WO2019057913A1 - Méthode et kits pour le pronostic du carcinome squameux pulmonaire (scc) - Google Patents

Méthode et kits pour le pronostic du carcinome squameux pulmonaire (scc) Download PDF

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WO2019057913A1
WO2019057913A1 PCT/EP2018/075641 EP2018075641W WO2019057913A1 WO 2019057913 A1 WO2019057913 A1 WO 2019057913A1 EP 2018075641 W EP2018075641 W EP 2018075641W WO 2019057913 A1 WO2019057913 A1 WO 2019057913A1
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prognosis
stc1
subject
expression levels
scc
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PCT/EP2018/075641
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Jackeline AGORRETA ARRAZUBI
Elena MARTÍNEZ TERROBA
Luis MONTUENGA BADÍA
María Josefa PAJARES VILLANDIEGO
Rubén PÍO OSÉS
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Fundación Para La Investigación Médica Aplicada
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407Specifically defined cancers
    • G01N33/57423Specifically defined cancers of lung
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00Detection or diagnosis of diseases
    • G01N2800/52Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • SCC squamous cell carcinoma
  • the invention relates to the field of cancer prognosis and more particularly, to methods for predicting the outcome of a lung squamous cell carcinoma (SCC) patient based on the expression levels of several proteins and to specific prognostic signatures and algorithms, as well as to methods for selecting those patients who would benefit from adjuvant treatment after tumor resection, and kits for implementing those methods. More specifically, said prognostic/predictive signature is based on the protein expression levels of cytoplasmic STC1 (STC1-C) and cytoplasmic RAEl (RAEl-C) in a biological sample of a subject having lung SCC.
  • STC1-C cytoplasmic STC1
  • RAEl-C cytoplasmic RAEl
  • Lung cancer remains the leading cause of cancer deaths worldwide with an overall 5 years survival of 10-15%. This low survival rate is mainly associated with the fact that about two thirds of lung cancer patients are diagnosed at advanced stages for which curative treatment is not feasible. These data could change very soon since the National Lung Screening Trial, a randomised study of over 53,000 high-risk individuals randomized between screening with low dose spiral computed tomography (LDCT) versus chest X-ray, has demonstrated that the LDCT screening is truly efficient in detecting more early stage lung cancers. After resection of these early stage tumors, they have shown a 20% relative mortality reduction in the LDCT group respect to the CXR one in seven years of follow up (Aberle DR et al, 2011). These data have led to the implementation of policies of coverage and reimbursement of low-dose CT (LDCT) lung cancer screening by the U.S. Centers for Medicare and Medicaid Services and other Health Providers.
  • LDCT low-dose CT
  • NSCLC Non-small cell lung cancer
  • stage IA patients adjuvant chemotherapy is not recommended since the LACE meta-analysis, which combines data from 5 large adjuvant cisplatin-based chemotherapy trials, suggested worse outcome in stage IA patients treated with adjuvant therapy (Pignon JP et al 2008). Moreover, in stage IB patients, adjuvant chemotherapy is recommended only in special circumstances (Pisters KM et al, 2007). All these data suggest that among stage I patients exist a subpopulation with poor prognosis which may potentially benefit from adjuvant treatment, whereas other stage I or II patients might not benefit or even could be harmed by postsurgical therapy.
  • NSCLC tumor-node-metastasis
  • q-PCR the gold standard assay for gene expression.
  • Q-PCR has significant advantages to microarray-based assays, including widespread availability, cost and reproducibility.
  • q-PCR-based molecular signatures with prognostic implications have been described (Lau SK et al, 2007; Bianchi F et al, 2007; Skrzypski M et al 2008; Raz DJ et al 2008).
  • One of the major logistic problems in order to implement q-PCR-based signatures in daily clinical management is the need of good quality RNA extracted from fresh frozen resected tissue.
  • the different variables of time, preservation protocol and sample transport logistics may have a profound effect in the quality of RNA of a fresh or frozen sample. This becomes an important limiting factor in order to build a robust, reproducible and feasible technical RNA-based tool of molecular classification.
  • RNA extracted from formalin fixed, paraffin embedded (FFPE) samples which require less sophistication in the procurement protocol.
  • FFPE formalin fixed, paraffin embedded
  • Kratz et al defined an 11 -gene prognostic profile for non-squamous NSCLC (Kratz JR et al, 2012).
  • Wistuba et al demonstrated the utility of a 31 genes prognostic signature (the cell cycle proliferation score) as predictor of survival in early stage lung adenocarcinoma (ADC) (Wistuba II et al, 2013).
  • Patent application No. CN106442991 discloses a method for predicting the prognosis of patients with lung adenocarcinoma and determining the benefit of adjuvant chemotherapy, which comprises determining the expression levels of a set of protein markers, namely c-Src, Cyclin El, TTF1, p65, CHK1 and JNK1, by immunohistochemical methods.
  • lung cancer e.g. lung adenocarcinoma and squamous cell carcinoma
  • help clinicians in the selection of the more appropriate treatment strategies.
  • prognostic methods are particularly needed in early stages of the disease where an adjuvant treatment may only be recommended for those patients having poor prognosis.
  • cytoplasmic STC1 STC1-C
  • cytoplasmic RAEl cytoplasmic RAEl
  • additional markers selected from the group consisting of: cytoplasmic RRM2 (RRM2-C), nuclear STC1 (STC1-N), nuclear LIG1 (LIG1-N), nuclear QKI (QKI-N), cytoplasmic RAD51 (RAD51-C), nuclear SRSF1 (SRSF1-N), nuclear SIRT2 (SIRT2-N), cytoplasmic SLC2A1 (SLC2A1-C), and cytoplasmic QKI (QKI-C).
  • the identified signatures preferably consist of an algorithm which combines the coefficient- weighted expression of the selected marker proteins analyzed by immunohistochemical methods.
  • the levels of the expression of each protein are preferably measured by H-Score and the algorithm assigns a coefficient to each protein H-Score to get a final combined score that in the context of this invention is called Prognostic Index (PI) value.
