CN115232877A

CN115232877A - Molecular typing diagnosis marker for esophageal squamous carcinoma and application thereof

Info

Publication number: CN115232877A
Application number: CN202210941019.0A
Authority: CN
Inventors: 刘芝华; 赵丫卉; 焦宇辰; 崔永萍; 詹启敏
Original assignee: Cancer Hospital and Institute of CAMS and PUMC
Current assignee: Cancer Hospital and Institute of CAMS and PUMC
Priority date: 2022-08-05
Filing date: 2022-08-05
Publication date: 2022-10-25

Abstract

The invention relates to a molecular diagnostic marker of esophageal squamous carcinoma and application thereof. The invention provides a group of genes for molecular typing of esophageal squamous cell carcinoma, which comprise: CCND1, CDKN2A/B, NFE2L2, SOX2, and ERBB2 (HER 2). The invention also provides a molecular typing detection kit for esophageal squamous cell carcinoma, which comprises the gene detection reagent, and a related application. The invention provides the molecular typing system of the esophageal squamous cell carcinoma, and provides a reliable basis for clinical accurate diagnosis and accurate targeted therapy of the esophageal squamous cell carcinoma.

Description

Molecular typing diagnosis marker for esophageal squamous carcinoma and application thereof

Technical Field

The invention relates to a molecular diagnostic marker of diseases and application thereof. In particular, the invention relates to molecular diagnostic markers of esophageal cancer such as esophageal squamous carcinoma, potential therapeutic targets and related diagnostic and therapeutic uses.

Background

Esophageal squamous carcinoma (ESCC) is one of the most common and most lethal tumors at present. Although VEGFR-2 antagonists have been approved for esophageal squamous carcinoma treatment [1], and PD-1/PD-L1 immunotherapy has achieved better effect in some cases [2], both methods have great heterogeneity, and for esophageal squamous carcinoma, an effective molecular therapeutic target or molecular typing mechanism still lacks at present, and the treatment of esophageal squamous carcinoma is mainly based on traditional radiotherapy and chemotherapy. While other common tumors such as breast cancer and lung cancer discover novel therapeutic targets and better biomarkers for early screening and diagnosis from molecular big data mining [3-5].

Current typing for esophageal squamous carcinoma is mainly based on pathology and a single set of chemical data. Arnold et al have analyzed 398000 esophageal squamous carcinomas and 52000 esophageal adenocarcinoma data and have typed esophageal carcinomas based on pathological rather than molecular characteristics [6]. Recently, a number of studies have been reported on the molecular typing of esophageal squamous cell carcinoma based on genomic data [7-10], and a small-scale (10 patients with esophageal squamous cell carcinoma) study on the epigenetic modification group [11]. However, the above studies are limited to single omics analysis in a single queue of esophageal squamous cell carcinoma, and there is no report on molecular characteristic difference analysis and molecular typing system studies based on multiple sets of chemical data.

Disclosure of Invention

Due to the complex pathogenesis of tumors, there is a high degree of heterogeneity in both histopathological and molecular biological characteristics, even between patients of the same tumor type, and even between different stages of disease in the same patient. The molecular typing is carried out on the tumor based on multiple groups of data, which is beneficial to realizing the accurate treatment of the cancer.

Therefore, in order to promote the development of the field of accurate treatment of esophageal squamous cell carcinoma, the invention establishes a molecular typing system for esophageal squamous cell carcinoma patients, particularly Chinese esophageal squamous cell carcinoma patients, and determines molecular treatment targets and biomarkers of different subtype esophageal squamous cell carcinomas through analysis of multiple sets of mathematical data, thereby providing a basis for individual treatment of esophageal squamous cell carcinoma and benefiting a great number of patients.

The present invention carried out proteome analysis on 73 samples of 155 patients with esophageal squamous carcinoma by performing Whole Genome Sequencing (WGS), whole genome sulfite sequencing (WGBS), RNA sequencing (RNA-seq) and small RNA sequencing (small RNA-seq) on tumor tissues and paracarcinoma tissues of 155 patients. The invention collates the obtained multigroup analysis data of esophageal squamous cell carcinoma, establishes the largest esophageal squamous cell carcinoma multiomic database (ESCC Genome and Epigenome Atlas, ECGEA) in China at present, and establishes a molecular typing system of esophageal squamous cell carcinoma.

In order to establish a molecular classification system of esophageal squamous cell carcinoma, first, a preliminary Cluster assignment (Cluster assignment) is generated by using single platform data of 155 cases through consensus clustering [12, 13], and then, cluster of Cluster Assignments (COCA) is applied to Cluster analysis of four types of COCA subtypes of esophageal squamous cell carcinoma, C1-C4 (figure 1B, C), which are obviously related to clinical TNM staging and lymph node metastasis (figure 1D). Meanwhile, the invention also identifies the diagnostic molecular marker and the potential therapeutic target aiming at each subtype. The four classes of COCA subtypes are:

1) C1: CCA, a cell cycle activation subtype (CCA), accounts for 25.2%, and is mainly characterized by the copy number variation of CCND1 and CDKN 2A/B;

2) C2: NRFA, NRF2 signaling pathway activation subtype (NRFA), accounts for 24.5%, and is mainly characterized by up-regulated expression of NFE2L2 and SOX2 genes;

3) C3: IS, an Immunosuppressive Subtype (IS), accounting for 19.3%, with high levels of immunoinfiltrating cells, low Tumor Mutational Burden (TMB), and high expression of ERBB2 (HER 2) protein as major features;

4) C4: IM, the immunoregulatory subtype (IM), accounts for 31.0% and is characterized mainly by high levels of immune infiltrating cells (especially tumor infiltrating macrophages, NK cells and CD8T cells), high Tumor Mutation Burden (TMB), and low expression of ERBB2 (HER 2) protein.

