots, radioimmunoassay (RIA), ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, e.g. latex agglutination, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, e.g.
  • FIA fluorescence -linked immunoassay
  • CLIA chemiluminescence immunoassays
  • ELIA electrochemiluminescence immunoassay
  • CIA counting immunoassay
  • LFIA lateral flow tests or immunoassay
  • MIA magnetic immunoassay
  • protein A immunoassays Methods for performing such assays are known in the art, provided an appropriate antibody reagent is available.
  • the immunoassay can be a quantitative or a semi-quantitative immunoassay.
  • Enzyme-linked immunosorbent assay also called ELISA, enzyme immunoassay or EIA
  • ELISA enzyme immunoassay
  • EIA enzyme immunoassay
  • an ELISA involving at least one antibody with specificity for the particular desired antigen can also be performed.
  • a known amount of sample and/or antigen is immobilized on a solid support (usually a polystyrene micro titer plate). Immobilization can be either non-specific (e.g., by adsorption to the surface) or specific (e.g. where another antibody immobilized on the surface is used to capture antigen or a primary antibody). After the antigen is immobilized, the detection antibody is added, forming a complex with the antigen.
  • a competitive ELISA is used.
  • Purified antibodies that are directed against a target polypeptide or fragment thereof are coated on the solid phase of multi-well plate, i.e., conjugated to a solid surface.
  • a second batch of purified antibodies that are not conjugated on any solid support is also needed.
  • These non-conjugated purified antibodies are labeled for detection purposes, for example, labeled with horseradish peroxidase to produce a detectable signal.
  • TMB (3, 3', 5, 5'-tetramethylbenzidene) color development substrate for localization of horseradish peroxidase-conjugated antibodies in the wells.
  • TMB 3, 3', 5, 5'-tetramethylbenzidene
  • TMB 3, 3', 5, 5'-tetramethylbenzidene
  • ELISA ELISA-Linked Immunodiagnosis
  • 2nd Edition Rose and Bigazzi, eds. John Wiley & Sons, 1980
  • Oellerich M. 1984, J. Clin. Chem. Clin. Biochem. 22:895-904.
  • LFIA lateral flow immunoassay test
  • LFIAs are a simple device intended to detect the presence (or absence) of antigen, e.g. a polypeptide, in a fluid sample.
  • antigen e.g. a polypeptide
  • LFIA tests are a form of immunoassay in which the test sample flows along a solid substrate via capillary action. After the sample is applied to the test strip it encounters a colored reagent (generally comprising antibody specific for the test target antigen) bound to microparticles which mixes with the sample and transits the substrate encountering lines or zones which have been pretreated with another antibody or antigen.
  • LFIAs are essentially immunoassays adapted to operate along a single axis to suit the test strip format or a dipstick format. Strip tests are extremely versatile and can be easily modified by one skilled in the art for detecting an enormous range of antigens from fluid samples such as urine, blood, water, and/or homogenized tissue samples etc. Strip tests are also known as dip stick tests, the name bearing from the literal action of "dipping" the test strip into a fluid sample to be tested. LFIA strip tests are easy to use, require minimum training and can easily be included as components of point-of-care test (POCT) diagnostics to be use on site in the field.
  • POCT point-of-care test
  • LFIA tests can be operated as either competitive or sandwich assays.
  • Sandwich LFIAs are similar to sandwich ELISA.
  • the sample first encounters colored particles which are labeled with antibodies raised to the target antigen.
  • the test line will also contain antibodies to the same target, although it may bind to a different epitope on the antigen.
  • the test line will show as a colored band in positive samples.
  • the lateral flow immunoassay can be a double antibody sandwich assay, a competitive assay, a quantitative assay or variations thereof.
  • Competitive LFIAs are similar to competitive ELISA.
  • the sample first encounters colored particles which are labeled with the target antigen or an analogue.
  • the test line contains antibodies to the target/its analogue.
  • Unlabeled antigen in the sample will block the binding sites on the antibodies preventing uptake of the colored particles.
  • the test line will show as a colored band in negative samples.
  • lateral flow technology It is also possible to apply multiple capture zones to create a multiplex test.
  • Detectably labeled enzyme-linked secondary or detection antibodies can then be used to detect and assess the amount of polypeptide in the sample tested.
  • a dot blot immobilizes a protein sample on a defined region of a support, which is then probed with antibody and labelled secondary antibody as in Western blotting.
  • the intensity of the signal from the detectable label in either format corresponds to the amount of enzyme present, and therefore the amount of polypeptide.
  • Levels can be quantified, for example by densitometry.
  • the level of a target can be measured, by way of non-limiting example, by Western blot; immunoprecipitation; enzyme-linked immunosorbent assay (ELISA); radioimmunological assay (RIA); sandwich assay; fluorescence in situ hybridization (FISH); immunohistological staining; radioimmunometric assay; immunofluoresence assay; mass spectroscopy and/or immunoelectrophoresis assay.
  • Western blot immunoprecipitation
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunological assay
  • FISH fluorescence in situ hybridization
  • immunohistological staining radioimmunometric assay
  • immunofluoresence assay immunofluoresence assay
  • mass spectroscopy and/or immunoelectrophoresis assay can be measured, by way of non-limiting example, by Western blot; immunoprecipitation; enzyme-linked immunosorbent assay (ELISA); radioimmunological assay (RIA); sandwich assay; flu
  • detectable labels can include labels that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifluoresence, or chemiluminescence, or any other appropriate means.
  • the detectable labels used in the methods described herein can be primary labels (where the label comprises a moiety that is directly detectable or that produces a directly detectable moiety) or secondary labels (where the detectable label binds to another moiety to produce a detectable signal, e.g., as is common in immunological labeling using secondary and tertiary antibodies).
  • the detectable label can be linked by covalent or non-covalent means to the reagent.
  • the detection reagent is label with a fluorescent compound.
  • a detectable label can be a fluorescent dye molecule, or fluorophore including, but not limited to fluorescein, phycoerythrin, phycocyanin, o-phthaldehyde, fluorescamine, Cy3TM, Cy5TM, allophy cocyanine, Texas Red, peridenin chlorophyll, cyanine, tandem conjugates such as phycoerythrin-Cy5TM, green fluorescent protein, rhodamine, fluorescein isothiocyanate (FITC) and Oregon GreenTM, rhodamine and derivatives (e.g., Texas red and tetrarhodimine isothiocynate (TRITC)), biotin, phycoerythrin,
  • a detectable label can be a radiolabel including, but not limited to 3 H, 125 1, 35 S, 14 C, 32 P, and 33 P.
  • a detectable label can be an enzyme including, but not limited to horseradish peroxidase and alkaline phosphatase.
  • An enzymatic label can produce, for example, a chemiluminescent signal, a color signal, or a fluorescent signal.
  • Enzymes contemplated for use to detectably label an antibody reagent include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • a detectable label is a chemiluminescent label, including, but not limited to lucigenin, luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • a detectable label can be a spectral colorimetric label including, but not limited to colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, and latex) beads.
  • detection reagents can also be labeled with a detectable tag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin.
  • a detectable tag such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin.
  • Other detection systems can also be used, for example, a biotin-streptavidin system.
  • the antibodies immunoreactive (i. e. specific for) with the biomarker of interest is biotinylated. Quantity of biotinylated antibody bound to the biomarker is determined using a streptavidin-peroxidase conjugate and a chromagenic substrate.
  • streptavidin peroxidase detection kits are commercially available, e. g.
  • a reagent can also be detectably labeled using fluorescence emitting metals such as 152 Eu, or others of the lanthanide series. These metals can be attached to the reagent using such metal chelating groups as diethylenetriaminepentaacetic acid (DTP A) or ethylenediaminetetraacetic acid (EDTA).
  • DTP A diethylenetriaminepentaacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • compositions and methods described herein can be administered to a subject having or diagnosed as having lung cancer.
  • the methods described herein comprise administering an effective amount of compositions described herein to a subject in order to alleviate a symptom of a lung cancer.
  • "alleviating a symptom” is ameliorating any condition or symptom associated with the lung cancer. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique.
  • a variety of means for administering the compositions described herein to subjects are known to those of skill in the art.
  • Such methods can include, but are not limited to oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, topical, injection, or intratumoral administration. Administration can be local or systemic.
  • effective amount refers to the amount of an agent needed to alleviate at least one or more symptom of the disease or disorder and relates to a sufficient amount of pharmacological composition to provide the desired effect.
  • therapeutically effective amount therefore refers to an amount of an agent that is sufficient to provide a particular anti-cancer effect when administered to a typical subject.
  • an effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not generally practicable to specify an exact “effective amount”. However, for any given case, an appropriate “effective amount” can be determined by one of ordinary skill in the art using only routine experimentation.
  • Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dosage can vary depending upon the dosage form employed and the route of administration utilized.
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50.
  • Compositions and methods that exhibit large therapeutic indices are preferred.
  • a therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (z.e., the concentration of the active agent, which achieves a half-maximal inhibition of symptoms) as determined in cell culture, or in an appropriate animal model.
  • IC50 z.e., the concentration of the active agent, which achieves a half-maximal inhibition of symptoms
  • Levels in plasma can be measured, for example, by high performance liquid chromatography.
  • the effects of any particular dosage can be monitored by a suitable bioassay, e.g., assay for tumor size or growth, among others. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.
  • an effective dose of a composition described herein can be administered to a patient once.
  • an effective dose of a composition can be administered to a patient repeatedly.
  • subjects can be administered a therapeutic amount of a composition such as, e.g. 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, or more.
  • the treatments can be administered on a less frequent basis.
  • a "subject” means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters.
  • a subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g. lung cancer) or one or more complications related to such a condition, and optionally, have already undergone treatment for the condition or the one or more complications related to the condition.
  • a subject can also be one who has not been previously diagnosed as having the condition or one or more complications related to the condition.
  • a subject can be one who exhibits one or more risk factors for the condition, or one or more complications related to the condition or a subject who does not exhibit risk factors.
  • a “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.
  • cancer relates generally to a class of diseases or conditions in which abnormal cells divide without control and can invade nearby tissues. Cancer cells can also spread to other parts of the body through the blood and lymph systems.
  • Carcinoma is a cancer that begins in the skin or in tissues that line or cover internal organs.
  • Adenocarcinoma is a carcinoma of epithelial tissue with a glandular origin and/or glandular characteristics.
  • Sarcoma is a cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue.
  • Leukemia is a cancer that starts in blood-forming tissue such as the bone marrow and causes large numbers of abnormal blood cells to be produced and enter the blood.
  • Lymphoma and multiple myeloma are cancers that begin in the cells of the immune system.
  • Central nervous system cancers are cancers that begin in the tissues of the brain and spinal cord.
  • the cancer is lung cancer. In some embodiments of any of the aspects, the cancer is non-small cell lung cancer. In some embodiments of any of the aspects, the cancer is lung adenocarcinoma. In some embodiments of any of the aspects, the cancer is stage I lung adenocarcinoma. In some embodiments of any of the aspects, the cancer is stage I lung cancer.
  • the cancer is a primary cancer. In some embodiments of any of the aspects, the cancer is a malignant cancer.
  • malignant refers to a cancer in which a group of tumor cells display one or more of uncontrolled growth (z.e., division beyond normal limits), invasion (z.e., intrusion on and destruction of adjacent tissues), and metastasis (z.e., spread to other locations in the body via lymph or blood).
  • metastasize refers to the spread of cancer from one part of the body to another.
  • a tumor formed by cells that have spread is called a “metastatic tumor” or a “metastasis.”
  • the metastatic tumor contains cells that are like those in the original (primary) tumor.
  • the term “benign” or “non-malignant” refers to tumors that may grow larger but do not spread to other parts of the body. Benign tumors are self-limited and typically do not invade or metastasize.
  • a “cancer cell” or “tumor cell” refers to an individual cell of a cancerous growth or tissue.
  • a tumor refers generally to a swelling or lesion formed by an abnormal growth of cells, which may be benign, pre-malignant, or malignant. Most cancer cells form tumors, but some, e.g., leukemia, do not necessarily form tumors. For those cancer cells that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably.
  • neoplasm refers to any new and abnormal growth of tissue, e.g., an abnormal mass of tissue, the growth of which exceeds and is uncoordinated with that of the normal tissues.
  • a neoplasm can be a benign neoplasm, premalignant neoplasm, or a malignant neoplasm.
  • a subject that has a cancer or a tumor is a subject having objectively measurable cancer cells present in the subject’s body. Included in this definition are malignant, actively proliferative cancers, as well as potentially dormant tumors or micrometastatses. Cancers which migrate from their original location and seed other vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs.
  • a “cancer cell” is a cancerous, pre-cancerous, or transformed cell, either in vivo, ex vivo, or in tissue culture, that has spontaneous or induced phenotypic changes that do not necessarily involve the uptake of new genetic material.
  • transformation can arise from infection with a transforming virus and incorporation of new genomic nucleic acid, or uptake of exogenous nucleic acid, it can also arise spontaneously or following exposure to a carcinogen, thereby mutating an endogenous gene.
  • Transformation/cancer is associated with, e.g., morphological changes, immortalization of cells, aberrant growth control, foci formation, anchorage independence, malignancy, loss of contact inhibition and density limitation of growth, growth factor or serum independence, tumor specific markers, invasiveness or metastasis, and tumor growth in suitable animal hosts such as nude mice.
  • protein and “polypeptide” are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alphaamino and carboxy groups of adjacent residues.
  • protein and “polypeptide” refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function.
  • modified amino acids e.g., phosphorylated, glycated, glycosylated, etc.
  • amino acid analogs regardless of its size or function.
  • Protein and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps.
  • Polypeptide -encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a variant protein or fragment thereof that retains the relevant biological activity relative to the reference protein, e.g., at least 50% relative to wildtype.
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage, (i.e. 5% or fewer, e.g.
  • 4% or fewer, or 3% or fewer, or 1% or fewer) of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. It is contemplated that some changes can potentially improve the relevant activity, such that a variant, whether conservative or not, has more than 100% of the activity of wildtype, e.g. 110%, 125%, 150%, 175%, 200%, 500%, 1000% or more.
  • the variant amino acid or DNA sequence can be at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to a native or reference sequence, or a nucleic acid encoding one of those amino acid sequences.
  • the degree of homology (percent identity) between a native and a mutant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web.
  • the variant amino acid or DNA sequence can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, similar to the sequence from which it is derived (referred to herein as an “original” sequence).
  • the degree of similarity (percent similarity) between an original and a mutant sequence can be determined, for example, by using a similarity matrix. Similarity matrices are well known in the art and a number of tools for comparing two sequences using similarity matrices are freely available online, e.g.
  • BLASTp or BLASTn available on the world wide web at blast.ncbi.nlm.nih.gov, with default parameters set.
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid and retains the desired activity of the polypeptide.
  • conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure.
  • a given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as He, Vai, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn).
  • Other such conservative substitutions e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known.
  • Polypeptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired activity and specificity of a native or reference polypeptide is retained.
  • a given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as He, Vai, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn).
  • Other such conservative substitutions e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known.
  • Polypeptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired activity of a native or reference polypeptide is retained.
  • Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure.
  • Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H).
  • Naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
  • Particular conservative substitutions include, for example; Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into His; Asp into Glu; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; He into Leu or into Vai; Leu into He or into Vai; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into He; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and/or Phe into Vai, into He or into Leu.
  • conservative substitutions for one another also include: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).
  • nucleic acid or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof.
  • the nucleic acid can be either single -stranded or double-stranded.
  • a single-stranded nucleic acid can be one nucleic acid strand of a denatured double- stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double -stranded DNA.
  • the nucleic acid can be DNA.
  • nucleic acid can be RNA.
  • Suitable DNA can include, e.g., genomic DNA or cDNA.
  • Suitable RNA can include, e.g., mRNA.
  • “Expression products” include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene.
  • the term “gene” means the nucleic acid sequence which is transcribed (DNA) to RNA in vitro or in vivo when operably linked to appropriate regulatory sequences.
  • the gene may or may not include regions preceding and following the coding region, e.g. 5’ untranslated (5’UTR) or “leader” sequences and 3’ UTR or “trailer” sequences, as well as intervening sequences (introns) between individual coding segments (exons).
  • prophylactic refers to the timing and intent of a treatment relative to a disease or symptom, that is, the treatment is administered prior to clinical detection or diagnosis of that particular disease or symptom in order to protect the patient from the disease or symptom.
  • Prophylactic treatment can encompass a reduction in the severity or speed of onset of the disease or symptom, or contribute to faster recovery from the disease or symptom. Accordingly, the methods described herein can be prophylactic relative to metastasis, vascular invasion, and/or spread to lymph nodes. In some embodiments of any of the aspects, prophylactic treatment is not prevention of all symptoms or signs of a disease.
  • contacting refers to any suitable means for delivering, or exposing, an agent to at least one protein or nucleic acid.
  • exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, perfusion, injection, or other delivery method well known to one skilled in the art.
  • contacting comprises physical human activity, e.g., an injection; an act of dispensing, mixing, and/or decanting; and/or manipulation of a delivery device or machine.
  • the contacting provides direct physical contact of the agent and the at least one protein or nucleic acid, e.g., the agent and the at least one protein or nucleic acid are touching, e.g., at least one surface of the agent is touching or forming a junction with at least one aspect of the at least one protein or nucleic acid.
  • the term “statistically significant” or “significantly” refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.
  • compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
  • the term "consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
  • the term “specific binding” refers to an interaction between two molecules, compounds, cells and/or particles wherein the first entity binds to the second entity with greater specificity and affinity than it binds to a third entity.
  • a first entity specifically bound to a second entity is not displaced by a non-similar competitor.
  • a first entity is said to specifically bind a second entity when it preferentially recognizes the second entity in a complex mixture of proteins and/or macromolecules.
  • specific binding can refer to an affinity of the first entity for the second entity which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or greater than the affinity for the third entity.
  • specific binding refers to the ability of a first entity to bind to a second entity with a K D ICT 5 M (10000 nM) or less, e.g., 1 6 M. 10 7 M. 10 2 M. 10 ' M. 10
  • a first entity e.g., an aptamer or an antibody described herein
  • a second entity e.g., an antigen or biomolecule
  • Specific binding can comprise ionic bonding, hydrogen bonds, ionic bonds, van der Waals interactions, and/or London dispersion forces. In some embodiments, specific binding does not refer to covalent bonding. In some embodiments, specific binding does not refer to a peptide bond. In some embodiments, specific binding does not refer to a phosphodiester bond. [00268]
  • the singular terms "a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise.
  • a sample is taken, obtained, or provided via minimally invasive methods and/or involves only a minor intervention.
  • a sample is taken, obtained, or provided by one or more of a blood draw or prick, an epidermal or mucus membrane swab, buccal sampling, saliva sample, a epidermal skin sampling technique, and/or collection of a secreted or expelled bodily fluid (e.g., mucus, urine, sweat, etc.), fecal sampling, semen/seminal fluid sampling, or clippings (e.g., of hair or nails).
  • a secreted or expelled bodily fluid e.g., mucus, urine, sweat, etc.
  • fecal sampling semen/seminal fluid sampling
  • clippings e.g., of hair or nails.
  • the sample comprises, consists of, or consists essentially of blood (or any fraction or component thereof), serum, urine, mucus, epithelial cells, saliva, buccal cells, a secreted or expelled bodily fluid, and/or hair or nail clippings.
  • the present technology may be defined in any of the following numbered paragraphs:
  • a method of treating or prognosing lung cancer comprising: administering adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and/or lymphadenectomy to a subject having decreased expression of at least one gene selected from the group consisting of:
  • SHR00M4 and NBEAL1 and/or increased expression of at least one gene selected from the group consisting of: MUC16; NOTCH3; H19; COL1A1; and GARSl .
  • SHR00M4 and NBEAL1 and/or not having increased expression of at least one gene selected from the group consisting of: MUC16; N0TCH3; H19; C0L1A1; and GARS1.
  • the at least one gene is SHR00M4 or
  • method of treating or prognosing lung cancer comprising determining the level of expression: a) at least one gene selected from the group consisting of:
  • BRCA1; HLTF; and FANCA at least one gene selected from the group consisting of:
  • PRKDC TPI1; PKM; GAPDH; AHNAK2; YWHAZ; ATP 13 A3; GARS1; CP;
  • PRKDC TPI1; PKM; GAPDH; AHNAK2; YWHAZ; ATP 13 A3; GARS1; CP; MUC16; TUBA1C; H19; PPFIA1; N0TCH3; ACLY; GCLC; CAD; and CCT6A; and h) decreased expression of at least one gene selected from the group consisting of: SPTBN1; SFTPB; LRRK2; AKAP13; CHD2; EPAS1; ATP11A; R0S1; MTUS1;
  • ANKHD1; NBEAL1; MPRIP; SHR00M4; and TET2 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and/or lymphadenectomy.
  • the increased expression and/or decreased expression is relative to an average for subjects with lung cancer.
  • the method of any one of the preceding paragraphs, wherein the increased expression and/or decreased expression is relative to an average for subjects with lung adenocarcinoma.
  • the increased expression and/or decreased expression is relative to an average for subjects with lung cancer that is not vascular invasive.
  • the method of any one of the preceding paragraphs, wherein the subject having increased expression of the at least one gene is determined to have increased expression of the at least one gene.
  • the method of any one of the preceding paragraphs, wherein the subject having increased expression of the at least one gene is determined to have increased expression of the at least one gene in a sample of tumor cells.
  • the level of expression is the level of mRNA.
  • the subject having increased expression of the at least one gene is not administered a lobectomy.
  • Lung cancer is the leading cause of cancer-related deaths in the United States.
  • Lung adenocarcinoma (LU AD) is the most common lung cancer subtype diagnosed in the US and represents about l/3 rd of total cases. With increasing incidence, there is a need for improved patient stratification to help guide appropriate treatment for high risk patients and to ensure that low risk patients are not subjected to complications from unnecessary treatments.
  • the invention described herein comprises a method for stratifying patients according to the aggressiveness of LU AD based on gene expression in the tumor.
  • the biomarker is predictive of the presence of vascular invasion (VI) within LUAD tumors.
  • VI vascular invasion
  • the gene expression biomarker can accurately classify patients with VI into a high-risk category. This distinguishes it from existing molecular biomarkers such as DetermaRx (Oncocyte) that predict outcomes in NSCLC. Biomarkers based on patient outcomes may be influenced by treatment response and standard of care.
  • VI is difficult to assess in resection specimens and not routinely reported, despite its prognostic significance. It requires a time-consuming procedure for a pathologist to inspect hundreds of consecutive sections of the tumor.
  • the molecular biomarker can accurately predict VI positive tumors from a small amount of resected material, streamlining pathology assessment. This may also guide appropriate adjuvant chemotherapy.
  • the molecular biomarker can be used to predict VI prior to surgery. While the standard of care for early-stage LUAD resection is complete lobectomy, the field is moving toward limited resection or tissue-sparing surgery, as the favorable approach. Predicting VI prior to surgery offers the clinical team with additional information that may influence moving to traditional lobectomy over limited resection to minimize chances of patient recurrence. Finally, predicting high-risk angioinvasive tumors prior to surgery also allows for an opportunity to guide future neoadjuvant treatment approaches. In patients who are not surgical candidates, VI may predict which ablative (non-surgical) therapies (RFA, MWA, cryoablation) are radiation therapies (SBRT) are likely to benefit the patient.
  • ablative (non-surgical) therapies RAA, MWA, cryoablation
  • SBRT radiation therapies
  • a VI biomarker score is calculated from quantitation of mRNA abundance in LUAD tumor samples. If the levels of mRNA from genes in Cluster 1, 2 and 3 are higher than reference levels observed in non-VI LUAD samples and the levels of mRNA from genes in Cluster 4 are lower than reference levels observed in non-VI LUAD samples, this is predictive of the presence of vascular invasion in the LU AD tumor. Specifically, the expression of 48 genes is used in the calculation of the VI biomarker score.
  • Cluster 1 (expression from 12 genes) contains the following that are increased in tumors with VI:"MKI67” “ASPM” "ATAD2” “TOP2A” "FANCI” "PLOD2” “NCAPG2” "CENPF” "KPNA2” "BRCA1” "HLTF” “ “FANCA”
  • Cluster 2 (expression from 4 genes) contains the following that are increased in tumors with VI: "COL1A2” "COL3A1” "COL1A1” "COL6A3"
  • Cluster 3 (expression from 18 genes) contains the following that are increased in tumors with VI: “PRKDC” “TPI1” “PKM” “GAPDH” “AHNAK2” “YWHAZ” “ATP13A3” “GARS1” “CP” ”MUC16” “TUBA1C” “H19” “PPFIA1” “NOTCH3” “ACLY” “GCLC” “CAD” “CCT6A” [00286]
  • Cluster 4 (expression from 14 genes) contains the following that are decreased in tumors with VI: “SPTBN1” “SFTPB” “LRRK2” “AKAP13” “CHD2” “EPAS1” “ATP11A” “ROS1” “MTUS1” “ANKHD1” “NBEAL1” “MPRIP” “SHROOM4" “TET2”
  • Cluster 2 (expression from 37 genes) contains the following that are increased in tumors with VI: "SULF1” "THBS2” "CSMD2” “CTHRC1” “COL1A1” “IGFBP3” "CILP” "MMP11” “COL11A1” “COL5A1” “MXRA5" “ADAM12” “COL5A2” “COL3A1” “VCAN” "ITGA11” “FAP” "COL12A1” “COL1A2” “COL6A3” “SPOCK1” "MMP14” “LRRC15” "DNM1” "WT1.AS” "COL10A1” “CCN4" "ADAM TS 12" "SFRP2” "POSTN” "OLFML2B” "THY1” "HTRA3” "INHBA” "ALDH1L2” “DIO2” “SH3PXD2 B” [00290] Cluster 3 (expression from 182 genes) contains the following that are increased in tumors with VI: "PO
  • Cluster 4 (expression from 140 genes) contains the following that are decreased in tumors with VI: "CYP4B1" "SCGB3A1” "ITGA10” “LRRK2” “GANC” “ACOXL” “REPS2” "CGNL1” “SNX30 " "GCNT4" "IVD” "PIGA” “MACROD2” “CTSH” “CELF2” “PDZD2” “SMAD4" “SHROOM4" "ARID4A” "MFSD2A” "CAPN3” "KIAA1328” "FLRT3” "ALDH2” "ROBO2” "T LR2” “CCDC68” “ACADSB” “TLE4" "BTG2” "ZNF540” “FILIP1” “ZNF385B” "C4BPA” "NOSTRIN” "CFTR” "VIPR1” "FBXO38” "CYP4X1” "BAIAP2” "RBPMS” "DNM3” “Fll” "PCDH20”
  • Example 2 Spatially informed profiling of stage I lung adenocarcinoma reveals a gene expression signature of vascular invasion
  • V Microscopic vascular invasion
  • LU AD stage I lung adenocarcinoma
  • new biomarkers are needed to identify this aggressive subset of stage I LU AD tumors.
  • RNA-seq To assess molecular and tumor microenvironment features associated with angioinvasive LU AD we analyzed 163 resected stage I tumors with and without VI by RNA-seq, including 15 samples by high-resolution spatial transcriptomics (stRNA-seq).
  • Lung adenocarcinoma is the most common lung cancer subtype and invasive LU AD represents 70-90% of surgically resected lung cancers 1 .
  • CT computed tomography
  • VI Microscopic vascular invasion (VI), defined as tumor invasion within the lumen of veins or arteries, is a well described route to metastatic dissemination and is consistently associated with higher rates of tumor recurrence among early stage LUAD 2 s .
  • VI is not included in the current World Health Organization (WHO) grading system for lung cancer but may be a better predictor of recurrence than the most severe WHO-2021 grade, leading to our proposal for VI+ LU AD to be reclassified as distinct angioinvasive LU AD 9 .
  • WHO World Health Organization
  • Patients with stage I VI+ LU AD may benefit from adjuvant therapy, but it is difficult to assess in resected tumor specimens 10 11 .
  • Elastic stains can be used to improve visualization of invaded blood vessels over hematoxylin and eosin (H&E), but comprehensive tumor histopathology review for small ( ⁇ lmm) VI foci is difficult and prone to false negatives.
  • biopsy specimens do not provide enough tissue material to evaluate for VI+ LUAD prior to surgery, preventing informed surgical or neoadjuvant treatment approaches.
  • Pathologists infrequently document VI as a separate entity from lymphatic invasion (LI), preferring to designate the presence of either type as lymphovascular invasion (LVI). This obfuscates the independent prognostic value of VI 16 and complicates downstream molecular approaches seeking to disentangle these modes of tumor spread, the differences of which are still incompletely understood 17 .
  • Molecular profiling technologies have become a staple of clinical pathology but prior molecular studies of LUAD have primarily focused on identifying signatures of poorly differentiated tumors, which often manifest with solid growth patterns.
  • New advances in spatial transcriptomics (stRNA-seq) allow for probing transcriptomic changes associated with tumor invasion within the geographic context of the tumor microenvironment and with complete preservation of pathological substructures 18 .
  • RNA-seq RNA-sequencing
  • Fig. 2C the independent spatial patterning of each cluster was apparent (Fig. 2C).
  • cluster 2 was not expressed directly within any of the invaded vessels in stRNA-seq capture areas.
  • cluster 3 was significantly enriched directly in invasive foci in addition to high-grade patterns, we were interested to see whether the expression of these genes was also higher in VI+ tumors independently of aggressive pattern.
  • Fig. 18 This conclusion is reinforced by Fig. 18, which demonstrates that cluster 1 is significantly independent form high grade growth patterns. This indicates that the biomarker signals are not exclusively due to proportion of aggressive histology in the tumor.
  • the VI signature is composed of both tumor-specific and tumor-microenvironment changes reflective of angioinvasion.
  • Our supervised analysis above relied upon detailed pathologic annotation of the tissue containing Visium spots, but spots were only labeled if there was a clear consensus on morphology, especially for the histologic pattern.
  • tumor areas may not have been annotated if they did not clearly contain canonical features of one of the six histologic patterns 25 .
  • the annotation may include admixed stromal or immune cells.
  • VI + samples also had a higher proportion of plasma cells and B cells.
  • VI cluster 2 was most spatially correlated with peribronchial fibroblasts and dividing stromal cells, as well as other stromal cell types including endothelial and smooth muscle cells.
  • VI cluster 3 showed the most correlation with tumor cells and a transitional club/AT2 phenotype.
  • VI cluster 4 showed expected spatial correlation with normal lung epithelial cell types including ATI and AT2 cells.
  • VI cluster 1 was tightly spatially correlated with dividing B and T cells, but not dividing stromal, plasma, or myeloid cells. Collectively, these results show that the VI gene clusters capture biological changes associated with angioinvasion from both tumor cells and the tumor microenvironment.
  • a VI predictor derived from the signature validates in an independent stage I LUAD cohort.
  • Our finding of biologically and spatially distinct gene expression clusters from bulk tumors associated with VI + LUAD suggest that all four clusters provide orthogonal information and might be combined to form a predictor of angioinvasive Stage I LUAD.
  • Predictors selected via a binomial logit generalized linear model (GLM) utilizing ridge regression performed the best on the cross-validation internal test sets within our discovery cohort (Fig. 11A).
  • a 48-gene predictor achieved an area under the receiver operating characteristic curve (AUROC) of 0.85 to separate VI + and VI’ LUAD in the training set (Fig. 4B).
  • AUROC receiver operating characteristic curve
  • the top genes in the predictor were enriched for cluster 3 membership and included MUC16. NOTCH 3. and Hl 9 (Fig. 4C).
  • the top features for clusters 1, 2, and 4 were MKI67. COL1A1 and CHD2, respectively.
  • the VI predictor is robust to intra-tumor heterogeneity.
  • Our stRNA-seq analysis of the expression of Vi-associated genes implies that each Vi-associated cluster may be spatially distinct. This suggests that a VI predictor combining genes from each cluster might overcome intra-tumor heterogeneity (ITH).
  • ITH intra-tumor heterogeneity
  • Overcoming intra-tumor heterogeneity is crucial for molecular biomarkers that sample only a portion of the tumor volume, such as those measured on tissue available from biopsies 32 . This is particularly important for detecting VI due to the small size of invaded vessels.
  • Cluster 2 was most strongly enriched in regions of desmoplastic but not normal stroma while cluster 4 was enriched in low-grade histologic patterns and normal pulmonary structures.
  • Desmoplastic stroma is a natural response of surrounding normal tissue to invasive tumor, and involves widespread tissue remodeling, which was also reflected in the cluster 2 enrichment results from the discovery cohort described above.
  • RNA-seq was performed on 108 tumors, with 103 passing quality control.
  • VI Vascular invasion
  • MIA minimally invasive adenocarcinoma
  • LMP tumors were non- mucinous adenocarcinoma measuring ⁇ 3 cm in total size, with >15% lepidic growth, and without nonpredominant high-grade patterns (>10% cribriform, >5% micropapillary, >5% solid), >1 mitosis per 2 mm 2 , vascular, lymphatic or visceral pleural invasion, STAS or necrosis.
  • AIS/MIA and LMP were analyzed together due to their identical outcome (100% 10-year DSS). No special type (NST) designation was given for all other tumors not classified as VI or LMP. Stage assignments were retrospectively made using the 8th edition of the AJCC.
  • RNA-Seq library preparation sequencing, and data processing.
  • Total RNA was extracted from FFPE tissue using AllPrepTM DNA/RNA Universal Kit (Qiagen) and exome-targeted sequencing libraries were prepared using Illumina TruSeqTM RNA Exome Library Prep Kit (formerly TruSeq RNA Access). Samples were sequenced on the Illumina HiSeq 2500 to generate paired-end 50-nucleotide reads. Basespace was used to demultiplex and generate FASTQ files.
  • Tissue sections cut by a microtome at 5 microns were placed in a 42°C water bath and transferred onto the 6.5 x 6.5 mm tissue capture areas on the VisiumTM Spatial Gene Expression Slide (PN-1000185, lOx Genomics).
  • Tissues were deparaffinized, H&E stained, imaged with a Leica AperioTM AT2, and decrosslinked according to the manufacturer’s recommended protocol, with the following user modifications made to deparaffinization to prevent tissue detachment: 1) the 15 min incubation step during deparaffinization was removed, 2) after the 96% ethanol immersion, one 85%, one 70% and one 50% ethanol immersion for 3 min each was added.
  • FIG. 1 Spatial transcriptomics data analysis. VisiumTM spatial transcriptomics data was processed using the SeuratTM R package, unless otherwise specified 55 . First, low-quality spots were filtered using a cutoff of ⁇ 250 features per spot. For the global sample analysis, samples were merged and normalized using SCTransform and scaled 56 . For individual stRNA-seq samples, spots were log- normalized. Visium spots were scored with gene signatures using the AddModule Score function from the SeuratTM R package. Spatial enrichment of pathology regions by VI cluster enrichment score was assessed with a generalized binomial linear mixed effect models with sample as a random effect after averaging scores across regions. Pathology regions with ⁇ 200 spots annotated were removed prior to analysis and all regions were downsampled to 200 spots each. Normal lung was used as the reference factor level.
  • [00331] Feature selection. Genes associated with VI were derived within each train split as described above. The final gene set used in each fold of the cross-validation was selected by taking the top genes within each VI cluster ranked by mean expression. A gene set size of 48 was arbitrarily selected to allow for proportional representation of the original cluster size with a scaling factor of 10. [00332] Models. Model selection was evaluated with 5 -fold cross-validation within an inner fold of the train split using the AutoML interface from the h2o.ai package v.3.40.0.4. The best performing model in the inner fold was retrained on the entire train set and then applied to the test hold out set. The final model was selected as the model with the highest mean and median AUROC across all 100 cross- validation iterations and was re-trained on the entire discovery cohort.
  • VI predictor validation After quality control and individual batch correction, the mean and variance of log -transformed cpm in the validation cohort was adjusted to match the discovery cohort using reference ComBat with the discovery cohort as the reference batch. After filtering the validation cohort to the final features, VI predictions were generated using the final trained model and performance was evaluated by AUROC.
  • RNA-Seq and matching clinical data from the Uppsala NSCUC cohort were downloaded using the Gene Expression Omnibus (GSE81089) and filtered to 44 stage IA LU AD samples with overall survival data. Analysis for both datasets was limited to stage IB samples because these included tumors > 5 cm in previous TNM editions and our predictor was derived in TNM 8 th edition stage I samples. Genes in both datasets were filtered to genes remaining after filtering in the discovery cohort and the mean and variance of log-transformed cpm was adjusted to match the discovery cohort using reference ComBat with the discovery cohort as the reference batch 58 . [00336] TRACERx intra-tumor heterogeneity analysis.
  • RNA-seq and clinical annotations from the TRACERx NSCLC cohort of multi-region tumor sampling data was downloaded from doi.org/10.5281/zenodo.7683605 and doi.org/10.5281/zenodo.7603386.
  • Samples were filtered to TNM 8 th edition stage I LU AD tumors with data at least two regions. Genes were filtered to those remaining after filtering in the discovery cohort and the mean and variance of log -transformed cpm was adjusted to match the discovery data using reference ComBat with the discovery cohort as the reference batch. VI predictions were generated as described above.
  • a Grading System for Invasive Pulmonary Adenocarcinoma A Proposal From the International Association for the Study of Lung Cancer Pathology Committee. J. Thorac. Oncol. 15, 1599-1610 (2020). . Tavemari, D. et al. Nongenetic Evolution Drives Lung Adenocarcinoma Spatial Heterogeneity and Progression. Cancer Di scov. (2021) doi: 10.1158/2159-8290. CD-20-1274.. Nguyen, T. T. et al. A lepidic gene signature predicts patient prognosis and sensitivity to immunotherapy in lung adenocarcinoma. Genome Med. 14, 5 (2022). . Subramanian, J. & Simon, R.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Hematology (AREA)
  • Medicinal Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Cell Biology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • General Physics & Mathematics (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Food Science & Technology (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The technology described herein is directed to methods of treating and prognosing lung cancer.