  • PI Prognostic Index
  • These protein scores and the prognostic indicator have been shown to provide independent prognostic information for recurrence free survival (DFS) and overall survival (OS) in patients with lung SCC. Indeed, as shown in Examples 1 to 3, the identified signatures are able to discriminate with high statistical significance a group of SCC patients of early stages of lung cancer who have higher risk of recurrence or worse survival prospects. This group of patients should be monitored more closely and managed more intensely, and may be potential candidates for adjuvant chemotherapy.
  • the inventors have also shown the prognostic value of these signatures in combination with the cancer clinico-pathological stage (i.e. using the TNM staging system), see Examples 4 and 5. Also, the signature which has been identified as having the highest prognostic value for SCC has been shown to be able to predict which patients would likely benefit from postsurgical therapy, in particular in early stage lung SCC patients. Indeed, in Example 6 is shown that in stage I SCC patients presenting higher PI who received platinum-based chemotherapy after surgical resection a statistically significant association was observed with longer overall survival. However this association was not observed in patients with lower PI.
  • the present invention relates to an in vitro method for determining the prognosis of a subject having lung squamous cell carcinoma (SCC), wherein said method comprises: a) determining in a biological sample isolated from said subject the protein expression levels of cytoplasmic STC1 (STC1-C) and cytoplasmic RAE1 (RAE1-C), optionally, further determining in said biological sample the protein expression levels of at least one, two, three, four, five, six, seven, eight, or nine additional markers selected from the group consisting of:
  • RRM2-C -cytoplasmic RRM2
  • LIG1-N -nuclear LIG1
  • RAD51-C -cytoplasmic RAD51
  • SRSF1-N -nuclear SRSF1
  • SIRT2-N -nuclear SIRT2
  • QKI-C -cytoplasmic QKI
  • the invention in another aspect, relates to a method for determining target protein expression levels by immunohistochemistry, wherein said method is suitable for carrying out the determination of the protein expression levels of the markers in the biological sample as defined in step a) of the prognosis method of the invention.
  • said method comprises incubating the biological sample isolated from said subject with an affinity reagent for STC1, an affinity reagent for RAEl, and optionally, an affinity reagent for each of the at least one, two, three, four, five, six or the seven additional protein markers defined in step a) of the method of prognosis of the invention.
  • the invention provides an w vitro method for selecting those subjects having lung SCC who are expected to benefit from an adjuvant treatment, wherein said method comprises: a) classifying said subject according to the prognosis method as defined herein;
  • step b) selecting for administration of an adjuvant treatment a subject classified in step a) as having poor prognosis.
  • the invention also relates to an in vitro method for predicting the efficacy of an adjuvant chemotherapy in a subject having lung SCC, wherein said method comprises:
  • classification of the subject in step a) as having poor prognosis is indicative of increased efficacy of the adjuvant chemotherapy.
  • the invention provides an in vitro method for selecting an adjuvant treatment for a subject having lung SCC, wherein said method comprises:
  • step a) wherein when the subject is classified in step a) as having poor prognosis then adjuvant chemotherapy is selected as adjuvant treatment.
  • the invention relates to a data-processing apparatus comprising means for carrying out one or more of the steps of the methods as described herein.
  • the invention pertains to a computer program comprising instructions which, when the program is executed by a computer, cause the computer to carry out one or more of the steps of the methods of the invention.
  • the invention also refers to a computer-readable storage medium having stored thereon such a computer program.
  • the present invention provides a method for treating a subject having lung SCC comprising administering to said patient a therapeutically effective amount of a treatment, typically an adjuvant treatment, wherein said treatment is selected according to the classification of said patient according to the prognosis method of the invention.
  • a treatment typically an adjuvant treatment
  • the administered treatment comprises a platinum anticancer agent.
  • the invention relates to a kit suitable for use in a method for the prognosis of a subject having lung SCC as defined herein, wherein said kit comprises:
  • step a) optionally, further comprising an affinity reagent for each of the at least one, two, three, four, five, six or the seven additional proteins of the additional protein markers defined in step a);
  • cancer cells to be used as low and/or high expression controls optionally, further comprising cancer cells to be used as low and/or high expression controls;
  • the invention relates to the use of a kit in a method for the prognosis of a subject having lung SCC as defined herein, wherein said kit comprises:
  • step a) optionally, further comprising a reagent for each of the at least one, two, three, four, five, six or the seven additional proteins of the additional protein markers defined in step a) for determining the protein expression levels thereof;
  • cancer cells to be used as low and/or high expression controls optionally, further comprising cancer cells to be used as low and/or high expression controls;
  • FIG. 1 Study of the specificity of QKI antibody.
  • FIG. 1 Study of the specificity of RAE1 antibody.
  • FIG. 1 Study of the specificity of RRM2 antibody.
  • FIG. 4 Study of the specificity of SLC2A1 antibody.
  • A. WB of total and fraction extracts (C: cytoplasmic and N nuclear).
  • B. Relative expression after densitometry quantification of the bands, standardized to GAPDH
  • C. Representative images of signal detection by ICQ in the cell lines. Scale bar: 20 ⁇ m..
  • FIG. 1 Study of the specificity of SRSF1 antibody.
  • FIG. 1 Study of the specificity of STC1 antibody.
  • FIG. 7 Study of the specificity of LIG1 antibody.
  • FIG. 8 Study of the specificity of SIRT2 antibody.
  • A. WB of total and fraction extracts (C: cytoplasmic and N nuclear).
  • B. Relative expression after densitometry quantification of the bands, standardized to GAPDH
  • C. Representative images of signal detection by ICQ in the cell lines. Scale bar: 20 ⁇ m..
  • FIG. 9 Study of the specificity of CDC6 antibody.
  • FIG. 10 Study of the specificity of RAD51 antibody .
  • FIG. 11 Study of the specificity of SNRPE antibody .
  • A. WB of total and fraction extracts (C: cytoplasmic and N nuclear).
  • B. Relative expression after densitometry quantification of the bands, standardized to GAPDH
  • C. Representative images of signal detection by ICQ in the cell lines. Scale bar: 20 ⁇ m..
  • FIG. 12 Study of the specificity of BRCA1 antibody.
  • Figure 13 Association between PI score obtained from ADC model 1 and survival in ADC patients from a MD Anderson Cancer Center (MDA) cohort.