Accordingly, the invention provides an invention described in one or more of the following aspects.

1. A group of genes for molecular typing of esophageal squamous cell carcinoma comprises: CCND1 (Genbank ID No.: NG _ 007375.1), CDKN2A (Genbank ID No.: NG _ 007485.1), CDKN2B (Genbank ID No.: NG _ 023297.1), NFE2L2 (Genbank ID No.: NC _ 000002.12), SOX2 (Genbank ID No.: NG _ 009080.1) and ERBB2 (HER 2, genbank ID No.: NG _ 007503.1).

2. The gene according to item 1, which further comprises one or more of the following genes: CTTN (Genbank ID No.: NM-005231.4), ANO1 (Genbank ID No.: NM-001378092.1), ORAOV1 (LTO 1, genbank ID No.: NM-001031186.2); KEAP1 (Genbank ID No.: NC-000019.10), CUL3 (Genbank ID No.: NG-032169.1).

3. The gene according to

item

1 or 2, which comprises the following combinations of genes: 1) CCND1 (Genbank ID number: NG _ 007375.1), CDKN2A (Genbank ID number: NG _ 007485.1), CDKN2B (Genbank ID number: NG — 023297.1), CTTN (Genbank ID number: NM — 005231.4), ANO1 (Genbank ID No.: NM _ 001378092.1), ORAOV1 (LTO 1, genbank ID No.: NM _ 001031186.2); 2) NFE2L2 (Genbank ID number: NC — 000002.12), KEAP1 (Genbank ID number: NC — 000019.10), CUL3 (Genbank ID number: NG — 032169.1), SOX2 (Genbank ID number: NG _ 009080.1); and 3) ERBB2 (HER 2, genbank ID accession: NG _ 007503.1).

4. An esophageal squamous carcinoma molecular typing detection kit, wherein the kit comprises a reagent for detecting the gene or the protein coded by the gene in any item 1-3.

5. The kit of item 4, wherein the reagents comprise primers or probes for detecting the genes.

6. The kit according to item 4 or 5, wherein the reagent comprises an antibody detecting a protein encoded by the gene.

7. The kit of item 6, wherein the antibody comprises a polyclonal antibody or a monoclonal antibody.

8. The kit according to any one of items 4 to 7, which further comprises a reagent for detecting immunoinfiltrated cells and/or tumor mutational burden.

9. Use of the gene according to any one of items 1-3 or the protein encoded by the gene in the preparation of an esophageal squamous cell carcinoma molecular typing detection kit and/or an esophageal squamous cell carcinoma individualized treatment kit.

10. The use according to item 9, wherein the gene or the protein encoded by the gene is used for the following molecular typing of esophageal squamous carcinoma:

1) CCA, a cell cycle activating subtype including copy number variations of CCND1 and CDKN2A, CDKN 2B;

2) NRFA, an NRF2 signaling pathway activating subtype, which includes upregulated expression of NFE2L2 and SOX2 genes;

3) IS, an immunosuppressive subtype including high ERBB2 (HER 2) protein expression;

4) IM, immunoregulatory subtype, including low expression of ERBB2 (HER 2) protein.

In some embodiments, the present invention relates to a combination of one or more genes, the use of said genes (or combinations thereof) as biomarkers (biomarkers), in particular as biomarkers for molecular typing, diagnosis and treatment of esophageal squamous carcinoma. In some embodiments, the genes include a combination of one or more, preferably all, of CCND1, CDKN2A, CDKN2B, NFE2L2, SOX2, and ERBB2 (HER 2); optionally, one or more of the following genes can be further included: CTTN, ANO1, ORAOV1; KEAP1, CUL3. In some embodiments, the invention includes one or more of the following specific combinations of genes: 1) CCND1, CDKN2A, CDKN2B, CTTN, ANO1, and ORAOV1; 2) NFE2L2, KEAP1, CUL3, SOX2; and 3) ERBB2 (HER 2). The genes and corresponding encoded products involved in the present invention can be readily obtained and detected from public databases (e.g., genebank).

In some embodiments, the present invention may suitably include a protein encoded by the above-described gene. In other words, CCND1, CDKN2A, CDKN2B, NFE2L2, SOX2, ERBB2 (HER 2) and optionally CTTN, ANO1, ORAOV1; one or more genes of KEAP1, CUL3 also include combinations involving one or more corresponding proteins encoded by the above genes, the use of said proteins as biomarkers, in particular as biomarkers for molecular typing, diagnosis and treatment of esophageal squamous carcinoma. In some embodiments, genes and/or proteins that are biomarkers may be used simultaneously. Thus, the invention also relates to a compound selected from CCND1, CDKN2A, CDKN2B, NFE2L2, SOX2, ERBB2 (HER 2) and optionally CTTN, ANO1, ORAOV1; the combination of one or more genes of KEAP1 and CUL3 and/or the combination of one or more proteins selected from the genes respectively encoded by the genes, the use of the combination as a biomarker, in particular as a biomarker for molecular typing, diagnosis and treatment of esophageal squamous cell carcinoma.