Description

METHODS AND COMPOSITIONS RELATING TO ANGIOINVASIVE LUNG ADENOCARCINOMA
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit under 35 U.S.C. § 119 of U.S. Provisional Application No. 63/570,598 filed March 27, 2024, the contents of which are incorporated herein by reference in their entirety.
GOVERNMENT SUPPORT
[0002] This invention was made with government support under Grant No. CA196408 awarded by the National Institutes of Health. The Government has certain rights in the invention.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created March 20, 2025, is named 701586-000130WOPT_SL.xml and is 678,312 bytes in size.
TECHNICAL FIELD
[0004] The technology described herein relates to the treatment and prognosis of lung cancer, e.g., particularly stage I lung adenocarcinoma.
BACKGROUND
[0005] Microscopic vascular invasion (VI) is predictive of recurrence in early-stage lung adenocarcinoma (LU AD) but is difficult to assess in resection specimens to guide adjuvant therapy. It also cannot be accurately predicted prior to surgery when it could be used to guide neoadjuvant, ablative, radiation-based therapy and/or surgical approaches to curative therapy. Thus, new biomarkers are needed to identify this aggressive subset of stage I LU AD tumors.
SUMMARY
[0006] Described herein are methods relating to gene expression biomarkers of vascular invasion that can accurately identify subjects with vascular invasion. This provides the ability to predict angioinvasive LU AD from small biopsy specimens, allowing for more tailored treatment prior to and after surgery.
[0007] In one aspect of any of the embodiments, described herein is a method comprising, determining the expression of at least one gene selected from the group consisting of: SHROOM4; NBEAL1; MUC16; NOTCH3; H19; COL1A1; and GARS1 in a subject with lung cancer.
[0008] In one aspect of any of the embodiments, described herein is a method of treating or prognosing lung cancer, the method comprising: administering adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and/or lymphadenectomy to a subject having decreased expression of at least one gene selected from the group consisting of: SHROOM4 and NBEAL1; and/or increased expression of at least one gene selected from the group consisting of: MUC16; N0TCH3; H19; C0L1A1; and GARS1.
[0009] In some embodiments of any of the aspects, the method described herein further comprises administering active surveillance, wedge resection of the lung, or a segmentectomy of the lung to a subject not having decreased expression of at least one gene selected from the group consisting of:
SHR00M4 and NBEAL1; and/or not having increased expression of at least one gene selected from the group consisting of: MUC16; NOTCH3; H19; COL1A1; and GARS1.
[0010] In some embodiments of any of the aspects, the at least one gene is SHR00M4 or NBEAL1. In some embodiments of any of the aspects, the at least one gene is SHR00M4. In some embodiments of any of the aspects, the at least one gene is NBEAL1. In some embodiments of any of the aspects, the at least one gene is MUC16; NOTCH3; H19; or COL1A1. In some embodiments of any of the aspects, the at least one gene is SHROOM4; NBEAL1; MUC16; NOTCH3; H19; and COL1A1.
[0011] In one aspect of any of the embodiments, described herein is a method of treating or prognosing lung cancer, the method comprising determining the level of expression: a) at least one gene selected from the group consisting of:
MKI67; ASPM; ATAD2; TOP2A; FANCI; PLOD2; NCAPG2; CENPF; KPNA2;
BRCA1; HLTF; and FANCA; b) at least one gene selected from the group consisting of:
COL1A2; COL3A1; COL1A1; and COL6A3; c) at least one gene selected from the group consisting of:
PRKDC; TPI1; PKM; GAPDH; AHNAK2; YWHAZ; ATP 13 A3; GARS1; CP;
MUC16; TUBA1C; H19; PPFIA1; NOTCH3; ACLY; GCLC; CAD; and CCT6A; d) at least one gene selected from the group consisting of:
SPTBN1; SFTPB; LRRK2; AKAP13; CHD2; EPAS1; ATP 11 A; ROS1; MTUS1;
ANKHD1; NBEAL1; MPRIP; SHROOM4; and TET2 wherein: e) increased expression of at least one gene selected from the group consisting of:
MKI67; ASPM; ATAD2; TOP2A; FANCI; PLOD2; NCAPG2; CENPF; KPNA2;
BRCA1; HLTF; and FANCA; f) increased expression of at least one gene selected from the group consisting of:
COL1A2; COL3A1; COL1A1; and COL6A3; g) increased expression of at least one gene selected from the group consisting of:
PRKDC; TPI1; PKM; GAPDH; AHNAK2; YWHAZ; ATP 13 A3; GARS1; CP;
MUC16; TUBA1C; H19; PPFIA1; NOTCH3; ACLY; GCLC; CAD; and CCT6A; and h) decreased expression of at least one gene selected from the group consisting of: SPTBN1; SFTPB; LRRK2; AKAP13; CHD2; EPAS1; ATP11A; R0S1; MTUS1;
ANKHD1; NBEAL1; MPRIP; SHR00M4; and TET2 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and/or lymphadenectomy.
[0012] In some embodiments of any of the aspects, the increased expression and/or decreased expression is relative to an average for subjects with lung cancer. In some embodiments of any of the aspects, the increased expression and/or decreased expression is relative to an average for subjects with lung adenocarcinoma. In some embodiments of any of the aspects, the increased expression and/or decreased expression is relative to an average for subjects with lung cancerthat is not vascular invasive. [0013] In some embodiments of any of the aspects, the subject having increased expression of the at least one gene is determined to have increased expression of the at least one gene. In some embodiments of any of the aspects, the subject having increased expression of the at least one gene is determined to have increased expression of the at least one gene in a sample of tumor cells. In some embodiments of any of the aspects, the level of expression is the level of mRNA.
[0014] In some embodiments of any of the aspects, the subject having increased expression of the at least one gene is not administered a lobectomy. In some embodiments of any of the aspects, the subject having increased expression of the at least one gene is not administered a sublobar resection. In some embodiments of any of the aspects, the subject having increased expression of the at least one gene is not administered a lobectomy or sublobar resection.
[0015] In some embodiments of any of the aspects, the lung cancer is non-small cell lung cancer (NSCLC). In some embodiments of any of the aspects, the lung cancer is lung adenocarcinoma. In some embodiments of any of the aspects, the lung cancer comprises vascular invasion. In some embodiments of any of the aspects, the lung cancer is vascular invasive lung adenocarcinoma. In some embodiments of any of the aspects, the lung cancer is stage I lung adenocarcinoma.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figs. 1A-1D: Distinct gene expression changes associated with VI in LUAD. Fig. 1A. Overview of cohorts, sequencing technologies, and analyses utilized in the study. BMC, Boston Medical Center; LHMC, Lahey Hospital and Medical Center. Fig. IB. Co-expression heatmap of 474 genes differentially expressed between VI and LMP (FDR < 0.01) in a discovery cohort of stage I LUAD tumors (n = 103) grouped into k=4 clusters. VI, vascular invasion; NST, no special type; LMP, low malignant potential; VPI, visceral pleural invasion; LI, lymphatic invasion. Figs. 1C-1D. Top 10 Biological enrichment terms of genes within each gene co-expression cluster. Pathways were ranked within each cluster by FDR values obtained from inputting cluster genes into the EnrichR database with queries for MSigDB Hallmark 2020 (H) and GO Biological Process 2021 (BP). [0017] Figs. 2A-2D. Spatial transcriptomics of early-stage LUAD reveals association of VI gene clusters with specific LUAD histopathology features. Fig. 2A. Pathology annotations present across spatial transcriptomics (stRNA-seq) capture areas that passed QC (n=15). Fig. 2B. Association between expression of Vi-associated clusters and pathology across all 15 samples. Only results with FDR < 0.01 are shown. FDR values were derived from a linear-mixed model predicting cluster expression with sample as random effect. Fig. 2C. StRNA-seq expression maps of VI gene expression clusters in representative samples from the VI+ and VI- LUAD. Fig. 2D. Spot-wise scoring of VP and VI+ stRNA- seq samples for expression of VI gene expression clusters in spots annotated with high-grade LUAD patterns (solid, micropapillary, and cribriform). P values were derived from a linear-mixed model predicting cluster expression with sample as a random effect.
[0018] Figs. 3A-3C: The VI signature is composed of both tumor-specific and tumormicroenvironment changes reflective of angioinvasion. Fig. 3A. Cell type proportions by VI status (with the VI focus excluded) in the stRNA-seq data, revealed by spot deconvolution. Fig. 3B. Peribronchial fibroblast and macrophage proportions by VI status in the bulk RNA-seq discovery cohort. Fig. 3C. Mean spatially weighted correlation of VI gene clusters and stage I LUAD cell type signatures from the Salcher et al. lung cancer atlas across all stRNA-seq samples.
[0019] Figs. 4A-4I: A VI predictor derived from the signature validates in an independent stage I LUAD cohort. Fig. 4A. Cross-validation approach for feature and model selection. Fig. 4B. Predictor performance for predicting VI in discovery cohort (n=103). The shaded region represents the 95% confidence interval (CI) of the sensitivity at different specificity points, computed with 2000 bootstrap replicates. Fig. 4C. Feature importance for the final VI predictor. Fig. 4D. Predictor performance in the validation cohort (n=60). Fig. 4E. Predictor performance in the validation cohort in non-LMP tumors (n=53). Fig. 4F. Predictor association with RFS in the full validation cohort. The Youden point from the ROC classifying RFS in the validation cohort (left) was used as the cutoff for the Kaplan Meir (KM) curve (right). Fig. 4G. Predictor association with RFS in the Vi-negative tumors. KM p values were calculated using the log-rank test. Fig. 4H. Predictor performance in the validation cohort by histologic pattern (n varies). Error bar shows bootstrapped 95% CI. Fig. 41. Predictor performance when classifying other LUAD invasion types in the validation cohort.
[0020] Figs. 5A-5E: The VI predictor is robust to intra-tumor heterogeneity. Fig. 5A. Examples of binning VI+ tumors into distal VI+ (spots > 1mm outside invaded foci boundary) and proximal VI+ (spots < 1mm from and including the invaded foci). Any VI+ tumors that did not contain VI foci in the capture area were considered as proximal VI+. Fig. 5B. Spot-wise expression of the VI predictor (predictor up gene enrichment minus predictor down gene enrichment) between distal VI+ and VI" tumors. P values are derived from a type II Anova of a linear mixed model predicting gene expression with sample as a random effect. Fig. 5C. Correlation between VI predictor scores from randomly sampled tumor-matched regions (n=136) of stage I LUAD cases from TRACERx, a study ofNSCLC in which RNA-seq was performed on multiple tumor regions for a subset of tumors. P value shown for Spearman rank coefficient. Fig. 5D. The enrichment of the 48 VI predictor genes among all filtered genes ranked by correlation between two randomly selected regions of the same tumor. P value calculated by gene set enrichment analysis (GSEA). Fig. 5E. Differences in scores between unmatched regions (inter-tumor heterogeneity) and matched regions (intra-tumor heterogeneity).
[0021] Figs. 6A-6K: VI is the LU AD invasion type most associated with recurrence. Fig. 6A. Association of novel grading system with RFS in the stage I LUAD clinical cohort (n=254 tumors). Figs. 6B-6E. Association of VI, STAS, VPI, and LI pathology, respectively, with RFS. Fig. 6F. Co-occurrence of invasion types in the stage I LUAD clinical cohort. Fig. 6G. Association of VI with RFS when controlling for common clinical variables, collection site (LHMC - Lahey Hospital & Medical Center, BMC - Boston Medical Center) and the other invasion types. Fig. 6H. Association between invasion types and LUAD growth patterns. Fig. 61. Proportion of VI- and VI+ cases that recurred at different locations. Fig. 6J. Recurrence site-specific sub-distribution hazard ratio (HR) for VI (n=235 tumors). Patients with unknown recurrence site were excluded from the analysis. HRs were adjusted for gender, age, pack years, surgical procedure, and collection site. Fig. 6K. Sub-distribution HR at the indicated sites of recurrence.
[0022] Figs. 7A-7B: The four VI gene expression clusters individually predict VI even in the absence of LMP tumors. Fig. 7A. ROC curves for predicting VI+ vs. VI- tumors using the mean z- score of each gene expression cluster. Fig. 7B. ROC curves for predicting VI vs. NST tumors using the mean z-score of each gene expression cluster. P values are reported for the Wilcoxon test where the null hypothesis is that the AUROC is equal to 0.5.
[0023] Figs. 8A-8C: VI but not LI is associated with expression of tissue remodeling genes and larger tumor invasive size. Fig. 8A. Co-expression heatmap of 139 genes differentially expressed between VI and LMP (FDR < 0.01) in a discovery cohort of stage I LUAD tumors (n = 103), with LI as a covariate. Genes are annotated by the clusters defined in Fig IB. VI, vascular invasion; NST, no special type; LMP, low malignant potential; VPI, visceral pleural invasion; LI, lymphatic invasion. Fig. 8B. Heatmap of 15 genes associated with LI contrasts (FDR < 0.01). Genes are annotated if they belong to one of the clusters defined in Fig IB. Fig. 8C. Gene-set enrichment analysis (GSEA) results of the 4 VI gene clusters against a ranked list of genes ordered by association with LI.
[0024] Figs. 9A-9E: Bulk RNA-seq and stRNA-seq data show strong concordance in gene expression. Fig. 9A. The association of spot-wise pathology annotations with tumor-level VI status in stRNA-seq data. FDR values were calculated by chi-square test. Fig. 9B. Features per spot and Fig. 9C. UMI counts per spot by sample in the stRNA-seq data. Sample 6 failed QC and was excluded from downstream analysis. Fig. 9D. Correlation of mean gene expression between pseudo bulked stRNA-seq data and tumor-matched bulk RNA-seq data. P value shown for Spearman rank coefficient. Correlation of spot-wise expression of VI cluster genes across all stRNA-seq samples (n=15). Fig. 9E. Mean spatially weighted correlation of spot-wise VI cluster enrichment scores across all stRNA-seq samples. [0025] Figs. 10A-10C: Cell type enrichment in stRNA-seq data. Fig. 10A. Representative images showing the association of cell type enrichment signatures with pathology. Fig. 10B. Proportions of plasma cells from deconvolution of the bulk RNA-seq discovery cohort, stratified by pathologist annotated plasma cell grade. Fig. 10C. Correlation of cell type markers (using the average expression of the top 50 differentially expressed marker genes) in the stage I LU AD samples from the Salcher lung cancer atlas.
[0026] Figs. 11A-1 IE: VI predictor development and validation. Fig. 11A. Cross-validation performance for the four AutoML models. Drl, distributed random forest; gbm, gradient boosting machine; glm, generalized linear model; xgboost, extreme gradient boosting. Error bars represent standard deviation (SD) of AUROCs across all cross-validation folds (n=100). Fig. 1 IB. VI predictor association with novel, International Association for the Study of Lung Cancer (IASLC) 2020, and World Health Organization (WHO) 2015 grading systems in the validation cohort (n=60 tumors). For IASLC and WHO analyses, mucinous tumors (n=2) were removed from grade 3 as these grading systems exclude mucinous tumors. P values were calculated by Wilcoxon test. Figs. 11C. Association of pathology features with VI predictor scores in VI- tumors within the validation cohort. A univariate linear model was used to calculate FDR values for each pathology feature, with predictor scores as the dependent variable. Figs. 1 ID-1 IE: Association of mean predictor scores per tumor with LVI+ (Fig. 1 ID) and ctDNA+ tumors (Fig. HE).
[0027] Figs. 12A-12E depict VI predictor genes that are most correlated with overall VI predictor score. Individual VI predictor genes ranked by their correlation with the overall cluster mean expression in the validation cohort for clusters 1 (Fig. 12A), 2 (Fig. 12B), 3 (Fig. 12C), and. 4 (Fig. 12D). (Fig. 12E) shows the location of genes (MKI67, COL1A1, PRKDC, CHD2) that have strong correlation with mean cluster expression in the validation cohort, are high ranking in the VI model, and have ISH probes readily available.
[0028] Figs. 13A-13B demonstrate that a minimal VI gene predictor built from 4 genes performs well in predicting VI and is robust to ITH. (Fig. 13A) A VI gene predictor built from the 4 genes with the highest GLM standardized coefficients per cluster in the validation cohort. (Fig. 13B) A VI gene predictor built from the 4 genes(l from each cluster) that had strong correlation with mean cluster expression in the validation cohort, are high ranking in the VI model, and have ISH probes readily available. Performance is shown for predicting VI in the discovery and validation cohorts, and also robustness to intra-tumor heterogeneity in the TRACERx cohort.
[0029] Figs. 14A-14B demonstrate that VI predictor score is associated with lymph node positivity in TRACERx stage I and stage II LU AD. (Fig. 14A) An unsupervised heatmap of the VI predictor genes’ expression in the TRACERx stage I and stage II LU AD cohort. (Fig. 14B) Mean VI predictor score across regions of the same tumors is significantly associated with lymph node status.
[0030] Figs. 15A-15B demonstrate that the VI gene cluster 2 may be important in LN metastasis. (Fig. 15A) Genes associated with presence of positive lymph nodes in TRACERx stage II LU AD. 671 genes up with LN positivity and 755 genes down at FDR < 0.05 were derived using a negative binomial model of ~1+Lymph Node+Stage+LVI. (Fig. 15B) When examining the enrichment of the VI gene clusters against the ranked list of these genes associated with LN positivity in TRACERx, VI gene cluster 2 was significantly enriched, despite not being enriched in genes associated with LI.
[0031] Fig. 16 demonstrates that VI gene cluster 2 and genes from VI gene cluster 2 are associated with myofibroblasts in scRNAseq LU AD data.
[0032] Figs. 17A-17D demonstrate that COL1A1, a key gene from VI gene cluster 2 that we demonstrated is significantly associated with predicted myofibroblast proportion within the stRNAseq data, shows a similar pattern within a VI+ tumor sample to THY-1 protein, a known marker of myofibroblasts/cancer associated fibroblasts. (Fig. 17A) Expression pattern of COL1A1 in Visium stRNAseq data. (Fig. 17B). Expression pattern of COL1A1 RNA by ISH RNAscope labeling. (Fig. 17C) Expression pattern of COL1A1 protein by IHC. (Fig. 17D) Expression pattern of THY-1 protein by IHC.
[0033] Fig. 18 demonstrates that spatial transcriptomics of early-stage LU AD reveals an association of VI gene clusters with specific LU AD histopathology features. Figure depicts spotwise scoring of VI- and VI+ stRNA-seq samples for expression of VI gene expression clusters in spots annotated with high-grade LU AD patterns (solid, micropapillary, and cribriform). P values are derived form a type II Anova of a linear mixed model predicting gene expression with sample as a random effect and high-grade pattern as a fixed effect.
[0034] Fig. 19 demonstrates that a VI predictor derived from the signature validates in an independent stage I LU AD cohort. Figure depicts the predictor association with outcome across Stage I LU AD patient cohorts (discovery (RFS), validation (RFS), TRACERx (RFS), TCGA (OS), Uppsala (OS)). P values were each calculated using univariate cox proportional hazards regression.
[0035] Figs. 20A-20B demonstrate that the predictor can identify patients with high risk as measured by 5-year RFS, with improvement over TNM staging.
DETAILED DESCRIPTION
[0036] As described herein, the inventors have identified characteristics of lung cancer which are indicative of vascular invasion. Because these characteristics are associated with a pathological feature of the cancer and not a specific prognosis or therapeutic response, the characteristics are more robust and broadly applicable as they are not influenced by patient comorbidities or variability in local treatment centers. This permits clinicians to reliably identify the patients whose tumors are aggressive by nature of vascular invasion. These patients are in need of aggressive treatment, while patients without vascular invasion can be treated with less aggressive approaches which carry less risk of side effects, morbidity, and mortality.
[0037] The relevant characteristics, e.g., a multi-gene expression based classifier, are therefore used to identify or predict angioinvasive lung adenocarcinoma. This biomarker can predict vascular invasion based on pre-surgical lung nodule biopsy and impact decision making about the extent of surgical resection, neo-adjuvant, or other ablative therapies. It can also be used on resected tumor tissue to detect occult VI or guide the use of adjuvant therapy. It is contemplated herein that the biomarker can also be applied to pre-surgical needle biopsy tissues from lung nodules, therefore being able to predict whether a tumor has vascular invasion prior to resection. Further variations can include measuring the biomarker in circulating tumor cells or from circulating tumor DNA or by staining of tumor material for biomarker components.
[0038] In some embodiments of any of the aspects, the methods described herein relate to determining tumor pathology (specifically, a novel grading approach to tumor pathology) rather than patient outcome, which distinguishes this method from existing molecular biomarkers that predict recurrence or overall survival in NSCLC, which may be influenced by patient comorbidities, treatment response and standard of care. In some embodiments of any of the aspects, the methods described herein determine gene expression in pre-surgical biopsies. Such determinations in pre-surgical biopsies permit decision making prior to curative therapy, as opposed to existing technologies that predict aggressiveness in an adjuvant setting.
[0039] In one aspect of any of the embodiments, described herein is a method comprising, determining the expression of at least one gene selected from the group consisting of: SHR00M4; NBEAL1; MUC16; NOTCH3; H19; COL1A1; and GARS1 in a subject with lung cancer.
[0040] In some embodiments of any of the aspects, the at least one gene comprises SHR00M4. In some embodiments of any of the aspects, the at least one gene comprises NBEAL1. In some embodiments of any of the aspects, the at least one gene comprises SHR00M4 and NBEAL1. In some embodiments of any of the aspects, the at least one gene comprises SHR00M4; NBEAL1; MUC16; NOTCH3; H19; COL1A1; and GARS1. In some embodiments of any of the aspects, the at least one gene comprises SHR00M4; NBEAL1; MUC16; NOTCH3; H19; and COL1A1. In some embodiments of any of the aspects, the at least one gene comprises SHR00M4; NBEAL1; and MUC16. In some embodiments of any of the aspects, the at least one gene comprises SHROOM4; NBEAL1; MUC16; and NOTCH3. In some embodiments of any of the aspects, the at least one gene comprises MUC16. [0041] In some embodiments of any of the aspects, the at least one gene is SHROOM4. In some embodiments of any of the aspects, the at least one gene is NBEAL1. In some embodiments of any of the aspects, the at least one gene is SHROOM4 and NBEAL1. In some embodiments of any of the aspects, the at least one gene is SHROOM4; NBEAL1; MUC16; NOTCH3; H19; COL1A1; and GARS1. In some embodiments of any of the aspects, the at least one gene is SHR00M4; NBEAL1; MUC16; N0TCH3; H19; and COL1A1. In some embodiments of any of the aspects, the at least one gene is SHR00M4; NBEAL1; and MUC16. In some embodiments of any of the aspects, the at least one gene is SHR00M4; NBEAL1; MUC16; and N0TCH3. In some embodiments of any of the aspects, the at least one gene is MUC16.
[0042] In one aspect of any of the embodiments, described herein is a method comprising, determining the expression of: at least one gene selected from the group consisting of: MKI67; ASPM; ATAD2; TOP2A; FANCI; PLOD2; NCAPG2; CENPF; KPNA2; BRCA1; HLTF; and FANCA; at least one gene selected from the group consisting of: COL1A2; COL3A1; COL1A1; and COL6A3; at least one gene selected from the group consisting of: PRKDC; TPI1; PKM; GAPDH;
AHNAK2; YWHAZ; ATP13A3; GARS1; CP; MUC16; TUBA1C; H19; PPFIA1; NOTCH3; ACLY; GCLC; CAD; and CCT6A; or at least one gene selected from the group consisting of: SPTBN1; SFTPB; LRRK2; AKAP13; CHD2; EPAS1; ATP 11 A; ROS1; MTUS1; ANKHD1; NBEAL1; MPRIP; SHROOM4; and TET2.
[0043] In one aspect of any of the embodiments, described herein is a method comprising, determining the expression of: at least one gene selected from the group consisting of: MKI67; ASPM; ATAD2; TOP2A; FANCI; PLOD2; NCAPG2; CENPF; KPNA2; BRCA1; HLTF; and FANCA; at least one gene selected from the group consisting of: COL1A2; COL3A1; COL1A1; and COL6A3; at least one gene selected from the group consisting of: PRKDC; TPI1; PKM; GAPDH;
AHNAK2; YWHAZ; ATP13A3; GARS1; CP; MUC16; TUBA1C; H19; PPFIA1; NOTCH3; ACLY; GCLC; CAD; and CCT6A; and at least one gene selected from the group consisting of: SPTBN1; SFTPB; LRRK2; AKAP13; CHD2; EPAS1; ATP 11 A; ROS1; MTUS1; ANKHD1; NBEAL1; MPRIP; SHROOM4; and TET2.
[0044] In one aspect of any of the embodiments, described herein is a method comprising, determining the expression of: at least one gene selected from the group consisting of: COL1A2; COL3A1; COL1A1; and COL6A3; at least one gene selected from the group consisting of: PRKDC; TPI1; PKM; GAPDH; AHNAK2; YWHAZ; ATP13A3; GARS1; CP; MUC16; TUBA1C; H19; PPFIA1; NOTCH3; ACLY; GCLC; CAD; and CCT6A; or at least one gene selected from the group consisting of: SPTBN1; SFTPB; LRRK2; AKAP13;
CHD2; EPAS1; ATP 11 A; ROS1; MTUS1; ANKHD1; NBEAL1; MPRIP; SHROOM4; and TET2.
[0045] In one aspect of any of the embodiments, described herein is a method comprising determining the expression of: at least one gene selected from the group consisting of: COL1A2; COL3A1; COL1A1; and COL6A3; at least one gene selected from the group consisting of: PRKDC; TPI1; PKM; GAPDH;
AHNAK2; YWHAZ; ATP13A3; GARS1; CP; MUC16; TUBA1C; H19; PPFIA1; NOTCH3;
ACLY; GCLC; CAD; and CCT6A; and at least one gene selected from the group consisting of: SPTBN1; SFTPB; LRRK2; AKAP13; CHD2; EPAS1; ATP 11 A; ROS1; MTUS1; ANKHD1; NBEAL1; MPRIP; SHROOM4; and TET2.
[0046] In one aspect of any of the embodiments, described herein is a method comprising determining the expression of: at least one gene selected from the group consisting of: MKI67; ASPM; ATAD2; TOP2A;
FANCI; and PLOD2; at least one gene selected from the group consisting of: COL1A2; COL3A1; COL1A1; and COL6A3; at least one gene selected from the group consisting of: PRKDC; TPI1; PKM; GAPDH;
AHNAK2; and YWHAZ; or at least one gene selected from the group consisting of: SPTBN1; SFTPB; LRRK2; AKAP13;
CHD2; and EPAS1.
[0047] In one aspect of any of the embodiments, described herein is a method comprising, determining the expression of: at least one gene selected from the group consisting of: MKI67; ASPM; ATAD2; and TOP2A; at least one gene selected from the group consisting of: COL1A2; COL3A1; COL1A1; and COL6A3; at least one gene selected from the group consisting of: PRKDC; TPI1; PKM; and GAPDH; or at least one gene selected from the group consisting of: SPTBN1; SFTPB; LRRK2; and AKAP13.
[0048] In one aspect of any of the embodiments, described herein is a method comprising determining the expression of: at least one gene selected from the group consisting of: MKI67; and ASPM; at least one gene selected from the group consisting of: COL1A2; and COL3A1; at least one gene selected from the group consisting of: PRKDC; and TPI1; or at least one gene selected from the group consisting of: SPTBN1; and SFTPB. [0049] In one aspect of any of the embodiments, described herein is a method comprising determining the expression of: at least one gene selected from the group consisting of: MKI67; and ASPM; at least one gene selected from the group consisting of: COL1A2; and COL3A1; at least one gene selected from the group consisting of: PRKDC; and TPI1; or at least one gene selected from the group consisting of: SHROOM4; and NBEAL1.
[0050] In one aspect of any of the embodiments, described herein is a method comprising determining the expression of: at least one gene selected from the group consisting of: MKI67; ASPM; ATAD2; TOP2A;
FANCI; and PLOD2; at least one gene selected from the group consisting of: COL1A2; COL3A1; COL1A1; and COL6A3; at least one gene selected from the group consisting of: PRKDC; TPI1; PKM; GAPDH;
AHNAK2; and YWHAZ; and at least one gene selected from the group consisting of: SPTBN1; SFTPB; LRRK2; AKAP13;
CHD2; and EPAS1.
[0051] In one aspect of any of the embodiments, described herein is a method comprising determining the expression of: at least one gene selected from the group consisting of: MKI67; ASPM; ATAD2; and TOP2A; at least one gene selected from the group consisting of: COL1A2; COL3A1; COL1A1; and COL6A3; at least one gene selected from the group consisting of: PRKDC; TPI1; PKM; and GAPDH; and at least one gene selected from the group consisting of: SPTBN1; SFTPB; LRRK2; and AKAPI3.
[0052] In one aspect of any of the embodiments, described herein is a method comprising determining the expression of: at least one gene selected from the group consisting of: MKI67; and ASPM; at least one gene selected from the group consisting of: COL1A2; and COL3A1; at least one gene selected from the group consisting of: PRKDC; and TPI1; and at least one gene selected from the group consisting of: SPTBN1; and SFTPB.
[0053] In one aspect of any of the embodiments, described herein is a method comprising determining the expression of: at least one gene selected from the group consisting of: MKI67; and ASPM; at least one gene selected from the group consisting of: COL1A2; and COL3A1; at least one gene selected from the group consisting of: PRKDC; and TPI1; and at least one gene selected from the group consisting of: SHROOM4; and NBEAL1. [0054] In one aspect of any of the embodiments, described herein is a method comprising determining the expression of: at least one gene selected from the group consisting of: COL1A2; COL3A1; COL1A1; and COL6A3; at least one gene selected from the group consisting of: PRKDC; TPI1; PKM; GAPDH;
AHNAK2; and YWHAZ; and at least one gene selected from the group consisting of: SPTBN1; SFTPB; LRRK2; AKAP13;
CHD2; and EPAS1.
[0055] In one aspect of any of the embodiments, described herein is a method comprising determining the expression of: at least one gene selected from the group consisting of: COL1A2; COL3A1; COL1A1; and COL6A3; at least one gene selected from the group consisting of: PRKDC; TPI1; PKM; and GAPDH; and at least one gene selected from the group consisting of: SPTBN1; SFTPB; LRRK2; and AKAPI3.
[0056] In one aspect of any of the embodiments, described herein is a method comprising determining the expression of: at least one gene selected from the group consisting of: COL1A2; and COL3A1; at least one gene selected from the group consisting of: PRKDC; and TPI1; and at least one gene selected from the group consisting of: SPTBN1; and SFTPB.
[0057] In one aspect of any of the embodiments, described herein is a method comprising determining the expression of: at least one gene selected from the group consisting of: COL1A2; and COL3A1; at least one gene selected from the group consisting of: PRKDC; and TPI1; and at least one gene selected from the group consisting of: SHROOM4 and NBEAL1.
[0058] In one aspect of any of the embodiments, described herein is a method comprising determining the expression of:
MKI67; ASPM; ATAD2; TOP2A; FANCI; and PLOD2;
COL1A2; COL3A1; COL1A1; and COL6A3;
PRKDC; TPI1; PKM; GAPDH; AHNAK2; and YWHAZ; and
SPTBN1; SFTPB; LRRK2; AKAP13; CHD2; and EPAS1.
[0059] In one aspect of any of the embodiments, described herein is a method comprising determining the expression of:
MKI67; ASPM; ATAD2; and TOP2A;
COL1A2; COL3A1; COL1A1; and COL6A3;
PRKDC; TPI1; PKM; and GAPDH; and
SPTBN1; SFTPB; LRRK2; and AKAP13. [0060] In one aspect of any of the embodiments, described herein is a method comprising determining the expression of:
MKI67; and ASPM;
COL1A2; and COL3A1;
PRKDC; and TPI1; and
SPTBN1; and SFTPB.
[0061] In one aspect of any of the embodiments, described herein is a method comprising determining the expression of:
MKI67; and ASPM;
COL1A2; and COL3A1;
PRKDC; and TPI1; and
SHR00M4 and NBEAL1.
[0062] In one aspect of any of the embodiments, described herein is a method comprising determining the expression of:
COL1A2; COL3A1; COL1A1; and COL6A3;
PRKDC; TPI1; PKM; GAPDH; AHNAK2; and YWHAZ; and
SPTBN1; SFTPB; LRRK2; AKAP13; CHD2; and EPAS1.
[0063] In one aspect of any of the embodiments, described herein is a method comprising determining the expression of:
COL1A2; COL3A1; COL1A1; and COL6A3;
PRKDC; TPI1; PKM; and GAPDH; and
SPTBN1; SFTPB; LRRK2; and AKAP13.
[0064] In one aspect of any of the embodiments, described herein is a method comprising determining the expression of:
COL1A2; and COL3A1;
PRKDC; and TPI1; and
SPTBN1; and SFTPB.
[0065] In one aspect of any of the embodiments, described herein is a method comprising determining the expression of:
COL1A2; and COL3A1;
PRKDC; and TPI1; and
SHROOM4 and NBEAL1.
[0066] The sequences for the genes described herein are known in the art, e.g., the human sequences for the genes described herein are available in the NCBI database, e.g., associated with the EntrezID provided in the tables herein, or the ENSEMBL database. The NCBI database provides the sequences of homologs and orthologs for numerous species. In some embodiments of any of the aspects, a gene, mRNA, or protein has the sequence provided in the NCBI database for that ID number as of March 24, 2025. Where reference is made to database entries, e.g., NCBI database entries, reference is made to the information and sequences available as of March 24, 2025 under the indicated ID and/or Accession Number.
[0067] In some embodiments of any of the aspects, a gene expression product has a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or greater sequence identity to a sequence provided in the NCBI database for that ID number. In some embodiments of any of the aspects, a gene expression product has a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or greater sequence identity to a sequence provided in the NCBI database forthat ID number and retaining the same activity as the reference sequence. In some embodiments of any of the aspects, a gene expression product has a sequence with at least at least 95% sequence identity to a sequence provided in the NCBI database for that ID number and retaining the same activity as the reference sequence. In some embodiments of any of the aspects, a gene expression product has a sequence with at least 95% sequence identity to a sequence provided in the NCBI database for that ID number and retaining the same activity as the reference sequence.
[0068] Table 4
[0069] In some embodiments of any of the aspects, a SHROOM4 expression product is a polynucleotide having the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, a SHROOM4 expression product is a polynucleotide having a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, a SHROOM4 expression product is a polynucleotide having a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, a SHROOM4 expression product is a polynucleotide having a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, a SHROOM4 expression product is a polynucleotide having a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, a SHROOM4 expression product is a polynucleotide having a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 1.
[0070] In some embodiments of any of the aspects, a SHR00M4 expression product is a polypeptide having the sequence of SEQ ID NO: 2. In some embodiments of any of the aspects, a SHR00M4 expression product is a polypeptide having a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 2. In some embodiments of any of the aspects, a SHROOM4 expression product is a polypeptide having a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 2. In some embodiments of any of the aspects, a SHR00M4 expression product is a polypeptide having a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 2. In some embodiments of any of the aspects, a SHROOM4 expression product is a polypeptide having a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 2. In some embodiments of any of the aspects, a SHROOM4 expression product is a polypeptide having a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 2.
[0071] In some embodiments of any of the aspects, a NBEAL1 expression product is a polynucleotide having the sequence of SEQ ID NO: 3 or 4. In some embodiments of any of the aspects, a NBEAL1 expression product is a polynucleotide having a sequence with at least 80% sequence identity to the sequence of SEQ ID NO:3 or 4. In some embodiments of any of the aspects, a NBEAL1 expression product is a polynucleotide having a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 3 or 4. In some embodiments of any of the aspects, a NBEAL1 expression product is a polynucleotide having a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 3 or 4. In some embodiments of any of the aspects, a NBEAL1 expression product is a polynucleotide having a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 3 or 4. In some embodiments of any of the aspects, a NBEAL1 expression product is a polynucleotide having a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 3 or 4.
[0072] In some embodiments of any of the aspects, a NBEAL1 expression product is a polypeptide having the sequence of SEQ ID NO: 5 or 6. In some embodiments of any of the aspects, a NBEAL1 expression product is a polypeptide having a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 5 or 6. In some embodiments of any of the aspects, a NBEAL1 expression product is a polypeptide having a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 5 or 6. In some embodiments of any of the aspects, a NBEAL1 expression product is a polypeptide having a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 5 or 6. In some embodiments of any of the aspects, a NBEAL1 expression product is a polypeptide having a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 5 or 6. In some embodiments of any of the aspects, a NBEAL1 expression product is a polypeptide having a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 5 or 6.
[0073] In some embodiments of any of the aspects, a MUC16 expression product is a polynucleotide having the sequence of one of SEQ ID NOs: 7-10. In some embodiments of any of the aspects, a MUC16 expression product is a polynucleotide having a sequence with at least 80% sequence identity to the sequence of one of SEQ ID NOs: 7-10. In some embodiments of any of the aspects, a MUC16 expression product is a polynucleotide having a sequence with at least 85% sequence identity to the sequence of one of SEQ ID NOs: 7-10. In some embodiments of any of the aspects, a MUC16 expression product is a polynucleotide having a sequence with at least 90% sequence identity to the sequence of one of SEQ ID NOs: 7-10. In some embodiments of any of the aspects, a MUC16 expression product is a polynucleotide having a sequence with at least 95% sequence identity to the sequence of one of SEQ ID NOs: 7-10. In some embodiments of any of the aspects, a MUC16 expression product is a polynucleotide having a sequence with at least 98% sequence identity to the sequence of one of SEQ ID NOs: 7-10.
[0074] In some embodiments of any of the aspects, a MUC16 expression product is a polypeptide having the sequence of one of SEQ ID NOs: 11-14. In some embodiments of any of the aspects, a MUC16 expression product is a polypeptide having a sequence with at least 80% sequence identity to the sequence of one of SEQ ID NOs: 11-14. In some embodiments of any of the aspects, a MUC16 expression product is a polypeptide having a sequence with at least 85% sequence identity to the sequence of one of SEQ ID NOs: 11-14. In some embodiments of any of the aspects, a MUC16 expression product is a polypeptide having a sequence with at least 90% sequence identity to the sequence of one of SEQ ID NOs: 11-14. In some embodiments of any of the aspects, a MUC16 expression product is a polypeptide having a sequence with at least 95% sequence identity to the sequence of one of SEQ ID NOs: 11-14. In some embodiments of any of the aspects, a MUC16 expression product is a polypeptide having a sequence with at least 98% sequence identity to the sequence of one of SEQ ID NOs: 11-14.
[0075] In some embodiments of any of the aspects, a NOTCH3 expression product is a polynucleotide having the sequence of SEQ ID NO: 15. In some embodiments of any of the aspects, a NOTCH3 expression product is a polynucleotide having a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 15. In some embodiments of any of the aspects, aNOTCH3 expression product is a polynucleotide having a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 15. In some embodiments of any of the aspects, a NOTCH3 expression product is a polynucleotide having a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 15. In some embodiments of any of the aspects, a NOTCH3 expression product is a polynucleotide having a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 15. In some embodiments of any of the aspects, a NOTCH3 expression product is a polynucleotide having a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 15. [0076] In some embodiments of any of the aspects, a NOTCH3 expression product is a polypeptide having the sequence of SEQ ID NO: 16. In some embodiments of any of the aspects, a NOTCH3 expression product is a polypeptide having a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 16. In some embodiments of any of the aspects, a NOTCH3 expression product is a polypeptide having a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 16. In some embodiments of any of the aspects, a NOTCH3 expression product is a polypeptide having a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 16. In some embodiments of any of the aspects, a NOTCH3 expression product is a polypeptide having a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 16. In some embodiments of any of the aspects, a NOTCH3 expression product is a polypeptide having a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 16.
[0077] In some embodiments of any of the aspects, a H19 expression product is a polynucleotide having the sequence of one of SEQ ID NOs: 17-23. In some embodiments of any of the aspects, a H19 expression product is a polynucleotide having a sequence with at least 80% sequence identity to the sequence of one of SEQ ID NOs: 17-23. In some embodiments of any of the aspects, a H19 expression product is a polynucleotide having a sequence with at least 85% sequence identity to the sequence of one of SEQ ID NOs: 17-23. In some embodiments of any of the aspects, a H19 expression product is a polynucleotide having a sequence with at least 90% sequence identity to the sequence of one of SEQ ID NOs: 17-23. In some embodiments of any of the aspects, a H19 expression product is a polynucleotide having a sequence with at least 95% sequence identity to the sequence of one of SEQ ID NOs: 17-23. In some embodiments of any of the aspects, a Hl 9 expression product is a polynucleotide having a sequence with at least 98% sequence identity to the sequence of one of SEQ ID NOs: 17-23.
[0078] In some embodiments of any of the aspects, a COL1A1 expression product is a polynucleotide having the sequence of SEQ ID NO: 24. In some embodiments of any of the aspects, a COL1A1 expression product is a polynucleotide having a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 24. In some embodiments of any of the aspects, a COL1A1 expression product is a polynucleotide having a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 24. In some embodiments of any of the aspects, a COL1A1 expression product is a polynucleotide having a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 24. In some embodiments of any of the aspects, a COL1A1 expression product is a polynucleotide having a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 24. In some embodiments of any of the aspects, a COL1A1 expression product is a polynucleotide having a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 24.
[0079] In some embodiments of any of the aspects, a COL1A1 expression product is a polypeptide having the sequence of SEQ ID NO:25. In some embodiments of any of the aspects, a COL1A1 expression product is a polypeptide having a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 25. In some embodiments of any of the aspects, a COL1A1 expression product is a polypeptide having a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 25. In some embodiments of any of the aspects, a C0L1A1 expression product is a polypeptide having a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 25. In some embodiments of any of the aspects, a COL1A1 expression product is a polypeptide having a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 25. In some embodiments of any of the aspects, a COL1A1 expression product is a polypeptide having a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 25.
[0080] In some embodiments of any of the aspects, a GARS 1 expression product is a polynucleotide having the sequence of SEQ ID NO: 26 or 27. In some embodiments of any of the aspects, a GARS1 expression product is a polynucleotide having a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 26 or 27. In some embodiments of any of the aspects, a GARS1 expression product is a polynucleotide having a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 26 or 27. In some embodiments of any of the aspects, a GARS 1 expression product is a polynucleotide having a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 26 or 27. In some embodiments of any of the aspects, a GARS1 expression product is a polynucleotide having a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 26 or 27. In some embodiments of any of the aspects, a GARS1 expression product is a polynucleotide having a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 26 or 27.