  • MDA MD Anderson Cancer Center
  • Figure 15 Association between PI score obtained from ADC model 3 and survival in ADC patients from MDA cohort.
  • A-B Kaplan-Meier curves and log rank statistics for disease free survival (A) and overall survival (B) in ADC patients from the training set (MDA cohort) divided in high and low prognostic index (PI) scores.
  • Figure 16 Association between PI score obtained from ADC model 4 and survival in ADC patients from MDA cohort.
  • A-B Kaplan-Meier curves and log rank statistics for disease free survival (A) and overall survival (B) in ADC patients from the training set (MDA cohort) divided in high and low prognostic index (PI) scores.
  • Figure 17 Association between PI score obtained from SCC model 1 and survival in SCC patients from MDA.
  • A-B Kaplan-Meier curves and log rank statistics for disease free survival (A) and overall survival (B) in SCC patients from the training set (MDA cohort) divided in high and low prognostic index (PI) scores.
  • Figure 18 Association between PI score obtained from SCC model 2 and survival in SCC patients from MDA.
  • A-B Kaplan-Meier curves and log rank statistics for disease free survival (A) and overall survival (B) in SCC patients from the training set (MDA cohort) divided in high and low prognostic index (PI) scores.
  • Figure 19 Association between PI score obtained from SCC model 3 and survival in SCC patients from MDA.
  • A-B Kaplan-Meier curves and log rank statistics for disease free survival (A) and overall survival (B) in SCC patients from the training set (MDA cohort) divided in high and low prognostic index (PI) scores.
  • Figure 20 Association between PI score obtained from SCC model 4 and survival in SCC patients from MDA.
  • A-B Kaplan-Meier curves and log rank statistics for disease free survival (A) and overall survival (B) in SCC patients from the training set (MDA cohort) divided in high and low prognostic index (PI) scores.
  • Figure 21 Association between PI score obtained from SCC model 5 and survival in SCC patients from MDA.
  • A-B Kaplan-Meier curves and log rank statistics for disease free survival (A) and overall survival (B) in SCC patients from the training set (MDA cohort) divided in high and low prognostic index (PI) scores.
  • FIG 22 ADC model 1 validation in an independent cohort of patients (CIBERES-CUN).
  • Figure 24 Association between PI score and survival in stage I-II ADC patients from MDA .
  • A-B Kaplan-Meier curves and log rank statistics for disease free survival (A) and overall survival (B) in stage I-II ADC patients from the training set (MDA cohort) divided in high and low prognostic index (PI) scores.
  • Figure 25 Association between PI score and survival in stage I-II SCC patients from MDA.
  • A-B Kaplan-Meier curves and log rank statistics for disease free survival (A) and overall survival (B) in stage I-II SCC patients from the training set (MDA cohort) divided in high and low prognostic index (PI) scores.
  • Figure 26 ADC model validation in an independent cohort of stage I-II patients (CIBERES-CUN). Kaplan-Meier curve and log rank statistics for overall survival (A) and disease free survival (B) in stage I-II ADC patients from the CIBERES-CUN (A) and CUN (B) cohort divided in high and low molecular prognostic index (PI) scores.
  • CIBERES-CUN stage I-II patients
  • PI molecular prognostic index
  • FIG. 27 SCC model validation in an independent cohort of stage I-II patients (CIBERES-CUN) Kaplan-Meier curve and log rank statistics for overall survival (A) and disease free survival (B) in stage I-II SCC patients from the CIBERES-CUN (A) and CUN (B) cohort divided in high and low molecular prognostic index (PI) scores.
  • Figure 28 Assessment of the clinical utility of the ADC prognostic signature.
  • A Prognostic value of the PI score and stage in univariate and bivariate models for ADC patients of MDA cohort.
  • B-C Kaplan-Meier curve and log rank statistics for disease-free survival (B) and overall survival (C) in ADC patients from the training set (MDAcohort) divided in high and low combined prognostic index (CPI) scores.
  • CPI combined prognostic index
  • Figure 29 Assessment of the clinical utility of the SCC prognostic signature.
  • A Prognostic value of the PI score and stage in univariate and bivariate models for SCC patients of MDA cohort.
  • B-C Kaplan-Meier curve and log rank statistics for disease-free survival (B) and overall survival (C) in SCC patients from the training set (MDA cohort) divided in high and low combined prognostic index (CPI) scores.
  • B Prognostic value of the PI score and stage in univariate and bivariate models for SCC patients of MDA cohort.
  • B-C Kaplan-Meier curve and log rank statistics for disease-free survival (B) and overall survival (C) in SCC patients from the training set (MDA cohort) divided in high and low combined prognostic index (CPI) scores.
  • C prognostic index
  • Figure 30 CPI validation for ADC in the CIBERES-CUN cohort.
  • A Kaplan-Meier curve and log rank statistics for overall survival in ADC patients from the validation set (CIBERES- CUN cohort) divided in high and low combined prognostic index (CPI) scores.
  • B Kaplan-Meier curve and log rank statistics for disease free survival in patients from the CUN cohort divided in high and low combined prognostic index (CPI) scores
  • FIG 31 CPI validation for SCC in the CIBERES-CUN cohort.
  • A Kaplan-Meier curve and log rank statistics for disease-free survival and overall survival in SCC patients from the validation set (CIBERES-CUN cohort) divided in high and low combined prognostic index (CPI) scores.
  • B Kaplan-Meier curve and log rank statistics for disease free survival in patients from the CUN cohort divided in high and low combined prognostic index (CPI) scores
  • Figure 32 The ADC model is predictive of differential benefit in survival for adjuvant post-operative therapy versus resection alone.
  • PI molecular prognostic index
  • the SCC model is predictive of differential benefit in survival for adjuvant postoperative therapy versus resection alone Kaplan-Meier curve and log rank statistics for overall survival (A) and disease-free survival (B) in SCC patients from the training set (MD Anderson cohort) after stratification of patients according to molecular prognostic index (PI) and status of adjuvant therapy receiver.
  • A overall survival
  • B disease-free survival
  • subject or “individual”' are used herein interchangeably to refer to all the animals classified as mammals and includes but is not limited to domestic and farm animals, primates and humans, for example, human beings, non- human primates, cows, horses, pigs, sheep, goats, dogs, cats, or rodents.