In some embodiments, the invention also relates to a kit comprising said genes and/or protein combinations, the use of said genes and/or protein combinations in diagnosis, grading, staging and prognosis of esophageal squamous carcinoma. In some embodiments, the kits of the invention comprise a means for detecting CCND1, CDKN2A, CDKN2B, NFE2L2, SOX2, ERBB2 (HER 2), and optionally CTTN, ANO1, ORAOV1; one or more genes in KEAP1 and CUL3 and/or one or more corresponding proteins coded by the genes. In some embodiments, the reagents include reagents that detect the presence, mutation, and/or an increase and/or decrease in the amount of expression of any one or more of the genes and/or proteins described above. In some embodiments, the mutations of genes referred to herein include any mutation known in the art, including, for example, alterations of insertions, deletions, inversions, copy numbers, and the like. Reagents for detecting the presence, mutation, expression level of a known gene and/or protein can be performed by any reagent known in the art. In some embodiments, the reagents to which the invention relates may include, for example, detection of CCND1, CDKN2A, CDKN2B, NFE2L2, SOX2, ERBB2 (HER 2), and optionally CTTN, ANO1, ORAOV1; agents for the presence, mutation and/or expression level of one or more genes of KEAP1, CUL3. In some embodiments, the agents contemplated by the present invention may include, for example, agents that detect copy number variation of CCND1 and CDKN2A, CDKN2B, up-regulated expression of NFE2L2 and SOX2 genes, high expression of ERBB2 (HER 2) protein, low expression of ERBB2 (HER 2) protein. Methods and reagents for the detection of specific genes and/or proteins are well known to those skilled in the art and can be performed, for example, using primers and/or probes specific for the corresponding genes, antibodies (including polyclonal or monoclonal antibodies) specific for the corresponding proteins. In some embodiments, the biomarkers can be detected, for example, using methods including, but not limited to, enzyme-linked immunosorbent assay (ELISA), mass spectrometry, radioimmunoassay, chemiluminescence, real-time PCR, nucleic acid hybridization methods, western blot analysis, immunodetection (e.g., immunoprecipitation and/or immunofluorescence), and the like. In some embodiments, for example, monoclonal antibodies may be used as detection reagents for biomarkers. Methods and reagents for designing corresponding primers and/or probes based on known genes and for preparing corresponding antibodies based on known proteins can be performed by any methods and reagents well known in the art.

In some embodiments, a biomarker of the invention (e.g., a combination of one or more genes and/or selected from the group consisting of one or more proteins encoded by each of CCND1, CDKN2A, CDKN2B, NFE2L2, SOX2, ERBB2 (HER 2), and optionally CTTN, ANO1, ORAOV1; KEAP1, CUL 3) refers to a molecule associated with a disease (e.g., cancer, e.g., esophageal squamous carcinoma) such as a molecule associated with the presence, stage, typing, prognosis, predicted response to treatment, etc., of esophageal squamous carcinoma. Esophageal squamous carcinoma biomarkers can include genes and/or proteins that are differentially expressed in esophageal squamous carcinoma subjects (e.g., full-length genes and/or proteins and corresponding mutant genes and/or proteins, as well as differences in their expression levels). In some embodiments, the methods of the invention comprise determining a combination of one or more genes and/or proteins described above in a biological sample (e.g., a biological sample from blood, such as whole blood, plasma, blood, and/or tissue, such as biopsy tissue) from a subject suspected of having a disease (e.g., cancer, e.g., esophageal squamous carcinoma). In some embodiments, the biomarker itself and/or a product of the biomarker, such as a fragment of the biomarker and/or other product directly associated with the biomarker, may be detected.