[0081] In some embodiments of any of the aspects, a GARS 1 expression product is a polypeptide having the sequence of SEQ ID NO:28 or 29. In some embodiments of any of the aspects, a GARS 1 expression product is a polypeptide having a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 28 or 29. In some embodiments of any of the aspects, a GARS1 expression product is a polypeptide having a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 28 or 29. In some embodiments of any of the aspects, a GARS1 expression product is a polypeptide having a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 28 or 29. In some embodiments of any of the aspects, a GARS1 expression product is a polypeptide having a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 28 or 29. In some embodiments of any of the aspects, a GARS1 expression product is a polypeptide having a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 28 or 29.
[0082] In some embodiments of any of the aspects, determining the expression is measuring the expression level. In some embodiments of any of the aspects, determining the expression is detecting the expression level. In some embodiments of any of the aspects, determining the expression is measuring the level or amount of at least one expression product. In some embodiments of any of the aspects, determining the expression is detecting the level or amount of at least one expression product. [0083] In some embodiments of the any of the aspects, the expression level is the mRNA level. In some embodiments of the any of the aspects, the expression level is the protein level. [0084] In some embodiments of any of the aspects, decreased expression is a level which is decreased or less than a reference level. A level which is less than a reference level can be a level which is less by at least about 10%, at least about 20%, at least about 50%, at least about 60%, at least about 80%, at least about 90%, or less relative to the reference level. In some embodiments of any of the aspects, a level which is less than a reference level can be a level which is statistically significantly less than the reference level.
[0085] In some embodiments of any of the aspects, increased expression is a level which is increased or more or greater than a reference level. A level which is more than a reference level can be a level which is greater by at least about 10%, at least about 20%, at least about 50%, at least about 60%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 500% or more than the reference level. In some embodiments of any of the aspects, a level which is more than a reference level can be a level which is statistically significantly greater than the reference level.
[0086] In some embodiments of any of the aspects, the reference can be a level of the target molecule in a population of subjects who do not have or are not diagnosed as having, and/or do not exhibit signs or symptoms of lung cancer. In some embodiments of any of the aspects, the reference can be a level of the target molecule in a population of subjects who do not have or are not diagnosed as having, and/or do not exhibit signs or symptoms of lung adenocarcinoma. In some embodiments of any of the aspects, the reference can be a level of the target molecule in a population of subjects who do not have or are not diagnosed as having, and/or do not exhibit signs or symptoms of stage I lung adenocarcinoma. In some embodiments of any of the aspects, the reference can be a level of the target molecule in a population of subjects who do not have or are not diagnosed as having, and/or do not exhibit signs or symptoms of vascular invasive lung adenocarcinoma. In some embodiments of any of the aspects, the reference can also be a level of expression of the target molecule in a control sample, a pooled sample of control individuals or a numeric value or range of values based on the same. In some embodiments of any of the aspects, the reference can be the level of a target molecule in a sample obtained from the same subject at an earlier point in time, e.g., the methods described herein can be used to determine if a subject’s sensitivity or response to a given therapy is changing over time.
[0087] In some embodiments of any of the aspects, an increase or decrease in expression is an increase or decrease relative to an average level for subjects with lung cancer. In some embodiments of any of the aspects, an increase or decrease in expression is an increase or decrease relative to an average level for subjects with non-small cell lung cancer (NSCLC). In some embodiments of any of the aspects, an increase or decrease in expression is an increase or decrease relative to an average level for subjects with lung adenocarcinoma. In some embodiments of any of the aspects, an increase or decrease in expression is an increase or decrease relative to an average level for subjects with stage I lung adenocarcinoma. [0088] In some embodiments of any of the aspects, an increase or decrease in expression is an increase or decrease relative to an average level for subjects with lung cancer which does not exhibit vascular invasion. In some embodiments of any of the aspects, an increase or decrease in expression is an increase or decrease relative to an average level for subjects with non-small cell lung cancer (NSCLC) which does not exhibit vascular invasion. In some embodiments of any of the aspects, an increase or decrease in expression is an increase or decrease relative to an average level for subjects with lung adenocarcinoma which does not exhibit vascular invasion. In some embodiments of any of the aspects, an increase or decrease in expression is an increase or decrease relative to an average level for subjects with stage I lung adenocarcinoma which does not exhibit vascular invasion.
[0089] In some embodiments of any of the aspects, the level of expression products of no more than 200 other genes is determined. In some embodiments of any of the aspects, the level of expression products of no more than 100 other genes is determined. In some embodiments of any of the aspects, the level of expression products of no more than 20 other genes is determined. In some embodiments of any of the aspects, the level of expression products of no more than 10 other genes is determined.
[0090] In some embodiments of the foregoing aspects, the expression level of a given gene can be normalized relative to the expression level of one or more reference genes or reference proteins.
[0091] In some embodiments, the reference level can be the level in a sample of similar cell type, sample type, sample processing, and/or obtained from a subject of similar age, sex and other demographic parameters as the sample/subject for which the level of the at least one gene is to be determined. In some embodiments, the test sample and control reference sample are of the same type, that is, obtained from the same biological source, and comprising the same composition, e.g. the same number and type of cells.
[0092] The term “sample” or “test sample” as used herein denotes a sample taken or isolated from a biological organism, e.g., cells or expression product samples from a subject. In some embodiments of any of the aspects, the present invention encompasses several examples of a biological sample. In some embodiments of any of the aspects, the biological sample is cells, or tissue, or peripheral blood, or bodily fluid. Exemplary biological samples include, but are not limited to, a biopsy, a tumor sample, biofluid sample; blood; serum; plasma; urine; sperm; mucus; tissue biopsy; organ biopsy; synovial fluid; bile fluid; cerebrospinal fluid; mucosal secretion; effusion; sweat; saliva; and/or tissue sample etc. The term also includes a mixture of the above-mentioned samples. The term “test sample” also includes untreated or pretreated (or pre-processed) biological samples. In some embodiments of any of the aspects, a test sample can comprise cells from a subject.
[0093] In some embodiments of any of the aspects, the expression is determined in a sample, or is the expression in a sample comprising material selected from the group consisting of: tumor cells, cancer cells from the airway, circulating tumor cells, biopsied lung tissue, a lung tissue section, circulating tumor DNA (ctDNA), bronchial brushing sample, nasal epithelial sample, bronchial biopsy, bronchial epithelium sample, airway epithelium sample, nasal brushing sample, and bronchoalveolar lavage sample. In some embodiments of any of the aspects, the expression is determined in a sample, or is the expression in a sample comprising material selected from the group consisting of: tumor cells, cancer cells from the airway, circulating tumor cells, biopsied lung tissue, a lung tissue section, and circulating tumor DNA (ctDNA). In some embodiments of any of the aspects, the expression is determined in a sample, or is the expression in a sample comprising biopsied lung tissue.
[0094] In some embodiments of any of the aspects, the sample is tumor cells, cancer cells from the airway, circulating tumor cells, biopsied lung tissue, a lung tissue section, circulating tumor DNA (ctDNA), bronchial brushing sample, nasal epithelial sample, bronchial biopsy, bronchial epithelium sample, airway epithelium sample, nasal brushing sample, or bronchoalveolar lavage sample. In some embodiments of any of the aspects, the sample is: tumor cells, cancer cells from the airway, circulating tumor cells, biopsied lung tissue, a lung tissue section, or circulating tumor DNA (ctDNA). In some embodiments of any of the aspects, the sample is biopsied lung tissue.
[0095] The test sample can be obtained by removing a sample from a subject but can also be accomplished by using a previously isolated sample (e.g. isolated at a prior timepoint and isolated by the same or another person).
[0096] In some embodiments of any of the aspects, the test sample can be an untreated test sample. As used herein, the phrase “untreated test sample” refers to a test sample that has not had any prior sample pre-treatment except for dilution and/or suspension in a solution. Exemplary methods for treating a test sample include, but are not limited to, centrifugation, fdtration, sonication, homogenization, heating, freezing and thawing, and combinations thereof. In some embodiments of any of the aspects, the test sample can be a frozen test sample, e.g., a frozen tissue. The frozen sample can be thawed before employing methods, assays and systems described herein. After thawing, a frozen sample can be centrifuged before being subjected to methods, assays and systems described herein. In some embodiments of any of the aspects, the test sample is a clarified test sample, for example, by centrifugation and collection of a supernatant comprising the clarified test sample. In some embodiments of any of the aspects, a test sample can be a pre-processed test sample, for example, supernatant or filtrate resulting from a treatment selected from the group consisting of centrifugation, filtration, thawing, purification, and any combinations thereof. In some embodiments of any of the aspects, the test sample can be treated with a chemical and/or biological reagent. Chemical and/or biological reagents can be employed to protect and/or maintain the stability of the sample, including biomolecules (e.g., nucleic acid and protein) therein, during processing. One exemplary reagent is a protease inhibitor, which is generally used to protect or maintain the stability of protein during processing. The skilled artisan is well aware of methods and processes appropriate for pre-processing of biological samples required for determination of the level of an expression product as described herein. [0097] In some embodiments of any of the aspects, the methods, assays, and systems described herein can further comprise a step of obtaining or having obtained a test sample from a subject. In some embodiments of any of the aspects, the subject can be a human subject. In some embodiments of any of the aspects, the subject can be a subject in need of treatment for (e.g. having or diagnosed as having) lung cancer or a subject at risk of or at increased risk of developing lung cancer as described elsewhere herein.
[0098] In some embodiments of any of the aspects, the sample obtained from a subject can be a biopsy sample. In some embodiments of any of the aspects, the sample obtained from a subject can be a blood or serum sample.
[0099] In some embodiments of any of the aspects, the lung cancer is non-small cell lung cancer (NSCLC). In some embodiments of any of the aspects, the lung cancer is stage I and/or stage II non- small cell lung cancer (NSCLC). In some embodiments of any of the aspects, the lung cancer is stage I non-small cell lung cancer (NSCLC). In some embodiments of any of the aspects, the lung cancer is stage II non-small cell lung cancer (NSCLC).
[00100] In some embodiments of any of the aspects, the lung cancer is lung adenocarcinoma. In some embodiments of any of the aspects, the lung cancer is vascular invasive lung adenocarcinoma. In some embodiments of any of the aspects, the lung cancer is early-stage lung adenocarcinoma. In some embodiments of any of the aspects, the lung cancer is early-stage/organ confined lung adenocarcinoma. In some embodiments of any of the aspects, the lung cancer is stage I lung adenocarcinoma and/or stage II lung adenocarcinoma. In some embodiments of any of the aspects, the lung cancer is stage I lung adenocarcinoma. In some embodiments of any of the aspects, the lung cancer is stage II lung adenocarcinoma. One of skill in the art is aware of lung cancer staging and how to perform it. An exemplary staging system can be found in Asamura, H. (Ed). (2016) The IASLC Staging Handbook in Thoracic Oncology (2nd Edition) includes 8th Edition Tumor, Node, and Metastasis Classification (TNM) information for Lung Cancer, Plural Mesothelioma, Thymic Malignancies, and Carcinoma of the Oesophagus and of Oesophagogastric Junction. International Association for the Study of Lung Cancer; which is incorporated by reference herein in its entirety. In some embodiments of any of the aspects, the lung cancer comprises vascular invasion.
[00101] In some embodiments of any of the aspects, the subject has or is diagnosed as having lung cancer. In some embodiments of any of the aspects, the subject has or is diagnosed as having non-small cell lung cancer (NSCLC). In some embodiments of any of the aspects, the subject has or is diagnosed as having lung adenocarcinoma. In some embodiments of any of the aspects, the subject has or is diagnosed as having stage I lung adenocarcinoma.
[00102] In some embodiments, the methods described herein relate to treating a subject having or diagnosed as having lung cancer. Subjects having lung cancer can be identified by a physician using current methods of diagnosing lung cancer. Symptoms and/or complications of lung cancer which characterize these conditions and aid in diagnosis are well known in the art and include but are not limited to, coughing, coughing blood, shortness of breath, chest pain, wheezing, hoarseness, difficulty breathing, unexplained weight loss, bone pain, and headaches. Tests that may aid in a diagnosis of, e.g. lung cancer include, but are not limited to, x-rays, CT scan, sputum cytology, or biopsies. A family history of lung cancer, prior history of lung cancer, presence of other chronic lung disease such as COPD, or exposure to risk factors for lung cancer (e.g. radon or asbestos exposure) can also aid in determining if a subject is likely to have lung cancer or in making a diagnosis of lung cancer.
[00103] As described herein, the characteristics, biomarkers, and/or signatures described herein identify those subjects experiencing or at risk of experiencing vascular invasion. If vascular invasion is present or likely to be present, the goal of treatment is to aggressively reduce tumor volume. Patients without vascular invasion can be provided less aggressive treatments.
[00104] In one aspect of any of the embodiments, described herein is a method of treating lung cancer, the method comprising administering adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and/or lymphadenectomy to a subject having decreased expression of at least one gene selected from the group consisting of: SHR00M4 and NBEAL1, and/or increased expression of at least one gene selected from the group consisting of: MUC16, NOTCH3, H19, COL1A1, and GARS1. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, and/or a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy and/or a lobectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy and/or a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, and/or lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy and/or lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy and/or lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, a lobectomy, and/or lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, a lobectomy, and/or lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy and ablation therapy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy and ablation therapy. In some embodiments of any of the aspects, the subject is administered a lobectomy and ablation therapy. In some embodiments of any of the aspects, the subject is administered lymphadenectomy and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered, neoadjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered ablation therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered radiation therapy and a lobectomy. In some embodiments of any of the aspects, the subject is administered radiation therapy and lymphadenectomy.
[00105] In one aspect of any of the embodiments, described herein is a method of treating lung cancer, the method comprising administering adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and/or lymphadenectomy to a subject having decreased expression of at least one gene selected from the group consisting of: SHROOM4 and NBEAL1, and/or increased expression of at least one gene selected from the group consisting of: MUC16, NOTCH3, H19, and COL1A1. In some embodiments of any of the aspects, the subject is administered adjuvant therapy and ablation therapy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy and ablation therapy. In some embodiments of any of the aspects, the subject is administered a lobectomy and ablation therapy. In some embodiments of any of the aspects, the subject is administered lymphadenectomy and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered, neoadjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered ablation therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered radiation therapy and a lobectomy. In some embodiments of any of the aspects, the subject is administered radiation therapy and lymphadenectomy. [00106] In one aspect of any of the embodiments, described herein is a method of treating lung cancer, the method comprising administering adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and/or lymphadenectomy to a subject having decreased expression of at least one gene selected from the group consisting of: SHR00M4 and NBEAL1, and increased expression of at least one gene selected from the group consisting of: MUC16, NOTCH3, H19, COL1A1, and GARS1. In some embodiments of any of the aspects, the subject is administered adjuvant therapy and ablation therapy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy and ablation therapy. In some embodiments of any of the aspects, the subject is administered a lobectomy and ablation therapy. In some embodiments of any of the aspects, the subject is administered lymphadenectomy and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered, neoadjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered ablation therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered radiation therapy and a lobectomy. In some embodiments of any of the aspects, the subject is administered radiation therapy and lymphadenectomy.
[00107] In one aspect of any of the embodiments, described herein is a method of treating lung cancer, the method comprising administering adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and/or lymphadenectomy to a subject having decreased expression of at least one gene selected from the group consisting of: SHROOM4 and NBEAL1, and increased expression of at least one gene selected from the group consisting of: MUC16, NOTCH3, H19, and COL1A1. In some embodiments of any of the aspects, the subject is administered adjuvant therapy and ablation therapy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy and ablation therapy. In some embodiments of any of the aspects, the subject is administered a lobectomy and ablation therapy. In some embodiments of any of the aspects, the subject is administered lymphadenectomy and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered, neoadjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered ablation therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered radiation therapy and a lobectomy. In some embodiments of any of the aspects, the subject is administered radiation therapy and lymphadenectomy.
[00108] In one aspect of any of the embodiments, described herein is a method of treating lung cancer, the method comprising administering adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and/or lymphadenectomy to a subject having increased expression of at least one gene selected from the group consisting of: MUC16, NOTCH3, H19, and COL1A1. In some embodiments of any of the aspects, the subject is administered adjuvant therapy and ablation therapy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy and ablation therapy. In some embodiments of any of the aspects, the subject is administered a lobectomy and ablation therapy. In some embodiments of any of the aspects, the subject is administered lymphadenectomy and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered, neoadjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered ablation therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered radiation therapy and a lobectomy. In some embodiments of any of the aspects, the subject is administered radiation therapy and lymphadenectomy.
[00109] In one aspect of any of the embodiments, described herein is a method of treating lung cancer, the method comprising administering adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and/or lymphadenectomy to a subject having increased expression of at least one gene selected from the group consisting of: MUC16, NOTCH3, H19, COL1A1, and GARS1. In some embodiments of any of the aspects, the subject is administered adjuvant therapy and ablation therapy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy and ablation therapy. In some embodiments of any of the aspects, the subject is administered a lobectomy and ablation therapy. In some embodiments of any of the aspects, the subject is administered lymphadenectomy and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered, neoadjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered ablation therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered radiation therapy and a lobectomy. In some embodiments of any of the aspects, the subject is administered radiation therapy and lymphadenectomy. [00110] In one aspect of any of the embodiments, described herein is a method of treating lung cancer, the method comprising administering adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and/or lymphadenectomy to a subject having increased expression of MUC16. In some embodiments of any of the aspects, the subject is administered adjuvant therapy and ablation therapy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy and ablation therapy. In some embodiments of any of the aspects, the subject is administered a lobectomy and ablation therapy. In some embodiments of any of the aspects, the subject is administered lymphadenectomy and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered, neoadjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered ablation therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered radiation therapy and a lobectomy. In some embodiments of any of the aspects, the subject is administered radiation therapy and lymphadenectomy.
[00111] In one aspect of any of the embodiments, described herein is a method of treating lung cancer, the method comprising administering adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and/or lymphadenectomy to a subject having decreased expression of at least one gene selected from the group consisting of: SHROOM4 and NBEAL1. In some embodiments of any of the aspects, the subject is administered adjuvant therapy and ablation therapy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy and ablation therapy. In some embodiments of any of the aspects, the subject is administered a lobectomy and ablation therapy. In some embodiments of any of the aspects, the subject is administered lymphadenectomy and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered, neoadjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered ablation therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered radiation therapy and a lobectomy. In some embodiments of any of the aspects, the subject is administered radiation therapy and lymphadenectomy.
[00112] In one aspect of any of the embodiments, described herein is a method of treating lung cancer, the method comprising administering adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and/or lymphadenectomy to a subject having decreased expression of SHROOM4 and NBEAL1. In some embodiments of any of the aspects, the subject is administered adjuvant therapy and ablation therapy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy and ablation therapy. In some embodiments of any of the aspects, the subject is administered a lobectomy and ablation therapy. In some embodiments of any of the aspects, the subject is administered lymphadenectomy and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered, neoadjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered ablation therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered radiation therapy and a lobectomy. In some embodiments of any of the aspects, the subject is administered radiation therapy and lymphadenectomy.
[00113] In one aspect of any of the embodiments, described herein is a method of treating lung cancer, the method comprising administering adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and/or lymphadenectomy to a subject having decreased expression of SHROOM4. In some embodiments of any of the aspects, the subject is administered adjuvant therapy and ablation therapy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy and ablation therapy. In some embodiments of any of the aspects, the subject is administered a lobectomy and ablation therapy. In some embodiments of any of the aspects, the subject is administered lymphadenectomy and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered, neoadjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered ablation therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered radiation therapy and a lobectomy. In some embodiments of any of the aspects, the subject is administered radiation therapy and lymphadenectomy.
[00114] In one aspect of any of the embodiments, described herein is a method of treating lung cancer, the method comprising administering adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and/or lymphadenectomy to a subject having decreased expression of NBEAL1. In some embodiments of any of the aspects, the subject is administered adjuvant therapy and ablation therapy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy and ablation therapy. In some embodiments of any of the aspects, the subject is administered a lobectomy and ablation therapy. In some embodiments of any of the aspects, the subject is administered lymphadenectomy and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered, neoadjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered ablation therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered radiation therapy and a lobectomy. In some embodiments of any of the aspects, the subject is administered radiation therapy and lymphadenectomy.
[00115] In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy and a lobectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, and a lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy and ablation therapy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy and ablation therapy. In some embodiments of any of the aspects, the subject is administered a lobectomy and ablation therapy. In some embodiments of any of the aspects, the subject is administered lymphadenectomy and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy.
[00116] In some embodiments of any of the aspects, the subject is administered adjuvant therapy and ablation therapy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy and ablation therapy. In some embodiments of any of the aspects, the subject is administered a lobectomy and ablation therapy. In some embodiments of any of the aspects, the subject is administered lymphadenectomy and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, a lobectomy, lymphadenectomy, and ablation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, ablation therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is radiation therapy, a lobectomy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is ablation therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered ablation therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered neoadjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and lymphadenectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, radiation therapy, and a lobectomy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy, neoadjuvant therapy, and radiation therapy. In some embodiments of any of the aspects, the subject is administered adjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered, neoadjuvant therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered ablation therapy and radiation therapy. In some embodiments of any of the aspects, the subject is administered radiation therapy and a lobectomy. In some embodiments of any of the aspects, the subject is administered radiation therapy and lymphadenectomy.
[00117] As used herein, “adjuvant therapy” refers to a therapy administered after surgery (e.g., lobectomy and/or lymphadenectomy). The goal of adjuvant therapy is to kill any remaining cancer cells with the goal of reducing the chances of recurrence. Adjuvant therapies are generally selected from chemotherapy, radiation therapy, immunotherapy or a combination thereof.
[00118] As used herein, “neo-adjuvant therapy” or “neoadjuvant therapy” refers to a therapy administered before surgery (e.g., lobectomy and/or lymphadenectomy). The goal of neoadjuvant therapy is shrinking a tumor or stopping the spread of cancer to make surgery less invasive and more effective. Neoadjuvant therapies are generally selected from chemotherapy, radiation therapy, immunotherapy or a combination thereof.
[00119] As used herein “ablation therapy” refers to a therapy or procedure that creates conditions that destroy a target tissue by minimally invasive means. Such means include a radio frequency, microwave, freezing, and/or laser, to a target tissue by minimally invasive means. Ablation therapy can include but is not limited to microwave ablation, radiofrequency ablation, and cryoablation. Ablation therapy is often used in patients that cannot tolerate surgery, e.g., heavy smokers with damaged lungs. [00120] In some embodiments of any of the aspects, an adjuvant therapy or neoadjuvant therapy is selected from the group consisting of: tegafur-uracil (UFT), nivolumab, ipilimumab, atezolizumab, pembrolizumab, durvalumab, oleclumab, monalizumab, alectinib, osimertinib, paclitaxel, carboplatin, and platinum doublet. Adjuvant therapy and neoadjuvant therapy are known in the art and discussed further in, e.g., Spicer et al. Journal of Thoracic Oncology 19( 10):P 1373- 1414 (2024); which is incorporated by reference herein in its entirety.
[00121] As used herein, “chemotherapy” refers to any chemical or biological agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth by inhibiting a cellular activity upon which the cancer cell depends for continued survival and/or proliferation. In some aspect of all the embodiments, a chemotherapeutic agent is a cell cycle inhibitor or a cell division inhibitor. Categories of chemotherapeutic agents that are useful in the methods of the invention include alkylating/alkaloid agents, antimetabolites, hormones or hormone analogs, and miscellaneous antineoplastic drugs. Most of these agents are directly or indirectly toxic to cancer cells. In one embodiment, a chemotherapeutic agent is a radioactive molecule. One of skill in the art can readily identify a chemotherapeutic agent of use ( e.g. see Edward Chu et al., Physicians' Cancer Chemotherapy Drug Manual, published by Jones & Bartlett Learning, 2024 (ISBN 1284000001, 978-1284000009); Joseph Loscalzo et al., Harrison's Principles of Internal Medicine, 21st edition, published by McGraw Hill/Medical, 2022 (ISBN 1264268505, 978-1264268504); John E. Neiderhuber et al, Abeloff s Clinical Oncology, 6th Edition, published by Elsevier, 2019 (ISBN 0323476740, 978-0323476744; Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al. , Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2nd ed. 2000 Churchill Livingstone, Inc; Baltzer L, Berkery R (eds): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer D S, Knobf M F, Durivage H J (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993, the contents of which are all incorporated by reference herein in their entireties). In some embodiments, the chemotherapeutic agent can be a cytotoxic chemotherapeutic. The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g. At211, 1131, 1125, Y90, Rel86, Rel88, Sml53, Bi212, P32 and radioactive isotopes of Lu), chemotherapeutic agents, and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof.
[00122] Exemplary chemotherapeutics include an anthracycline (e.g., doxorubicin (e.g., liposomal doxorubicin)), a vinca alkaloid (e.g., vinblastine, vincristine, vindesine, vinorelbine), an alkylating agent (e.g., cyclophosphamide, decarbazine, melphalan, ifosfamide, temozolomide), an antibody (e.g., alemtuzamab, bevacizumab (A vastin®), gemtuzumab, nivolumab (Opdivo®), pembrolizumab (Keytruda®), rituximab (Rituxan®), traztuzumab (Herceptin®) tositumomab), an antimetabolite (including, e.g., folic acid antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors (e.g., fludarabine)), an mTOR inhibitor, a TNFR glucocorticoid induced TNFR related protein (GITR) agonist, a proteasome inhibitor (e.g., aclacinomycin A, gliotoxin or bortezomib), an immunomodulator such as thalidomide or a thalidomide derivative (e.g., lenalidomide (Revlimid®)), a kinase inhibitor (e.g., palbociclib (Ibrance®), or a hormone therapy (e.g., abiraterone acetate (Zytiga®)). General chemotherapeutic agents include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5- deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin (Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine®), daunorubicin citrate liposome injection (DaunoXome®), dexamethasone, docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®, Etopophos®, Toposar®), fludarabine phosphate (Fludara®), 5- fluorouracil (Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, gemcitabine (difluorodeoxycitidine), hydroxyurea (Hydrea®), ibrutinib (Imbruvica®), Idarubicin (Idamycin®), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®), leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine (Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®), mylotarg, paclitaxel (Taxol®), phoenix (Yttrium90/MX-DTPA), pentostatin, polifeprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), topotecan hydrochloride for injection (Hycamptin®), vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine (Navelbine®).
Exemplary alkylating agents include, without limitation, nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes): uracil mustard (Aminouracil Mustard®, Chlorethaminacil®, Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracil nitrogen mustard®, Uracillost®, Uracilmostaza®, Uramustin®, Uramustine®), chlormethine (Mustargen®), cyclophosphamide (Cytoxan®, Neosar®, Clafen®, Endoxan®, Procytox®, Revimmune™), ifosfamide (Mitoxana®), melphalan (Alkeran®), Chlorambucil (Leukeran®), pipobroman (Amedel®, Vercyte®), triethylenemelamine (Hemel®, Hexalen®, Hexastat®), triethylenethiophosphoramine, Temozolomide (Temodar®), thiotepa (Thioplex®, Tepadina®), busulfan (Busilvex®, Myleran®), improsulfan, piposulfan, carmustine (BiCNU®), lomustine (CeeNU®), streptozocin (Zanosar®), and Dacarbazine (DTIC-Dome®). Additional exemplary alkylating agents include, without limitation, Oxaliplatin (Eloxatin®); Temozolomide (Temodar® and Temodal®); Dactinomycin (also known as actinomycin-D, Cosmegen®); Melphalan (also known as L-PAM, L- sarcolysin, and phenylalanine mustard, Alkeran®); Altretamine (also known as hexamethylmelamine (HMM), Hexalen®); Carmustine (BiCNU®); Bendamustine (Treanda®); Busulfan (Busulfex® and Myleran®); carboplatin (Paraplatin®); Lomustine (also known as CCNU, CeeNU®); Cisplatin (also known as CDDP, Platinol® and Platinol®-AQ); Chlorambucil (Leukeran®); Cyclophosphamide (Cytoxan® and Neosar®); Dacarbazine (also known as DTIC, DIC and imidazole carboxamide, DTIC- Dome®); Altretamine (also known as hexamethylmelamine (HMM), Hexalen®); Ifosfamide (Ifex®); Prednumustine; Procarbazine (Matulane®); Mechlorethamine (also known as nitrogen mustard, mustine and mechloroethamine hydrochloride, Mustargen®); Streptozocin (Zanosar®); Thiotepa (also known as thiophosphoamide, TESPA and TSPA, Thioplex®); Cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune®); and Bendamustine HC1 (Treanda®). Exemplary mTOR inhibitors include, e.g., temsirolimus; ridaforolimus (formally known as deferolimus, (lR,2R,45)-4- [(2R)-2 [(lR,95,I25,I5R,I6E,I8R,I9R,2IR,235,24E,26E,28Z,305,325,35R)-l,I8-dihydroxy-I9,30- dimethoxy-15,17,21,23, 29,35- hexamethyl-2,3,10,14,20-pentaoxo-l l,36-dioxa-4- azatricyclo [30.3.1.04'9] hexatriaconta- 16,24,26,28-tetraen- 12-yl] propyl] -2 -methoxy cyclohexyl dimethylphosphinate, also known as AP23573 and MK8669, and described in PCT Publication No. WO 03/064383); everolimus (Afmitor® or RADOO1); rapamycin (AY22989, Sirolimus®); simapimod (CAS 164301-51-3); emsirolimus, (5-{2,4-Bis[(35,)-3-methylmorpholin-4-yl]pyrido[2,3-(i]pyrimidin-7- yl}-2- methoxyphenyl)methanol (AZD8055); 2-Amino-8-[iraw5,-4-(2-hydroxyethoxy)cyclohexyl]-6- (6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-JJpyrimidin-7(8H)-one (PF04691502, CAS 1013101-36- 4); and N2-[l,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-l-benzopyran-2- yl)morpholinium-4- yl]methoxy]butyl]-L-arginylglycyl-L-a-aspartylL-serine- (SEQ ID NO: 39), inner salt (SF1126, CAS 936487-67-1), and XL765. Exemplary immunomodulators include, e.g., afutuzumab (available from Roche®); pegfdgrastim (Neulasta®); lenalidomide (CC-5013, Revlimid®); thalidomide (Thalomid®), actimid (CC4047); and IRX-2 (mixture of human cytokines including interleukin 1, interleukin 2, and interferon y, CAS 951209-71-5, available from IRX Therapeutics). Exemplary anthracy clines include, e.g., doxorubicin (Adriamycin® and Rubex®); bleomycin (lenoxane®); daunorubicin (dauorubicin hydrochloride, daunomycin, and rubidomycin hydrochloride, Cerubidine®); daunorubicin liposomal (daunorubicin citrate liposome, DaunoXome®); mitoxantrone (DHAD, Novantrone®); epirubicin (Ellence™); idarubicin (Idamycin®, Idamycin PFS®); mitomycin C (Mutamycin®); geldanamycin; herbimycin; ravidomycin; and desacetylravidomycin. Exemplary vinca alkaloids include, e.g., vinorelbine tartrate (Navelbine®), Vincristine (Oncovin®), and Vindesine (Eldisine®)); vinblastine (also known as vinblastine sulfate, vincaleukoblastine and VLB, Alkaban-AQ® and Velban®); and vinorelbine (Navelbine®). Exemplary proteosome inhibitors include bortezomib (Velcade®); carfilzomib (PX- 171-007, (5)-4-Methyl-N-((5)-l-(((5)-4-methyl-l-((R)-2-methyloxiran-2-yl)-l- oxopentan-2- yl)amino)-l-oxo-3-phenylpropan-2-yl)-2-((5,)-2-(2-morpholinoacetamido)-4- phenylbutanamido)-pentanamide); marizomib (NPT0052); ixazomib citrate (MLN-9708); delanzomib (CEP-18770); and O-Methyl-N-[(2-methyl-5-thiazolyl)carbonyl]-L-seryl-O- methyl-N-[(HS')-2-[(2R)-2- methyl-2-oxiranyl]-2-oxo-l-(phenylmethyl)ethyl]- L-serinamide (ONX-0912). Additional exemplary anti-cancer agents also include AMG479, vorinostat, ABT-737, PI-103; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew, Chem. Inti. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2, 2', 2"- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE® Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone -Poulenc Rorer, Antony, France); chloranbucil; GEMZAR® gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine;
NAVELBINE™, vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb.RTM.); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and VEGF-A that reduce cell proliferation.
[00123] Platinum-based chemotherapeutics include cisplatin, carboplatin, oxaliplatin, nedaplatin, and lobaplatin. Cisplatin-based combination chemotherapy comprises one or more cisplatin-based chemotherapeutic agent and one or more adjuvants. Exemplary cisplatin-based neoadjuvants (or adjuvants) include, but are not limited to, (1) methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC); (2) dose-dense, or accelerated, MVAC (ddMVAC); (3) gemcitabine and cisplatin (GC); (4) paclitaxel/gemcitabine/cisplatin (PGC); (5) cisplatin/methotrexate/vinblastine (CMV); (6) a combination thereof, such as ddMVAC/GC/MVAC.
[00124] As used herein, the term “immunotherapy” refers to refers to any chemical or biological agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth by promoting, preserving, or increasing the activity of immune cells. Immunotherapies include immune checkpoint inhibitors, T-cell transfer therapy (e.g., CAR-T therapies), antibody therapies, treatment vaccines, and immune system modulators.
[00125] Immune checkpoint inhibitors inhibit one or more immune checkpoint proteins. The immune system has multiple inhibitory pathways that are critical for maintaining self-tolerance and modulating immune responses. For example, in T-cells, the amplitude and quality of response is initiated through antigen recognition by the T-cell receptor and is regulated by immune checkpoint proteins that balance co-stimulatory and inhibitory signals. In some embodiments of any of the aspects, a subject or patient is treated with at least one inhibitor of an immune checkpoint protein. As used herein, “immune checkpoint protein” refers to a protein which, when active, exhibits an inhibitory effect on immune activity, e.g., T cell activity. Exemplary immune checkpoint proteins can include PD-1 (e.g., NCBI Gene ID: 5133); PD-L1 (e.g., NCBI Gene ID: 29126); PD-L2 (e g., NCBI Gene ID: 80380); TIM-3 (e g., NCBI Gene ID: 84868); CTLA4 (e.g., NCBI Gene ID: 1493); TIGIT (e.g., NCBI Gene ID: 201633); KIR (e.g., NCBI Gene ID: 3811); LAG3 (e g., NCBI Gene ID: 3902); DDl-a (e.g., NCBI Gene ID: 64115); A2AR (e.g., NCBI Gene ID: 135); B7-H3 (e.g., NCBI Gene ID: 80381); B7- H4 (e g., NCBI Gene ID: 79679); BTLA (e.g., NCBI Gene ID: 151888); IDO (e g., NCBI Gene ID: 3620); TDO (e.g., NCBI Gene ID: 6999); HVEM (e g., NCBI Gene ID: 8764); GAL9 (e.g., NCBI Gene ID: 3965); 2B4 (belongs to the CD2 family of molecules and is expressed on all NK, y5, and memory CD8+ (aP) T cells) (e.g., NCBI Gene ID: 51744); CD160 (also referred to as BY55) (e.g., NCBI Gene ID: 11126); and various B-7 family ligands. B7 family ligands include, but are not limited to, B7- 1, B7- 2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6 and B7-H7.
[00126] Non-limiting examples of immune checkpoint inhibitors (with checkpoint targets and manufacturers noted in parentheses) can include :MGA271 (B7-H3: MacroGenics); ipilimumab (CTLA- 4; Bristol Meyers Squibb); pembrolizumab (PD-1; Merck); nivolumab (PD-1; Bristol Meyers Squibb) ; atezolizumab (PD-L1; Genentech); galiximab (B7.1; Biogen); IMP321 (LAG3: Immuntep); BMS- 986016 (LAG3; Bristol Meyers Squibb); SMB-663513 (CD137; Bristol-Meyers Squibb); PF-05082566 (CD137; Pfizer); IPH2101 (KIR; Innate Pharma); KW-0761 (CCR4; Kyowa Kirin); CDX-1127 (CD27; CellDex); MEDI-6769 (0x40; Medlmmune); CP-870,893 (CD40; Genentech); tremelimumab (CTLA- 4; Medimmune); pidilizumab (PD-1; Medivation); MPDL3280A (PD-L1; Roche); MEDI4736 (PD-L1; AstraZeneca); MSB0010718C (PD-L1; EMD Serono); AUNP12 (PD-1; Aurigene); avelumab (PD-L1; Merck); durvalumab (PD-L1; Medimmune); IMP321, a soluble Ig fusion protein (Brignone et al., 2007, J. Immunol. 179:4202-4211); the anti-B7-H3 antibody MGA271 (Loo et al., 2012, Clin. Cancer Res. July 15 (18) 3834); TIM3 (T-cell immunoglobulin domain and mucin domain 3) inhibitors (Fourcade et al., 2010, J. Exp. Med. 207:2175-86 and Sakuishi et al., 2010, J. Exp. Med. 207:2187-94); anti-CTLA-4 antibodies described in US Patent Nos: 5,811,097; 5,811,097; 5,855,887; 6,051,227; 6,207,157;
6,682,736; 6,984,720; and 7,605,238; tremelimumab, (ticilimumab, CP-675,206); ipilimumab (also known as 10D1, MDX-D010); PD-1 and PD-L1 blockers described in US Patent Nos. 7,488,802;
7,943,743; 8,008,449; 8,168,757; 8,217,149, and PCT Published Patent Application Nos: W003042402, WO2008156712, W02010089411, W02010036959, WO2011066342, WO2011159877, WO2011082400, and WO2011161699; nivolumab (MDX 1106, BMS 936558, ONO 4538); lambrolizumab (MK-3475 or SCH 900475); CT-011; AMP-224; and BMS-936559 (MDX- 1105-01). The foregoing references are incorporated by reference herein in their entireties.
[00127] As used herein, “lobectomy” refers to removing at least one full lobe of the five lobes of a lung.
[00128] As used herein, “lymphadenectomy” refers to the dissection and removal of at least one lymph node. A regional or selective lymphadenectomy removes a sample of the lymph node which is closest to the tumor. A radical, complete, or total lymphadenectomy removes all lymph nodes at a given location. In some embodiments of any of the aspects, the lymphadenectomy is a regional lymphadenectomy. In some embodiments of any of the aspects, the lymphadenectomy is a radical lymphadenectomy .
[00129] In some embodiments of any of the aspects, the method further comprises administering active surveillance, wedge resection of the lung, or a segmentectomy of the lung to a subject not having decreased expression of at least one gene selected from the group consisting of: SHR00M4 and NBEAL1; and/or not having increased expression of at least one gene selected from the group consisting of: MUC16, NOTCH3, H19, COL1A1, and GARS1. In some embodiments of any of the aspects, the subject is administered active surveillance. In some embodiments of any of the aspects, the subject is administered a wedge resection of the lung. In some embodiments of any of the aspects, the subject is administered a segmentectomy of the lung. In some embodiments of any of the aspects, the subject is administered a wedge resection of the lung and is not administered an adjuvant or neoadjuvant. In some embodiments of any of the aspects, the subject is administered a segmentectomy of the lung and is not administered an adjuvant or neoadjuvant. In some embodiments of any of the aspects, the subject is administered a wedge resection of the lung and is not administered an adjuvant therapy, a neoadjuvant therapy, an ablation therapy, radiation thearpy, a lobectomy, and/or a lymphadenectomy. In some embodiments of any of the aspects, the subject is administered a segmentectomy of the lung and is not administered an adjuvant therapy, a neoadjuvant therapy, an ablation therapy, radiation therapy, lobectomy, and/or a lymphadenectomy.