  • the subject is a male or female human being of any age or race.
  • subject suspected of having cancer refers to a subject that presents one or more signs or symptoms indicative of a cancer and is being screened for cancer.
  • a subject suspected of having cancer encompasses for instance an individual who has received a preliminary diagnosis (e.g., an X-ray computed tomography scan showing a mass) but for whom a confirmatory test (e.g., biopsy and/or histology) has not been done or for whom the stage of cancer is not known or not known in enough detail.
  • a confirmatory test e.g., biopsy and/or histology
  • prognosis refers to predicting disease progression or outcome.
  • prognostic markers may refer to patient or tumor characteristics that predict outcome (usually survival) independent of the treatment. Thus, they are usually identified and validated in patients who receive no therapy or surgical therapy only. The goal of identifying prognostic markers is to define patient subpopulations with significantly different anticipated outcomes, which might benefit from different therapies. Good prognostic patients may not require additional treatment beyond the primary surgical resection, while poor prognostic patients may derive improved survival benefit from adjuvant therapy or other closer clinical follow up or therapeutic strategy.
  • Predictive markers may refer to patient or tumor characteristics that predict benefit from specific treatments (either in terms of tumor shrinkage or survival). In other words, the differences in tumor response or survival benefit between treated versus untreated patients will be significantly different in those positive or negative for the predictive marker (Zhu CQ and Tsao MS, 2014).
  • therapeutically effective amount refers to an amount that is effective, upon single or multiple dose administration to a subject (such as a human patient) in the prophylactic or therapeutic treatment of a disease, disorder or pathological condition.
  • substantially identical sequence refers to a sequence which is at least about 90%, preferably at least about 95%, 96%, 97%, 98%, or 99% identical to a reference sequence. Identity percentage between the two sequences can be determined by any means known in the art, for example the Needleman and Wunsch global alignment algorithm.
  • antibody refers to an immunoglobulin or an antigen-binding fragment thereof. Unless otherwise specified, the term includes monoclonal and polyclonal antibodies, as well as recombinant antibodies. The term “antibody” also refers to fragments and derivatives of all of the foregoing, and may further comprise any variants thereof that retain the ability to specifically bind an epitope.
  • Antibodies may include, but are not limited to monoclonal antibodies (mAbs), camelid antibodies, single-chain antibodies (scFvs), Fab fragments, F(ab')2 fragments, disulphide- linked Fvs (sdFv) fragments, anti-idiotypic (anti-Id) antibodies, intra- bodies, synthetic antibodies, and epitope-binding fragments of any of the above.
  • mAbs monoclonal antibodies
  • scFvs single-chain antibodies
  • Fab fragments fragments
  • F(ab')2 fragments fragments
  • disulphide- linked Fvs sdFv fragments
  • anti-Id anti-idiotypic antibodies
  • intra- bodies intra- bodies
  • synthetic antibodies and epitope-binding fragments of any of the above.
  • antibody also refers to a fusion protein that includes a region equivalent to the Fc region of an immunoglobulin.
  • recombinant antibody refers to an antibody produced or expressed using a recombinant expression vector, where the expression vector comprises a nucleic acid encoding the recombinant antibody, such that introduction of the expression vector into an appropriate host cell results in the production or expression of the recombinant antibody.
  • Recombinant antibodies may be chimeric or humanized antibodies, mono- or multi-specific antibodies.
  • an antibody mimetic refers to single-domain scaffolds, which have been engineered to bind therapeutic targets with affinity and specificity that match that of natural antibodies.
  • Antibody mimetics have been developed utilizing an immunoglobulin-like fold, for example, fibronectin type III, NCAM and CTLA-4.
  • Other mimetics scaffolds bearing no similarity to immunoglobulin folds have also been obtained.
  • Non-limiting examples of said scaffolds are DARPins, anticalins, affibodies, adnectins, fynomers, etc. (see for instance, Weidle et al. Cancer Genomics & Proteomics. 2013, 10: 1-18; Lofblom, J. et al, Curr. Opin. Biotechnol. 2011, 22: 843-848; Banta, S. et al, Annu. Rev. Biomed. Eng., 2010, 15: 93-113).
  • marker refers to markers of disease, prognostic or predictive markers which are typically substances found in a bodily sample that can be easily measured.
  • Said bodily sample can be for instance a blood, plasma or feces sample.
  • biomarker encompasses biophysical and biochemical determinations, including genetic and serological markers.
  • combination therapy is meant to comprise the administration of the referred therapeutic agents to a subject suffering from cancer, in the same or separate pharmaceutical formulations, and at the same time or at different times. If the therapeutic agents are administered at different times they should be administered sufficiently close in time to provide for the combined effect (e.g. potentiating or synergistic response) to occur.
  • the particular combination of therapies to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and/or the desired therapeutic effect to be achieved. It will be appreciated that the therapies employed may achieve a desired effect for the same disorder (for example, anticancer effects), and/or they may achieve different effects (e.g., control of any adverse effects).
  • the present invention provides an in vitro method for determining the prognosis of a subject having lung squamous cell carcinoma (SCC), wherein said method comprises: a) determining in a biological sample isolated from said subject the protein expression levels of cytoplasmic STC1 (STC1-C) and cytoplasmic RAE1 (RAE1-C), optionally, further determining in said biological sample the protein expression levels of at least one, two, three, four, five, six, seven, eight, or nine additional markers selected from the group consisting of:
  • RRM2 cytoplasmic RRM2 (RRM2-C)
  • cytoplasmic RAD51 (RAD51 -C), nuclear SRSF 1 (SRSF 1 -N),
  • SIRT2-N nuclear SIRT2
  • cytoplasmic QKI QKI-C
  • lung cancer refers to an in vitro method for obtaining useful data for the prognosis of a subject having lung SCC, said method comprising the steps defined above.
  • lung cancer is used for cancer that starts in the lung.
  • Non-small cell lung carcinoma (NSCLC) accounts for 80-85% of all cases and includes the two most frequent lung cancer types: adenocarcinomas (ADC) and squamous cell carcinomas (SCC).