In some embodiments, the invention provides compositions and/or kits for assessing, diagnosing, typing (including molecular typing) and/or monitoring esophageal cancer (e.g., esophageal squamous carcinoma) in a subject, wherein the compositions or kits comprise detection reagents for biomarkers described herein (combinations of genes and/or proteins described herein, e.g., CCND1, CDKN2A, CDKN2B, NFE2L2, SOX2, ERBB2 (HER 2), and optionally CTTN, ANO1, ORV 1; KEAP1, CUL3 in combination with one or more genes and/or selected from combinations of one or more proteins each encoded therein). In some embodiments, the biomarker detection reagent may include various buffers for biomarker analysis such as PBS, running buffer, and the like. In some embodiments, the detection reagents may include protein and/or nucleic acid detection reagents. In some embodiments, the biomarker detection reagent may comprise an antibody and/or primer, probe, directed to a specific biomarker. In some embodiments, the kit may include a plurality of antibodies, antibody derivatives, or antibody fragments that specifically bind to the biomarker and the protein or fragment thereof. Nucleic acid biomarkers can include genomic DNA, mRNA, spliced mRNA, cDNA, and the like. The detection reagent may comprise a complementary nucleic acid. For example, labeled or unlabeled oligonucleotides immobilized on a substrate, oligonucleotides not bound to a substrate, PCR primers, molecular beacons, and the like. In some embodiments, the detection reagents for the biomarkers can include nucleic acid and/or protein analysis reagents such as nucleic acid and/or western blots, ELISA analysis reagents, proteome analysis reagents such as protein spectrometry and antibody chips, and the like. In some embodiments, the detection reagent for the biomarker may comprise a tracer reagent, for example, to link the biomarker to a detectable label. In some embodiments, the biomarker detection reagent may comprise a sequence analysis reagent such as a high throughput sequencing reagent. In some embodiments, a kit may include a capture reagent that binds a biomarker of the present invention and a container that includes at least one biomarker. In some embodiments, the capture reagent can bind to multiple biomarkers, and can also bind to at least one known biomarker. In some embodiments, the kit may further comprise a second or more capture reagents. In some embodiments, the kit comprises a buffer. In some embodiments, the kit comprises instructions for use. In some embodiments, the kit comprises a chip or microarray. In some embodiments, the kit comprises one or more substrates with an adsorbent attached.

In some embodiments, the invention can assess, diagnose, classify (including molecularly classify) and/or monitor esophageal cancer (e.g., esophageal squamous carcinoma), e.g., a biological fluid sample from the patient can be extracted and the presence and/or concentration of a biomarker detected (e.g., using a kit of the invention) can be detected, wherein an alteration in the level of the biomarker as compared to a normal subject or control patient not having esophageal cancer (e.g., esophageal squamous carcinoma) is indicative of the patient having esophageal cancer (e.g., esophageal squamous carcinoma) or classifying esophageal cancer (e.g., esophageal squamous carcinoma) based on the detection of the biomarker.

In some embodiments, the patient has an increased or decreased level of a biomarker 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or more compared to a biomarker level not having the cancer, or has a genetic mutation, including a change in copy number, or the like, compared to a control not having the cancer. In some embodiments, the invention includes a combination of two or more biomarkers of the invention. In some embodiments, the level of a biomarker (e.g., nucleic acid expression level, protein activity level, RNA level, levels of related metabolites, etc.) can be used alone for the assessment, diagnosis, typing, and/or prognosis of esophageal cancer (e.g., esophageal squamous carcinoma). In some embodiments, the biomarkers can be used in combination with esophageal cancer (e.g., esophageal squamous carcinoma) biomarkers known in the art for the assessment, diagnosis, typing, and/or prognosis of esophageal cancer (e.g., esophageal squamous carcinoma).

The methods, compositions, kits of the invention can be used to assess whether a subject has esophageal cancer (e.g., esophageal squamous carcinoma); assessing the stage of esophageal cancer (e.g., esophageal squamous carcinoma) in the subject; assessing the grade of esophageal cancer (e.g., esophageal squamous carcinoma) in the subject; assessing the stage of esophageal cancer (e.g., esophageal squamous carcinoma) in the subject; assessing the typing (e.g., molecular typing) of esophageal cancer (e.g., esophageal squamous carcinoma) in a subject, assessing the benign or malignant nature of esophageal cancer (e.g., esophageal squamous carcinoma) in a subject, assessing the metastatic potential of esophageal cancer (e.g., esophageal squamous carcinoma) in a subject, assessing the presence of cells of esophageal cancer (e.g., esophageal squamous carcinoma) in a subject, assessing the efficacy of one or more candidate compounds for inhibiting esophageal cancer (e.g., esophageal squamous carcinoma) in a subject, assessing the efficacy of a therapeutic method, monitoring the progression of esophageal cancer (e.g., esophageal squamous carcinoma) in a subject, screening a composition or therapeutic method for inhibiting esophageal cancer (e.g., esophageal squamous carcinoma) in a subject, assessing the esophageal carcinogenic (e.g., esophageal squamous carcinoma) potency of a test compound, and preventing the onset of esophageal cancer (e.g., esophageal squamous carcinoma) in a subject at risk of developing esophageal cancer (e.g., esophageal squamous carcinoma).

According to the experimental result, the invention establishes a multigroup chemical database of the esophageal squamous cell carcinoma and a molecular classification system of the esophageal squamous cell carcinoma, and simultaneously, the invention determines molecular treatment targets and biomarkers of different subtype esophageal squamous cell carcinomas through multigroup chemical data analysis, thereby providing a basis for individual treatment of the esophageal squamous cell carcinoma and enabling vast patients to benefit from the treatment.

1) The invention completes the analysis and the arrangement of multiomic data in a large-scale clinical sample for the first time, establishes an ECGEA (ensemble spectroscopic database) of the esophageal squamous carcinoma and an esophageal squamous carcinoma molecular classification system based on multigroup chemical data analysis, and changes the current situation that the esophageal squamous carcinoma only has a histological classification system or a single omic data classification system;

2) The molecular typing system of the esophageal squamous carcinoma built by the invention divides the esophageal squamous carcinoma into four subtypes with obvious molecular difference: CCA, NRFA, IS and IM, and specifically analyzes the main characteristics of four subtypes in detail, thereby being beneficial to accurate diagnosis in clinic;

3) The invention also explains the heterogeneity reason of the esophageal squamous carcinoma effect according to the proposed molecular classification system of the esophageal squamous carcinoma and related research results, discusses molecular treatment targets corresponding to four subtypes, and provides reliable basis for the precise targeted treatment of the esophageal squamous carcinoma;

4) Aiming at the high sensitivity of the IM subtype to ICB therapy, the invention also establishes a screening system specially for diagnosing the IM subtype, screens 28 molecular characteristics from the obtained multigroup data and tests the sensitivity and the accuracy of the molecular characteristics.