[00130] In some embodiments of any of the aspects, the method further comprises administering active surveillance, wedge resection of the lung, or a segmentectomy of the lung to a subject not having decreased expression of at least one gene selected from the group consisting of: SHR00M4 and NBEAL1; and not having increased expression of at least one gene selected from the group consisting of: MUC16, NOTCH3, H19, COL1A1, and GARS1. In some embodiments of any of the aspects, the subject is administered active surveillance. In some embodiments of any of the aspects, the subject is administered a wedge resection of the lung. In some embodiments of any of the aspects, the subject is administered a segmentectomy of the lung. In some embodiments of any of the aspects, the subject is administered a wedge resection of the lung and is not administered an adjuvant or neoadjuvant. In some embodiments of any of the aspects, the subject is administered a segmentectomy of the lung and is not administered an adjuvant or neoadjuvant. In some embodiments of any of the aspects, the subject is administered a wedge resection of the lung and is not administered an adjuvant therapy, a neoadjuvant therapy, an ablation therapy, radiation therapy, a lobectomy, and/or a lymphadenectomy. In some embodiments of any of the aspects, the subject is administered a segmentectomy of the lung and is not administered an adjuvant therapy, a neoadjuvant therapy, an ablation therapy, radiation therapy, a lobectomy, and/or a lymphadenectomy. [00131] In some embodiments of any of the aspects, the method further comprises administering active surveillance, wedge resection of the lung, or a segmentectomy of the lung to a subject not having decreased expression of at least one gene selected from the group consisting of: SHR00M4 and NBEAL1; and/or not having increased expression of at least one gene selected from the group consisting of: MUC16, NOTCH3, H19, COL1A1. In some embodiments of any of the aspects, the subject is administered active surveillance. In some embodiments of any of the aspects, the subject is administered a wedge resection of the lung. In some embodiments of any of the aspects, the subject is administered a segmentectomy of the lung. In some embodiments of any of the aspects, the subject is administered a wedge resection of the lung and is not administered an adjuvant or neoadjuvant. In some embodiments of any of the aspects, the subject is administered a segmentectomy of the lung and is not administered an adjuvant or neoadjuvant. In some embodiments of any of the aspects, the subject is administered a wedge resection of the lung and is not administered an adjuvant therapy, a neoadjuvant therapy, an ablation therapy, radiation therapy, a lobectomy, and/or a lymphadenectomy. In some embodiments of any of the aspects, the subject is administered a segmentectomy of the lung and is not administered an adjuvant therapy, a neoadjuvant therapy, an ablation therapy, radiation therapy, a lobectomy, and/or a lymphadenectomy.
[00132] In some embodiments of any of the aspects, the method further comprises administering active surveillance, wedge resection of the lung, or a segmentectomy of the lung to a subject not having decreased expression of at least one gene selected from the group consisting of: SHR00M4 and NBEAL1; and not having increased expression of at least one gene selected from the group consisting of: MUC16, NOTCH3, H19, and COL1A1. In some embodiments of any of the aspects, the subject is administered active surveillance. In some embodiments of any of the aspects, the subject is administered a wedge resection of the lung. In some embodiments of any of the aspects, the subject is administered a segmentectomy of the lung. In some embodiments of any of the aspects, the subject is administered a wedge resection of the lung and is not administered an adjuvant or neoadjuvant. In some embodiments of any of the aspects, the subject is administered a segmentectomy of the lung and is not administered an adjuvant or neoadjuvant. In some embodiments of any of the aspects, the subject is administered a wedge resection of the lung and is not administered an adjuvant therapy, a neoadjuvant therapy, an ablation therapy, radiation therapy, a lobectomy, and/or a lymphadenectomy. In some embodiments of any of the aspects, the subject is administered a segmentectomy of the lung and is not administered an adjuvant therapy, a neoadjuvant therapy, an ablation therapy, radiation therapy, a lobectomy, and/or a lymphadenectomy.
[00133] In some embodiments of any of the aspects, the method further comprises administering active surveillance, wedge resection of the lung, or a segmentectomy of the lung to a subject not having increased expression of at least one gene selected from the group consisting of: MUC16, NOTCH3, H19, COL1A1, and GARS1. In some embodiments of any of the aspects, the subject is administered active surveillance. In some embodiments of any of the aspects, the subject is administered a wedge resection of the lung. In some embodiments of any of the aspects, the subject is administered a segmentectomy of the lung. In some embodiments of any of the aspects, the subject is administered a wedge resection of the lung and is not administered an adjuvant or neoadjuvant. In some embodiments of any of the aspects, the subject is administered a segmentectomy of the lung and is not administered an adjuvant or neoadjuvant. In some embodiments of any of the aspects, the subject is administered a wedge resection of the lung and is not administered an adjuvant therapy, a neoadjuvant therapy, an ablation therapy, radiation therapy, a lobectomy, and/or a lymphadenectomy. In some embodiments of any of the aspects, the subject is administered a segmentectomy of the lung and is not administered an adjuvant therapy, a neoadjuvant therapy, an ablation therapy, radiation therapy, a lobectomy, and/or a lymphadenectomy.
[00134] In some embodiments of any of the aspects, the method further comprises administering active surveillance, wedge resection of the lung, or a segmentectomy of the lung to a subject not having increased expression of at least one gene selected from the group consisting of: MUC16, NOTCH3, H19, and COL1A1. In some embodiments of any of the aspects, the subject is administered active surveillance. In some embodiments of any of the aspects, the subject is administered a wedge resection of the lung. In some embodiments of any of the aspects, the subject is administered a segmentectomy of the lung. In some embodiments of any of the aspects, the subject is administered a wedge resection of the lung and is not administered an adjuvant or neoadjuvant. In some embodiments of any of the aspects, the subject is administered a segmentectomy of the lung and is not administered an adjuvant or neoadjuvant. In some embodiments of any of the aspects, the subject is administered a wedge resection of the lung and is not administered an adjuvant therapy, a neoadjuvant therapy, an ablation therapy, radiation therapy, a lobectomy, and/or a lymphadenectomy. In some embodiments of any of the aspects, the subject is administered a segmentectomy of the lung and is not administered an adjuvant therapy, a neoadjuvant therapy, an ablation therapy, radiation therapy, a lobectomy, and/or a lymphadenectomy.
[00135] In some embodiments of any of the aspects, the method further comprises administering active surveillance, wedge resection of the lung, or a segmentectomy of the lung to a subject not having increased expression of MUC16. In some embodiments of any of the aspects, the subject is administered active surveillance. In some embodiments of any of the aspects, the subject is administered a wedge resection of the lung. In some embodiments of any of the aspects, the subject is administered a segmentectomy of the lung. In some embodiments of any of the aspects, the subject is administered a wedge resection of the lung and is not administered an adjuvant or neoadjuvant. In some embodiments of any of the aspects, the subject is administered a segmentectomy of the lung and is not administered an adjuvant or neoadjuvant. In some embodiments of any of the aspects, the subject is administered a wedge resection of the lung and is not administered an adjuvant therapy, a neoadjuvant therapy, an ablation therapy, radiation therapy, a lobectomy, and/or a lymphadenectomy. In some embodiments of any of the aspects, the subject is administered a segmentectomy of the lung and is not administered an adjuvant therapy, a neoadjuvant therapy, an ablation therapy, radiation therapy, a lobectomy, and/or a lymphadenectomy.
[00136] In some embodiments of any of the aspects, the method further comprises administering active surveillance, wedge resection of the lung, or a segmentectomy of the lung to a subject not having decreased expression of at least one gene selected from the group consisting of: SHR00M4 and NBEAL1. In some embodiments of any of the aspects, the subject is administered active surveillance. In some embodiments of any of the aspects, the subject is administered a wedge resection of the lung. In some embodiments of any of the aspects, the subject is administered a segmentectomy of the lung. In some embodiments of any of the aspects, the subject is administered a wedge resection of the lung and is not administered an adjuvant or neoadjuvant. In some embodiments of any of the aspects, the subject is administered a segmentectomy of the lung and is not administered an adjuvant or neoadjuvant. In some embodiments of any of the aspects, the subject is administered a wedge resection of the lung and is not administered an adjuvant therapy, a neoadjuvant therapy, an ablation therapy, radiation therapy, a lobectomy, and/or a lymphadenectomy. In some embodiments of any of the aspects, the subject is administered a segmentectomy of the lung and is not administered an adjuvant therapy, a neoadjuvant therapy, an ablation therapy, radiation therapy, a lobectomy, and/or a lymphadenectomy.
[00137] In some embodiments of any of the aspects, the method further comprises administering active surveillance, wedge resection of the lung, or a segmentectomy of the lung to a subject not having decreased expression of SHR00M4 and NBEAL1. In some embodiments of any of the aspects, the subject is administered active surveillance. In some embodiments of any of the aspects, the subject is administered a wedge resection of the lung. In some embodiments of any of the aspects, the subject is administered a segmentectomy of the lung. In some embodiments of any of the aspects, the subject is administered a wedge resection of the lung and is not administered an adjuvant or neoadjuvant. In some embodiments of any of the aspects, the subject is administered a segmentectomy of the lung and is not administered an adjuvant or neoadjuvant. In some embodiments of any of the aspects, the subject is administered a wedge resection of the lung and is not administered an adjuvant therapy, a neoadjuvant therapy, an ablation therapy, radiation therapy, a lobectomy, and/or a lymphadenectomy. In some embodiments of any of the aspects, the subject is administered a segmentectomy of the lung and is not administered an adjuvant therapy, a neoadjuvant therapy, an ablation therapy, radiation therapy, a lobectomy, and/or a lymphadenectomy.
[00138] In some embodiments of any of the aspects, the method further comprises administering active surveillance, wedge resection of the lung, or a segmentectomy of the lung to a subject not having decreased expression of SHR00M4. In some embodiments of any of the aspects, the subject is administered active surveillance. In some embodiments of any of the aspects, the subject is administered a wedge resection of the lung. In some embodiments of any of the aspects, the subject is administered a segmentectomy of the lung. In some embodiments of any of the aspects, the subject is administered a wedge resection of the lung and is not administered an adjuvant or neoadjuvant. In some embodiments of any of the aspects, the subject is administered a segmentectomy of the lung and is not administered an adjuvant or neoadjuvant. In some embodiments of any of the aspects, the subject is administered a wedge resection of the lung and is not administered an adjuvant therapy, a neoadjuvant therapy, an ablation therapy, radiation therapy, a lobectomy, and/or a lymphadenectomy. In some embodiments of any of the aspects, the subject is administered a segmentectomy of the lung and is not administered an adjuvant therapy, a neoadjuvant therapy, an ablation therapy, radiation therapy, a lobectomy, and/or a lymphadenectomy.
[00139] In some embodiments of any of the aspects, the method further comprises administering active surveillance, wedge resection of the lung, or a segmentectomy of the lung to a subject not having decreased expression of NBEAL1. In some embodiments of any of the aspects, the subject is administered active surveillance. In some embodiments of any of the aspects, the subject is administered a wedge resection of the lung. In some embodiments of any of the aspects, the subject is administered a segmentectomy of the lung. In some embodiments of any of the aspects, the subject is administered a wedge resection of the lung and is not administered an adjuvant or neoadjuvant. In some embodiments of any of the aspects, the subject is administered a segmentectomy of the lung and is not administered an adjuvant or neoadjuvant. In some embodiments of any of the aspects, the subject is administered a wedge resection of the lung and is not administered an adjuvant therapy, a neoadjuvant therapy, an ablation therapy, radiation therapy, a lobectomy, and/or a lymphadenectomy. In some embodiments of any of the aspects, the subject is administered a segmentectomy of the lung and is not administered an adjuvant therapy, a neoadjuvant therapy, an ablation therapy, radiation therapy, a lobectomy, and/or a lymphadenectomy.
[00140] As used herein, “active surveillance” refers to repeated exams, scans, and/or screens without administration of a drug or surgical intervention. In some embodiments, active surveillance comprises administration of a CT scan at least twice at intervals of 1-3 months. In some embodiments, active surveillance comprises administration of a CT scan at least twice at intervals of 1-3 months, during which time the subject is not administered a drug or surgical intervention. In some embodiments, active surveillance comprises administration of a CT scan at least twice at intervals of 1-18 months. In some embodiments, active surveillance comprises administration of a CT scan at least twice at intervals of 1-18 months, during which time the subject is not administered a drug or surgical intervention. In some embodiments, active surveillance comprises administration of a CT scan at least twice at intervals of 3-6 months. In some embodiments, active surveillance comprises administration of a CT scan at least twice at intervals of 3-6 months, during which time the subject is not administered a drug or surgical intervention. In some embodiments, active surveillance comprises administration of a CT scan at least twice at intervals of 1-12 months. In some embodiments, active surveillance comprises administration of a CT scan at least twice at intervals of 1-12 months, during which time the subject is not administered a drug or surgical intervention. In some embodiments, active surveillance comprises administration of a CT scan at least twice at intervals of 3-12 months. In some embodiments, active surveillance comprises administration of a CT scan at least twice at intervals of 3-12 months, during which time the subject is not administered a drug or surgical intervention. In some embodiments, active surveillance comprises administration of a CT scan at least twice at intervals of 3-18 months. In some embodiments, active surveillance comprises administration of a CT scan at least twice at intervals of 3-18 months, during which time the subject is not administered a drug or surgical intervention. In some embodiments, active surveillance comprises administration of a CT scan at least twice at intervals of 6-18 months. In some embodiments, active surveillance comprises administration of a CT scan at least twice at intervals of 6- 18 months, during which time the subject is not administered a drug or surgical intervention.
[00141] As used herein, “wedge resection of the lung” refers to removing a small section of the lung.
[00142] As used herein, “segmentectomy of the lung” refers to removing a segment of the lung. [00143] In one aspect of any of the embodiments, the method comprises a first step of determining the level of at least one gene as described above herein in a sample obtained from a subject. In some embodiments of any of the aspects, the method comprises administering the treatment to a subject previously determined to have the indicated level of the at least one gene.
[00144] In some embodiments of any of the aspects, the step of determining the expression level comprises i) obtaining or having obtained a sample from the subject and ii) performing or having performed an assay on the sample obtained from the subject to determine/measure the expression level in the subject. In some embodiments of any of the aspects, the step of determining the expression level comprises performing or having performed an assay on a sample obtained from the subject to determine/measure the expression level in the subject. In some embodiments of any of the aspects, the step of determining the expression level comprises comprise ordering or requesting an assay on a sample obtained from the subject to determine/measure the expression level in the subject. In some embodiments of any of the aspects, the step of determining the expression level can comprise receiving the results of an assay on a sample obtained from the subject to determine/measure the expression level in the subject. In some embodiments of any of the aspects, the step of determining if the expression level can comprise receiving a report, results, or other means of identifying the subject as a subject with certain expression level(s).
[00145] In one aspect of any of the embodiments, described herein is a method of treating lung cancer in a subject in need thereof, the method comprising: a) determining the expression level of one or more genes as described herein; and b) instructing or directing that the subject be administered the treatment described herein. In some embodiments of any of the aspects, the step of instructing or directing that the subject be administered a particular treatment can comprise providing a report of the assay results. In some embodiments of any of the aspects, the step of instructing or directing that the subject be administered a particular treatment can comprise providing a report of the assay results and/or treatment recommendations in view of the assay results. [00146] In one aspect of any of the embodiments, described herein is a method of treating or prognosing lung cancer, the method comprising determining the level of expression: a) at least one gene selected from the group consisting of:
MKI67; ASPM; ATAD2; TOP2A; FANCI; PLOD2; NCAPG2; CENPF; KPNA2;
BRCA1; HLTF; and FANCA; b) at least one gene selected from the group consisting of:
COL1A2; COL3A1; COL1A1; and COL6A3; c) at least one gene selected from the group consisting of:
PRKDC; TPI1; PKM; GAPDH; AHNAK2; YWHAZ; ATP 13 A3; GARS1; CP;
MUC16; TUBA1C; H19; PPFIA1; NOTCH3; ACLY; GCLC; CAD; and CCT6A; and/or d) at least one gene selected from the group consisting of:
SPTBN1; SFTPB; LRRK2; AKAP13; CHD2; EPAS1; ATP11A; ROS1; MTUS1;
ANKHD1; NBEAL1; MPRIP; SHROOM4; and TET2 wherein: e) increased expression of at least one gene selected from the group consisting of:
MKI67; ASPM; ATAD2; TOP2A; FANCI; PLOD2; NCAPG2; CENPF; KPNA2;
BRCA1; HLTF; and FANCA; f) increased expression of at least one gene selected from the group consisting of:
COL1A2; COL3A1; COL1A1; and COL6A3; g) increased expression of at least one gene selected from the group consisting of:
PRKDC; TPI1; PKM; GAPDH; AHNAK2; YWHAZ; ATP 13 A3; GARS1; CP;
MUC16; TUBA1C; H19; PPFIA1; NOTCH3; ACLY; GCLC; CAD; and CCT6A; and h) decreased expression of at least one gene selected from the group consisting of:
SPTBN1; SFTPB; LRRK2; AKAP13; CHD2; EPAS1; ATP11A; ROS1; MTUS1;
ANKHD1; NBEAL1; MPRIP; SHROOM4; and TET2 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
[00147] In one aspect of any of the embodiments, described herein is a method of treating or prognosing lung cancer, the method comprising determining the level of expression: a) at least one gene selected from the group consisting of:
COL1A2; COL3A1; COL1A1; and COL6A3; b) at least one gene selected from the group consisting of:
PRKDC; TPI1; PKM; GAPDH; AHNAK2; YWHAZ; ATP 13 A3; GARS1; CP;
MUC16; TUBA1C; H19; PPFIA1; NOTCH3; ACLY; GCLC; CAD; and CCT6A; and/or c) at least one gene selected from the group consisting of: SPTBN1; SFTPB; LRRK2; AKAP13; CHD2; EPAS1; ATP11A; R0S1; MTUS1;
ANKHD1; NBEAL1; MPRIP; SHR00M4; and TET2 wherein: d) increased expression of at least one gene selected from the group consisting of:
C0L1A2; C0L3A1; C0L1A1; and COL6A3; e) increased expression of at least one gene selected from the group consisting of:
PRKDC; TPI1; PKM; GAPDH; AHNAK2; YWHAZ; ATP 13 A3; GARS1; CP;
MUC16; TUBA1C; H19; PPFIA1; N0TCH3; ACLY; GCLC; CAD; and CCT6A; and f) decreased expression of at least one gene selected from the group consisting of:
SPTBN1; SFTPB; LRRK2; AKAP13; CHD2; EPAS1; ATP11A; ROS1; MTUS1;
ANKHD1; NBEAL1; MPRIP; SHR00M4; and TET2 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
[00148] In one aspect of any of the embodiments, described herein is a method of treating or prognosing lung cancer, the method comprising determining the level of expression: a) at least one gene selected from the group consisting of:
MUC16; NOTCH3; H19; COL1A1; GARS1; NBEAL1; and SHR00M4 wherein: b) increased expression of at least one gene selected from the group consisting of:
MUC16; NOTCH3; H19; COL1A1; and GARS1; and/or c) decreased expression of at least one gene selected from the group consisting of:
NBEAL1 and SHR00M4 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
[00149] In one aspect of any of the embodiments, described herein is a method of treating or prognosing lung cancer, the method comprising determining the level of expression: a. at least one gene selected from the group consisting of:
MUC16; NOTCH3; H19; COL1A1; GARS1; NBEAL1; and SHR00M4 wherein: b. increased expression of at least one gene selected from the group consisting of:
MUC16; NOTCH3; H19; COL1A1; and GARS1; and/or c. decreased expression of at least one gene selected from the group consisting of:
NBEAL1 and SHR00M4 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
[00150] In one aspect of any of the embodiments, described herein is a method of treating or prognosing lung cancer, the method comprising determining the level of expression: a) at least one gene selected from the group consisting of:
MUC16; NOTCH3; H19; COL1A1; NBEAL1; and SHROOM4 wherein: b) increased expression of at least one gene selected from the group consisting of:
MUC16; NOTCH3; H19; and COL1A1; and/or c) decreased expression of at least one gene selected from the group consisting of:
NBEAL1 and SHROOM4 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
[00151] In one aspect of any of the embodiments, described herein is a method of treating or prognosing lung cancer, the method comprising determining the level of expression: a. at least one gene selected from the group consisting of:
MUC16; NOTCH3; H19; COL1A1; NBEAL1; and SHROOM4 wherein: b. increased expression of at least one gene selected from the group consisting of: MUC16; NOTCH3; H19; and COL1A1; and/or c. decreased expression of at least one gene selected from the group consisting of: NBEAL1 and SHROOM4 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
[00152] In one aspect of any of the embodiments, described herein is a method of treating or prognosing lung cancer, the method comprising determining the level of expression: a) at least one gene selected from the group consisting of:
MUC16; NOTCH3; H19; and COL1A1 wherein: b) increased expression of at least one gene selected from the group consisting of:
MUC16; NOTCH3; H19; and COL1A1 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
[00153] In one aspect of any of the embodiments, described herein is a method of treating or prognosing lung cancer, the method comprising determining the level of expression: a. at least one gene selected from the group consisting of:
MUC16; NOTCH3; H19; and COL1A1; wherein: b. increased expression of at least one gene selected from the group consisting of:
MUC16; NOTCH3; H19; and COL1A1 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
[00154] In one aspect of any of the embodiments, described herein is a method of treating or prognosing lung cancer, the method comprising determining the level of expression of MUC16, wherein increased expression of MUC16 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
[00155] In one aspect of any of the embodiments, described herein is a method of treating or prognosing lung cancer, the method comprising determining the level of expression of MUC16, wherein increased expression of MUC16 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
[00156] In one aspect of any of the embodiments, described herein is a method of treating or prognosing lung cancer, the method comprising determining the level of expression of MUC16, NBEAL1, and SHR00M4 wherein: a) increased expression of MUC16; and b) decreased expression of at least one gene selected from the group consisting of:
NBEAL1 and SHR00M4 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
[00157] In one aspect of any of the embodiments, described herein is a method of treating or prognosing lung cancer, the method comprising determining the level of expression of MUC16, NBEAL1, and SHR00M4, wherein: a. increased expression MUC 16; and b. decreased expression of at least one gene selected from the group consisting of: NBEAL1 and SHR00M4 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
[00158] In one aspect of any of the embodiments, described herein is a method of treating or prognosing lung cancer, the method comprising determining the level of expression of NBEAL1 and SHR00M4 wherein decreased expression of at least one gene selected from the group consisting of: NBEAL1 and SHR00M4 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
[00159] In one aspect of any of the embodiments, described herein is a method of treating or prognosing lung cancer, the method comprising determining the level of expression of NBEAL1 and SHR00M4, wherein decreased expression of at least one gene selected from the group consisting of: NBEAL1 and SHR00M4 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
[00160] In one aspect of any of the embodiments, described herein is a method of treating or prognosing lung cancer, the method comprising determining the level of expression of NBEAL1 wherein decreased expression of NBEAL1 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
[00161] In one aspect of any of the embodiments, described herein is a method of treating or prognosing lung cancer, the method comprising determining the level of expression of NBEAL1, wherein decreased expression of NBEAL1 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
[00162] In one aspect of any of the embodiments, described herein is a method of treating or prognosing lung cancer, the method comprising determining the level of expression of SHR00M4 wherein decreased expression of SHR00M4 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
[00163] In one aspect of any of the embodiments, described herein is a method of treating or prognosing lung cancer, the method comprising determining the level of expression of SHR00M4, wherein decreased expression of SHR00M4 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
[00164] In one aspect of any of the embodiments, described herein is a method of treating or prognosing lung cancer, the method comprising determining the level of expression: a) at least one gene selected from the group consisting of:
MKI67; ASPM; ATAD2; TOP2A; FANCI; and PLOD2; b) at least one gene selected from the group consisting of:
COL1A2; COL3A1; COL1A1; and COL6A3; c) at least one gene selected from the group consisting of:
PRKDC; TPI1; PKM; GAPDH; AHNAK2; and YWHAZ; or d) at least one gene selected from the group consisting of:
SPTBN1; SFTPB; LRRK2; AKAP13; CHD2; and EPAS1; wherein: e) increased expression of at least one gene selected from the group consisting of:
MKI67; ASPM; ATAD2; TOP2A; FANCI; and PLOD2; f) increased expression of at least one gene selected from the group consisting of: C0L1A2; C0L3A1; C0L1A1; and COL6A3; g) increased expression of at least one gene selected from the group consisting of:
PRKDC; TPI1; PKM; GAPDH; AHNAK2; and YWHAZ; and/or h) decreased expression of at least one gene selected from the group consisting of:
SPTBN1; SFTPB; LRRK2; AKAP13; CHD2; and EPAS1; indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
[00165] In one aspect of any of the embodiments, described herein is a method of treating or prognosing lung cancer, the method comprising determining the level of expression: a) at least one gene selected from the group consisting of:
MKI67; ASPM; ATAD2; TOP2A; FANCI; and PLOD2; b) at least one gene selected from the group consisting of:
COL1A2; COL3A1; COL1A1; and COL6A3; c) at least one gene selected from the group consisting of:
PRKDC; TPI1; PKM; GAPDH; AHNAK2; and YWHAZ; and d) at least one gene selected from the group consisting of:
SPTBN1; SFTPB; LRRK2; AKAP13; CHD2; and EPAS1; wherein: e) increased expression of at least one gene selected from the group consisting of:
MKI67; ASPM; ATAD2; TOP2A; FANCI; and PLOD2; f) increased expression of at least one gene selected from the group consisting of:
COL1A2; COL3A1; COL1A1; and COL6A3; g) increased expression of at least one gene selected from the group consisting of:
PRKDC; TPI1; PKM; GAPDH; AHNAK2; and YWHAZ; and/or h) decreased expression of at least one gene selected from the group consisting of:
SPTBN1; SFTPB; LRRK2; AKAP13; CHD2; and EPAS1; indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
[00166] In one aspect of any of the embodiments, described herein is a method of treating or prognosing lung cancer, the method comprising determining the level of expression: a) at least one gene selected from the group consisting of:
MKI67; ASPM; ATAD2; and TOP2A; b) at least one gene selected from the group consisting of:
COL1A2; COL3A1; COL1A1; and COL6A3; c) at least one gene selected from the group consisting of:
PRKDC; TPI1; PKM; and GAPDH; or d) at least one gene selected from the group consisting of: SPTBN1; SFTPB; LRRK2; and AKAP13; wherein: e) increased expression of at least one gene selected from the group consisting of:
MKI67; ASPM; ATAD2; and TOP2A; f) increased expression of at least one gene selected from the group consisting of:
C0L1A2; C0L3A1; C0L1A1; and COL6A3; g) increased expression of at least one gene selected from the group consisting of:
PRKDC; TPI1; PKM; and GAPDH; and/or h) decreased expression of at least one gene selected from the group consisting of:
SPTBN1; SFTPB; LRRK2; and AKAP13; indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
[00167] In one aspect of any of the embodiments, described herein is a method of treating or prognosing lung cancer, the method comprising determining the level of expression: a) at least one gene selected from the group consisting of:
MKI67; ASPM; ATAD2; and TOP2A; b) at least one gene selected from the group consisting of:
COL1A2; COL3A1; COL1A1; and COL6A3; c) at least one gene selected from the group consisting of:
PRKDC; TPI1; PKM; and GAPDH; and d) at least one gene selected from the group consisting of:
SPTBN1; SFTPB; LRRK2; and AKAP13; wherein: e) increased expression of at least one gene selected from the group consisting of:
MKI67; ASPM; ATAD2; and TOP2A; f) increased expression of at least one gene selected from the group consisting of:
COL1A2; COL3A1; COL1A1; and COL6A3; g) increased expression of at least one gene selected from the group consisting of:
PRKDC; TPI1; PKM; and GAPDH; and/or h) decreased expression of at least one gene selected from the group consisting of:
SPTBN1; SFTPB; LRRK2; and AKAP13; indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
[00168] In one aspect of any of the embodiments, described herein is a method of treating or prognosing lung cancer, the method comprising determining the level of expression: a) at least one gene selected from the group consisting of:
MKI67; and ASPM; b) at least one gene selected from the group consisting of:
COL1A2; and COL3A1; c) at least one gene selected from the group consisting of:
PRKDC; and TPIl; or d) at least one gene selected from the group consisting of:
SPTBN1; and SFTPB; wherein: e) increased expression of at least one gene selected from the group consisting of:
MKI67; and ASPM; f) increased expression of at least one gene selected from the group consisting of:
COL1A2; and COL3A1; g) increased expression of at least one gene selected from the group consisting of:
PRKDC; and TPI1; and/or h) decreased expression of at least one gene selected from the group consisting of:
SPTBN1; and SFTPB; indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
[00169] In one aspect of any of the embodiments, described herein is a method of treating or prognosing lung cancer, the method comprising determining the level of expression: a) at least one gene selected from the group consisting of:
MKI67; and ASPM; b) at least one gene selected from the group consisting of:
COL1A2; and COL3A1; c) at least one gene selected from the group consisting of:
PRKDC; and TPI1; and d) at least one gene selected from the group consisting of:
SPTBN1; and SFTPB; wherein: e) increased expression of at least one gene selected from the group consisting of:
MKI67; and ASPM; f) increased expression of at least one gene selected from the group consisting of:
COL1A2; and COL3A1; g) increased expression of at least one gene selected from the group consisting of:
PRKDC; and TPI1; and/or h) decreased expression of at least one gene selected from the group consisting of:
SPTBN1; and SFTPB; indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
[00170] In one aspect of any of the embodiments, described herein is a method of treating or prognosing lung cancer, the method comprising determining the level of expression of: a) MKI67; and ASPM; b) COL1A2; and COL3A1; c) PRKDC; and TPI1; and d) SPTBN1; and SFTPB; wherein: e) increased expression of MKI67; and ASPM; f) increased expression of COL1A2; and COL3A1; g) increased expression of PRKDC; and TPI 1 ; and/or h) decreased expression of SPTBN 1 ; and SFTPB; indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, and/or suitability for treatment with lobectomy.
[00171] In one aspect of any of the embodiments, described herein is a method of determining the likelihood of vascular invasion in a subject with lung cancer, the method comprising determining the level of expression: a) at least one gene selected from the group consisting of:
MKI67; ASPM; ATAD2; TOP2A; FANCI; PLOD2; NCAPG2; CENPF; KPNA2;
BRCA1; HLTF; and FANCA; b) at least one gene selected from the group consisting of:
COL1A2; COL3A1; COL1A1; and COL6A3; c) at least one gene selected from the group consisting of:
PRKDC; TPI1; PKM; GAPDH; AHNAK2; YWHAZ; ATP 13 A3; GARS1; CP;
MUC16; TUBA1C; H19; PPFIA1; NOTCH3; ACLY; GCLC; CAD; and CCT6A; d) at least one gene selected from the group consisting of:
SPTBN1; SFTPB; LRRK2; AKAP13; CHD2; EPAS1; ATP11A; ROS1; MTUS1;
ANKHD1; NBEAL1; MPRIP; SHR00M4; and TET2 wherein: e) increased expression of at least one gene selected from the group consisting of:
MKI67; ASPM; ATAD2; TOP2A; FANCI; PLOD2; NCAPG2; CENPF; KPNA2;
BRCA1; HLTF; and FANCA; f) increased expression of at least one gene selected from the group consisting of:
COL1A2; COL3A1; COL1A1; and COL6A3; g) increased expression of at least one gene selected from the group consisting of: PRKDC; TPI1; PKM; GAPDH; AHNAK2; YWHAZ; ATP 13 A3; GARS1; CP;
MUC16; TUBA1C; H19; PPFIA1; N0TCH3; ACLY; GCLC; CAD; and CCT6A; and h) decreased expression of at least one gene selected from the group consisting of:
SPTBN1; SFTPB; LRRK2; AKAP13; CHD2; EPAS1; ATP11A; ROS1; MTUS1;
ANKHD1; NBEAL1; MPRIP; SHR00M4; and TET2 indicates increased likelihood of vascular invasion in a subject with lung cancer.
[00172] In one aspect of any of the embodiments, described herein is a method of determining the likelihood of vascular invasion in a subject with lung cancer, the method comprising determining the level of expression: a) at least one gene selected from the group consisting of:
COL1A2; COL3A1; COL1A1; and COL6A3; b) at least one gene selected from the group consisting of:
PRKDC; TPI1; PKM; GAPDH; AHNAK2; YWHAZ; ATP 13 A3; GARS1; CP;
MUC16; TUBA1C; H19; PPFIA1; NOTCH3; ACLY; GCLC; CAD; and CCT6A; c) at least one gene selected from the group consisting of:
SPTBN1; SFTPB; LRRK2; AKAP13; CHD2; EPAS1; ATP 11 A; ROS1; MTUS1;
ANKHD1; NBEAL1; MPRIP; SHR00M4; and TET2 wherein: d) increased expression of at least one gene selected from the group consisting of:
COL1A2; COL3A1; COL1A1; and COL6A3; e) increased expression of at least one gene selected from the group consisting of:
PRKDC; TPI1; PKM; GAPDH; AHNAK2; YWHAZ; ATP 13 A3; GARS1; CP;
MUC16; TUBA1C; H19; PPFIA1; NOTCH3; ACLY; GCLC; CAD; and CCT6A; and f) decreased expression of at least one gene selected from the group consisting of:
SPTBN1; SFTPB; LRRK2; AKAP13; CHD2; EPAS1; ATP 11 A; ROS1; MTUS1;
ANKHD1; NBEAL1; MPRIP; SHR00M4; and TET2 indicates increased likelihood of vascular invasion in a subject with lung cancer.
[00173] In one aspect of any of the embodiments, described herein is a method of determining the likelihood of vascular invasion in a subject with lung cancer, the method comprising determining the level of expression of at least one gene selected from the group consisting of:
MUC16, NOTCH3, H19, COL1A1, GARS1; NBEAL, and SHR00M4 wherein: increased expression of at least one gene selected from the group consisting of:
MUC16, NOTCH3, H19, COL1A1, and GARS1; and/or decreased expression of at least one gene selected from the group consisting of:
NBEAL 1 and SHR00M4 indicates increased likelihood of vascular invasion in a subject with lung cancer.
[00174] In one aspect of any of the embodiments, described herein is a method of determining the likelihood of vascular invasion in a subject with lung cancer, the method comprising determining the level of expression of at least one gene selected from the group consisting of:
MUC16, NOTCH3, H19, COL1A1, NBEAL, and SHR00M4 wherein: increased expression of at least one gene selected from the group consisting of:
MUC16, NOTCH3, H19, and COL1A1; and/or decreased expression of at least one gene selected from the group consisting of: NBEAL 1 and SHR00M4 indicates increased likelihood of vascular invasion in a subject with lung cancer.
[00175] In one aspect of any of the embodiments, described herein is a method of determining the likelihood of vascular invasion in a subject with lung cancer, the method comprising determining the level of expression of at least one gene selected from the group consisting of:
MUC16, NOTCH3, H19, COL1A1, and GARS1 wherein: increased expression of at least one gene selected from the group consisting of: MUC16, NOTCH3, H19, COL1A1, and GARS1; indicates increased likelihood of vascular invasion in a subject with lung cancer.
[00176] In one aspect of any of the embodiments, described herein is a method of determining the likelihood of vascular invasion in a subject with lung cancer, the method comprising determining the level of expression of at least one gene selected from the group consisting of:
MUC16, NOTCH3, H19, and COL1A1 wherein: increased expression of at least one gene selected from the group consisting of: MUC16, NOTCH3, H19, and COL1A1; indicates increased likelihood of vascular invasion in a subject with lung cancer.
[00177] In one aspect of any of the embodiments, described herein is a method of determining the likelihood of vascular invasion in a subject with lung cancer, the method comprising determining the level of expression of MUC16 wherein increased expression of MCU16 indicates increased likelihood of vascular invasion in a subject with lung cancer.
[00178] In one aspect of any of the embodiments, described herein is a method of determining the likelihood of vascular invasion in a subject with lung cancer, the method comprising determining the level of expression of at least one gene selected from the group consisting of:
MUC16, NBEAL, and SHR00M4 wherein: increased expression of MUC16; and/or decreased expression of at least one gene selected from the group consisting of:
NBEAL1 and SHR00M4 indicates increased likelihood of vascular invasion in a subject with lung cancer.
[00179] In one aspect of any of the embodiments, described herein is a method of determining the likelihood of vascular invasion in a subject with lung cancer, the method comprising determining the level of expression of MUC16, NBEAL, and SHR00M4 wherein: increased expression of MUC16; and/or decreased expression of at least one gene selected from the group consisting of: NBEAL 1 and SHR00M4 indicates increased likelihood of vascular invasion in a subject with lung cancer.
[00180] In one aspect of any of the embodiments, described herein is a method of determining the likelihood of vascular invasion in a subject with lung cancer, the method comprising determining the level of expression of at least one gene selected from the group consisting of:
NBEAL, and SHR00M4 wherein decreased expression of at least one gene selected from the group consisting of:
NBEAL 1 and SHR00M4 indicates increased likelihood of vascular invasion in a subject with lung cancer.
[00181] In one aspect of any of the embodiments, described herein is a method of determining the likelihood of vascular invasion in a subject with lung cancer, the method comprising determining the level of expression of NBEAL, and SHR00M4 wherein decreased expression of at least one of NBEAL 1 and SHR00M4 indicates increased likelihood of vascular invasion in a subject with lung cancer.
[00182] In one aspect of any of the embodiments, described herein is a method of determining the likelihood of vascular invasion in a subject with lung cancer, the method comprising determining the level of expression of NBEAL, and SHR00M4 wherein decreased expression ofNBEALl and SHR00M4 indicates increased likelihood of vascular invasion in a subject with lung cancer.
[00183] In one aspect of any of the embodiments, described herein is a method of determining the likelihood of vascular invasion in a subject with lung cancer, the method comprising determining the level of expression of NBEAL wherein decreased expression ofNBEALl indicates increased likelihood of vascular invasion in a subject with lung cancer.
[00184] In one aspect of any of the embodiments, described herein is a method of determining the likelihood of vascular invasion in a subject with lung cancer, the method comprising determining the level of expression of SHR00M4 wherein decreased expression of SHR00M4 indicates increased likelihood of vascular invasion in a subject with lung cancer. [00185] In one aspect of any of the embodiments, described herein is a method of determining the likelihood of vascular invasion in a subject with lung cancer, the method comprising determining the level of expression: a) at least one gene selected from the group consisting of:
MKI67; ASPM; ATAD2; TOP2A; FANCI; and PLOD2; b) at least one gene selected from the group consisting of:
COL1A2; COL3A1; COL1A1; and COL6A3; c) at least one gene selected from the group consisting of:
PRKDC; TPI1; PKM; GAPDH; AHNAK2; and YWHAZ; or d) at least one gene selected from the group consisting of:
SPTBN1; SFTPB; LRRK2; AKAP13; CHD2; and EPAS1; wherein: e) increased expression of at least one gene selected from the group consisting of:
MKI67; ASPM; ATAD2; TOP2A; FANCI; and PLOD2; f) increased expression of at least one gene selected from the group consisting of:
COL1A2; COL3A1; COL1A1; and COL6A3; g) increased expression of at least one gene selected from the group consisting of:
PRKDC; TPI1; PKM; GAPDH; AHNAK2; and YWHAZ; and/or h) decreased expression of at least one gene selected from the group consisting of:
SPTBN1; SFTPB; LRRK2; AKAP13; CHD2; and EPAS1; indicates increased likelihood of vascular invasion.
[00186] In one aspect of any of the embodiments, described herein is a method of determining the likelihood of vascular invasion in a subject with lung cancer, the method comprising determining the level of expression: a) at least one gene selected from the group consisting of:
MKI67; ASPM; ATAD2; TOP2A; FANCI; and PLOD2; b) at least one gene selected from the group consisting of:
COL1A2; COL3A1; COL1A1; and COL6A3; c) at least one gene selected from the group consisting of:
PRKDC; TPI1; PKM; GAPDH; AHNAK2; and YWHAZ; and d) at least one gene selected from the group consisting of:
SPTBN1; SFTPB; LRRK2; AKAP13; CHD2; and EPAS1; wherein: e) increased expression of at least one gene selected from the group consisting of:
MKI67; ASPM; ATAD2; TOP2A; FANCI; and PLOD2; f) increased expression of at least one gene selected from the group consisting of:
COL1A2; COL3A1; COL1A1; and COL6A3; g) increased expression of at least one gene selected from the group consisting of:
PRKDC; TPI1; PKM; GAPDH; AHNAK2; and YWHAZ; and/or h) decreased expression of at least one gene selected from the group consisting of:
SPTBN1; SFTPB; LRRK2; AKAP13; CHD2; and EPAS1; indicates increased likelihood of vascular invasion.
[00187] In one aspect of any of the embodiments, described herein is a method of determining the likelihood of vascular invasion in a subject with lung cancer, the method comprising determining the level of expression: a) at least one gene selected from the group consisting of:
MKI67; ASPM; ATAD2; and TOP2A; b) at least one gene selected from the group consisting of:
COL1A2; COL3A1; COL1A1; and COL6A3; c) at least one gene selected from the group consisting of:
PRKDC; TPI1; PKM; and GAPDH; or d) at least one gene selected from the group consisting of:
SPTBN1; SFTPB; LRRK2; and AKAP13; wherein: e) increased expression of at least one gene selected from the group consisting of:
MKI67; ASPM; ATAD2; and TOP2A; f) increased expression of at least one gene selected from the group consisting of:
COL1A2; COL3A1; COL1A1; and COL6A3; g) increased expression of at least one gene selected from the group consisting of:
PRKDC; TPI1; PKM; and GAPDH; and/or h) decreased expression of at least one gene selected from the group consisting of:
SPTBN1; SFTPB; LRRK2; and AKAP13; indicates increased likelihood of vascular invasion.
[00188] In one aspect of any of the embodiments, described herein is a method of determining the likelihood of vascular invasion in a subject with lung cancer, the method comprising determining the level of expression: a) at least one gene selected from the group consisting of:
MKI67; ASPM; ATAD2; and TOP2A; b) at least one gene selected from the group consisting of:
COL1A2; COL3A1; COL1A1; and COL6A3; c) at least one gene selected from the group consisting of:
PRKDC; TPI1; PKM; and GAPDH; and d) at least one gene selected from the group consisting of:
SPTBN1; SFTPB; LRRK2; and AKAP13; wherein: e) increased expression of at least one gene selected from the group consisting of:
MKI67; ASPM; ATAD2; and TOP2A; f) increased expression of at least one gene selected from the group consisting of:
C0L1A2; C0L3A1; C0L1A1; and COL6A3; g) increased expression of at least one gene selected from the group consisting of:
PRKDC; TPI1; PKM; and GAPDH; and/or h) decreased expression of at least one gene selected from the group consisting of:
SPTBN1; SFTPB; LRRK2; and AKAP13; indicates increased likelihood of vascular invasion.
[00189] In one aspect of any of the embodiments, described herein is a method of determining the likelihood of vascular invasion in a subject with lung cancer, the method comprising determining the level of expression: a) at least one gene selected from the group consisting of:
MKI67; and ASPM; b) at least one gene selected from the group consisting of:
COL1A2; and COL3A1; c) at least one gene selected from the group consisting of:
PRKDC; and TPI1; or d) at least one gene selected from the group consisting of:
SPTBN1; and SFTPB; wherein: e) increased expression of at least one gene selected from the group consisting of:
MKI67; and ASPM; f) increased expression of at least one gene selected from the group consisting of:
COL1A2; and COL3A1; g) increased expression of at least one gene selected from the group consisting of:
PRKDC; and TPI1; and/or h) decreased expression of at least one gene selected from the group consisting of:
SPTBN1; and SFTPB; indicates increased likelihood of vascular invasion.
[00190] In one aspect of any of the embodiments, described herein is a method of determining the likelihood of vascular invasion in a subject with lung cancer, the method comprising determining the level of expression: a) at least one gene selected from the group consisting of:
MKI67; and ASPM; b) at least one gene selected from the group consisting of: C0L1A2; and C0L3A1; c) at least one gene selected from the group consisting of:
PRKDC; and TPI1; and d) at least one gene selected from the group consisting of:
SPTBN1; and SFTPB; wherein: e) increased expression of at least one gene selected from the group consisting of:
MKI67; and ASPM; f) increased expression of at least one gene selected from the group consisting of:
COL1A2; and COL3A1; g) increased expression of at least one gene selected from the group consisting of:
PRKDC; and TPI1; and/or h) decreased expression of at least one gene selected from the group consisting of:
SPTBN1; and SFTPB; indicates increased likelihood of vascular invasion.
[00191] In one aspect of any of the embodiments, described herein is a method of determining the likelihood of vascular invasion in a subject with lung cancer, the method comprising determining the level of expression of: a) MKI67; and ASPM; b) COL1A2; and COL3A1; c) PRKDC; and TPI1; and d) SPTBN1; and SFTPB; wherein: e) increased expression of MKI67; and ASPM; f) increased expression of COL1A2; and COL3A1; g) increased expression of PRKDC; and TPI 1 ; and/or h) decreased expression of SPTBN 1 ; and SFTPB; indicates increased likelihood of vascular invasion.
[00192] In some embodiments of any of the aspects, measurement of the level of a target and/or detection of the level or presence of a target, e.g. of an expression product (nucleic acid or polypeptide of one of the genes described herein) can comprise a transformation. As used herein, the term “transforming” or “transformation” refers to changing an object or a substance, e.g., biological sample, nucleic acid or protein, into another substance. The transformation can be physical, biological or chemical. Exemplary physical transformation includes, but is not limited to, pre-treatment of a biological sample, e.g., from whole blood to blood serum by differential centrifugation. A biological/chemical transformation can involve the action of at least one enzyme and/or a chemical reagent in a reaction. For example, a DNA sample can be digested into fragments by one or more restriction enzymes, or an exogenous molecule can be attached to a fragmented DNA sample with a ligase. In some embodiments of any of the aspects, a DNA sample can undergo enzymatic replication, e.g., by polymerase chain reaction (PCR).
[00193] Transformation, measurement, and/or detection of a target molecule, e.g. a mRNA or polypeptide can comprise contacting a sample obtained from a subject with a reagent (e.g. a detection reagent) which is specific for the target, e.g., a target-specific reagent. In some embodiments of any of the aspects, the target-specific reagent is detectably labeled. In some embodiments of any of the aspects, the target-specific reagent is capable of generating a detectable signal. In some embodiments of any of the aspects, the target-specific reagent generates a detectable signal when the target molecule is present.
[00194] In certain embodiments, the gene expression products as described herein can be determined by determining the level of messenger RNA (mRNA) expression of the genes described herein. Such molecules can be isolated, derived, or amplified from a biological sample, such as a biopsy sample. Techniques for the detection of mRNA expression are known by persons skilled in the art, and can include but are not limited to, PCR procedures, RT-PCR, quantitative RT-PCR Northern blot analysis, differential gene expression, RNAse protection assay, microarray based analysis, next-generation sequencing; hybridization methods, single cell RNA sequencing (bead based and plate based), spatial transcriptomics, etc.
[00195] In general, the PCR procedure describes a method of gene amplification which is comprised of (i) sequence-specific hybridization of primers to specific genes or sequences within a nucleic acid sample or library, (ii) subsequent amplification involving multiple rounds of annealing, elongation, and denaturation using a thermostable DNA polymerase, and (iii) screening the PCR products for a band of the correct size. The primers used are oligonucleotides of sufficient length and appropriate sequence to provide initiation of polymerization, i.e. each primer is specifically designed to be complementary to a strand of the genomic locus to be amplified. In an alternative embodiment, mRNA level of gene expression products described herein can be determined by reverse-transcription (RT) PCR and by quantitative RT-PCR (QRT-PCR) or real-time PCR methods. Methods of RT-PCR and QRT-PCR are well known in the art.
[00196] In some embodiments of any of the aspects, the level of an mRNA can be measured by a quantitative sequencing technology, e.g. a quantitative next-generation sequence technology. Methods of sequencing a nucleic acid sequence are well known in the art. Briefly, a sample obtained from a subject can be contacted with one or more primers which specifically hybridize to a single-strand nucleic acid sequence flanking the target gene sequence and a complementary strand is synthesized. In some next-generation technologies, an adaptor (double or single-stranded) is ligated to nucleic acid molecules in the sample and synthesis proceeds from the adaptor or adaptor compatible primers. In some third-generation technologies, the sequence can be determined, e.g. by determining the location and pattern of the hybridization of probes or measuring one or more characteristics of a single molecule as it passes through a sensor (e.g. the modulation of an electrical field as a nucleic acid molecule passes through a nanopore). Exemplary methods of sequencing include, but are not limited to, Sanger sequencing, dideoxy chain termination, high-throughput sequencing, next generation sequencing, 454 sequencing, SOLiD sequencing, polony sequencing, Illumina sequencing, Ion Torrent sequencing, sequencing by hybridization, nanopore sequencing, Helioscope sequencing, single molecule real time sequencing, RNAP sequencing, long read sequencing, and the like. Methods and protocols for performing these sequencing methods are known in the art, see, e.g. “Next Generation Genome Sequencing” Ed. Michal Janitz, Wiley-VCH; “High-Throughput Next Generation Sequencing” Eds. Kwon and Rieke, Humana Press, 2011; and Sambrook et al., Molecular Cloning: A Laboratory Manual (4 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012); which are incorporated by reference herein in their entireties.
[00197] The nucleic acid sequences of the genes described herein have been assigned NCBI accession numbers for different species such as human, mouse and rat. Accordingly, a skilled artisan can design an appropriate primer based on the known sequence for determining the mRNA level of the respective gene.
[00198] Nucleic acid and ribonucleic acid (RNA) molecules can be isolated from a particular biological sample using any of a number of procedures, which are well-known in the art, the particular isolation procedure chosen being appropriate for the particular biological sample. For example, freeze-thaw and alkaline lysis procedures can be useful for obtaining nucleic acid molecules from solid materials; heat and alkaline lysis procedures can be useful for obtaining nucleic acid molecules from urine; and proteinase K extraction can be used to obtain nucleic acid from blood (Roiff, A et al. PCR: Clinical Diagnostics and Research, Springer (1994)).
[00199] Methods to measure gene expression products are known to a skilled artisan. Such methods to measure gene expression products, e.g., protein level, include ELISA (enzyme linked immunosorbent assay), western blot, immunoprecipitation, and immunofluorescence using detection reagents such as an antibody or protein binding agents. Alternatively, a peptide can be detected in a subject by introducing into a subject a labeled anti -peptide antibody and other types of detection agent. For example, the antibody can be labeled with a detectable marker whose presence and location in the subject is detected by standard imaging techniques.
[00200] For example, antibodies for the various targets described herein are commercially available and can be used for the purposes of the invention to measure protein expression levels. Alternatively, since the amino acid sequences for the targets described herein are known and publicly available at the NCBI website, one of skill in the art can raise their own antibodies against these polypeptides of interest for the purpose of the methods described herein. The amino acid sequences of the polypeptides described herein have been assigned NCBI accession numbers for different species such as human, mouse and rat. [00201] In some embodiments of any of the aspects, immunohistochemistry (“IHC”) and immunocytochemistry (“ICC”) techniques can be used. IHC is the application of immunochemistry to tissue sections, whereas ICC is the application of immunochemistry to cells or tissue imprints after they have undergone specific cytological preparations such as, for example, liquid-based preparations. Immunochemistry is a family of techniques based on the use of an antibody, wherein the antibodies are used to specifically target molecules inside or on the surface of cells. The antibody typically contains a marker that will undergo a biochemical reaction, and thereby experience a change of color, upon encountering the targeted molecules. In some instances, signal amplification can be integrated into the particular protocol, wherein a secondary antibody, that includes the marker stain or marker signal, follows the application of a primary specific antibody.
[00202] In some embodiments of any of the aspects, the assay can be a Western blot analysis. Alternatively, proteins can be separated by two-dimensional gel electrophoresis systems. Two- dimensional gel electrophoresis is well known in the art and typically involves iso-electric focusing along a first dimension followed by SDS-PAGE electrophoresis along a second dimension. These methods also require a considerable amount of cellular material. The analysis of 2D SDS-PAGE gels can be performed by determining the intensity of protein spots on the gel or can be performed using immune detection. In other embodiments, protein samples are analyzed by mass spectroscopy.
[00203] Immunological tests can be used with the methods and assays described herein and include, for example, competitive and non-competitive assay systems using techniques such as Western blots, radioimmunoassay (RIA), ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, e.g. latex agglutination, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, e.g. FIA (fluorescence -linked immunoassay), chemiluminescence immunoassays (CLIA), electrochemiluminescence immunoassay (ECLIA, counting immunoassay (CIA), lateral flow tests or immunoassay (LFIA), magnetic immunoassay (MIA), and protein A immunoassays. Methods for performing such assays are known in the art, provided an appropriate antibody reagent is available. In some embodiments of any of the aspects, the immunoassay can be a quantitative or a semi-quantitative immunoassay.
[00204] An immunoassay is a biochemical test that measures the concentration of a substance in a biological sample, typically a fluid sample such as blood or serum, using the interaction of an antibody or antibodies to its antigen. The assay takes advantage of the highly specific binding of an antibody with its antigen. For the methods and assays described herein, specific binding of the target polypeptides with respective proteins or protein fragments, or an isolated peptide, or a fusion protein described herein occurs in the immunoassay to form a target protein/peptide complex. The complex is then detected by a variety of methods known in the art. An immunoassay also often involves the use of a detection antibody. [00205] Enzyme-linked immunosorbent assay, also called ELISA, enzyme immunoassay or EIA, is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries.
[00206] In one embodiment, an ELISA involving at least one antibody with specificity for the particular desired antigen (e.g., any of the targets as described herein) can also be performed. A known amount of sample and/or antigen is immobilized on a solid support (usually a polystyrene micro titer plate). Immobilization can be either non-specific (e.g., by adsorption to the surface) or specific (e.g. where another antibody immobilized on the surface is used to capture antigen or a primary antibody). After the antigen is immobilized, the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme or can itself be detected by a secondary antibody which is linked to an enzyme through bio-conjugation. Between each step the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. After the final wash step the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample. Older ELISAs utilize chromogenic substrates, though newer assays employ Anorogenic substrates with much higher sensitivity.
[00207] In another embodiment, a competitive ELISA is used. Purified antibodies that are directed against a target polypeptide or fragment thereof are coated on the solid phase of multi-well plate, i.e., conjugated to a solid surface. A second batch of purified antibodies that are not conjugated on any solid support is also needed. These non-conjugated purified antibodies are labeled for detection purposes, for example, labeled with horseradish peroxidase to produce a detectable signal. A sample (e.g., a blood sample) from a subject is mixed with a known amount of desired antigen (e.g., a known volume or concentration of a sample comprising a target polypeptide) together with the horseradish peroxidase labeled antibodies and the mixture is then added to coated wells to form competitive combination. After incubation, if the polypeptide level is high in the sample, a complex of labeled antibody reagent-antigen will form. This complex is free in solution and can be washed away. Washing the wells will remove the complex. Then the wells are incubated with TMB (3, 3', 5, 5'-tetramethylbenzidene) color development substrate for localization of horseradish peroxidase-conjugated antibodies in the wells. There will be no color change or little color change if the target polypeptide level is high in the sample. If there is little or no target polypeptide present in the sample, a different complex in formed, the complex of solid support bound antibody reagents-target polypeptide. This complex is immobilized on the plate and is not washed away in the wash step. Subsequent incubation with TMB will produce significant color change. Such a competitive ELSA test is specific, sensitive, reproducible and easy to operate.
[00208] There are other different forms of ELISA, which are well known to those skilled in the art. The standard techniques known in the art for ELISA are described in "Methods in Immunodiagnosis", 2nd Edition, Rose and Bigazzi, eds. John Wiley & Sons, 1980; and Oellerich, M. 1984, J. Clin. Chem. Clin. Biochem. 22:895-904. These references are hereby incorporated by reference in their entirety. [00209] In one embodiment, the levels of a polypeptide in a sample can be detected by a lateral flow immunoassay test (LFIA), also known as the immunochromatographic assay, or strip test. LFIAs are a simple device intended to detect the presence (or absence) of antigen, e.g. a polypeptide, in a fluid sample. There are currently many LFIA tests used for medical diagnostics, either for home testing, point of care testing, or laboratory use. LFIA tests are a form of immunoassay in which the test sample flows along a solid substrate via capillary action. After the sample is applied to the test strip it encounters a colored reagent (generally comprising antibody specific for the test target antigen) bound to microparticles which mixes with the sample and transits the substrate encountering lines or zones which have been pretreated with another antibody or antigen. Depending upon the level of target polypeptides present in the sample the colored reagent can be captured and become bound at the test line or zone. LFIAs are essentially immunoassays adapted to operate along a single axis to suit the test strip format or a dipstick format. Strip tests are extremely versatile and can be easily modified by one skilled in the art for detecting an enormous range of antigens from fluid samples such as urine, blood, water, and/or homogenized tissue samples etc. Strip tests are also known as dip stick tests, the name bearing from the literal action of "dipping" the test strip into a fluid sample to be tested. LFIA strip tests are easy to use, require minimum training and can easily be included as components of point-of-care test (POCT) diagnostics to be use on site in the field. LFIA tests can be operated as either competitive or sandwich assays. Sandwich LFIAs are similar to sandwich ELISA. The sample first encounters colored particles which are labeled with antibodies raised to the target antigen. The test line will also contain antibodies to the same target, although it may bind to a different epitope on the antigen. The test line will show as a colored band in positive samples. In some embodiments of any of the aspects, the lateral flow immunoassay can be a double antibody sandwich assay, a competitive assay, a quantitative assay or variations thereof. Competitive LFIAs are similar to competitive ELISA. The sample first encounters colored particles which are labeled with the target antigen or an analogue. The test line contains antibodies to the target/its analogue. Unlabeled antigen in the sample will block the binding sites on the antibodies preventing uptake of the colored particles. The test line will show as a colored band in negative samples. There are a number of variations on lateral flow technology. It is also possible to apply multiple capture zones to create a multiplex test.
[00210] The use of "dip sticks" or LFIA test strips and other solid supports have been described in the art in the context of an immunoassay for a number of antigen biomarkers. U.S. Pat. Nos. 4,943,522; 6,485,982; 6,187,598; 5,770,460; 5,622,871; 6,565,808, U. S. patent applications Ser. No. 10/278,676; U.S. Ser. No. 09/579,673 and U.S. Ser. No. 10/717,082, which are incorporated herein by reference in their entirety, are non-limiting examples of such lateral flow test devices. Examples of patents that describe the use of "dip stick" technology to detect soluble antigens via immunochemical assays include, but are not limited to US Patent Nos. 4,444,880; 4,305,924; and 4,135,884; which are incorporated by reference herein in their entireties. The apparatuses and methods of these three patents broadly describe a first component fixed to a solid surface on a "dip stick" which is exposed to a solution containing a soluble antigen that binds to the component fixed upon the "dip stick," prior to detection of the component-antigen complex upon the stick. It is within the skill of one in the art to modify the teachings of this "dip stick" technology for the detection of polypeptides using antibody reagents as described herein.
[00211] Other techniques can be used to detect the level of a polypeptide in a sample. One such technique is the dot blot, an adaptation of Western blotting (Towbin et at., Proc. Nat. Acad. Sci. 76:4350 (1979)). In a Western blot, the polypeptide or fragment thereof can be dissociated with detergents and heat and separated on an SDS-PAGE gel before being transferred to a solid support, such as a nitrocellulose or PVDF membrane. The membrane is incubated with an antibody reagent specific for the target polypeptide or a fragment thereof. The membrane is then washed to remove unbound proteins and proteins with non-specific binding. Detectably labeled enzyme-linked secondary or detection antibodies can then be used to detect and assess the amount of polypeptide in the sample tested. A dot blot immobilizes a protein sample on a defined region of a support, which is then probed with antibody and labelled secondary antibody as in Western blotting. The intensity of the signal from the detectable label in either format corresponds to the amount of enzyme present, and therefore the amount of polypeptide. Levels can be quantified, for example by densitometry.
[00212] In some embodiments of any of the aspects, the level of a target can be measured, by way of non-limiting example, by Western blot; immunoprecipitation; enzyme-linked immunosorbent assay (ELISA); radioimmunological assay (RIA); sandwich assay; fluorescence in situ hybridization (FISH); immunohistological staining; radioimmunometric assay; immunofluoresence assay; mass spectroscopy and/or immunoelectrophoresis assay.
[00213] In some embodiments of any of the aspects, one or more of the reagents (e.g. an antibody reagent and/or nucleic acid probe) described herein can comprise a detectable label and/or comprise the ability to generate a detectable signal (e.g. by catalyzing reaction converting a compound to a detectable product). Detectable labels can comprise, for example, a light-absorbing dye, a fluorescent dye, or a radioactive label. Detectable labels, methods of detecting them, and methods of incorporating them into reagents (e.g. antibodies and nucleic acid probes) are well known in the art.
[00214] In some embodiments of any of the aspects, detectable labels can include labels that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifluoresence, or chemiluminescence, or any other appropriate means. The detectable labels used in the methods described herein can be primary labels (where the label comprises a moiety that is directly detectable or that produces a directly detectable moiety) or secondary labels (where the detectable label binds to another moiety to produce a detectable signal, e.g., as is common in immunological labeling using secondary and tertiary antibodies). The detectable label can be linked by covalent or non-covalent means to the reagent. Alternatively, a detectable label can be linked such as by directly labeling a molecule that achieves binding to the reagent via a ligand-receptor binding pair arrangement or other such specific recognition molecules. Detectable labels can include, but are not limited to radioisotopes, bioluminescent compounds, chromophores, antibodies, chemiluminescent compounds, fluorescent compounds, metal chelates, and enzymes.
[00215] In other embodiments, the detection reagent is label with a fluorescent compound. When the fluorescently labeled reagent is exposed to light of the proper wavelength, its presence can then be detected due to fluorescence. In some embodiments of any of the aspects, a detectable label can be a fluorescent dye molecule, or fluorophore including, but not limited to fluorescein, phycoerythrin, phycocyanin, o-phthaldehyde, fluorescamine, Cy3™, Cy5™, allophy cocyanine, Texas Red, peridenin chlorophyll, cyanine, tandem conjugates such as phycoerythrin-Cy5™, green fluorescent protein, rhodamine, fluorescein isothiocyanate (FITC) and Oregon Green™, rhodamine and derivatives (e.g., Texas red and tetrarhodimine isothiocynate (TRITC)), biotin, phycoerythrin, AMCA, CyDyes™, 6- carboxyfhiorescein (commonly known by the abbreviations FAM and F), 6-carboxy-2',4',7',4,7- hexachlorofiuorescein (HEX), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfiuorescein (JOE or J), N,N,N',N'-tetramethyl-6carboxyrhodamine (TAMRA or T), 6-carboxy-X-rhodamine (ROX or R), 5- carboxyrhodamine-6G (R6G5 or G5), 6-carboxyrhodamine-6G (R6G6 or G6), and rhodamine 110; cyanine dyes, e.g. Cy3, Cy5 and Cy7 dyes; coumarins, e.g umbelliferone; benzimide dyes, e.g. Hoechst 33258; phenanthridine dyes, e.g. Texas Red; ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes; polymethine dyes, e.g. cyanine dyes such as Cy3, Cy5, etc; BODIPY dyes and quinoline dyes. In some embodiments of any of the aspects, a detectable label can be a radiolabel including, but not limited to 3H, 1251, 35S, 14C, 32P, and 33P. In some embodiments of any of the aspects, a detectable label can be an enzyme including, but not limited to horseradish peroxidase and alkaline phosphatase. An enzymatic label can produce, for example, a chemiluminescent signal, a color signal, or a fluorescent signal. Enzymes contemplated for use to detectably label an antibody reagent include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. In some embodiments of any of the aspects, a detectable label is a chemiluminescent label, including, but not limited to lucigenin, luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester. In some embodiments of any of the aspects, a detectable label can be a spectral colorimetric label including, but not limited to colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, and latex) beads.
[00216] In some embodiments of any of the aspects, detection reagents can also be labeled with a detectable tag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin. Other detection systems can also be used, for example, a biotin-streptavidin system. In this system, the antibodies immunoreactive (i. e. specific for) with the biomarker of interest is biotinylated. Quantity of biotinylated antibody bound to the biomarker is determined using a streptavidin-peroxidase conjugate and a chromagenic substrate. Such streptavidin peroxidase detection kits are commercially available, e. g. from DAKO; Carpinteria, CA. A reagent can also be detectably labeled using fluorescence emitting metals such as 152Eu, or others of the lanthanide series. These metals can be attached to the reagent using such metal chelating groups as diethylenetriaminepentaacetic acid (DTP A) or ethylenediaminetetraacetic acid (EDTA).
[00217] The compositions and methods described herein can be administered to a subject having or diagnosed as having lung cancer. In some embodiments, the methods described herein comprise administering an effective amount of compositions described herein to a subject in order to alleviate a symptom of a lung cancer. As used herein, "alleviating a symptom" is ameliorating any condition or symptom associated with the lung cancer. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique. A variety of means for administering the compositions described herein to subjects are known to those of skill in the art. Such methods can include, but are not limited to oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, topical, injection, or intratumoral administration. Administration can be local or systemic. [00218] The term “effective amount" as used herein refers to the amount of an agent needed to alleviate at least one or more symptom of the disease or disorder and relates to a sufficient amount of pharmacological composition to provide the desired effect. The term "therapeutically effective amount" therefore refers to an amount of an agent that is sufficient to provide a particular anti-cancer effect when administered to a typical subject. An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not generally practicable to specify an exact “effective amount". However, for any given case, an appropriate “effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.
[00219] Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50. Compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (z.e., the concentration of the active agent, which achieves a half-maximal inhibition of symptoms) as determined in cell culture, or in an appropriate animal model. Levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay, e.g., assay for tumor size or growth, among others. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.
[00220] In certain embodiments, an effective dose of a composition described herein can be administered to a patient once. In certain embodiments, an effective dose of a composition can be administered to a patient repeatedly. For systemic administration, subjects can be administered a therapeutic amount of a composition such as, e.g. 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, or more. [00221] In some embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after treatment biweekly for three months, treatment can be repeated once per month, for six months or a year or longer. Treatment according to the methods described herein can reduce levels of a marker or symptom of a condition, e.g. tumor size or growth rate by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 % or at least 90% or more.
[00222] The dosage of a composition as described herein can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume treatment, or make other alterations to the treatment regimen. The dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to a therapy. The desired dose or amount of activation can be administered at one time or divided into subdoses, e.g., 2-4 subdoses and administered over a period of time, e.g., at appropriate intervals through the day or other appropriate schedule. In some embodiments, administration can be chronic, e.g., one or more doses and/or treatments daily over a period of weeks or months. Examples of dosing and/or treatment schedules are administration daily, twice daily, three times daily or four or more times daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, or more. A composition can be administered over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period.
[00223] The dosage ranges for the administration of a therapy according to the methods described herein depend upon, for example, the form of the active ingredient(s), its potency, and the extent to which symptoms, markers, or indicators of a condition described herein are desired to be reduced, for example the percentage reduction desired for tumor growth/size or the extent to which, for example, immune activity, are desired to be induced. The dosage should not be so large as to cause adverse side effects. Generally, the dosage will vary with the age, condition, and sex of the patient and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of any complication. [00224] The efficacy of a therapy in, e.g. the treatment of a condition described herein, or to induce a response as described herein can be determined by the skilled clinician. However, a treatment is considered “effective treatment," as the term is used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 10% following treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and/or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate, e.g. tumor size, tumor growth, cancer staging, immune responses. Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions (i.e., progression of the disease is halted). Methods of measuring these indicators are known to those of skill in the art and/or are described herein. Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the disease, e.g., preventing a worsening of symptoms (e.g. pain or inflammation); or (2) relieving the severity of the disease, e.g., causing regression of symptoms. An effective amount for the treatment of a disease means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that disease. Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response. It is well within the ability of one skilled in the art to monitor efficacy of administration and/or treatment by measuring any one of such parameters, or any combination of parameters. Efficacy can be assessed in animal models of a condition described herein, for example treatment of murine lung cancer.
[00225] In one respect, the present invention relates to the herein described compositions, methods, and respective component(s) thereof, as essential to the technology, yet open to the inclusion of unspecified elements, essential or not ("comprising). In some embodiments of any of the aspects, other elements to be included in the description of the composition, method or respective component thereof are limited to those that do not materially affect the basic and novel characteristic(s) of the technology (e.g., the composition, method, or respective component thereof “consists essentially of’ the elements described herein). This applies equally to steps within a described method as well as compositions and components therein. In other embodiments of any of the aspects, the compositions, methods, and respective components thereof, described herein are intended to be exclusive of any element not deemed an essential element to the component, composition or method (e.g., the composition, method, or respective component thereof “consists of’ the elements described herein). This applies equally to steps within a described method as well as compositions and components therein.
[00226] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.
[00227] The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction" or “decrease" or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.
[00228] The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statistically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5 -fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, an “increase” is a statistically significant increase in such level.
[00229] As used herein, a "subject" means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “individual,” “patient” and “subject” are used interchangeably herein.
[00230] Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of lung cancer. A subject can be male or female.
[00231] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g. lung cancer) or one or more complications related to such a condition, and optionally, have already undergone treatment for the condition or the one or more complications related to the condition. Alternatively, a subject can also be one who has not been previously diagnosed as having the condition or one or more complications related to the condition. For example, a subject can be one who exhibits one or more risk factors for the condition, or one or more complications related to the condition or a subject who does not exhibit risk factors.
[00232] A “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.
[00233] As used herein, the term “cancer” relates generally to a class of diseases or conditions in which abnormal cells divide without control and can invade nearby tissues. Cancer cells can also spread to other parts of the body through the blood and lymph systems. There are several main types of cancer. Carcinoma is a cancer that begins in the skin or in tissues that line or cover internal organs. Adenocarcinoma is a carcinoma of epithelial tissue with a glandular origin and/or glandular characteristics. Sarcoma is a cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue. Leukemia is a cancer that starts in blood-forming tissue such as the bone marrow and causes large numbers of abnormal blood cells to be produced and enter the blood. Lymphoma and multiple myeloma are cancers that begin in the cells of the immune system. Central nervous system cancers are cancers that begin in the tissues of the brain and spinal cord.
[00234] In some embodiments of any of the aspects, the cancer is lung cancer. In some embodiments of any of the aspects, the cancer is non-small cell lung cancer. In some embodiments of any of the aspects, the cancer is lung adenocarcinoma. In some embodiments of any of the aspects, the cancer is stage I lung adenocarcinoma. In some embodiments of any of the aspects, the cancer is stage I lung cancer.
[00235] In some embodiments of any of the aspects, the cancer is a primary cancer. In some embodiments of any of the aspects, the cancer is a malignant cancer. As used herein, the term “malignant” refers to a cancer in which a group of tumor cells display one or more of uncontrolled growth (z.e., division beyond normal limits), invasion (z.e., intrusion on and destruction of adjacent tissues), and metastasis (z.e., spread to other locations in the body via lymph or blood). As used herein, the term “metastasize” refers to the spread of cancer from one part of the body to another. A tumor formed by cells that have spread is called a “metastatic tumor” or a “metastasis.” The metastatic tumor contains cells that are like those in the original (primary) tumor. As used herein, the term “benign” or “non-malignant” refers to tumors that may grow larger but do not spread to other parts of the body. Benign tumors are self-limited and typically do not invade or metastasize. [00236] A “cancer cell” or “tumor cell” refers to an individual cell of a cancerous growth or tissue. A tumor refers generally to a swelling or lesion formed by an abnormal growth of cells, which may be benign, pre-malignant, or malignant. Most cancer cells form tumors, but some, e.g., leukemia, do not necessarily form tumors. For those cancer cells that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably.
[00237] As used herein the term "neoplasm" refers to any new and abnormal growth of tissue, e.g., an abnormal mass of tissue, the growth of which exceeds and is uncoordinated with that of the normal tissues. Thus, a neoplasm can be a benign neoplasm, premalignant neoplasm, or a malignant neoplasm. [00238] A subject that has a cancer or a tumor is a subject having objectively measurable cancer cells present in the subject’s body. Included in this definition are malignant, actively proliferative cancers, as well as potentially dormant tumors or micrometastatses. Cancers which migrate from their original location and seed other vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs.
[00239] A “cancer cell” is a cancerous, pre-cancerous, or transformed cell, either in vivo, ex vivo, or in tissue culture, that has spontaneous or induced phenotypic changes that do not necessarily involve the uptake of new genetic material. Although transformation can arise from infection with a transforming virus and incorporation of new genomic nucleic acid, or uptake of exogenous nucleic acid, it can also arise spontaneously or following exposure to a carcinogen, thereby mutating an endogenous gene. Transformation/cancer is associated with, e.g., morphological changes, immortalization of cells, aberrant growth control, foci formation, anchorage independence, malignancy, loss of contact inhibition and density limitation of growth, growth factor or serum independence, tumor specific markers, invasiveness or metastasis, and tumor growth in suitable animal hosts such as nude mice.
[00240] As used herein, the term “vascular invasion” refers to the presence of tumor cells within the lumen of blood and/or lymph vessel.
[00241] As used herein, the terms “protein" and “polypeptide" are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alphaamino and carboxy groups of adjacent residues. The terms "protein", and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. "Protein" and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing. The terms also refer to fragments or variants of the polypeptide that maintain at least 50% of the activity or effect of the full length polypeptide. Conservative substitution variants that maintain the activity of wildtype protein will include a conservative substitution as defined herein. The identification of amino acids most likely to be tolerant of conservative substitution while maintaining at least 50% of the activity of the wildtype is guided by, for example, sequence alignment with homologs or paralogs from other species. Amino acids that are identical between homologs are less likely to tolerate change, while those showing conservative differences are obviously much more likely to tolerate conservative change in the context of an artificial variant. Similarly, positions with non-conservative differences are less likely to be critical to function and more likely to tolerate conservative substitution in an artificial variant.
[00242] In some embodiments, a polypeptide can be a variant of a sequence described herein. In some embodiments, the variant is a conservative substitution variant. Variants can be obtained by mutations of native nucleotide sequences, for example. A “variant,” as referred to herein, is a polypeptide substantially homologous to a native or reference polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions or substitutions. Polypeptide -encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a variant protein or fragment thereof that retains the relevant biological activity relative to the reference protein, e.g., at least 50% relative to wildtype. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage, (i.e. 5% or fewer, e.g. 4% or fewer, or 3% or fewer, or 1% or fewer) of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. It is contemplated that some changes can potentially improve the relevant activity, such that a variant, whether conservative or not, has more than 100% of the activity of wildtype, e.g. 110%, 125%, 150%, 175%, 200%, 500%, 1000% or more.
[00243] One method of identifying amino acid residues which can be substituted is to align, for example, a human sequence to a homolog from one or more non-human species. Alignment can provide guidance regarding not only residues likely to be necessary for function but also, conversely, those residues likely to tolerate change. Where, for example, an alignment shows two identical or similar amino acids at corresponding positions, it is more likely that that site is important functionally. Where, conversely, alignment shows residues in corresponding positions to differ significantly in size, charge, hydrophobicity, etc., it is more likely that that site can tolerate variation in a functional polypeptide. The variant amino acid or DNA sequence can be at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to a native or reference sequence, or a nucleic acid encoding one of those amino acid sequences. The degree of homology (percent identity) between a native and a mutant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web. The variant amino acid or DNA sequence can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, similar to the sequence from which it is derived (referred to herein as an “original” sequence). The degree of similarity (percent similarity) between an original and a mutant sequence can be determined, for example, by using a similarity matrix. Similarity matrices are well known in the art and a number of tools for comparing two sequences using similarity matrices are freely available online, e.g. BLASTp or BLASTn (available on the world wide web at blast.ncbi.nlm.nih.gov), with default parameters set. [00244] In the various embodiments described herein, it is further contemplated that variants (naturally occurring or otherwise), alleles, homologs, conservatively modified variants, and/or conservative substitution variants of any of the particular polypeptides described are encompassed. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid and retains the desired activity of the polypeptide. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure. [00245] A given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as He, Vai, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known. Polypeptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired activity and specificity of a native or reference polypeptide is retained.
[00246] A given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as He, Vai, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known. Polypeptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired activity of a native or reference polypeptide is retained. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure.
[00247] Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Particular conservative substitutions include, for example; Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into His; Asp into Glu; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; He into Leu or into Vai; Leu into He or into Vai; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into He; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and/or Phe into Vai, into He or into Leu. Typically conservative substitutions for one another also include: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).