  • SCLC Small Cell lung cancer comprises 10-15% of lung cancer cases.
  • Signs or symptoms which may be indicative of lung cancer include for instance one or more of the following: weight loss, loss of appetite, malaise, fever, cough, dyspnea, wheezing, stridor, hoarseness, shortness of breath, weakness, haemoptisis, chest and or back pain, obstructive pneumonia and pleural effusion (see for instance, WHO classification of Tumors of the Lung, Pleura, Thymus and Heart. Edited by WD Travis, E Brambilla, A.P. Burke, A. Marx and A.G. Nicholson (2015).
  • SCC is a malignant epithelial tumor that either shows keratinization and/or intercellular bridges, or is a morphologically undifferenciated non small cell carcinoma that expresses immunohistochemical markers of squamous cell differentiation, (see for instance, WHO classification of Tumors of the Lung, Pleura, Thymus and Heart. Edited by WD Travis, E Brambilla, A.P. Burke, A. Marx and A.G. Nicholson (2015).
  • a subject having lung SCC may refer to a subject which has been suspected to have or has been diagnosed with lung cancer and wherein further to histological analysis (e.g. from a pre-surgery biopsy or a biopsy from the resected tumor) it has been determined that said cancer is SCC.
  • said subject has been submitted to tumor resection surgery and has not received any neoadjuvant treatment.
  • the combined score determined by the methods of the present invention may be predictive and/or prognostic.
  • Disease progression or outcome may be measured using different parameters, including but not limited to, tumor growth, tumor growth delay, increase/decrease of tumor size, increase/decrease in tumor markers, and patient's survival.
  • the clinical outcome of a subject is expressed as overall survival and/or disease-free survival.
  • Survival of cancer patients is generally suitably expressed by Kaplan-Meier curves, named after Edward L. Kaplan and Paul Meier who first described it (Kaplan, Meier: Amer. Statist. Assn. 53:457-481).
  • the Kaplan-Meier estimator is also known as the product limit estimator. It serves for estimating the survival function from lifetime data.
  • a plot of the Kaplan-Meier estimate of the survival function is a series of horizontal steps of declining magnitude which, when a large enough sample is taken, approaches the true survival function for that population. The value of the survival function between successive distinct sampled observations is assumed to be constant.
  • the Kaplan-Meier estimator may be used to measure the fraction of patients living for a certain amount of time after beginning a therapy (e.g. after tumor resection).
  • the clinical outcome predicted may be the (overall/disease-free) survival in months/years from the time point of taking the sample. It may be survival for a certain period from taking the sample, such as of six months or more, one year or more, two years or more, three years or more, four years or more, five years or more, six years or more.
  • “survival” may refer to "overall survival” or "disease free survival”.
  • disease free survival is defined as the interval of time from start of treatment (e.g., date of surgery) to the first measurement of cancer growth.
  • all survival or “OS” as used herein, is defined as the interval of time from the start of treatment (e.g., date of surgery) to death from any cause.
  • poor prognosis refers to a high risk of recurrence and/or death. In preferred embodiments, the term “poor prognosis” means a survival (i.e.
  • DFS and/or OS of six months or less, one year or less, two years or less, three years or less, four years or less, five years or less, six years or less, etc.
  • the term poor prognosis refers to a DFS and/or OS of less than 5 years.
  • Step (a) of the method under the first aspect of the invention comprises determining in said biological sample the expression levels of the protein markers defined above at the target subcellular location [i.e., nuclear (N), cytoplasmic (C) and/or membrane (MB)].
  • N nuclear
  • C cytoplasmic
  • MB membrane
  • Stanniocalcin-1 is a protein which may be expressed in the nucleus or cytoplasm and has been reported stimulate renal phosphate reabsorption, and could therefore prevent hypercalcemia.
  • STC1 Stanniocalcin-1
  • SEQ ID NO: 11 UniProtKB Accession Number P52823-1 of the entry version 135 of 7 Jun 2017, sequence version 1 of 1 Oct 1996):
  • STC1 refers to human STC1 protein with SEQ ID NO: 11 and to sequences substantially identical thereto. Preferably, said sequence is SEQ ID NO: 11.
  • RNA export 1 homo log (RAEl) is a nuclear export protein involved in m NA transport from the nucleus to the cytoplasm. RAEl also plays a critical role in the maintenance of spindle bipolarity during cell division. RAEl mRNA and protein levels decrease upon inhibition of neuroblastoma cell proliferation, and its overexpression prevents retinoic acid-induced cell cycle arrest and differentiation; however, a previous study demonstrated that RAE1/NUP98 mutant mice are more susceptible to DMBA-induced lung tumors compared to wild-type mice, indicating that combined RAE1/NUP98 haplo-insufficiency potentially promotes tumorigenesis.
  • SEQ ID NO:6 UniProtKB Accession Number P78406-1 of the entry version 168 of 30 Aug 2017, sequence version 1 of 1 May 1997):
  • RAEl refers to human RAEl protein with SEQ ID NO:6 and to sequences substantially identical thereto. Preferably, said sequence is SEQ ID NO:6.
  • Ribonucleoside-diphosphate reductase subunit M2 (RRM2) is an enzyme that catalyzes the formation of deoxyribonucleotides from ribonucleotides, provides the precursors necessary for DNA synthesis and catalyzes the biosynthesis of deoxyribonucleotides from the corresponding ribonucleotides.
  • This protein is involved in the pathway DNA replication, which is part of genetic information processing.
  • the canonical sequence of human RRM2 is referred as SEQ ID NO:7 (UniProtKB Accession Number P31350-1 of the entry version 183 of 05 Jul 2017, sequence version 1 of 1 Jul 1993):
  • RRM2 refers to human RRM2 protein with SEQ ID NO:7 and to sequences substantially identical thereto. Preferably, said sequence is SEQ ID NO:7.
  • DNA ligase 1 is an enzyme that seals nicks in double-stranded DNA during DNA replication, DNA recombination and DNA repair.
  • DNA ligase 1 utilizes adenosine triphosphate (ATP) to catalyze the energetically favorable ligation events in both DNA replication and repair.
  • ATP adenosine triphosphate
  • S-phase synthesis phase of the eukaryotic cell cycle
  • DNA replication occurs.