Drawings

FIG. 1 characteristics of the COCA subtypes. (A) Schematic of each molecular typing performed on 155 ESCC queues. Columns represent individual tumors, clinical features are summarized in the top panel. The subsequent panels show statistics for the multi-layer changes: the proportion of mutant feature contributions; mutational burden of coding and non-coding regions; gene copy number variation rate (CNA); differentially Expressed Genes (DEGs); differentially expressed mirnas and Differentially Methylated Regions (DMR). (B) Four subtypes determined were clustered with integrated COCA of single set of chemistry data. Heat maps showing the distribution of a single set of chemical subtypes among the four subtypes. multiple sets of chemical alterations in mRNA, miRNA, promoter methylation, and CNA and COCA subtypes. (C) consensus clustering heatmaps of the four COCA subtypes. (D) Correlation between TNM staging (left panel) and lymphatic metastasis (right panel) and COCA subtypes. The P value was determined by Fisher's exact test.

Acc subtype correlates with activated cell cycle signalling. (A) Distribution of 11q13.3 and CDKN2A/B genomic alterations in ACC and non-ACC subtypes. (B and C) comparison of (B) 11q13.3 differentially expressed genes (CCND 1, CTTN, ANO1 and ORAOV 1) and CDKN2A/B (C) expression between tumor tissues for each COCA subtype. P values were determined by one-way analysis of variance, F test. (D and E) Bright field images (D) and cell viability (E) of patient organoids (PDO) in CDKN2A/2B deleted or wild type state after palbociclib treatment (mean. + -. Sem; two-sided t-test; ruler =200 μm).

Figure 3 multigroup chemical alterations of nrfa subtype tumors. (A) The genomic altered distribution of NRF2 pathway genes and SOX2 in NRFA and non-NRFA subtypes. (B and C) expression of NFE2L2 and SOX2 genes in the respective COCA subtypes. P values were determined by one-way anova F-test. (D) The correlation between NFE2L2 and SOX2 expression was examined by Spearman correlation analysis. (E) ESCC cell lines ZEC145 and ZEC166 were enriched with anti-SOX 2 antibody, analyzed by ChIP-qPCR to detect specific binding of SOX2 to the NFE2L2 promoter region, and analyzed by a two-sided t-test. (F) ChIP-seq data in public databases confirmed the binding peak of SOX2 in the NFE2L2 promoter region. (G) IHC staining of NFE2L2 in three groups of ESCC specimens.

FIG. 4 tumor infiltrating immune cells in ESCC. (A) mRNA expression for each COCA subtype was immuno-scored and Mann-Whitney U was subjected to follow-up assay analysis. (B) Number heatmap of 27 microenvironment cell subsets calculated for each COCA subtype of ESCC based on ssGSEA. (C) TME patterns of the IS (upper panel) and IM (lower panel) subtypes. Different cell modules and immune cell types are represented by regular and irregular circles of different colors. The red line connects the cells to positive correlation. The numbers in the legend represent the weights of the corresponding cellular modules, calculated from the relationship of the different immune cell types, describing the distribution and weights of the immune cell clusters in the ESCC. (D) comparison of the number of mutations between IS and IM tumors. Mann-Whitney U was subjected to follow-up assay. (E) protein expression of ERBB2 in each of the COCA subtypes. Mann-Whitney U was subjected to follow-up assay.

FIG. 5 is a classifier that identifies patients who respond to immunotherapy in an IM subtype of esophageal squamous carcinoma. (A) The correlation between the subtypes of esophageal squamous cell carcinoma COCA and the immunotherapy response group is shown based on the dendrograms of the genes differentially expressed between the subtypes of COCA. And (B) a sample selection and model training flow chart. (C) The ROC curves show the performance of the classifier on the training set, the validation set, and the test set, respectively.

Detailed Description

[ Collection of specimens ]

155 patients with esophageal squamous carcinoma related in the invention are diagnosed in Shanxi tumor hospital from 5 months to 2018 months in 2017, and are added into the clinical research queue. All patients were treated for the first time, and the tumor classification was judged as esophageal squamous carcinoma according to WHO criteria. Clinical staging of tumors was based on International Union Admission Cancer (UICC)/American Cancer Association (AJCC)/TNM staging (eighth edition) International Union Admission Cancer (UICC)/American Joint Commission for Cancer (AJCC) TNM staging system (the eigenh edition)

The pathology department of Shanxi tumor hospital is responsible for collecting and processing samples. In addition to collecting the tumor from each patient, the tissue adjacent to the tumor, i.e., normal or non-tumor tissue, was also collected (greater than 3cm from the resection site). The average weight of each tissue sample was 200mg and was uniquely marked (including patient ID number, time, disease name and sample type), and stored at-80 ℃ after flash freezing for 30 minutes in liquid nitrogen. Meanwhile, in order to perform pathological diagnosis and quality evaluation to judge whether the study conditions are satisfied, the Shanxi tumor Hospital Pathology department subjects the remaining tumor tissues and the para-carcinoma tissues to Formalin Fixation and Paraffin Embedding (FFPE), sectioning (thickness 4 μm), and H & E staining.