[00248] In some embodiments, the polypeptide described herein (or a nucleic acid encoding such a polypeptide) can be a functional fragment of one of the amino acid sequences described herein. As used herein, a “functional fragment” is a fragment or segment of a peptide which retains at least 50% of the wildtype reference polypeptide’s activity according to the assays described below herein. A functional fragment can comprise conservative substitutions of the sequences disclosed herein.
[00249] In some embodiments, the polypeptide described herein can be a variant of a sequence described herein. In some embodiments, the variant is a conservatively modified variant. Conservative substitution variants can be obtained by mutations of native nucleotide sequences, for example. A “variant," as referred to herein, is a polypeptide substantially homologous to a native or reference polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions or substitutions. Variant polypeptide- encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a variant protein or fragment thereof that retains activity.
[00250] As used herein, the term “nucleic acid” or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single -stranded or double-stranded. A single-stranded nucleic acid can be one nucleic acid strand of a denatured double- stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double -stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable DNA can include, e.g., genomic DNA or cDNA. Suitable RNA can include, e.g., mRNA.
[00251] The term "expression" refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. Expression can refer to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid fragment or fragments of the invention and/or to the translation of mRNA into a polypeptide.
[00252] In some embodiments, the expression of a biomarker(s), target(s), or gene/polypeptide described herein is/are tissue-specific. In some embodiments, the expression of a biomarker(s), target(s), or gene/polypeptide described herein is/are global. In some embodiments, the expression of a biomarker(s), target(s), or gene/polypeptide described herein is systemic.
[00253] "Expression products" include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" means the nucleic acid sequence which is transcribed (DNA) to RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. The gene may or may not include regions preceding and following the coding region, e.g. 5’ untranslated (5’UTR) or "leader" sequences and 3’ UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).
[00254] Marker" in the context of the present invention refers to an expression product, e.g., nucleic acid or polypeptide which is differentially present in a sample taken from subjects having lung cancer (e.g., vascular invasive lung adenocarcinoma), as compared to a comparable sample taken from control subjects (e.g., a healthy subject). The term "biomarker" is used interchangeably with the term "marker."
[00255] In some embodiments, the methods described herein relate to measuring, detecting, or determining the level of at least one marker. As used herein, the term "detecting" or “measuring” refers to observing a signal from, e.g. a probe, label, or target molecule to indicate the presence of an analyte in a sample. Any method known in the art for detecting a particular label moiety can be used for detection. Exemplary detection methods include, but are not limited to, spectroscopic, fluorescent, photochemical, biochemical, immunochemical, electrical, optical or chemical methods. In some embodiments of any of the aspects, measuring can be a quantitative observation.
[00256] As used herein, the terms "treat,” "treatment," "treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder, e.g. lung cancer. The term “treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with a lung cancer. Treatment is generally “effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective" if the progression of a disease is reduced or halted. That is, “treatment" includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (z.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality, whether detectable or undetectable. The term "treatment" of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).
[00257] In some embodiments of any of the aspects, treatment is effective if the size of the tumor is reduced. In some embodiments of any of the aspects, treatment is effective if the risk of metastasis is reduced. In some embodiments of any of the aspects, treatment is effective if the rate of metastasis is reduced. In some embodiments of any of the aspects, treatment is effective if the risk of vascular invasion is reduced. In some embodiments of any of the aspects, treatment is effective if the rate of vascular invasion is reduced. In some embodiments of any of the aspects, treatment is effective if the spread to lymph nodes is reduced. In some embodiments of any of the aspects, treatment is effective if the rate of spread to lymph nodes is reduced. In some embodiments of any of the aspects, treatment is effective if the spread to lymph nodes is prevented.
[00258] In some embodiments of any of the aspects, described herein is a prophylactic method of treatment. As used herein “prophylactic” refers to the timing and intent of a treatment relative to a disease or symptom, that is, the treatment is administered prior to clinical detection or diagnosis of that particular disease or symptom in order to protect the patient from the disease or symptom. Prophylactic treatment can encompass a reduction in the severity or speed of onset of the disease or symptom, or contribute to faster recovery from the disease or symptom. Accordingly, the methods described herein can be prophylactic relative to metastasis, vascular invasion, and/or spread to lymph nodes. In some embodiments of any of the aspects, prophylactic treatment is not prevention of all symptoms or signs of a disease.
[00259] As used herein, the term "administering," refers to the placement of a compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject. In some embodiments, administration comprises physical human activity, e.g., an injection, act of ingestion, an act of application, and/or manipulation of a delivery device or machine. Such activity can be performed, e.g., by a medical professional and/or the subject being treated.
[00260] As used herein, “contacting" refers to any suitable means for delivering, or exposing, an agent to at least one protein or nucleic acid. Exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, perfusion, injection, or other delivery method well known to one skilled in the art. In some embodiments, contacting comprises physical human activity, e.g., an injection; an act of dispensing, mixing, and/or decanting; and/or manipulation of a delivery device or machine. In some embodiments, the contacting provides direct physical contact of the agent and the at least one protein or nucleic acid, e.g., the agent and the at least one protein or nucleic acid are touching, e.g., at least one surface of the agent is touching or forming a junction with at least one aspect of the at least one protein or nucleic acid. [00261] The term “statistically significant" or “significantly" refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.
[00262] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%.
[00263] As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation.
[00264] The term "consisting of refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
[00265] As used herein the term "consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
[00266] As used herein, the term “corresponding to” refers to an amino acid or nucleotide at the enumerated position in a first polypeptide or nucleic acid, or an amino acid or nucleotide that is equivalent to an enumerated amino acid or nucleotide in a second polypeptide or nucleic acid. Equivalent enumerated amino acids or nucleotides can be determined by alignment of candidate sequences using degree of homology programs known in the art, e.g., BLAST.
[00267] As used herein, the term “specific binding” refers to an interaction between two molecules, compounds, cells and/or particles wherein the first entity binds to the second entity with greater specificity and affinity than it binds to a third entity. A first entity specifically bound to a second entity is not displaced by a non-similar competitor. In certain embodiments, a first entity is said to specifically bind a second entity when it preferentially recognizes the second entity in a complex mixture of proteins and/or macromolecules. In some embodiments, specific binding can refer to an affinity of the first entity for the second entity which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or greater than the affinity for the third entity. In some embodiments, specific binding refers to the ability of a first entity to bind to a second entity with a KD ICT5 M (10000 nM) or less, e.g., 1 6 M. 10 7 M. 10 2 M. 10 ' M. 10 |U M. 10 1 1 M, 10 12 M. or less. The person of ordinary skill in the art can determine appropriate conditions under which a first entity (e.g., an aptamer or an antibody described herein) selectively binds a second entity (e.g., an antigen or biomolecule) using any suitable methods, such as titration of an entity in a suitable binding assay. Specific binding can comprise ionic bonding, hydrogen bonds, ionic bonds, van der Waals interactions, and/or London dispersion forces. In some embodiments, specific binding does not refer to covalent bonding. In some embodiments, specific binding does not refer to a peptide bond. In some embodiments, specific binding does not refer to a phosphodiester bond. [00268] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a nonlimiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."
[00269] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
[00270] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978- 0911910421); Bruce Alberts et al., Molecular Biology of the Cell, published by W.W. Norton & Company, 2022 (ISBN 0393884821, 978-0393884821); John M. Lackie eat al. (eds.), The Dictionary of Cell and Molecular Biology, 5th Edition, published by Academic Press, 2013 (ISBN 0123849314, 978- 0123849311); Nalini Chandar et al., Lippincott Illustrated Reviews: Cell and Molecular Biology, 3rd Edition, published by LWW, 2023 (ISBN 1975180895, 978-1975180898); Teresa Atwood et al., Oxford Dictionary of Biochemistry and Molecular Biology, 2nd Edition, published by Oxford University Press, 2006; Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); Johnathan Law et al., (eds.), A Dictionary of Chemistry, 8th Edition, published by Oxford University Press, 2020 (ISBN 9780198841227, 9780191876783); Robert C. King et al. (eds.), A Dictionary of Genetics, 8th Edition, published by Oxford University Press, 2013 (ISBN 9780199766444, 9780199376865); Richard Cammack et al. (eds.), Oxford Dictionary of Biochemistry and Molecular Biology, 2nd Edition, published by Oxford University Press, 2006 (ISBN 9780198529170, 9780191727641); John Lackie et al. (eds.), A Dictionary of Biomedicine, 2nd Edition, published by Oxford University Press, 2019 (ISBN 9780191829116); Lodish et al., Molecular Cell Biology, 8th Edition, published by W.H. Freeman, 2016 (ISBN 1464183392, 978-1464183393); Abul K. Abbas et al., Cellular and Molecular Immunology, 10th Edition, published by Elsevier, 2021 (ISBN 0323757480, 978-0323757485); Kenneth M. Murphy et al., Janeway's Immunobiology, 10th Edition, published by W. W. Norton & Company, 2022 (ISBN 0393884899, 978-0393884890); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 0444569464); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Frederick M. Ausubel (ed.), Current Protocols in Molecular Biology (CPMB), John Wiley and Sons, 1987-2010 (ISBN 047150338X, 9780471503385); Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties. [00271] One of skill in the art can readily identify a therapeutic agent of use (e.g. see Edward Chu et al., Physicians' Cancer Chemotherapy Drug Manual, published by Jones & Bartlett Learning, 2024 (ISBN 1284000001, 978-1284000009); Joseph Loscalzo et al., Harrison's Principles of Internal Medicine, 21st edition, published by McGraw Hill/Medical, 2022 (ISBN 1264268505, 978- 1264268504); and John E. Neiderhuber et al, Abeloff s Clinical Oncology, 6th Edition, published by Elsevier, 2019 (ISBN 0323476740, 978-0323476744), the contents of which are all incorporated by reference herein in their entireties.
[00272] In all embodiments where a sample is obtained or has been obtained or provided, the sample can be sample taken, obtained, or provided via minimally invasive methods and/or involves only a minor intervention. In some embodiments of any of the aspects, a sample is taken, obtained, or provided by one or more of a blood draw or prick, an epidermal or mucus membrane swab, buccal sampling, saliva sample, a epidermal skin sampling technique, and/or collection of a secreted or expelled bodily fluid (e.g., mucus, urine, sweat, etc.), fecal sampling, semen/seminal fluid sampling, or clippings (e.g., of hair or nails). In some embodiments of any of the aspects, the sample comprises, consists of, or consists essentially of blood (or any fraction or component thereof), serum, urine, mucus, epithelial cells, saliva, buccal cells, a secreted or expelled bodily fluid, and/or hair or nail clippings. [00273] Other terms are defined herein within the description of the various aspects of the invention. [00274] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.
[00275] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.
[00276] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.
[00277] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.
[00278] In some embodiments, the present technology may be defined in any of the following numbered paragraphs:
1. A method comprising, determining the expression of at least one gene selected from the group consisting of:
SHR00M4; NBEAL1; MUC16; NOTCH3; H19; COL1A1; and GARS1; in a subject with lung cancer.
2. A method of treating or prognosing lung cancer, the method comprising: administering adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and/or lymphadenectomy to a subject having decreased expression of at least one gene selected from the group consisting of:
SHR00M4 and NBEAL1; and/or increased expression of at least one gene selected from the group consisting of: MUC16; NOTCH3; H19; COL1A1; and GARSl . he method of any one of the preceding paragraphs, further comprising administering active surveillance, wedge resection of the lung, or a segmentectomy of the lung to a subject not having decreased expression of at least one gene selected from the group consisting of:
SHR00M4 and NBEAL1; and/or not having increased expression of at least one gene selected from the group consisting of: MUC16; N0TCH3; H19; C0L1A1; and GARS1. he method of any one of the preceding paragraphs, wherein the at least one gene is SHR00M4 or
NBEAL1. he method of any one of the preceding paragraphs, wherein the at least one gene is SHR00M4 or
NBEAL1. he method of any one of the preceding paragraphs, wherein the at least one gene is MUC16;
N0TCH3; H19; or C0L1A1. he method of any one of the preceding paragraphs, wherein the at least one gene is SHR00M4;
NBEAL1; MUC16; N0TCH3; H19; and C0L1A1. method of treating or prognosing lung cancer, the method comprising determining the level of expression: a) at least one gene selected from the group consisting of:
MKI67; ASPM; ATAD2; TOP2A; FANCI; PLOD2; NCAPG2; CENPF; KPNA2;
BRCA1; HLTF; and FANCA; b) at least one gene selected from the group consisting of:
C0L1A2; C0L3A1; C0L1A1; and COL6A3; c) at least one gene selected from the group consisting of:
PRKDC; TPI1; PKM; GAPDH; AHNAK2; YWHAZ; ATP 13 A3; GARS1; CP;
MUC16; TUBA1C; H19; PPFIA1; N0TCH3; ACLY; GCLC; CAD; and CCT6A; d) at least one gene selected from the group consisting of:
SPTBN1; SFTPB; LRRK2; AKAP13; CHD2; EPAS1; ATP 11 A; ROS1; MTUS1;
ANKHD1; NBEAL1; MPRIP; SHR00M4; and TET2 wherein: e) increased expression of at least one gene selected from the group consisting of:
MKI67; ASPM; ATAD2; TOP2A; FANCI; PLOD2; NCAPG2; CENPF; KPNA2; BRCA1; HLTF; and FANCA; f) increased expression of at least one gene selected from the group consisting of:
COL1A2; COL3A1; COL1A1; and COL6A3; g) increased expression of at least one gene selected from the group consisting of:
PRKDC; TPI1; PKM; GAPDH; AHNAK2; YWHAZ; ATP 13 A3; GARS1; CP; MUC16; TUBA1C; H19; PPFIA1; N0TCH3; ACLY; GCLC; CAD; and CCT6A; and h) decreased expression of at least one gene selected from the group consisting of: SPTBN1; SFTPB; LRRK2; AKAP13; CHD2; EPAS1; ATP11A; R0S1; MTUS1;
ANKHD1; NBEAL1; MPRIP; SHR00M4; and TET2 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and/or lymphadenectomy. he method of any one of the preceding paragraphs, wherein the increased expression and/or decreased expression is relative to an average for subjects with lung cancer. The method of any one of the preceding paragraphs, wherein the increased expression and/or decreased expression is relative to an average for subjects with lung adenocarcinoma. The method of any one of the preceding paragraphs, wherein the increased expression and/or decreased expression is relative to an average for subjects with lung cancer that is not vascular invasive. The method of any one of the preceding paragraphs, wherein the subject having increased expression of the at least one gene is determined to have increased expression of the at least one gene. The method of any one of the preceding paragraphs, wherein the subject having increased expression of the at least one gene is determined to have increased expression of the at least one gene in a sample of tumor cells. The method of any one of the preceding paragraphs, wherein the level of expression is the level of mRNA. The method of any one of the preceding paragraphs, wherein the subject having increased expression of the at least one gene is not administered a lobectomy. The method of any one of the preceding paragraphs, wherein the subject having increased expression of the at least one gene is not administered a sublobar resection. The method of any one of the preceding paragraphs, wherein the subject having increased expression of the at least one gene is not administered a lobectomy or sublobar resection. The method of any one of the preceding paragraphs, wherein the lung cancer is non-small cell lung cancer (NSCLC). The method of any one of the preceding paragraphs, wherein the lung cancer is lung adenocarcinoma. The method of any one of the preceding paragraphs, wherein the lung cancer comprises vascular invasion. The method of any one of the preceding paragraphs, wherein the lung cancer is vascular invasive lung adenocarcinoma. The method of any one of the preceding paragraphs, wherein the lung cancer is stage I lung adenocarcinoma.
EXAMPLES Example 1
[00279] Lung cancer is the leading cause of cancer-related deaths in the United States. Currently, over 250,000 cases of lung cancer are diagnosed annually in the US and the incidence of early-stage disease is increasing due to earlier detection through implementation of CT-based screening programs. Lung adenocarcinoma (LU AD) is the most common lung cancer subtype diagnosed in the US and represents about l/3rd of total cases. With increasing incidence, there is a need for improved patient stratification to help guide appropriate treatment for high risk patients and to ensure that low risk patients are not subjected to complications from unnecessary treatments.
[00280] The invention described herein comprises a method for stratifying patients according to the aggressiveness of LU AD based on gene expression in the tumor. The biomarker is predictive of the presence of vascular invasion (VI) within LUAD tumors. Using the methods of this invention, the gene expression biomarker can accurately classify patients with VI into a high-risk category. This distinguishes it from existing molecular biomarkers such as DetermaRx (Oncocyte) that predict outcomes in NSCLC. Biomarkers based on patient outcomes may be influenced by treatment response and standard of care.
[00281] VI is difficult to assess in resection specimens and not routinely reported, despite its prognostic significance. It requires a time-consuming procedure for a pathologist to inspect hundreds of consecutive sections of the tumor. In one aspect of the invention, the molecular biomarker can accurately predict VI positive tumors from a small amount of resected material, streamlining pathology assessment. This may also guide appropriate adjuvant chemotherapy. There is currently no established way to predict VI in tumors prior to surgery and the complete pathologic assessment of the fully resected tumor. While pre-surgical biopsy material such as transthoracic needle biopsies are routinely collected during LUAD clinical work-up, it is insufficient for a pathologist to rule out the presence of angioinvasion. Thus, in another aspect of the invention, the molecular biomarker can be used to predict VI prior to surgery. While the standard of care for early-stage LUAD resection is complete lobectomy, the field is moving toward limited resection or tissue-sparing surgery, as the favorable approach. Predicting VI prior to surgery offers the clinical team with additional information that may influence moving to traditional lobectomy over limited resection to minimize chances of patient recurrence. Finally, predicting high-risk angioinvasive tumors prior to surgery also allows for an opportunity to guide future neoadjuvant treatment approaches. In patients who are not surgical candidates, VI may predict which ablative (non-surgical) therapies (RFA, MWA, cryoablation) are radiation therapies (SBRT) are likely to benefit the patient.
[00282] A VI biomarker score is calculated from quantitation of mRNA abundance in LUAD tumor samples. If the levels of mRNA from genes in Cluster 1, 2 and 3 are higher than reference levels observed in non-VI LUAD samples and the levels of mRNA from genes in Cluster 4 are lower than reference levels observed in non-VI LUAD samples, this is predictive of the presence of vascular invasion in the LU AD tumor. Specifically, the expression of 48 genes is used in the calculation of the VI biomarker score.
[00283] Cluster 1 (expression from 12 genes) contains the following that are increased in tumors with VI:"MKI67" "ASPM" "ATAD2" "TOP2A" "FANCI" "PLOD2" "NCAPG2" "CENPF" "KPNA2" "BRCA1" "HLTF " "FANCA”
[00284] Cluster 2 (expression from 4 genes) contains the following that are increased in tumors with VI: "COL1A2" "COL3A1" "COL1A1" "COL6A3"
[00285] Cluster 3 (expression from 18 genes) contains the following that are increased in tumors with VI: "PRKDC" "TPI1" "PKM" "GAPDH" "AHNAK2" "YWHAZ" "ATP13A3" "GARS1" "CP" ”MUC16" "TUBA1C" "H19" "PPFIA1" "NOTCH3" "ACLY" "GCLC" "CAD" "CCT6A" [00286] Cluster 4 (expression from 14 genes) contains the following that are decreased in tumors with VI: "SPTBN1" "SFTPB" "LRRK2" "AKAP13" "CHD2" "EPAS1" "ATP11A" "ROS1" "MTUS1" "ANKHD1" "NBEAL1" "MPRIP" "SHROOM4" "TET2"
[00287] These 48 genes were selected from a larger list of 474 genes that were found to be differentially expressed between tumors with VI and tumors of low malignant potential (LMP). Each of the four clusters thus contains the following genes (which include the 48 listed above):
[00288] Cluster 1 (expression from 115 genes) contains the following that are increased in tumors with VI: "TOP2A" "MCM4" "TROAP" "ASPM" " CENPF" "HJURP" "POLQ" " CENPI" "RRM2" "KIF4A" "UBE2T" " CEP55" "UBE2C" "FOXM 1" "ANLN" "TPX2" " CCNB 1" "BUB 1" "CDC45" "KIF 14" "DTL" "NUF2" "RAD51AP1" "PLOD2" "KPNA2" "SPAG5" "BUB 1B" "CDC6" "TTK" "UHRF1" "IQGAP3" "ZWINT" "CCNA2" "MKI67" "BRCA1" "KIF11" "EXO1" "NCAPG" "PRR11" "SGO 1" "KIF 15" "NEK2" "DLGAP5" "KIF23" "NUSAP1" "CDK1" "KIF18B" "FANCB" "CCNB2" "DEPDC1" "KIF20A" "WDHD1" "ARHGAP11A" "CENPE" "MCM10" "TYMS" "EZH2" "PLK1" "ESPL1" "HMMR" "PLK4" "CLSPN" "ERCC6L" "ECT2" "CENPU" "CIP2A" "ATAD5" "CDCA3" "NDC80" "NCAPH" "CHEK1" "DEPDC1B" "CCNE2" "KNL1" "RACGAP 1" "RFC4" "KIFC 1" "PBK" "RAD54L" "PCLAF" "TICRR" "CDCA5" "PTTG1" "BRIP 1" "CCDC 150" "MCM6" "ATAD2" " STIL" " SGO2" "FANCI" "SHCBP 1" "CDCA2" "FANCA" "MELK" "E2F8" "HROB" "FAM 1 1 1B" "POLE2" "MCM2" "AURKA" "CENPA" "CDC25C" "CDKN3" " SPC25" "ESCO2" "CKAP2L" "FAM83D" "ORC6" "PRC1" "ORC1" "XRCC2" "NCAPG2" "TIMELESS" "HELLS" "HLTF"
[00289] Cluster 2 (expression from 37 genes) contains the following that are increased in tumors with VI: "SULF1" "THBS2" "CSMD2" "CTHRC1" "COL1A1" "IGFBP3" "CILP" "MMP11" "COL11A1" "COL5A1" "MXRA5" "ADAM12" "COL5A2" "COL3A1" "VCAN" "ITGA11" "FAP" "COL12A1" "COL1A2" "COL6A3" "SPOCK1" "MMP14" "LRRC15" "DNM1" "WT1.AS" "COL10A1" "CCN4" "ADAM TS 12" "SFRP2" "POSTN" "OLFML2B" "THY1" "HTRA3" "INHBA" "ALDH1L2" "DIO2" "SH3PXD2 B" [00290] Cluster 3 (expression from 182 genes) contains the following that are increased in tumors with VI: "POTEE" "RECQL4" "AHNAK2" "MYBL2" "ABCC2" "NEIL3" "SLC2A1" "UBE2S" "COL7A 1" "EGLN3" "CERCAM" "GCLC" "GAPDH" "GARS1" "GRIN2D" "CDT1" "CDC20" "T ONSL" "FAM83F" "GTSE1" "ZNF850" "USB1" "IGF2BP3" "KIF2C" "ZDHHC18" "ADGRF4 " "MUC16" "SPP1" "KIF5A" "CASTOR2" "SLC6A8" "NGEF" "SRPX2" "MED12L" "TK1 " "RHOV" "SYNJ2" "KIF1A" "POTEF" "TUBB3" "SNCG" "GNPNAT1" "HOXB6" "FBXO32" "PAICS" "SRXN1" "ENTPD7" "CYP4F3" "AK4" "TPH" "TMPRSS11E" "SLC25A39" "ZYG11A" "IGHV3.73" "GALNT6" "TNS4" "DYRK2" "IGF2BP1" "PFKP" "ABCA12" "EMEI" "DGCR5.1" "H2BC13" "FIBP" "TMEM65" "ARNTL2" "H2AC4" "PSMB5" "IRAKI" "S LC16A3" "MTBP" "CPS1" "CGREF1" "MEIOC" "ATG9B" "TWF1" "DTYMK" "ASF1B" "AFAP1L2" "PPAT" "NUP62CL" "C1QTNF6" "GPC1" "OPN3" "GINS1" ITGB5" "ERO1A" "PRKDC" "VSIG10L" "PSAT1" "TNNC2" "FAM83A" "UCK2" "RAB5IF" "UPK3A" "KIAA03 19" "AZINI" "LGALS9B" "CDH17" "METTL1" "FLAD1" "HOXB5" FER1L4" "MAD2L1" "IT PKA" "RAB15" "FAM3C" "RUVBL1" "SHKBP1" "H2BC3" "ACLY" "SLC12A8" "PKM" "MAP7D2" "GPR87" "TDRD5" "POLR2H" "HMGA1" "ZNF229" "AKR1B15" "MEGF10" "P FKFB4" "Hl.5" "PLAU" "TOMM40" "DHRS11" "RN7SL3" "MELTF" "ERAL1" "FHL2" "PRAME" "NOTCH3" "FADS1" "CASTOR3" "PPFIA1" "PKMYT1" "GRIK5" "GALNT14" "CD C25A" "UGDH" "DCAF13" "YWHAZ" "FBL" "P3H4" "CCT6A" "LIMK1" "GAPDHP1" "ARHGEF39" "H2BC17" "PLEKHG6" "ALYREF" "PFN2" "H4C14" "LYG1" "LMNB2" "G6PD" "SLC7A5" "CAD" "PKD1L2" "CP" "RPLP0P2" "AC010329.5" "H2AC12" "H19" "TUBA1C" "C17orf58" "ALG1L2" "CMSS1" "TMEM250" "HOXA10" SRM" "CDK4" "H4C15" "GGH" "CLDN8" "NCBP2" "EPYC" "CARS1" "ATP13A3" "UPK1B" "FAAP24" "PAC SIN1"
[00291] Cluster 4 (expression from 140 genes) contains the following that are decreased in tumors with VI: "CYP4B1" "SCGB3A1" "ITGA10" "LRRK2" "GANC" "ACOXL" "REPS2" "CGNL1" "SNX30 " "GCNT4" "IVD" "PIGA" "MACROD2" "CTSH" "CELF2" "PDZD2" "SMAD4" "SHROOM4" "ARID4A" "MFSD2A" "CAPN3" "KIAA1328" "FLRT3" "ALDH2" "ROBO2" "T LR2" "CCDC68" "ACADSB" "TLE4" "BTG2" "ZNF540" "FILIP1" "ZNF385B" "C4BPA" "NOSTRIN" "CFTR" "VIPR1" "FBXO38" "CYP4X1" "BAIAP2" "RBPMS" "DNM3" "Fll" "PCDH20" "PLA2G4B" "PRSS12" "CHD2" "MIR29B2CHG" "CACNB4" "MTRR" "RIC3" "CREBRF" "CD44"
"TSPYL2" "L3MBTL4" "SMARCA2" "ANKHD1" "AKAP13" "AC012651.1" "EDNRB" "ZNF397" "HSDL2" "EPAS1" "GK" "SLC5A9" "JAM2" "CLEC16A" "SWAP70" "AKAP1" "MAN1C1" "ATP11A" "MTMR10" "TET2" "SNX25" "BMPER" "CACNA2D2" "BANK1 " "FHL1" "TFPI" "UST" "ERBB4" "PPP3CC" "DGKE" "VAMP2" "RPL5P9" "MTUS1" "DDX24" "RBMS3" "AL663070.1" "PIK3R1" "PCDH9" "PEBP4" "NDNF" "INSYN1" "ROS1" "SPATA18" "COL19A1" "ZNF609" "SAT2" "BMP2" "SUSD2" "ARHGEF2" "PPM1D" "ABCC4" "BCL11A" "TNS2" "SLC6A4" "HIRIP3" "CRIM1" "COL6A5" "PREX2" "ARNT2" "RAPGEF3" "ITPRID2" "SLC22A3" "NFIA" "ARHGAP24" "CYBRD1" "CYB5A" "SNORD61" "CISH" "FCER2" "NBEAL1" "SOCS2" "DRAM1" "TOX" "FGD4" "MASP1" "ALDH3A2" "KIDINS220" "GCOM1" "ARHGAP31" "ABCA10" "SPTBN1" "SFTPB" "CYFIP2" "AGL" "CALCOCO2" "MPRIP" "EPHX2"
[00292] Only the expression of the 48 genes is used for the biomarker score, but the derivation of these 474 genes was an important step in the process of developing the VI biomarker.
Example 2: Spatially informed profiling of stage I lung adenocarcinoma reveals a gene expression signature of vascular invasion
[00293] Microscopic vascular invasion (VI) is predictive of recurrence and benefit from lobectomy in stage I lung adenocarcinoma (LU AD) but is difficult to assess in resection specimens and cannot be accurately predicted prior to surgery. Thus, new biomarkers are needed to identify this aggressive subset of stage I LU AD tumors. To assess molecular and tumor microenvironment features associated with angioinvasive LU AD we analyzed 163 resected stage I tumors with and without VI by RNA-seq, including 15 samples by high-resolution spatial transcriptomics (stRNA-seq). Despite the small size of invaded blood vessels, we identified a molecular signature of VI from the bulk RNA-seq discovery cohort (n=I03) and found that it was associated with VI foci, desmoplastic stroma, and high-grade patterns in our stRNA-seq data. We observed a stronger association with high-grade patterns from VI+ compared with VI’ tumors. Using a discovery cohort, we developed a transcriptomic predictor of VI, that in an independent validation cohort (n = 60) was associated with VI (AUROC=0.86; p=5.4xl0’6) and predictive of recurrence -free survival (HR=1.98; p=0.02), even in VI’ LU AD (HR=2.76; p =0.003). To demonstrate our VI predictor’s robustness to intra-tumor heterogeneity in a larger cohort, we used RNA-seq data from multi-region sampling of stage I LU AD cases in TRACERx, where the predictor scores showed high correlation (R=0.88, p<2.2xl0 16) between two randomly sampled regions of the same tumor. Our study suggests that Vi-associated gene expression changes are detectable beyond the site of intravasation and can be used to predict the presence of VI. This may enable the prediction of angioinvasive LU AD from biopsy specimens, allowing for more tailored treatment prior to surgery.
[00294] Lung adenocarcinoma (LU AD) is the most common lung cancer subtype and invasive LU AD represents 70-90% of surgically resected lung cancers1. Improved early detection through computed tomography (CT)-based screening programs is expected to increase the proportion of LU AD diagnosed at an early stage. Microscopic vascular invasion (VI), defined as tumor invasion within the lumen of veins or arteries, is a well described route to metastatic dissemination and is consistently associated with higher rates of tumor recurrence among early stage LUAD2 s. VI is not included in the current World Health Organization (WHO) grading system for lung cancer but may be a better predictor of recurrence than the most severe WHO-2021 grade, leading to our proposal for VI+ LU AD to be reclassified as distinct angioinvasive LU AD9.
[00295] Patients with stage I VI+ LU AD may benefit from adjuvant therapy, but it is difficult to assess in resected tumor specimens10 11. Elastic stains can be used to improve visualization of invaded blood vessels over hematoxylin and eosin (H&E), but comprehensive tumor histopathology review for small (<lmm) VI foci is difficult and prone to false negatives. Similarly, biopsy specimens do not provide enough tissue material to evaluate for VI+ LUAD prior to surgery, preventing informed surgical or neoadjuvant treatment approaches. Recent evidence from large clinical trials including JCOG0802/WJOG4607L and CALGB 140503 suggests that lobectomy provides no clinical benefit over sublobar resection for patients with stage IA disease12 13; although post-hoc analyses are beginning to show subsets of patients for which lobectomy might be preferred14. While no post-hoc analysis for vascular invasion has yet to be published, retrospective analysis shows that VI+ patients that receive sublobar resection have worse outcomes, highlighting an emerging need to identify if patients with more aggressive disease will benefit from precision surgical approaches15.
[00296] Pathologists infrequently document VI as a separate entity from lymphatic invasion (LI), preferring to designate the presence of either type as lymphovascular invasion (LVI). This obfuscates the independent prognostic value of VI16 and complicates downstream molecular approaches seeking to disentangle these modes of tumor spread, the differences of which are still incompletely understood17. Molecular profiling technologies have become a staple of clinical pathology but prior molecular studies of LUAD have primarily focused on identifying signatures of poorly differentiated tumors, which often manifest with solid growth patterns. New advances in spatial transcriptomics (stRNA-seq) allow for probing transcriptomic changes associated with tumor invasion within the geographic context of the tumor microenvironment and with complete preservation of pathological substructures18. This represents an opportunity to elucidate VI+ LUAD biology with the aim to improve detection of these tumors. In this study, we profiled 163 tumors by RNA-sequencing (RNA-seq), including 15 by stRNA- seq, from a diverse multi-institutional cohort of stage I LUAD patients to comprehensively define the transcriptional landscape of angioinvasive LUAD. We derived a novel molecular signature associated with VI+ LUAD using bulk RNA-seq, described its association with histopathology in situ using stRNA-seq, developed and validated a machine learning based predictor of VI and demonstrated its robustness to LUAD intra-tumor heterogeneity (ITH).
[00297] RESULTS
[00298] VI is the invasion type most associated with stage I LUAD recurrence. We assembled retrospective discovery (n=193) and validation (n=61) cohorts of stage I LUAD tumors from patients across two institutions, Boston Medical Center (BMC), a large urban safety-net hospital, and Lahey Hospital and Medical Center (LHMC), a suburban hospital9 19. Tumors were graded using both World Health Organization (WHO) grading system and our proposed novel grading system, which classifies tumors into low malignant potential (LMP), no special type (NST), or VI, is associated with outcome Fig. 6A) and has been validated by us and others9 15,20’21. In a meta-analysis across institutions, VI was the strongest predictor (hazard ratio (HR)=5.47, 95% CI 2.92-10.24, p=1.16xl007) of 7-year recurrence-free survival (RFS) among the four observed modes of tumor spread (vascular invasion (VI), lymphatic invasion (LI), visceral pleural invasion (VPI), and spread through air spaces (STAS)), in agreement with previous reports of its prognostic value in stage I LUAD (Fig. 6B-6E)5-9’22’23. VI was present in 29.5% of tumors, and 85.3% of VI+ tumors contained multiple types of invasion (Fig. 6F). Regardless, VI remained a significant predictor of recurrence in multivariate models that included clinical covariates and the other invasion types (HR = 6.31, 95% CI 2.66-14.95, p=2.92xlO 05; Fig. 6G). When scoring tumors based on the proportion of LUAD growth patterns, VI was associated with increased solid proportion (p.adj (Bonferroni adjusted P value)=3.3OxlO 06, Wilcoxon test) but not micropapillary (p.adj=0.41) or cribriform proportion (p.adj=0.22) (Fig. 6H). In contrast, STAS was most strongly associated with increased micropapillary proportion (p.adj=4.13xlO 08), as previously reported.24 Among the most aggressive growth patterns (solid, micropapillary, cribriform), only micropapillary proportion was significantly associated with recurrence (HR=1.02, 95% CI 1.00 - 1.03, p=0.03), but not after controlling for VI (p=0.10) (Fig. 61). VI+ tumors were more strongly associated with distant recurrence (sub-distribution HR=1.82, standard error (SE) 1.29-2.36, p=0.00067; multivariable Fine- Gray regression) than loco-regional recurrence only (sub-distribution HR=1.24, 95% CI 0.67-1.81, p= 0.03; multivariable Fine-Gray regression), consistent with tumor spread through the vascular system (Figs. 6 J and 6K). We did not observe any preference for distant vs. loco-regional recurrence among the other modes of tumor invasion (data not shown). We next sought to distinguish the molecular signature of this aggressive early-stage LUAD pathology.
[00299] Gene expression changes in stage I LUAD with VI. To identify gene expression changes in tumors with VI, we profiled (n = 103, post-QC) surgically resected stage I LUAD tumors from the discovery cohort by bulk RNA-sequencing (RNA-seq) (Fig. 1A, Table 1). Among tumors that passed quality control, 78 were VI- and 25 were VI+. We identified 474 genes differentially expressed between VI+ tumors and tumors of low malignant potential (LMP) using a significance threshold of FDR < 0.01 after controlling for NST, which we clustered into four gene expression clusters via hierarchical clustering (Fig. IB). Genes in three of the clusters had increased expression in LUAD with VI (clusters 1, 2 and 3) and genes in the other cluster had decreased expression (cluster 4). Pathway enrichment analysis using EnrichR revealed that genes involved in different biological processes were enriched in each cluster (Fig. 1C). Cluster 1 (n=l 15 genes) was enriched for genes involved in cell cycle. In contrast, cluster 2 (n=37 genes) was enriched for genes involved in tissue remodeling and vasculogenesis, including the epithelial to mesenchymal transition (EMT), PDGF binding, and angiogenesis. The genes in cluster 3 (n=182 genes) were enriched for some pathways that overlapped with cluster 1 like mTORC 1 signaling and E2F targets but was distinguished by enrichment for pathways related to response to reactive oxygen species and hypoxia, implying tumor metabolic reprogramming under oxidative stress. Finally, the genes with decreased expression in VI tumors (cluster 4; n=140 genes) were enriched for pathways indicating reduced malignant progression (p53 pathway, regulation of cell growth), control of TGF-J3 signaling (regulation of pathway -restricted SMAD protein phosphorylation) and increased immune surveillance (IL-2/STAT5 signaling) in VI tumors. In the discovery cohort, the mean z-score of each of the four clusters was a significant predictor of VI (Fig. 7A) and was also able to distinguish between NST VI+ and NST VT tumors (Fig. 7B), supporting that the differentially expressed genes reflect Vi-specific biology. Given that VI and LI are routinely reported as combined LVI9 16, we sought to compare the VI signature with gene expression differences associated with LI in stage I LUAD. When we added LI as a covariate to our model, the VI gene cluster co-expression was preserved, with 134/139 genes also recovered in the model without LI (Fig. 8A). At the same significance threshold of FDR < 0.01, we recovered only 15 genes associated with LI, with 2/15 belonging to the VI signature (Fig. 8B), suggesting the biological signal for LI is much weaker. Gene-set enrichment analysis (GSEA) of each of the 4 VI gene clusters against a ranked list of genes associated with LI revealed that cluster 2 genes, which were enriched for EMT/angiogenesis, were not associated with LI (Fig. 8C). Taken together, we have identified a robust gene expression signature of VI+ LUAD from bulk RNA-seq.
[00300] VI gene clusters are associated with specific LUAD histopathology features in stRNA- seq data. Although the genes differentially expressed in tumors with VI were enriched for biological processes implicated in tumor intravasation, the RNA for sequencing was isolated from histologic sections selected to be representative of the predominant histologic pattern and were not selected to include invaded blood vessel(s). Given that VI foci represent such a small percentage of tumor volume (diameter < 1 mm), we hypothesized that the VI signature we identified reflected molecular changes extending beyond the site of intravasation. To assess the spatial architecture of the expression of the VI signature and its association with LUAD histopathology features we profiled 15 (post-QC) resected LUAD samples (n=8 VI’, n=7 VI+) from 13 patient tumors in the validation cohort by high-resolution stRNA-seq using the 10X Genomics Visium platform, including 3 instances of invaded vessels captured directly in the 6.5 mm2 Visium area (Table 2). Of the VI’ tumors, 7/8 were NST. All six non- mucinous LUAD growth patterns (solid, cribriform, micropapillary, acinar, papillary, lepidic) were represented across the stRNA-seq capture areas along with three invasion types (VI, VPI, and STAS) (Fig. 2A). Adjacent normal appearing lung was present in 12/16 capture areas. The histopathology associated with VI+ tumors in the stRNA-seq data was generally representative of trends observed in our larger clinical cohort, although the micropapillary pattern tended to be underrepresented in VI+ tumors (p.adj=8.62xl0 14) and the papillary pattern was overrepresented (p.adj=1.01xl0-11) (Fig. 9A). A solid growth pattern and the presence of desmoplastic stroma were both associated with angioinvasion (p.adj=6.38xl0-31; p.adj=2.26xl0 18). While VI co-occurred with other invasion types frequently in our clinical cohorts, we did not find more than one invasion type per capture area in the stRNA-seq dataset. In summary, our stRNA-seq dataset captures many of the pathology features that are known to occur in stage I LU AD and co-occur with VI.
[00301] After stRNA-seq quality control and filtering, we obtained expression estimates from 43,421 spots (50pm diameter) with a medium sequencing depth of 8,548 counts/spot (Fig. 9B) and 3,996 genes/spot (Fig. 9C). The low depth of sample 6 was likely due to tissue detachment that occurred during the stRNA-seq workflow, and this sample was removed prior to downstream analysis. Strong concordance was observed between pseudo-bulked stRNA-seq gene expression counts and bulk RNA- Seq counts of matched tumor tissues (spearman R=0.85) (Fig. 9D). We next scored individual spots to create an enrichment score representing the activity of the genes from each of the four clusters in our bulk VI signature. Across all capture areas, spatially weighted correlation analysis of enrichment scores revealed that individual VI clusters were spatially distinct from one another (Fig. 9E). These data suggest the distinct patterns of gene co-expression represented by the four clusters are also spatially distinct indicating that the biological processes that these gene expression patterns represent are spatially variable and are unlikely to represent redundant biology.
[00302] To investigate if the VI gene clusters are more strongly expressed in regions with different histopathologic feature annotations in the stRNA-seq data, we modeled the enrichment score of each cluster as a function of histopathologic annotation using a linear mixed model with the section as a random effect. Unexpectedly, despite the VI gene clusters being derived in bulk RNA-seq, we observed strong increased expression directly in VI foci of both cluster 1 (p.adj=9.02xl0-28) and cluster 3 (p.adj=8.92xl0-73). Cluster 1 and cluster 3 were also increased in regions annotated as high-grade patterns (solid (p.adj=8.87xl0 15; 7.90'75) micropapillary (p.adj=3.94xlO 07; 1.40xl0-23) and cribriform (p.adj=4.23xlO 08; 2.39xl0-58)) (Fig. 2B). Cluster 2 was most noticeably expressed in desmoplastic stroma (p.adj=1.22 122) with a greater than 2.95-fold higher mean enrichment score than in stroma (p.adj=1.87xl0-25). Conversely, expression of cluster 4 was strongly decreased in VI foci (p.adj=5.79xl0-79), followed by desmoplastic stroma (p.adj=6.53xlO-50) and showed strong positive enrichment for adjacent normal appearing lung (p.adj=2.57xlO 04), stroma (p.adj=8.90xl0 12), and papillary (1.68xl0 13). When enrichment scores were visualized directly on tissue sections, the independent spatial patterning of each cluster was apparent (Fig. 2C). As suggested by our linear model results, cluster 2 was not expressed directly within any of the invaded vessels in stRNA-seq capture areas. Given that cluster 3 was significantly enriched directly in invasive foci in addition to high-grade patterns, we were interested to see whether the expression of these genes was also higher in VI+ tumors independently of aggressive pattern. When we analyzed only spots annotated with high-grade LU AD patterns, we found that cluster 2 (p=0.024) and 3 (p=0.022) were both higher in spots annotated with high-grade patterns belonging to VI+ tumors, suggesting that our VI signature is capturing an angioinvasive phenotype that is independent of aggressive pattern and the invasive focus (Fig. 2D). This conclusion is reinforced by Fig. 18, which demonstrates that cluster 1 is significantly independent form high grade growth patterns. This indicates that the biomarker signals are not exclusively due to proportion of aggressive histology in the tumor. In total, these findings suggest that gene expression clusters of angioinvasive LU AD identified by bulk RNA-seq are associated with distinct histopathologic features in situ, show increased expression in the same tumor patterns if it is a VI+ tumor, and are not solely expressed in invaded blood vessels.
[00303] The VI signature is composed of both tumor-specific and tumor-microenvironment changes reflective of angioinvasion. Our supervised analysis above relied upon detailed pathologic annotation of the tissue containing Visium spots, but spots were only labeled if there was a clear consensus on morphology, especially for the histologic pattern. In some cases, tumor areas may not have been annotated if they did not clearly contain canonical features of one of the six histologic patterns25. Additionally, for poorly differentiated patterns such as solid, the annotation may include admixed stromal or immune cells. With the aim to further delineate the spatial biology of the VI clusters, we estimated the cell type composition of each stRNA-seq spot with CytoSPACE using a published singlecell RNAseq atlas of 295,813 cells from 124 stage I LU AD samples (primary tumor and normal adjacent lung) and segmenting nuclei per spot with StarDist2" 2 '. Per-spot estimates of cell types within the stRNA-seq data showed estimated cell types localizing with expected pathology structures (e.g., B cells with lymphoid aggregates) (Fig. 10A). We found a higher proportion of peribronchial fibroblasts belonging to VI+ samples (Fig. 3A), consistent with the tissue remodeling biology of VI cluster 2 described above. VI+ samples also had a higher proportion of plasma cells and B cells. To validate these findings, we deconvoluted the bulk RNA-seq discovery cohort data using the same atlas. Although the predicted plasma cell proportions were associated with pathologist-annotated plasma cell grade on case- matched H&E images (p=1.4xl0-8) (Fig. 10B), we did not observe an association with VI+ tumors (p.adj= 1 ), suggesting that plasma cell infiltration may be overrepresented in the stRNA-seq data. In contrast, we found associations between peribronchial fibroblasts (p.adj=0.005) and macrophages (p.adj=0.03) with VI+ tumors (Fig. 3B). To probe individual cell type associations with the VI gene clusters, we generated cell type specific gene signatures from the reference atlas (Fig. IOC). We then used spatially weighted regression to identify correlation between cell type signatures and our VI gene clusters across the stRNA-seq data. Unsupervised clustering of the average spatially weighted correlation across all tumors revealed co-localization of cluster expression with distinct cell types (Fig. 3C). As expected from our previous observation of enrichment in desmoplastic stroma, VI cluster 2 was most spatially correlated with peribronchial fibroblasts and dividing stromal cells, as well as other stromal cell types including endothelial and smooth muscle cells. VI cluster 3 showed the most correlation with tumor cells and a transitional club/AT2 phenotype. VI cluster 4 showed expected spatial correlation with normal lung epithelial cell types including ATI and AT2 cells.