  • DNA ligase 1 is responsible for joining Okazaki fragments formed during discontinuous DNA synthesis on the DNA's lagging strand after DNA polymerase ⁇ has replaced the RNA primer nucleotides with DNA nucleotides.
  • SEQ ID NO: 3 UniProtKB Accession Number P18858-1 of the entry version 190 of 30 Aug 2017, sequence version 1 of 1 Nov 1990:
  • LIG1 refers to human LIG1 protein with SEQ ID NO:3 and to sequences substantially identical thereto. Preferably, said sequence is SEQ ID NO:3.
  • the protein quaking also referred as Hqk, is an RNA-binding protein which may be expressed in the nucleus or cytoplasm and has been reported to play a central role in myelinization (Aberg K. Proc Natl Acad Sci U S A. 2006, 103(19):7482-7).
  • SEQ ID NO:4 UniProtKB Accession Number Q96PU8-1 of the entry entry version 140 of 30 Aug 2017, sequence version 1 of 1 Dec 2001
  • QKI refers to human QKI protein with SEQ ID NO:4 and to sequences substantially identical thereto. Preferably, said sequence is SEQ ID NO:4.
  • DNA repair protein RAD51 homo log 1 is a protein that plays an important role in DNA repair.
  • Breaks in DNA can be caused by natural and medical radiation or other environmental exposures, and also occur when chromosomes exchange genetic material in preparation for cell division.
  • the RAD51 protein binds to the DNA at the site of a break and encases it in a protein sheath, which is an essential first step in the repair process.
  • RAD51 has a critical role in the maintenance of genomic integrity by functioning in the repair of DNA double-strand breaks.
  • RAD51 mediates homologous pairing and strand exchange in recombinatory structures known as RAD51 foci in the nucleus.
  • the canonical sequence of human RAD51 is referred as SEQ ID NO: 1
  • RAD51 refers to human RAD51 protein with SEQ ID NO:5 and to sequences substantially identical thereto. Preferably, said sequence is SEQ ID NO:5.
  • Serine/arginine-rich splicing factor 1 is a protein involved in pre-mRNA splicing. SRSF1 is necessary for all splicing reactions to occur, and influences splice site selection in a concentration-dependent manner, resulting in alternative splicing. In addition to being involved in the splicing process, SRSF1 also mediates post-splicing activities, such as mRNA nuclear export and translation. .
  • SEQ ID NO: 10 UniProtKB Accession Number Q07955-1 of the entry version 211 of 30 Aug 2017, sequence version 2 of 23 Jan 2007
  • SRSF1 refers to human SRSF1 protein with SEQ ID NO: 10 and to sequences substantially identical thereto. Preferably, said sequence is SEQ ID NO: 10.
  • SIRT2 NAD-dependent protein deacetylase sirtuin-2
  • SIRT2 Regulatory protein SIR2 homolog 2 or SIR2-like protein 2
  • SIR2 Regulatory protein SIR2 homolog 2
  • SIR2-like protein 2 is an NAD-dependent protein deacetylase, which deacetylates internal lysines on histone and alpha-tubulin as well as many other proteins such as key transcription factors. It has been reported to participate in the modulation of multiple and diverse biological processes such as cell cycle control, genomic integrity, microtubule dynamics, cell differentiation, metabolic networks, and autophagy. It plays a major role in the control of cell cycle progression and genomic stability. This protein may be expressed in nucleus or the cytoplasm.
  • SEQ ID NO: 8 UniProtKB Accession Number Q8IXJ6-1 of the entry version 167 of 30 Aug 2017, sequence version 2 of 31 Oct 2003
  • SIRT2 refers to human SIRT2 protein with SEQ ID NO: 8 and to sequences substantially identical thereto. Preferably, said sequence is SEQ ID NO: 8.
  • the Solute carrier family 2 facilitated glucose transporter member 1 (SLC2A1), also referred as GLUT-1 or HepG2 glucose transporter, is a facilitative glucose transporter which may be located in the cell membrane or in the cytoplasm. This isoform may be responsible for constitutive or basal glucose uptake. It has been described to have very broad substrate specificity; and to be able to transport a wide range of aldoses including both pentoses and hexoses.
  • SEQ ID NO:9 UniProtKB Accession Number PI 1166-1 of the entry version 210 of 30 Aug 2017, sequence version 2 of 3 Oct 2006
  • SLC2A1 refers to human SLC2A1 protein with SEQ ID NO:9 and to sequences substantially identical thereto. Preferably, said sequence is SEQ ID NO:9.
  • the prognosis method of the invention comprises determining in step a) the protein expression levels of cytoplasmic STC1 (STC1-C) and cytoplasmic RAE1 (RAEl- C), and further comprises:
  • a. l determining in said biological sample the protein expression levels of at least one, two, three, four, five, six, seven, or eight additional markers selected from the group consisting of:
  • RRM2-C -cytoplasmic RRM2
  • LIG1-N -nuclear LIG1
  • RAD51-C -cytoplasmic RAD51
  • SRSF1-N -nuclear SRSF1
  • SIRT2-N -nuclear SIRT2
  • SLC2A1-C -cytoplasmic SLC2A1
  • the prognosis method of the invention comprises determining in step a) the protein expression levels of:
  • the prognosis method of the invention comprises determining in step a) the protein expression levels of:
  • the prognosis method of the invention comprises determining in step a) the protein expression levels of STC1-C, RAEl-C, RRM2-C, STC1-N, SRSF1-N, and SLC2A1-C.
  • the prognosis method of the invention comprises determining in step a) the protein expression levels of cytoplasmic STC1 (STC1-C), nuclear SRSF1 (SRSF1- N), and cytoplasmic RAEl (RAE1-C), optionally, further determining in said biological sample the protein expression levels of at least one, two, three, four, five, six, or seven additional markers selected from the group consisting of:
  • SIRT2-N nuclear SIRT2
  • Preferred marker combinations of this embodiment are as described for other embodiments herein above.