The sample conditions that met the requirements were: tumor samples must meet the requirements for the type of pathology (esophageal squamous carcinoma), tumor purity (greater than 70%), and cell necrosis rate (less than 20%); paracancerous specimens must be free of tumor cell infiltration. H & E sections of each tissue must be examined independently by at least 3 pathologists. If the requirements are met, the microneedle punctures the paraffin-embedded tissue sample and the corresponding cryopreserved tissue to be transported to a plain code (Shanghai) biological technology company Limited, the tissue is stored in a dry ice container during transportation, and a temperature recorder is used for tracking so as to ensure the transportation conditions. After arriving at the destination, the tissue sample is stored in liquid nitrogen again until the next processing after confirming that the transport temperature and other conditions are correct.

[ DNA extraction, whole genome sequencing library construction and sequencing ]

Genomic DNA (gDNA) was extracted from 10mg of tissue using the DNeasy 96 blood and tissue Kit (Qiagen, germanown, MD, USA). About 300ng of high quality DNA was required for the construction of the sequencing library (OD 260/OD280= 1.8-2.0). The integrity and concentration of genomic DNA was determined by agarose gel electrophoresis and the Qubit dsDNA HS Assay (ThermoFisher Scientific, waltham, MA, USA), and measured for OD260/OD280 values using NanoDrop2000 (ThermoFisher Scientific).

300ng of gDNA was fragmented into sequence fragments 150-200bp long using a sonicator (Covaris LE 220). DNA Library construction TruSeq Nano DNA LT Library Prep Kit (Illumina, san Diego, calif., USA) was used. End repair was done using End repair mix for the 5 'and 3' overhanging ends and purified using AMPure XP Beads (Beckman, brea, CA, USA). The purified fragment was terminally adenylated using A labeling Mix and ligated to a linker with DNA ligase to complete subsequent amplification and purification, and finally quantified with the Qubit dsDNA HS Assay (Thermo Fisher Scientific). After the library was completed, the sequence fragment length distribution was analyzed using Agilent Bioanalyzer 2100 (Agilent, santa Clara, calif., USA). 2X 150 paired-end sequencing was then performed using Illumina NovaSeq6000 according to the protocol provided by Illumina Inc.

[ WGBS library establishment and sequencing ]

Tissue whole genome DNA was extracted (same procedure as above), random fragments of 300bp insert size were generated by Covaris LE220 sonication from 200ng gDNA (spiked with 1% unmethylated Lambda DNA), followed by completion of end repair and end adenylation. The methylated linker is ligated to the DNA fragment. Sulfite treatment was performed according to the instruction procedure of EZ DNA Methylation-Gold Kit (Zymo Research, irvine, calif., USA). The single stranded DNA fragment was then PCR amplified (using KAPA HiFi HotStart Uracil + ReadyMix (2X)) and purified. Quantification was performed using the Qubit dsDNA HS Assay (Thermo Fisher Scientific). After the library was completed, the sequence fragment length distribution was analyzed using Agilent Bioanalyzer 2100 (Agilent, santa Clara, calif., USA). 2X 150 paired-end sequencing was then performed using Illumina NovaSeq6000 according to the protocol provided by Illumina Inc.

[ RNA extraction, transcriptome library construction and sequencing ]

10mg of tissue was prepared into homogenate (Scientz, zhejiang, china) using a homogenizer and used according to the manufacturer's instructions

Reagent (Invitrogen) and RNeasy MinElute spin column (Qiagen) extracted total RNA. Total RNA integrity was assessed using an Agilent bioanalyzer 2100 (Agilent, santa Clara, calif., USA) and quantified using a Nanodrop (Thermo Fisher Scientific). About 1. Mu.g of RNA of higher or moderate quality (OD 260/OD280=1.9-2.0, RIN ≧ 4) was required for construction of the sequencing library.

RNA purification, reverse transcription, library construction and sequencing are all completed according to corresponding operation guidance (Illumina). An Illumina TruSeq Standard Total RNA Gold prediction Kit was used to construct a Total RNA sequencing library with rRNA removed. The starting total RNA amount was approximately 1. Mu.g, and cytoplasmic rRNA and mitochondrial rRNA were removed using Ribo-Zero Gold Kit. rRNA was removed for purification and RNA was fragmented into small fragments using divalent cations at higher temperatures. The fragmented RNA fragments were reverse transcribed into the first strand of cDNA with reverse transcriptase and random primers, and end repair, phosphorylation and adenylation were done sequentially (experimental procedure was constructed according to Illumina library). The obtained product is enriched by using PCR, and AMPure XP Beads (Beckmen) are used for purifying the enriched target segment to construct a final cDNA library. The concentration was determined using a Qubit dsDNA HS Assay (Thermo Fisher Scientific) and the sequence fragment length distribution was analyzed using an Agilent bioanalyzer 2100 (Agilent, santa Clara, calif., USA). 2X 150 paired-end sequencing was then performed using Illumina NovaSeq6000 according to the protocol provided by Illumina Inc.