[00304] VI cluster 1 was tightly spatially correlated with dividing B and T cells, but not dividing stromal, plasma, or myeloid cells. Collectively, these results show that the VI gene clusters capture biological changes associated with angioinvasion from both tumor cells and the tumor microenvironment.
[00305] A VI predictor derived from the signature validates in an independent stage I LUAD cohort. Our finding of biologically and spatially distinct gene expression clusters from bulk tumors associated with VI+ LUAD suggest that all four clusters provide orthogonal information and might be combined to form a predictor of angioinvasive Stage I LUAD. To test this hypothesis, we proceeded with a standard machine learning cross validation pipeline utilizing all genes with Vi-associated expression (FDR<0.01) as inputs to feature selection (Fig. 4A). Training was performed in the discovery cohort (n=108) where the differentially expressed genes were first identified, using a cross- validation approach (Fig. 4A). Predictors selected via a binomial logit generalized linear model (GLM) utilizing ridge regression performed the best on the cross-validation internal test sets within our discovery cohort (Fig. 11A). When we re-trained this model on the full discovery cohort, a 48-gene predictor achieved an area under the receiver operating characteristic curve (AUROC) of 0.85 to separate VI+ and VI’ LUAD in the training set (Fig. 4B). When ranked by feature importance in the model, the top genes in the predictor were enriched for cluster 3 membership and included MUC16. NOTCH 3. and Hl 9 (Fig. 4C). The top features for clusters 1, 2, and 4 were MKI67. COL1A1 and CHD2, respectively.
[00306] The predictor retained a similar performance in predicting VI+ LUAD when applied to our independent validation cohort (n=43 VI", n=17 VI+) with an AUROC of 0.86 (Fig. 4D). It retained equivalent performance even after excluding LMP tumors (NST vs. VI only, AUROC 0.86) (Fig 4E). It was associated with histologic grade and 7-year recurrence free survival (HR=1.98, 95% CI 1.09-3.60, p=0.02) (Fig. 1 IB and Fig. 4F). Additionally, it was also a significant prognostic predictor among VI’ LUAD tumors (Fig. 4G; HR=2.76, 95% CI 1.41- 5.41, p=0.003). When we analyzed pathology features associated with predictor scores in the VI" tumors, only necrosis was significant in a univariate linear model (p.adj < 0.001) (Fig. 11C). In our validation set, the VI predictor retained good performance across tumor subsets that varied by histological growth patterns, with the best performance in samples that contained no lepidic pattern (0% lepidic, n=22, mean AUROC 0.96) and the worst performance in samples that contained no solid pattern (0% solid, n=35, mean AUROC 0.69) (Fig. 4H). It also showed variable performance in predicting other invasion types, and notably was not significantly better than a random classifier at detecting LI, regardless of whether VI was co-occurring or not, supporting our findings that a different molecular process may differentiate angioinvasion from lymphatic invasion (Fig. 41). Although we did not have separate VI annotations for previously published LUAD RNA-seq datasets, in TRACERx, a longitudinal study of NSCLC, we observed a difference in predictor scores between LVI+ and LVF LUAD (p<0.00I) (Fig. 1 ID). Interestingly, there was also an association in VI predictor scores with reported patient preoperative ctDNA status (Fig. 5G; p<0.01) (Fig. 1 IE). Finally, the predictor score was significantly associated with 5-year disease-specific survival (DSS) (HR=1.47, 95% CI 1.02-2.12, p<0.05) among stage I LUAD and approached significance for 5-year RFS (HR=1.56, 95% CI 1.00-2.45, p=0.05). It was associated with overall survival in stage IA LUAD tumors from both The Cancer Genome Atlas (TCGA) (n=127) (HR=1.72, 95% CI 1.05-2.81, p=0.031) and Uppsala (n=44) (HR=2.11, 95% CI 1.05- 4.27, p=0.037) cohorts30,31. The predictor was shown to correlate with outcome in numerous stage I LUAD patient cohorts (Fig. 19).
[00307] The VI predictor is robust to intra-tumor heterogeneity. Our stRNA-seq analysis of the expression of Vi-associated genes implies that each Vi-associated cluster may be spatially distinct. This suggests that a VI predictor combining genes from each cluster might overcome intra-tumor heterogeneity (ITH). Overcoming intra-tumor heterogeneity is crucial for molecular biomarkers that sample only a portion of the tumor volume, such as those measured on tissue available from biopsies32. This is particularly important for detecting VI due to the small size of invaded vessels. When we divided stRNA-seq spots from VI+ samples into distal VI+ (spots >Imm beyond the edge of invaded vessels) and proximal VI+ (spots < 1mm from and including invaded vessels) (Fig. 5A), we found a significant difference in the enrichment score between Vi and distal VI+ tumors for the predictor genes (p=0.028) (Fig. 5B). This further supports that the predictor may be able to detect VI away from the invasive focus. Next, we used RNA-seq from multi -region sampling of stage I LUAD in TRACERx, a longitudinal study of NSCLC, to evaluate the variability of the VI predictor to random tissue sampling in a larger cohort24.
[00308] Although a label for VI was not available for the TRACERx tumors, we observed strong correlation between predictor scores from two randomly selected paired regions across all tumors (R=0.88, p<2.2xl0 16) (Fig. 5C). When we filtered the dataset to remove lowly expressed genes and ranked the remaining 15,800 genes in the dataset by correlation between paired regions, the 48 genes present in our predictor were significantly enriched at the top of the list (p=l .13xlO 06) (Fig. 5D). Intertumor heterogeneity (as measured by the absolute difference of scores from regions of two randomly selected tumors) was significantly higher than intra-tumor heterogeneity (as measured by the absolute difference of scores from two randomly selected regions within the same tumor) (p<2.2xl0 16) (Fig. 5E). Taken together, these results support our observations made from the stRNA-seq data that our VI signature represents a global shift in molecular aggressiveness toward an angioinvasive phenotype and that these changes can be leveraged to predict VI+ LU AD from any region of the tumor.
[00309] DISCUSSION
[00310] In this study we profiled new patient cohorts of stage I LU AD tumors by RNA-seq and stRNA-seq, with a focus on gene expression changes associated with VI, a hallmark of tumor progression and a strong independent predictor of recurrence. Stage I LU AD tumors with VI have a clear signature of gene expression that is replicated across datasets, patient cohorts, and sequencing technologies. While tissue remodeling is not the only molecular process implicated in VI, it may not be as much of a requirement for lymphatic dissemination. In situ, components of the VI gene signature can be localized to histopathologic structures outside of the invaded vessel. A VI predictor developed from this signature performs well in an independent validation cohort and is independent of tumor histologic pattern. We demonstrated that the VI predictor is robust to ITH, and that this property has the potential to be exploited to develop a new biomarker for this patient population.
[00311] VI+ tumors contained four major gene expression clusters reflective of tumors with an aggressive phenotype. Clusters associated with the presence of VI were enriched for cancer hallmarks including cell cycle, EMT, angiogenesis, and hypoxic tumor metabolism. Tumor hypoxia, which characterized cluster 3, is well documented as a tumor phenotype with poor prognosis across multiple cancer types and has been shown to be moderately associated with solid histopathology in LU AD33-34. Tumors without VI exhibited upregulated genes responsible for a tighter control over cell-cell adhesion and immune responses. We provided molecular evidence to support previous reports9 16 that stage I LU AD VI and LI should be graded as separate histologic categories, which may further aid validation of VI alone for patient risk stratification. Specifically, tissue remodeling may not be as much of a requirement for lymphatic dissemination in stage I LUAD tumors. Lymphatics are thin walled with less surrounding connective tissue than blood vessels. The absence of a molecular signal of EMT or other extracellular matrix modifications associated with LUAD LI may reflect these microstructural differences that when compared to VI require less extensive tissue remodeling to invade or may rely upon alternate mechanisms of invasion. Despite a large spatial transcriptomics dataset containing many different histologic features, we were not able to profile an adequate area of LI. Future efforts may seek to separate additional biological signals present within the tumor that are associated with LU AD lymph node spread, given that tumor LI is not a reliable indicator of lymph node metastasis.
[00312] Spatial transcriptomics enables direct linkage between gene expression, morphology, and outcome33. Within the stRNA-seq data, cluster 2 was most strongly enriched in regions of desmoplastic but not normal stroma while cluster 4 was enriched in low-grade histologic patterns and normal pulmonary structures. Desmoplastic stroma is a natural response of surrounding normal tissue to invasive tumor, and involves widespread tissue remodeling, which was also reflected in the cluster 2 enrichment results from the discovery cohort described above. We were not able to examine the co-occurrence of VI and desmoplastic stroma within our larger clinical cohort as the latter is not routinely assessed in LU AD, but this association has been documented in thyroid carcinoma35. In LU AD, desmoplastic stroma is associated with higher rates of distant metastasis or other prognostically unfavorable histologic features36,37. Cluster 1 and 3 were both detected within invaded blood vessels, but cluster 3 showed enrichment in aggressive patterns including solid and cribriform. The percentages of these poorly differentiated LUAD patterns are known to be associated with poor outcome38, and solid percentage was also associated with VI in our data. But within the aggressive patterns, especially areas annotated as solid, individual cluster expression was significantly higher in patterns from VI+ tumors, suggesting that VI- associated gene expression is not just a proxy for poorly differentiated tumors, but a signature of the most aggressive early-stage tumors. Previous studies have identified gene expression signatures associated with solid LUAD, but grading based on the presence of poorly differentiated morphology alone may be less prognostic than grading that takes into account VI9,39,40.
[00313] An inherent limitation of spot-based stRNA-seq data is the lack of single cell resolution, preventing detailed cell -cell communication or higher resolution spatial neighborhood analyses26. Although this did not prevent us from spatial association of our bulk-derived VI signature in the ‘bulk’ stRNA-seq data with morphology, spot deconvolution with stRNA-seq data allowed for more detailed and pathologist-agnostic insights. Tumor regions away from the invasive focus showed higher proportions of peribronchial fibroblasts, which we validated in our bulk RNA-seq discovery cohort. While cluster 3 was most spatially correlated with tumor cells and transitional club/AT2 cells, the other clusters showed tighter correlation with stromal and endothelial cell subsets (cluster 2), dividing immune cells (cluster 1), and normal alveolar cells (cluster 4). The distinct cell type associations of the individual VI gene clusters likely reflects a broad remodeling of the tumor microenvironment associated with angioinvasion.
[00314] The size of our profiled cohorts allowed us to develop a VI predictor that performed well in an independent validation cohort and was independent of tumor histologic pattern. Furthermore, it was associated with RFS even in presumed VI- tumors. Historically, prognostic gene signatures that reportedly predict lung cancer progression are notorious for poor reproducibility and lack of independence from known risk factors41,42. Our approach to augment histopathological grading of stage I LU AD with a reproducible gene expression predictor of VI seeks to emulate the successful Oncoty e Dx assay, a signature of proliferation that reflects breast cancer tumor grade43. New spatial transcriptomics profding studies and studies that have leveraged deep learning to predict gene expression from tumor morphology alone provide further evidence that tumor histopathology and transcriptional output are tightly linked44,45. Although we did not derive a VI signature from our stRNA-seq data directly due to low sample size, the strong performance of the predictor in our bulk RNA-seq validation cohort suggests that a tumor’s representative molecular phenotype is strongly associated with the presence of angioinvasion.
[00315] We anticipate that molecular prediction of VI on resected LU AD tumors may improve reproducibility of histopathological grading, but performance on routinely collected preoperative biopsy tissue is of paramount importance for guiding precision lung cancer surgery46. Concordance of LU AD IASLC and WHO tumor grade between pre-surgical biopsies and resected specimens is poor47,48. Prospective studies should assess the agreement of VI estimation from biopsy and resected tumor material using both histopathology grading and molecular prediction. Here, as a proof of concept, we demonstrated that the VI predictor is robust to ITH in multi -region sampling data from the longitudinal TRACERx cohort. Additionally, the association of VI predictor scores with preoperative ctDNA status further supports that our predictor is indeed measuring angioinvasion. The ability to measure gene expression in 50 micron stRNA-seq tissue spots also highlights the sensitivity of our predictor. Although none of the cohorts we analyzed received neoadjuvant therapy, preoperative detection of VI+ tumors may in the future provide the opportunity to also guide these treatments, which are increasingly being evaluated in earlier stages of resectable NSCLC49.
[00316] Overall, our study elucidated new insights into the molecular signature of VI, which previously was not explored in LU AD. It further emphasized the prognostic power of VI in this disease and developed a new VI predictor that is resistant to LU AD ITH. Integration of the VI predictor with other biomarker modalities such as radiomics and pathomics is likely to further improve risk stratification of stage I LU AD.
[00317] METHODS
[00318] Clinical cohorts. Discovery cohort. A discovery cohort of 193 stage 1/0 LU AD tumors measuring < 4 cm total size and from patients not treated with neoadjuvant therapy were included in this study. Cases were from Boston Medical Center (BMC) and Lahey Hospital & Medical Center (LHMC) after IRB approval (BU/BMC IRB H-37859; Lahey Clinic IRB-518308). Relevant clinical
I l l information on all patients included can be found in Table 2. RNA-seq was performed on 108 tumors, with 103 passing quality control.
[00319] Validation cohort. A validation cohort consisting of 64 tumors from 63 patients with stage I LU AD measuring and not treated with neoadjuvant therapy were included in this study. Cases were from LHMC. Relevant clinical information on all patients included can be found in Table 3. RNA-seq was performed on 63 tumors from 62 patients, with all passing quality control. StRNA-seq was performed on a subset of these 63 tumors and included 16 samples taken from 14 tumors. Only tumors measuring < 4 cm total size were included in the combined clinical cohorts (n=61) and RNA-seq (n=60) analysis
[00320] Pathology review. An experienced thoracic pathologist (E.B.) reviewed all pathology cases. Vascular invasion (VI) was defined as luminal invasion of a vein or muscular artery either within or adjacent to the tumor. Tumor proportions of lepidic, acinar, papillary, micropapillary, and solid patterns were assessed in 5% increments with distinction of simple tubular acinar from complex and cribriform acinar patterns. Adenocarcinoma in situ (AIS) was assigned to purely lepidic tumors <3 cm whereas minimally invasive adenocarcinoma (MIA) was diagnosed when non-lepidic foci measured <0.5 cm as per WHO criteria. WHO-2021 grade was defined as Gl, lepidic predominant with <20% high-grade patterns; G2, acinar or papillary predominant with <20% high-grade patterns; and G3, >20% high-grade patterns (solid, micropapillary and/or complex glands)50,51. Low malignant potential adenocarcinoma (LMP) was assigned as previously described.20 LMP tumors were non- mucinous adenocarcinoma measuring <3 cm in total size, with >15% lepidic growth, and without nonpredominant high-grade patterns (>10% cribriform, >5% micropapillary, >5% solid), >1 mitosis per 2 mm2, vascular, lymphatic or visceral pleural invasion, STAS or necrosis. AIS/MIA and LMP were analyzed together due to their identical outcome (100% 10-year DSS). No special type (NST) designation was given for all other tumors not classified as VI or LMP. Stage assignments were retrospectively made using the 8th edition of the AJCC.
[00321] Statistical analysis. All statistical analysis was performed with R version 4.2.1. Tables were created with the tableone package. P values were corrected for multiple hypothesis testing using Bonferroni adjustment where appropriate.
[00322] RNA-Seq library preparation, sequencing, and data processing. Total RNA was extracted from FFPE tissue using AllPrep™ DNA/RNA Universal Kit (Qiagen) and exome-targeted sequencing libraries were prepared using Illumina TruSeq™ RNA Exome Library Prep Kit (formerly TruSeq RNA Access). Samples were sequenced on the Illumina HiSeq 2500 to generate paired-end 50-nucleotide reads. Basespace was used to demultiplex and generate FASTQ files. A Nextflow™ v21.10.6 RNA-Seq pipeline aligned samples using hg38 and STAR v2.6.0c. Counts were calculated using RSEM vl .3. 1 using Ensembl vlO8 annotation. Quality metrics were calculated with STAR and RSeQC. EdgeR was used to compute normalized data (library sizes normalized using trimmed mean of M-values). Genes were fdtered based on count per million (cpm) > 1 and expression in at least 10% of samples. Outlier samples were excluded if the corresponding transcript integrity number (TIN) values calculated by RSeQC was were > 2 standard deviations from the mean. Combat-seq was used to correct a batch effect between collection sites in the discovery cohort52.
[00323] Derivation of VI signature and biological annotation. Genes associated with the presence of VI were derived from 103 tumors in the discovery cohort after quality control and batch correction using a negative binomial generalized linear model from edgeR53. Our previously published novel grading system (LMP, NST, VI) was the independent variable and we compared VI and LMP groups using glmLRT()9 15. Up and downregulated genes were selected using a false-discovery rate (FDR) threshold of 0.01. Four gene expression clusters were identified using the Ward2 method of hierarchical clustering of genes and samples on the Euclidean distance. The top ten biological processes and pathways enriched in each of the four VI clusters were identified using Enrichr with queries to the following databases: MSigDB Hallmark 2020 and GO Biological Process 202154.
[00324] Spatial transcriptomics library preparation, sequencing, and data processing. 16 samples from 14 patient tumors within the validation cohort were selected for lOx Genomics Visium for FFPE spatial whole -transcriptome profiling. All samples were 8 th TNM edition stage IA/IB except for one stage IIA. Samples were chosen for the presence of pathological features of interest (e.g., VI foci, representative LU AD histologic patterns) with a percent of RNA fragments greater than 200 nucleotides (DV200) above 50 after extraction with the AllPrep™ DNA/RNA Universal Kit (Qiagen). Tissue sections cut by a microtome at 5 microns were placed in a 42°C water bath and transferred onto the 6.5 x 6.5 mm tissue capture areas on the Visium™ Spatial Gene Expression Slide (PN-1000185, lOx Genomics). Tissues were deparaffinized, H&E stained, imaged with a Leica Aperio™ AT2, and decrosslinked according to the manufacturer’s recommended protocol, with the following user modifications made to deparaffinization to prevent tissue detachment: 1) the 15 min incubation step during deparaffinization was removed, 2) after the 96% ethanol immersion, one 85%, one 70% and one 50% ethanol immersion for 3 min each was added. Probe hybridization was performed with the Visium™ Human Transcriptome Probe Kit (PN-1000364), followed by probe ligation, extension and elution for downstream qPCR cycle determination and lOx library construction with the Visium™ FFPE Reagent Kit (PN-1000362). Sequencing was done on an Illumina NextSeq2000™. The lOx SpaceRanger pipeline was used to demultiplex and generate FASTQ files. FASTQ files were input into the SpaceRanger™ FFPE count algorithm along with matching H&E .tiff images for alignment to the reference probe set and generation of count matrices for each Visium™ capture area. [00325] Spatial transcriptomics pathology annotations. H&E images of sections captured for Visium analysis were annotated for pathological features of interest by an experienced thoracic pathologist (EB) using Loupe Browser™ v6 (lOx Genomics).
[00326] Spatial transcriptomics data analysis. Visium™ spatial transcriptomics data was processed using the Seurat™ R package, unless otherwise specified55. First, low-quality spots were filtered using a cutoff of < 250 features per spot. For the global sample analysis, samples were merged and normalized using SCTransform and scaled56. For individual stRNA-seq samples, spots were log- normalized. Visium spots were scored with gene signatures using the AddModule Score function from the Seurat™ R package. Spatial enrichment of pathology regions by VI cluster enrichment score was assessed with a generalized binomial linear mixed effect models with sample as a random effect after averaging scores across regions. Pathology regions with < 200 spots annotated were removed prior to analysis and all regions were downsampled to 200 spots each. Normal lung was used as the reference factor level.
[00327] Spot deconvolution with scRNA-seq data. Nuclei segmentation of high-resolution H&E images of stRNA-seq samples was performed with the StarDist algorithm using default settings with a prob_thresh set to 0.3 and per-spot cell number estimates were generated within Squidpy.
Segmentation accuracy was confirmed by visual inspection. Spot deconvolution was performed with the cytospace algorithm using the per-spot cell numbers as input and the Salcher stage I LU AD atlas as the reference scRNA-seq dataset.
[00328] Deconvolution of bulk scRNA-seq data. Deconvolution of the bulk RNA-seq discovery cohort was performed with CIBERSORTx57™ using the Salcher stage I LU AD atlas as input to the signature genes matrix. The signature genes matrix was created by taking the average expression for each cell type per sample in the atlas.
[00329] Spatially weighted correlation analysis. Spatial transcriptomic spots cannot be assumed to be independent geographically; therefore, spatially weighted regression analysis is used to calculate correlation between gene signatures. Unlike simple linear regression, which contains a single coefficient per variable, spatially weighted regression allows for coefficients to change locally with tissue coordinates. Models are weighted so that data points closer to the coordinates at a given location within a set window are prioritized. This window (bandwidth) can then slide over the whole tissue to calculate per-spot relationships between variables. First, we used Seurat’s AddModule Score to generate per-spot scores for our VI gene cluster signatures and cell type signatures from the Salcher atlas. Cell type signatures were generated from the atlas’ pre-annotated cell types using Seurat’s FindAllMarkers function with default settings. We selected the top 50 most significantly differentially expressed genes for each cell type. Spatially weighted regression was performed for each sample using the gwss function from the GWmodel package 2.3.1 with default settings and a bandwidth set to five. The mean Spearman’s rho of each correlation pair was then taken for each sample and averaged across all samples.
[00330] VI predictor derivation. A nested cross-validation pipeline was used to evaluate performance of different machine learning models. Samples from the discovery cohort were divided into train and test sets using a 70/30 split 100 times.
[00331] Feature selection. Genes associated with VI were derived within each train split as described above. The final gene set used in each fold of the cross-validation was selected by taking the top genes within each VI cluster ranked by mean expression. A gene set size of 48 was arbitrarily selected to allow for proportional representation of the original cluster size with a scaling factor of 10. [00332] Models. Model selection was evaluated with 5 -fold cross-validation within an inner fold of the train split using the AutoML interface from the h2o.ai package v.3.40.0.4. The best performing model in the inner fold was retrained on the entire train set and then applied to the test hold out set. The final model was selected as the model with the highest mean and median AUROC across all 100 cross- validation iterations and was re-trained on the entire discovery cohort.
[00333] VI predictor validation. After quality control and individual batch correction, the mean and variance of log -transformed cpm in the validation cohort was adjusted to match the discovery cohort using reference ComBat with the discovery cohort as the reference batch. After filtering the validation cohort to the final features, VI predictions were generated using the final trained model and performance was evaluated by AUROC.
[00334] Survival analysis. The association of VI predictions with recurrence-free survival was assessed using log-rank test on Kaplan-Meier survival curves generated by binning predictions into low and high risk using the Youden point for classifying recurrence as a cutoff in each respective dataset. Univariate Cox proportional hazards regression was performed using the survival R package. [00335] Additional datasets for survival analysis. Pre-processed RNA-Seq and matching clinical data were downloaded using a Genomic Data Commons (GDC) query for TCGA UUAD using the TCGAbiolinks R package and filtered to 127 stage IA samples with data on overall survival. Pre- processed RNA-Seq and matching clinical data from the Uppsala NSCUC cohort were downloaded using the Gene Expression Omnibus (GSE81089) and filtered to 44 stage IA LU AD samples with overall survival data. Analysis for both datasets was limited to stage IB samples because these included tumors > 5 cm in previous TNM editions and our predictor was derived in TNM 8th edition stage I samples. Genes in both datasets were filtered to genes remaining after filtering in the discovery cohort and the mean and variance of log-transformed cpm was adjusted to match the discovery cohort using reference ComBat with the discovery cohort as the reference batch58. [00336] TRACERx intra-tumor heterogeneity analysis. Processed RNA-seq and clinical annotations from the TRACERx NSCLC cohort of multi-region tumor sampling data was downloaded from doi.org/10.5281/zenodo.7683605 and doi.org/10.5281/zenodo.7603386. Samples were filtered to TNM 8 th edition stage I LU AD tumors with data at least two regions. Genes were filtered to those remaining after filtering in the discovery cohort and the mean and variance of log -transformed cpm was adjusted to match the discovery data using reference ComBat with the discovery cohort as the reference batch. VI predictions were generated as described above. To determine intratumor heterogeneity in predictor scores, we calculated the absolute difference between regions of the same tumor, when all tumors were randomly downsampled to two regions each. For inter-tumor heterogeneity, the absolute difference was calculated between two regions that were randomly sampled from two different tumors. This was repeated n times equivalent to the number of tumor regions used in the intratumor heterogeneity calculation. Spearman correlation between predictor scores from regions of the same tumor were also calculated. Predictor gene enrichment among all genes ranked by region-region correlation was calculated using GSEA.
[00337] References
1. Travis, W. D. et al. International Association for the Study of Lung Cancer/American Thoracic
Society/European Respiratory Society International Multidisciplinary Classification of Lung Adenocarcinoma. J. Thorac. Oncol. Off. Publ. Int. Assoc. Study Lung Cancer 6, 244-285 (2011).
2. Mineo, T. C. et al. Prognostic impact of VEGF, CD31, CD34, and CD105 expression and tumour vessel invasion after radical surgery for IB-IIA non-small cell lung cancer. J. Clin. Pathol. 57, 591— 597 (2004).
3. Tsuchiya, T. et al. Upstaging by Vessel Invasion Improves the Pathology Staging System of Non-Small Cell Lung Cancer. Chest 132, 170-177 (2007).
4. Shimada, Y. et al. Extratumoral Vascular Invasion Is a Significant Prognostic Indicator and a Predicting Factor of Distant Metastasis in Non-small Cell Lung Cancer. J. Thorac. Oncol. 5, 970-975 (2010).
5. Shimada, Y. et al. Pathological Vascular Invasion and Tumor Differentiation Predict Cancer Recurrence in Stage ia Non- Small-Cell Lung Cancer After Complete Surgical Resection. J. Thorac. Oncol. 7, 1263-1270 (2012).
6. Ito, M. et al. Prognostic Impact of Cancer-Associated Stromal Cells in Patients With Stage I Lung Adenocarcinoma. Chest 142, 151-158 (2012).
7. Shimada, Y. et al. The Frequency and Prognostic Impact of Pathological Microscopic Vascular Invasion According to Tumor Size in Non-Small Cell Lung Cancer. Chest 149, 775-785 (2016). 8. Suaiti, L. et al. Vascular Invasion Predicts Recurrence in Stage IA2-IB Lung Adenocarcinoma but not Squamous Cell Carcinoma. Clin. Lung Cancer S 1525-7304(22)00294-7 (2022) doi: 10.1016/j .cllc.2022.12.006.
9. Yambayev, I. et al. Vascular invasion identifies the most aggressive histologic subset of stage I lung adenocarcinoma: Implications for adjuvant therapy. Lung Cancer 171, 82-89 (2022).
10. Tsuchiya, T. et al. Stage IA non-small cell lung cancer: Vessel invasion is a poor prognostic factor and a new target of adjuvant chemotherapy. Lung Cancer 56, 341-348 (2007).
11. Tsuchiya, T. et al. A single-arm, phase 2 study of adjuvant chemotherapy with oral tegafur- uracil for pathologically lymphovascular invasion positive stage IA non-small cell lung cancer: LOGIK0602 study. BMC Cancer 20, 1192 (2020).
12. Saji, H. et al. Segmentectomy versus lobectomy in small-sized peripheral non-small-cell lung cancer (JCOG0802/WJOG4607L): a multicentre, open-label, phase 3, randomised, controlled, noninferiority trial. The Lancet 399, 1607-1617 (2022).
13. Altorki, N. et al. Lobar or Sublobar Resection for Peripheral Stage IA Non- Small-Cell Lung Cancer. N. Engl. J. Med. 388, 489-498 (2023).
14. Hattori, A. et al. Segmentectomy versus lobectomy in small-sized peripheral non-small-cell lung cancer with radiologically pure-solid appearance in Japan (JCOG0802/WJOG4607L): a post- hoc supplemental analysis of a multicentre, open-label, phase 3 trial. Lancet Respir. Med. 12, 105— 116 (2024).
15. Ma, L. et al. Vascular Invasion Predicts the Subgroup of Lung Adenocarcinoma <2.0 cm at Risk of Poor Outcome Treated by Wedge Resection Compared to Lobectomy. JTCVS Open (2023) doi: 10.1016/j.xjon.2023.11.003.
16. Sugita, Y. et al. Lymphatic permeation and vascular invasion should not be integrated as lymphovascular invasion in lung adenocarcinoma. Gen. Thorac. Cardiovasc. Surg. 69, 1070- 1078 (2021).
17. Leong, S. P., Naxerova, K., Keller, L., Pantel, K. & Witte, M. Molecular mechanisms of cancer metastasis via the lymphatic versus the blood vessels. Clin. Exp. Metastasis 39, 159- 179 (2022).
18. Hunter, M. V., Moncada, R., Weiss, J. M., Yanai, I. & White, R. M. Spatially resolved transcriptomics reveals the architecture of the tumor-microenvironment interface. Nat. Commun. 12, 6278 (2021).
19. Burks, E. J. et al. Pathologic and gene expression comparison of CT- screen detected and routinely detected stage 1/0 lung adenocarcinoma in NCCN risk-matched cohorts. Cancer Treat. Res. Commun. 29, 100486 (2021).
20. Yambayev, I. et al. Pulmonary Adenocarcinomas of Low Malignant Potential. Am J Surg Pathol 00, 10 (2020). . Pitaro, A. et al. Pulmonary Low Malignant Potential Adenocarcinoma: A Validation of the Proposed Criteria for This Novel Subtype. Am. J. Surg. Pathol.
10.1097/PAS.0000000000002151 doi: 10.1097/PAS.000000000000215 L . Kagimoto, A. et al. Utility of Newly Proposed Grading System From International Association for the Study of Lung Cancer for Invasive Lung Adenocarcinoma. JTO Clin. Res. Rep. 2, 100126 (2021). . Lee, G. et al. Blood Vessel Invasion Predicts Postoperative Survival Outcomes and Systemic Recurrence Regardless of Location or Blood Vessel Type in Patients with Lung Adenocarcinoma. Ann. Surg. Oncol. (2021) doi: 10.1245/sl0434-021-10122-x. . Karasaki, T. et al. Evolutionary characterization of lung adenocarcinoma morphology in TRACERx. Nat. Med. 1-13 (2023) doi: 10.1038/s41591-023-02230-w. Travis, W. D. et al. The 2015 World Health Organization Classification of Lung Tumors: Impact of Genetic, Clinical and Radiologic Advances Since the 2004 Classification. J. Thorac. Oncol. 10, 1243-1260 (2015). Vahid, M. R. et al. High-resolution alignment of single-cell and spatial transcriptomes with CytoSPACE. Nat. Biotechnol. 41, 1543-1548 (2023). Salcher, S. et al. High-resolution single-cell atlas reveals diversity and plasticity of tissueresident neutrophils in non-small cell lung cancer. 67. Schmidt, U., Weigert, M., Broaddus, C. & Myers, G. Cell Detection with Star-Convex Polygons, in Medical Image Computing and Computer Assisted Intervention MICCAI 2018 (eds. Frangi, A. F., Schnabel, J. A., Davatzikos, C., Alberola-Lopez, C. & Fichtinger, G.) 265-273 (Springer International Publishing, Cham, 2018). doi: 10.1007/978-3-030-00934-2_30. Weigert, M., Schmidt, U., Haase, R., Sugawara, K. & Myers, G. Star-convex Polyhedra for 3D Object Detection and Segmentation in Microscopy, in 2020 IEEE Winter Conference on Applications of Computer Vision (WACV) 3655-3662 (2020). doi: 10.1109/WACV45572.2020.9093435. Collisson, E. A. et al. Comprehensive molecular profiling of lung adenocarcinoma. Nature 511, 543-550 (2014). Djureinovic, D. et al. Profiling cancer testis antigens in non-small-cell lung cancer. JCI Insight 1, (2016). Dotson, T. et al. Feasibility of lung cancer RNA acquisition from a single transbronchial or transthoracic needle pass (FASTT trial). Lung Cancer 127, 6-11 (2019). . Zheng, Y ., Carrillo-Perez, F., Pizurica, M., Heiland, D. H. & Gevaert, O. Spatial cellular architecture predicts prognosis in glioblastoma. Nat. Commun. 14, 4122 (2023). . Wang, Y. et al. Spatial transcriptomics delineates molecular features and cellular plasticity in lung adenocarcinoma progression. Cell Dis cov. 9, 1-19 (2023). . Koperek, O., Asari, R., Niederle, B. & Kaserer, K. Desmoplastic stromal reaction in papillary thyroid microcarcinoma. Histopathology 58, 919-924 (2011). . Xu, L., Tavora, F. & Burke, A. Histologic Features Associated With Metastatic Potential in Invasive Adenocarcinomas of the Lung. dm. J. Surg. Pathol. 37, 1100 (2013). . Soltermann, A. et al. Prognostic Significance of Epithelial -Mesenchymal and Mesenchymal-Epithelial Transition Protein Expression in Non- Small Cell Lung Cancer. Clin. Cancer Res. 14, 7430-7437 (2008). . Moreira, A. L. et al. A Grading System for Invasive Pulmonary Adenocarcinoma: A Proposal From the International Association for the Study of Lung Cancer Pathology Committee. J. Thorac. Oncol. 15, 1599-1610 (2020). . Tavemari, D. et al. Nongenetic Evolution Drives Lung Adenocarcinoma Spatial Heterogeneity and Progression. Cancer Di scov. (2021) doi: 10.1158/2159-8290. CD-20-1274.. Nguyen, T. T. et al. A lepidic gene signature predicts patient prognosis and sensitivity to immunotherapy in lung adenocarcinoma. Genome Med. 14, 5 (2022). . Subramanian, J. & Simon, R. Gene Expression-Based Prognostic Signatures in Lung Cancer: Ready for Clinical Use? JNCI J. Natl. Cancer Inst. 102, 464-474 (2010). . Subramanian, J. & Simon, R. What should physicians look for in evaluating prognostic gene-expression signatures? Nat. Rev. Clin. Oncol. 7, 327-334 (2010). Michiels, S., Temes, N. & Rotolo, F. Statistical controversies in clinical research: prognostic gene signatures are not (yet) useful in clinical practice. Ann. Oncol. 27, 1-9 (2016). Schmauch, B. et al. A deep learning model to predict RNA-Seq expression of tumours from whole slide images. Nat. Commun. 11, 3877 (2020). Janesick, A. et al. High resolution mapping of the tumor microenvironment using integrated single-cell, spatial and in situ analysis. Nat. Commun. 14, 8353 (2023). Rusch, V. W. Initiating the Era of “Precision” Lung Cancer Surgery. N. Engl. J. Med. 388, 557-558 (2023). Kim, T. H. et al. Utility of Core Biopsy Specimen to Identify Histologic Subtype and Predict Outcome for Lung Adenocarcinoma. Ann. Thorac. Surg. 108, 392-398 (2019). Tsai, P.-C. et al. CT-Guided Core Biopsy for Peripheral Sub-solid Pulmonary Nodules to Predict Predominant Histological and Aggressive Subtypes of Lung Adenocarcinoma. Ann. Surg. Oncol. 27, 4405-4412 (2020). Desai, A. P., Adashek, J. J., Reuss, J. E., West, H. (Jack) & Mansfield, A. S. Perioperative Immune Checkpoint Inhibition in Early-Stage Non- Small Cell Lung Cancer: A Review. JAMA Oncol. (2022) doi: 10.1001/jamaoncol.2022.5389. Thoracic Tumours, vol. 5 (International agency for research on cancer, Lyon, 2021). 51. Kadota, K. et al. A grading system combining architectural features and mitotic count predicts recurrence in stage I lung adenocarcinoma. Mod. Pathol. Off. J. U. S. Can. Acad. Pathol. Inc 25, 1117-1127 (2012).
52. Zhang, Y ., Parmigiani, G. & Johnson, W. E. ComBat-seq: batch effect adjustment for RNA-seq count data. NAR Genomics Bioinforma. 2, lqaa078 (2020).
53. Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139-140 (2010).
54. Chen, E. Y. et al. Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool. BMC Bioinformatics 14, 128 (2013).
55. Satija, R., Farrell, J. A., Gennert, D., Schier, A. F. & Regev, A. Spatial reconstruction of single-cell gene expression data. Nat. Biotechnol. 33, 495-502 (2015).
56. Hafemeister, C. & Satija, R. Normalization and variance stabilization of single-cell RNA-seq data using regularized negative binomial regression. Genome Biol. 20, 296 (2019).
57. Newman, A. M. et al. Determining cell type abundance and expression from bulk tissues with digital cytometry. Nat. Biotechnol. 31, T13-T 2 (2019).
58. Leek, J. T., Johnson, W. E., Parker, H. S., Jaffe, A. E. & Storey, J. D. The sva package for removing batch effects and other unwanted variation in high-throughput experiments. Bioinformatics 28, 882-883 (2012).
[00338] Table 1. Clinical characteristics of resected stage I LUAD tumors with RNA-seq (post- QC) from the discovery cohort (n=103 tumors) using the novel histopathology classification.
LMP NST VI p value n 23 55 25
Age (mean (SD)) 67.76 (11.39) 65.73 (10.11) 66.23 (7.92) 0.715
Gender (%) 0.099
Female 15 (65.2) 40 (72.7) 12 (48.0)
Male 8 (34.8) 15 (27.3) 13 (52.0)
Race (%) 0.177
Asian 2 ( 8.7) 2 ( 3.6) 1 ( 4.0)
Black / African American 2 ( 8.7) 11 (20.0) 7 (28.0)
Hispanic / Latino 2 ( 8.7) 0 ( 0.0) 0 ( 0.0)
Unknown 0 ( 0.0) 1 ( 1.8) 1 ( 4.0)
White 17 (73.9) 41 (74.5) 16 (64.0)
Pack years (mean (SD)) 35.50 (36.81) 43.77 (34.45) 36.37 (20.05) 0.494
Smoking status (%) 0.221
Current 4 (17.4) 18 (32.7) 12 (48.0)
Former 13 (56.5) 30 (54.5) 11 (44.0)
Never 6 (26.1) 6 (10.9) 2 ( 8.0)
Unknown 0 ( 0.0) 1 ( 1.8) 0 ( 0.0)
Total size (cm) (mean (SD)) 1.34 (0.43) 1.75 (0.75) 2.00 (0.70) 0.005
Invasive size (cm) (mean (SD)) 0.63 (0.46) 1.28 (0.60) 1.63 (0.57) <0.001
% Lepidic (mean (SD)) 63.48 (25.91) 25.27 (27.48) 16.60 (20.55) <0.001
% Acinar (mean (SD)) 23.48 (22.69) 26.45 (23.47) 36.00 (28.72) 0.169
% Papillary (mean (SD)) 12.83 (24.90) 16.09 (23.23) 8.40 (9.76) 0.323
% Solid (mean (SD)) 0.00 (0.00) 15.45 (30.60) 21.60 (32.04) 0.021
% Micropapillary (mean (SD)) 0.00 (0.00) 6.00 (13.69) 8.60 (17.11) 0.068
% Cribriform (mean (SD)) 0.22 (1.04) 10.55 (20.22) 8.80 (13.01) 0.038
LI 0 (0.0) 14 (25.5) 13 (52.0) <0.001
VPI 0 (0.0) 11 (20.0) 13 (52.0) <0.001
STAS 0 (0.0) 28 (50.9) 12 (48.0) <0.001
Institution (%) 0.093
LHMC 6 (26.1) 5 ( 9.1) 6 (24.0)
BMC 17 (73.9) 50 (90.9) 19 (76.0)
Procedure (%) 0.23
Lobe 9 (39.1) 35 (63.6) 12 (48.0)
Segment 2 ( 8.7) 1 ( 1.8) 2 ( 8.0)
Wedge 12 (52.2) 19 (34.5) 11 (44.0)
Note: The data are shown as the number and (%) unless otherwise indicated. Abbreviations: LMP, low malignant potential; NST, no special type; VI, vascular invasion; LI, lymphatic invasion; VPI, visceral pleural invasion; STAS, spread through air spaces; LHMC, Lahey Hospital and Medical Center; BMC, Boston Medical Center.
[00339] Table 2. Clinical characteristics of resected early-stage LUAD tumors with stRNA-seq (post- QC) from the discovery cohort (n=15 samples).
Patient ID Sample ID Batch Sth Stage Novel Grade Age Sex Smoking Status RNA-seq VI (Capture Frame)
1A LM SD 1216 1 1 IB VI 68 M Former Yes 1 IB LM SD 16 2 IB VI 68 M Former Yes 0
2B LM SD l 1 2 IA3 VI 68 F Current Yes 0
2A LM SD 2 2 IA3 VI 68 F Current Yes 0
3 LM SD 3 2 IA1 NST 71 M Former Yes 0
4 LM SD 4 2 IB VI 66 M Former Yes 1
5 LM _SD_5 2 IA1 NST 72 M Former Yes 0
7 LM SD 7 2 IA1 LMP 52 F Current Yes 0
8 LM SD 1216 8 1 IB VI 66 M Former Yes 0
9 LM SD 9 2 IA2 VI 63 F Current Yes 1
10 LM SD 10 2 IA2 NST 69 M Former Yes 0
11 LM SD 1216 12 1 ILA NST 63 F Former Yes 0
12 LM SD 13 2 IB NST 65 F Former Yes 0
13 LM SD 1216 14 1 IA1 NST 72 F Former Yes 0
14 LM SD 15 2 IA2 NST 75 F Former Yes 0
Note: Abbreviations: LMP, low malignant potential; NST, no special type: VI, vascular invasion; LI, lymphatic invasion; VPI, visceral pleural invasion; STAS, spread through air spaces; LHMC, Lahey Hospital and Medical Center; BMC, Boston Medical Center.
[00340] Table 3. Clinical characteristics of resected stage I LU AD tumors with RNA-seq (post-QC) from the validation cohort (n=60 tumors) using the novel histopathology classification.
LMP NST VI _ p value n 7 36 17
Age (mean (SD)) 65.57 (8.50) 66.31 (7.30) 61.76(6.44) 0.107 Gender (%) 0.205
Female 6 (85.7) 19 (52.8) 8 (47.1) Male 1 ( 14.3) 17(47.2) 9 ( 52.9)
Pack years (mean (SD)) 31.43 (11.80) 49.53 (25.19) 51.65(17.91) 0.114 Smoker (%) 0.408
Current 2 (28.6) 13 (36.1) 9 (52.9) Former 5 ( 71.4) 23 ( 63.9) 8 (47.1)
Total size (cm) (mean (SD)) 1.90 (0.76) 1.93 (0.87) 2.22 (0.90) 0.484 Invasive size (cm) (mean (SD)) 0.81 (0.38) 1.68 (0.95) 2.06 (0.84) 0.01 % Lepidic (mean (SD)) 55.71 (22.25) 22.78 (29.46) 9.12(14.71) 0.001 % Acinar (mean (SD)) 26.43 (22.12) 29.31 (26.68) 20.29 (24.46) 0.494 % Papillary (mean (SD)) 17.86 (30.80) 19.44(28.63) 8.82(10.97) 0.361 % Solid (mean (SD)) 0.00 (0.00) 5.00(11.46) 46.47(37.03) <0.001 % Micropapillary (mean (SD)) 0.00 (0.00) 10.28(20.91) 4.71 (10.23) 0.267 % Cribriform (mean (SD)) 0.00 (0.00) 13.33 (27.67) 10.59(20.30) 0.415 LI (%) 0 (0.0) 17 ( 47.2) 9 ( 52.9) 0.045 VPI (%) 0(0.0) 5( 13.9) 7(41.2) 0.025 STAS (%) 0(0.0) 14(38.9) 10 (58.8) 0.027 Procedure (%) 0.79 Lobe 6 ( 85.7) 27 ( 79.4) 14 ( 82.4) Segment 1 ( 14.3) 2 ( 5.9) 1 ( 5.9) Wedge 0 ( 0.0) 5 ( 14.7) 2 ( 11.8)
Note: Abbreviations: LMP, low malignant potential; NST, no special type; VI, vascular invasion; LI, lymphatic invasion; VPI, visceral pleural invasion; STAS, spread through air spaces; all cases were from Lahey Hospital and Medical Center.
Example 3
[00341] Tire correlation of each VI predictor gene with the overall VI predictor score was determined. Individual VI predictor genes were ranked by their correlation with the overall cluster mean expression in the validation cohort for each cluster (Figs. 12A-12E). MKI67. COL1A1, PRKDC, CHD2 that have strong correlation with mean cluster expression in the validation cohort, are high ranking in the VI model, and have ISH probes readily available.
[00342] Minimal VI gene predictors using 4 genes performed well in predicting VI and is robust to ITH. (Fig. 13A-13B). Performance in predicting VI was examined in the discovery and validation cohorts, and also robustness to intra-tumor heterogeneity in the TRACERx cohort. [00343] The VI predictor scores are also associated with lymph node positivity in TRACERx stage I and stage II LUAD. (Fig. 14A-14B). The mean VI predictor score across regions of the same tumors is significantly associated with lymph node status. The VI gene cluster 2 is also correlated with LN metastasis (Figs. 15A-15B) and myofibroblasts in scRNAseq LUAD data (Fig. 16). COL1A1, a key gene from VI gene cluster 2. is significantly associated with predicted myofibroblast proportion within the stRNAseq data and shows a similar pattern within a VI+ tumor sample to THY-1 protein, a known marker of myofibroblasts/cancer associated fibroblasts. (Fig. 17A-17D).
Example 4
[00344] Higher VI predictor scores were significantly associated with decreased 5-year RFS in TRACERx (HR=1.69. 95% CI 1.04-2.75, Cox regression p=0.036) (Figs. 20A-20B). When TRACERx patients were stratified into low and high-risk VI predictor groups using the 75th percentile of VI predictor scores in the discovery cohort, the predictor significantly improved discrimination of 5-year RFS by TNM stage (log-rank p=0.04). Among TRACERx stage I LUAD patients, 16% of stage IA patients were reclassified as high-risk.