  • determining the levels of the marker or “determining the protein expression levels”, as used herein, refers to ascertaining the absolute or relative amount or concentration of the biomarker in the sample. Techniques to assay levels of individual biomarkers from test samples are well known to the skilled technician, and the invention is not limited by the means by which the components are assessed. Expression levels may be absolute or relative. When the expression levels are normalized, normalization can be performed with respect to different measures in the sample. These procedures are well known to one skilled in the art. Typically, expression levels are normalized with respect to an "endogenous control". An "endogenous control” as used herein relates to a gene expression product whose expression levels do not change or change only in limited amounts in tumor cells with respect to non-tumorigenic cells.
  • Endogenous control is usually the expression product from a housekeeping gene and which codes for a protein which is constitutive ly expressed and carries out essential cellular functions.
  • Housekeeping genes that can be used as endogenous control include for example ⁇ -2-microglobulin, ubiquitin, 18-S ribosomal protein, cyclophilin, GAPDH, actin and HPRT.
  • the expression levels of the above-mentioned markers may be determined by any method enabling to determine the specific protein expression levels at the target subcellular localization (i.e., nucleus (N), cytoplasm (C) or membrane (MB).
  • N nucleus
  • C cytoplasm
  • MB membrane
  • protein extracts of the specific subcellular location of the target marker may be obtained.
  • Cell lysis, fractionation and protein extraction methods are well known in the art and may be found in protein preparation handbooks (see for instance, Walker JM (2009) The Protein Protocols Handbook. Third Edition. New York (NY): Springer- Verlag New York, LLC). Subsequently, protein determination from the protein extracts can be performed by a suitable method.
  • protein expression levels are determined by a method comprising: a) incubating nuclear protein extracts with an affinity reagent for STC1,
  • step a) incubating membrane protein extracts with an affinity reagent for RAE1 ; and c) optionally, incubating target protein extracts with an affinity reagent for each of the at least one, two, three, or the four additional proteins defined in step a).
  • Suitable methods for determining the levels of a given protein include, without limitation, those described herein below.
  • Preferred methods for determining the protein expression levels in the methods of the present invention are immunoassays.
  • Various types of immunoassays are known to one skilled in the art for the quantitation of proteins of interest. These methods are based on the use of affinity reagents, which may be any antibody or ligand specifically binding to the target protein or to a fragment thereof, wherein said affinity reagent is preferably labeled.
  • affinity reagent may be enzymatically labelled, or labeled with a radioactive isotope or with a fluorescent agent.
  • Affinity reagents may be any antibody or ligand specifically binding to the target protein or to a fragment thereof.
  • Affinity ligands may include proteins, peptides, peptide aptamers, affimers and other target specific protein scaffolds, like antibody-mimetics.
  • Preferred affinity reagents for use in the determination of the protein biomarkers of the invention are antibodies, for example the antibodies defined in Table 3.
  • Specific antibodies against the protein markers used in the methods of the invention may be produced for example by immunizing a host with a protein of the present invention or a fragment thereof.
  • peptides specific against the protein markers used in the methods of the invention may be produced by screening synthetic peptide libraries.
  • Western blot or immunoblotting techniques allow comparison of relative abundance of proteins separated by an electrophoretic gel (e.g., native proteins by 3-D structure or denatured proteins by the length of the polypept ide).
  • Immunoblotting techniques use antibodies (or other specific ligands in related techniques) to identify target proteins among a number of unrelated protein species. They involve identification of protein target via antigen-antibody (or protein- ligand) specific reactions. Proteins are typically separated by electrophoresis and transferred onto a sheet of polymeric material (generally nitrocellulose, nylon, or polyvinylidene difluoride). Dot and slot blots are simplified procedures in which protein samples are not separated by electrophoresis but immobilized directly onto a membrane.
  • Said immunoassay may be for example an enzyme-linked immunosorbent assay (ELISA), a fluorescent immunosorbent assay (FIA), a chemiluminescence immunoassay (CIA), or a radioimmunoassay (RIA), an enzyme multiplied immunoassay, a solid phase radioimmunoassay (SPROA), a fluorescence polarization (FP) assay, a fluorescence resonance energy transfer (FRET) assay, a time-resolved fluorescence resonance energy transfer (TR- FRET) assay, a surface plasmon resonance (SPR) assay.
  • ELISA enzyme-linked immunosorbent assay
  • FFA fluorescent immunosorbent assay
  • CIA chemiluminescence immunoassay
  • RIA radioimmunoassay
  • an enzyme multiplied immunoassay a solid phase radioimmunoassay (SPROA)
  • FP fluorescence polarization
  • FRET fluorescence resonance energy transfer
  • any next generation versions of any of the above such as bead-based flow-cytometry immunoassays (e.g., based on the Luminex xMAP technology) are specifically encompassed.
  • said immunoassay is an ELISA assay or any multiplex version thereof.
  • MS mass spectrometry
  • LC / MS liquid chromatography coupled to mass spectrometry
  • immunohistochemical and in-situ hybridization analysis are usually preferred.
  • protein quantification and subcellular location is performed by immunohistochemistry.
  • protein expression levels are determined by a method comprising incubating the biological sample isolated from said subject with:
  • step a) optionally, an affinity reagent for each of the at least one, two, three, four, five, six or the seven additional proteins defined in step a).
  • Immunohistochemistry (IHC) analysis is typically conducted using thin sections of the biological sample immobilized on coated slides. These sections, when derived from paraffin-embedded tissue samples, are deparaffinised and preferably treated so as to retrieve the antigen. The detection can be carried out in individual samples or in tissue microarrays. This procedure, although is subjectively determined by the pathologist, is the standard method of measurement of IHC results, and well known in the art.
  • the use of this technique entails the determination of the Histological score value.
  • H-Score Histological score
  • H-Score ⁇ (intensity grade x % stained cells)
  • the staining intensity of tumor cells may be scored in different intensity grades, for example the following 4 grades:
  • the percentage of positive staining cells for each intensity grade may be scored from 0 to 100.
  • the final score, called "H-Score”, is preferably calculated by adding the products of the percentage cells stained with a given intensity grade (0-100) by the corresponding staining intensity grade value (0-3).
  • the following formula may be applied:
  • the assessment of the staining intensity and the percentage of positive staining tumor cells can be determined by any means known to the skilled person including but not limited to a panel of at least two independent pathologists with no knowledge about clinical data scoring all immunohistochemical stainings. In case, the panel of pathologist were to disagree in the scores it is convenient to expand the panel of independent pathologists to at least 3, 4, or 5.