[ Small RNA library construction and sequencing ]

RNA purification, reverse transcription, library construction and sequencing were performed according to the corresponding operating instructions (Illumina). Small RNA sequencing libraries were constructed from total RNA using the Illumina TruSeq Small RNA Library Preparation Kit. About 1. Mu.g of total RNA was ligated to 3 '-and 5' -linkers in this order, and first and second strands of cDNA were synthesized using reverse transcriptase and a specific RT primer using the linker-ligated RNA fragment as a template. Illumina sequencing primers containing index were used to amplify cDNA with adaptors at both ends to create cDNA libraries. The 145-160bp fragment was purified using agarose gel electrophoresis to complete the final library creation. The concentration was quantified using a Qubit dsDNA HS Assay (Thermo Fisher Scientific) and the sequence fragment length distribution was analyzed using an Agilent bioanalyzer 2100 (Agilent, santa Clara, calif., USA). Cluster generation (Cluster formation) was done on Illumina cBOT Cluster generation system using HiSeq PE Cluster Kit (Illumina), followed by double-ended sequencing using Illumina HiSeq system according to the protocol provided by Illumina.

This is done in the clear (Shanghai) Biotechnology Ltd.

[ molecular typing of esophageal squamous carcinoma ]

According to the experimental results, four subtypes are established, and the specific description is as follows.

Both CCA and NRFA subtypes are characterized by CpG island methylation (E-CIMP +) and are frequently associated with lymph node metastasis and late clinical stages, with CCA subtypes appearing as alterations in cell cycle-associated genes and NRFA subtypes appearing as NRF2 pathway activation due to mutations in NRF2 pathway-associated genes.

Specifically, approximately 84.6% of CCA subtype patients had variations in cell cycle regulatory-associated genes and were characterized primarily by amplification of the 11q13.3 chromosomal segment (74.4% CCA subtype vs 28.4% non-CCA subtype, P value < 0.001) and homozygous deletion of the 9p21.3 chromosomal segment (43.6% CCA subtype vs 24.1% non-CCA subtype, P value = 0.026). Wherein the amplified 11q13.3 segment comprises the oncogenic genes CCND1, CTTN, ANO1 and ORAOV1, which are significantly up-regulated in patients with CCA subtypes (fig. 2B); whereas the segment 9p21.3 of the homozygous deletion contains the cancer suppressor gene CDKN2A/B. And CDKN2A/B can be inhibited by other means besides homozygous deletion, such as mutation and promoter hypermethylation, 59% of CCA subtype patients have CDKN2A/B variation, and thus the CDKN2A/B expression level is significantly down-regulated in CCA subtype patients (fig. 2C). To explore the sensitivity of CCA subtypes to CDK4/6 inhibitors that target the cell cycle [14, 15], pabociclib (palbociclib), we constructed CDKN2A knockout and wild-type tumor organoids (PDOs), and found that CDKN 2A-deficient ESCC is more sensitive to pabociclib (fig. 2d, e), so CCA subtype patients could be treated with CDK4/6 inhibitors.

While the NRFA subtypes are mainly characterized by NRF2 pathway activation (NRFA subtype accounts for 50.0% vs non-NRFA subtype accounts for 11.1%, P value < 0.001), and SOX2 pathway activation. Samples with NRF2 pathway-associated gene mutations and/or increased SOX2 copy number account for 73.7% of NRFA subtypes and 29.1% of non-NRFA subtypes, both of which are associated with a type of esophageal squamous cell carcinoma subtype previously reported in caucasian populations [7]. Among them, NRF2 pathway-related gene variation includes NFE2L2, KEAP1 and CUL3 (fig. 3A), and the three have mutually exclusive mutation phenomena (i.e. only one gene is mutated in 3 genes, and P value is less than 0.001). In the research queue of the invention, 38 cases of NFRA subtypes are involved, and the related gene variation comprises 5 cases of NFE2L2 copy number amplification; 7 cases of NFE2L2 activating mutations; 9 cases of KEAP1/CLU3 functional deletion mutation; KEAP1 hypermethylation 1 case; the copy number of SOX2/TP63 was 21 cases. The present invention, based on the type of genetic variation, has designed IHC to compare NFE2L2 gene expression levels in three sub-populations: 1) NFE2L2 gene variation-related (including NFE2L2 copy number amplification and activation mutations); 2) NFE2L2 regulatory gene mutations (KEAP 1/CLU3 loss of function mutation, KEAP1 hypermethylation and SOX2/TP63 copy number amplification); 3) Does not have NRF2 pathway-associated gene mutation. As a result, it was found that the NFE2L2 expression levels of

subpopulations

1 and 2 were comparable, both significantly higher than subpopulation 3 (fig. 3G). Therefore, all NRFA subtypes expressed NFE2L2 in the sample significantly higher than the other 3 subtypes.