Claims

What is claimed herein is:
1. A method comprising, determining the expression of at least one gene selected from the group consisting of:
SHR00M4; NBEAL1; MUC16; N0TCH3: H19; COL1A1; and GARSk in a subject with lung cancer.
2. A method of treating or prognosing lung cancer, the method comprising: administering adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and/or lymphadenectomy to a subject having decreased expression of at least one gene selected from the group consisting of:
SHR00M4 and NBEAL1; and/or increased expression of at least one gene selected from the group consisting of: MUC16; N0TCH3; Hl 9; COL1A1; and GARS1.
3. The method of any one of tire preceding claims, further comprising administering active surveillance, wedge resection of the lung, or a segmentectomy of the lung to a subject not having decreased expression of at least one gene selected from the group consisting of:
SHR00M4 and NBEAL1; and/or not having increased expression of at least one gene selected from the group consisting of: MUC16; N0TCH3; Hl 9; C0L1A1; and GARSl .
4. The method of any one of tire preceding claims, wherein the at least one gene is SHR00M4 or
NBEAL1.
5. Tire method of any one of the preceding claims, wherein the at least one gene is SHR00M4 or
NBEAL1.
6. The method of any one of the preceding claims, wherein the at least one gene is MUC16; N0TCH3;
H19; or COL1A1.
7. The method of any one of tire preceding claims, wherein the at least one gene is SHR00M4;
NBEAL1; MUC16; N0TCH3; H19; and C0L1A1.
8. A method of treating or prognosing lung cancer, the method comprising determining the level of expression: a) at least one gene selected from the group consisting of:
MKI67; ASPM; ATAD2; TOP2A; FANCI; PLOD2; NCAPG2; CENPF; KPNA2;
BRCA1; HLTF; and FANCA; b) at least one gene selected from the group consisting of:
C0L1A2; C0L3A1; C0L1A1; and COL6A3; c) at least one gene selected from the group consisting of:
PRKDC; TPI1; PKM: GAPDH: AHNAK2; YWHAZ; ATP13A3; GARS1; CP; MUC16;
TUBA1C; H19; PPFIA1; N0TCH3; ACLY; GCLC; CAD; and CCT6A; d) at least one gene selected from the group consisting of:
SPTBN1; SFTPB: LRRK2; AKAP13: CHD2: EPAS1; ATP11A; ROS1; MTUS1;
ANKHD1; NBEAL1; MPRIP; SHR00M4; and TET2 wherein: e) increased expression of at least one gene selected from the group consisting of:
MKI67; ASPM; ATAD2; TOP2A; FANCI; PLOD2; NCAPG2; CENPF; KPNA2;
BRCA1; HLTF; and FANCA; f) increased expression of at least one gene selected from the group consisting of:
COL1A2; COL3A1; COL1A1; and COL6A3; g) increased expression of at least one gene selected from the group consisting of:
PRKDC; TPI1; PKM: GAPDH: AHNAK2; YWHAZ; ATP13A3; GARS1; CP; MUC16;
TUBA1C; H19: PPFIA1; N0TCH3; ACLY; GCLC; CAD; and CCT6A; and h) decreased expression of at least one gene selected from the group consisting of:
SPTBN1; SFTPB; LRRK2; AKAP13; CHD2; EPAS1; ATP11A; ROS1; MTUS1;
ANKHD1; NBEAL1; MPRIP; SHR00M4; and TET2 indicates increased likelihood of vascular invasion, increased likelihood of metastasis, suitability for treatment with adjuvant therapy, neoadjuvant therapy, ablation therapy, radiation therapy, a lobectomy, and/or lymphadenectomy.
9. Tire method of any one of the preceding claims, wherein the increased expression and/or decreased expression is relative to an average for subjects with lung cancer.
10. The method of any one of the preceding claims, wherein the increased expression and/or decreased expression is relative to an average for subjects with lung adenocarcinoma.
11. The method of any one of the preceding claims, wherein the increased expression and/or decreased expression is relative to an average for subjects with lung cancerthat is not vascular invasive.
12. The method of any one of the preceding claims, wherein the subject having increased expression of the at least one gene is determined to have increased expression of the at least one gene.
13. The method of any one of the preceding claims, wherein the subject having increased expression of the at least one gene is determined to have increased expression of the at least one gene in a sample of tumor cells.
14. The method of any one of the preceding claims, wherein the level of expression is the level of mRNA.
15. The method of any one of the preceding claims, wherein the subject having increased expression of the at least one gene is not administered a lobectomy.
16. The method of any one of the preceding claims, wherein the subject having increased expression of the at least one gene is not administered a sublobar resection.
17. The method of any one of the preceding claims, wherein the subject having increased expression of the at least one gene is not administered a lobectomy or sublobar resection.
18. The method of any one of the preceding claims, wherein the lung cancer is non-small cell lung cancer (NSCLC).
19. The method of any one of the preceding claims, wherein the lung cancer is lung adenocarcinoma.
20. The method of any one of the preceding claims, wherein the lung cancer comprises vascular invasion.
21. The method of any one of the preceding claims, wherein the lung cancer is vascular invasive lung adenocarcinoma.
22. The method of any one of the preceding claims, wherein the lung cancer is stage I lung adenocarcinoma.
PCT/US2025/021084 2024-03-27 2025-03-24 Methods and compositions relating to angioinvasive lung adenocarcinoma Pending WO2025207474A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202463570598P 2024-03-27 2024-03-27
US63/570,598 2024-03-27

Publications (1)

Publication Number Publication Date
WO2025207474A1 true WO2025207474A1 (en) 2025-10-02

Family

ID=97216940

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2025/021084 Pending WO2025207474A1 (en) 2024-03-27 2025-03-24 Methods and compositions relating to angioinvasive lung adenocarcinoma

Country Status (1)

Country Link
WO (1) WO2025207474A1 (en)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11579140B2 (en) * 2018-08-20 2023-02-14 Trustees Of Boston University Methods related to bronchial premalignant lesion severity and progression

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11579140B2 (en) * 2018-08-20 2023-02-14 Trustees Of Boston University Methods related to bronchial premalignant lesion severity and progression

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
COCO SIMONA, BONFIGLIO SILVIA, CITTARO DAVIDE, VANNI IRENE, MORA MARCO, GENOVA CARLO, DAL BELLO MARIA GIOVANNA, BOCCARDO SIMONA, A: "Integrated Somatic and Germline Whole-Exome Sequencing Analysis in Women with Lung Cancer after a Previous Breast Cancer", CANCERS, vol. 11, no. 4, 1 January 2019 (2019-01-01), CH , pages 1 - 18, XP093362976, ISSN: 2072-6694, DOI: 10.3390/cancers11040441 *
ZHOU DAN, TANG WEIWEI, LIU XINLI, AN HAN-XIANG, ZHANG YUN: "Clinical verification of plasma messenger RNA as novel noninvasive biomarker identified through bioinformatics analysis for lung cancer", ONCOTARGET, vol. 8, no. 27, 4 July 2017 (2017-07-04), United States , pages 43978 - 43989, XP093362975, ISSN: 1949-2553, DOI: 10.18632/oncotarget.16701 *

Similar Documents

Publication Publication Date Title
US20250376733A1 (en) Methods for diagnosing and treating cancer by means of the expression status and mutational status of nrf2 and downstream target genes of said gene
EP2949764B1 (en) Methods for prediction of clinical outcome to epidermal growth factor receptor inhibitors by cancer patients
JP2021536434A (en) Methods for severity and progression of prebronchial malignancies
US20150225800A1 (en) Methods and assays for treatment of bladder cancer
CN105611940A (en) Diagnostic methods and compositions for treating glioblastoma
US20210363593A1 (en) CXCL13 Marker For Predicting Immunotherapeutic Responsiveness In Patient With Lung Cancer And Use Thereof
JP2020040959A (en) Use of egfr biomarkers for treatment of gastric cancer with anti-egfr agents
EP3788167A1 (en) Methods of diagnosing and treating patients with cutaneous squamous cell carcinoma
US20230113705A1 (en) Methods for diagnosing, prognosing and managing treatment of breast cancer
US20240377400A1 (en) Methods for treating a subtype of colorectal cancer
US20150017210A1 (en) Gene Signature Predicts Adenocarcinoma Prognosis and Therapeutic Response
WO2025207474A1 (en) Methods and compositions relating to angioinvasive lung adenocarcinoma
US20240182984A1 (en) Methods for assessing proliferation and anti-folate therapeutic response
US11092603B2 (en) Keratin 17 as a prognostic marker for pancreatic cancer
WO2021063972A1 (en) Cthrc1 as biomarker for a tgfbeta-activated tumor microenvironment
US20250356947A1 (en) Genome wide tumor derived gene expression based signatures associated with poor prognosis for melanoma patients with early stage disease
US20240301497A1 (en) Methods for diagnosing nasal intestinal type adenocarcinomas
JP2010535523A (en) Predictive marker for EGFR inhibitor treatment
Zambelli Analisi molecolare di forme aggressive di neoplasia endocrina per la identificazione di meccanismi di progressione e di potenziali nuovi bersagli terapeutici
WO2026015087A1 (en) A method for determining a subject&#39;s responsiveness to treatment with a vegf inhibitor
HK40012059A (en) Methods for diagnosing and treating cancer by means of the expression status and mutational status of nrf2 and downstream target genes of said gene.
HK40012059B (en) Methods for diagnosing and treating cancer by means of the expression status and mutational status of nrf2 and downstream target genes of said gene.

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 25777401

Country of ref document: EP

Kind code of ref document: A1