In addition to NRF 2-associated gene mutations, the copy number and expression level of SOX2 were significantly increased in NRFA subtype samples (fig. 3a,3 c), and according to the clinical cohort data we obtained, the RNA expression level of NFE2L2 was positively correlated with the expression level of SOX2 (fig. 3D). To explore whether SOX2 mediates transcription of NFE2L2, we designed ChIP experiments on both ZEC145 and ZEC166 ESCC cell lines and found that SOX2 can specifically bind to the promoter region of NFE2L2 (fig. 3E), with reference to ChIP-Seq data [16] (fig. 3F).

The IS and IM subtypes share a high immune score (immunescore) (fig. 4A) (and higher expression levels of the immune checkpoint egg CD276 as the main features IM and IS vs CCA and NRFA, P value < 0.05). Single sample gene set enrichment analysis (ssGSEA) is an assay based on cell-specific gene expression [17, 18]. The invention uses ssGSEA to analyze the cell type distribution characteristics of main tumor infiltration immune cell population and stromal cell population in IS subtype (n = 30) and IM subtype (n = 48) tumor microenvironment in a research queue, including 25 immune cells, endothelial cells and fibroblasts; the 27 cell types were analyzed for abundance in 155 samples using ssGSEA Normalized Enrichment Score (NES) (fig. 4B). The results show that both IS and IM subtypes have higher content of tumor immune infiltration cells, but the distribution of the immune cell subsets of the two subtypes IS different, the content of fibroblasts, CD8T cells and innate immune cells (including NK cells, macrophages and the like) of the IM subtype IS higher, and the abundance of B cells and CD56 bright NK cells of the IS subtype IS higher.

Based on the heterogeneity of esophageal squamous cell carcinoma in the curative effect of PD-1 monoclonal antibody [19-22], the invention researches the relevant characteristics of immunotherapy of IS subtype and IM subtype. Using TME module algorithm, tumor microenvironment cell maps (TME maps) were generated for patients with IS subtype and IM subtype (fig. 4C), as shown, the TME map of IM subtype mainly consists of CD8T cells (CD 8T-macro), myeloid suppressor cells (MDSCs) and macrophages, and secondly dendritic cells (including iDCs and pDCs) are also abundant; whereas the major module of the IS subtype (CD 4 Tcm-Tem) comprises CD4 central memory T cells (Tcm) and CD4 effector memory T cells (Tem). Immune cells [23-26], namely dendritic cells, macrophages and NK cells, which are related to the activation of the anti-tumor activity of T cells and the promotion of the curative effect of PD-1 monoclonal antibody immunotherapy are higher in the abundance of IM subtypes compared with IS subtypes (P value IS less than 0.01).

In addition to differences in cell types in the tumor microenvironment, the IM and IS subtypes also differ in tumor mutational burden (tumor mutational burden) and protein expression levels. IM subtype tumors had higher mutation load, while the IS subtype tumor has higher expression of ERBB2 (HER 2) (fig. 4d,4 e). Thus, IM subtype patients may be more suitable for immunotherapy, whereas IS subtype patients are more suitable for targeted therapy against ERBB2 (HER 2).

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Claims

1. a group of genes for molecular typing of esophageal squamous carcinoma comprises: CCND1, CDKN2A, CDKN2B, NFE2L2, SOX2 and ERBB2 (HER 2).

2. The gene of claim 1, further comprising one or more of the following genes: CTTN, ANO1, ORAOV1; KEAP1, CUL3.

3. The gene according to claim 1 or 2, which comprises the following combinations of genes: 1) CCND1, CDKN2A, CDKN2B, CTTN, ANO1, and ORAOV1; 2) NFE2L2, KEAP1, CUL3, SOX2; and 3) ERBB2 (HER 2).

4. An esophageal squamous carcinoma molecular typing detection kit, wherein the kit comprises a reagent for detecting the gene or the protein coded by the gene according to any one of claims 1-3.

5. The kit of claim 4, wherein the reagents comprise primers or probes for detecting the genes.

6. The kit of claim 4 or 5, wherein the reagent comprises an antibody that detects a protein encoded by the gene.

7. The kit of claim 6, wherein the antibody comprises a polyclonal antibody or a monoclonal antibody.

8. The kit of any one of claims 4-7, further comprising reagents for detecting immunoinfiltrated cells and/or tumor mutational burden.

9. Use of the gene or the encoded protein thereof according to any one of claims 1-3 in preparation of an esophageal squamous carcinoma molecular typing detection kit and/or an esophageal squamous carcinoma individualized treatment kit.

10. The use according to claim 9, wherein the gene or the encoded protein thereof is used for the following molecular typing of esophageal squamous carcinoma:

1) C1, cell cycle activation subtype, wherein the copy number variation of CCND1, CDKN2A and CDKN2B is included, the expression of CCDN1 is up-regulated, and the expression of CDKN2A and CDKN2B is down-regulated;

2) C2, NRF2 signal path activation subtypes, wherein the subtypes comprise NFE2L2, KEAP1 and CUL3 gene mutation, SOX2 copy number variation, NFE2L2 and SOX2 gene up-regulation expression and KEAP1 expression down-regulation;

3) C3, an immunosuppressive subtype including high ERBB2 (HER 2) protein expression;

4) C4, immunoregulatory subtype, including low ERBB2 (HER 2) protein expression and high TMB.