  • the value of the H-Score may be obtained by applying the above formula.
  • the resulting value of the H-Score determines the level of expression of the protein marker.
  • the prognostic method of the invention can be applied to any type of biological sample from a patient, such as a biopsy sample, tissue, cell or fluid (serum, saliva, semen, sputum, cerebral spinal fluid (CSF), tears, mucus, sweat, milk, brain extracts and the like).
  • a biological sample from the cancer patient is preferably a sample containing tumor cells. Tumors or portions thereof may be surgically resected from the patient or obtained by routine biopsy.
  • a tumor sample is obtained from the primary tumor.
  • said biological sample isolated from the subject is a tumor biopsy sample, preferably obtained from a resected tumor.
  • a tumor biopsy sample preferably obtained from a resected tumor.
  • Step (b) of the prognosis method under the first aspect of the invention comprises calculating a combined score.
  • the combined score is a value obtained according to a given mathematical algorithm wherein the expression values of each of the protein markers used in the methods of the invention are variables of said mathematical algorithm.
  • this is proportional to the expression levels of one or more of STC1-C, RRM2-C, QKI-N, RAD51-C, SRSF1-N, SIRT2-N, and SLC2A1-C, and QKI-C; and is inversely proportional to the expression levels of the RAE1- C, STC1-N, LIG1-N, wherein the higher the score, the worst the prognosis and/or the higher the risk of relapse.
  • said combined score is calculated as the sum of the products of the standardized beta coefficients obtained in a regression analysis for each marker and the protein expression values (e.g. expressed as H-Score value).
  • the combined score obtained in this way is also named Prognostic Index (PI).
  • said beta coefficients are positive for markers selected from the group consisting of STC1-C, RRM2-C, QKI-N, RAD51-C, SRSF1-N, SIRT2-N, SLC2A1-Cand QKI-C and negative for markers selected from the group consisting of RAE1-C, STC1-N, LIG1- N.
  • a positive sign is indicative that an increase of the individual marker is associated to poor prognosis, whereas a negative sign means that an increase of the individual marker is associated to good prognosis.
  • the combined score is a Prognostic Index obtained by using a formula selected from the group consisting of:
  • step (c) of the method under the first aspect of the invention comprises classifying the subject as having good prognosis or poor prognosis based on the combined score.
  • step c) said method comprises comparing the combined score in the subject sample with a reference combined score; and an increase of the combined score in the subject sample with regard to said reference combined score is indicative of poor prognosis.
  • reference combined score is a reference value obtained according to a given mathematical algorithm wherein reference expression values of each of the protein markers used in the prognosis method of the invention are variables of said mathematical algorithm.
  • reference value relates to a predetermined criteria used as a reference for evaluating the values or data obtained from the samples collected from a subject. This "reference value” may also be referred as “cut-off value” or “threshold value”.
  • the reference value or reference level can be an absolute value, a relative value, a value that has an upper or a lower limit, a range of values, an average value, a median value, a mean value, a tertile value, or a value as compared to a particular control or baseline value.
  • said reference value is the mean value or the tertile value.
  • a reference value can be based on an individual sample value but is generally based on a large number of samples, including or excluding the sample to be tested.
  • this reference value may be derived from a collection of tumor tissue samples from a reference lung squamous cell carcinoma (SCC) patients' population for whom historical information relating to the actual clinical outcome for the corresponding cancer patient is available.
  • Said reference lung SCC patient's population may for instance be from subjects suffering from lung SCC, from patients' suffering from resectable lung SCC (e.g., from stages I to III; or I to Ilia), or from subjects suffering from early stage lung SCC (e.g., from stage I or II).
  • said combined reference value is determined by a method comprising: a) determining, for each lung SCC patient in a reference population, the protein expression levels of the protein markers as defined in step a) and calculating the combined score as defined in step b) of the method of the invention; b) selecting as provisional reference value an arbitrary combined score from the ones obtained in step a); c) classifying the patients in the reference SCC population in two groups according to the provisional reference value selected combined score obtained in b), wherein:
  • the first group comprises lung SCC patients that exhibit a combined score that is lower than the provisional reference value
  • the second group comprises lung SCC patients that exhibit a combined score that is higher than the provisional reference value
  • the combined score obtained in step b) is considered "decreased" when said combined score is lower than a reference combined score.
  • the combined score is considered to be lower than a reference combined score when it is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, or more lower than the reference combined score.
  • the combined score obtained in step b) is considered "increased" when said combined score is higher than a reference value.
  • the combined score is considered to be higher than a reference combined score when it is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, or more higher than a reference combined score.
  • Prognosis or outcome prediction in the method of the invention does not claim to be correct in 100% of the analyzed samples. However, it requires that a statistically significant amount of the analyzed samples are classified correctly.
  • the amount that is statistically significant can be established by a person skilled in the art by means of using different statistical tools; illustrative, non-limiting examples of said statistical tools include determining confidence intervals, determining the p-value, the Chi-Square test discriminating functions, etc.
  • Preferred confidence intervals are at least 90%, at least 95%, at least 97%), at least 98%>, at least 99%>.
  • the p-values are, preferably less than 0.1, less than 0.05, less than 0.01, less than 0.005 or less than 0.0001.
  • the teachings of the present invention preferably allow correctly classifying at least 60%>, at least 70%>, at least 80%>, or at least 90%> of the subjects of a determining group or population analyzed.
  • tumors can be further stratified according to its stage of development.
  • the tumor-node-metastasis (TNM) staging system is the standard method for clinically predicting survival of patients with lung cancer. This system is based on tumor size, tumor location and involved structures, and presence of nodal and distant metastasis to categorize lung cancer patients in different clinical stages. With the continuous flow of new data and the increasing knowledge of the disease, the staging system requires continuous adjustment.
  • the seventh edition of the TNM classification of lung tumors is been used worldwide, although the eight edition has been published; the implementation of the new system has been delayed until January 1, 2018 (Goldstraw P et al, 2007; Goldstraw P et al, 2016). Further details on the TNM method, as well as descriptors and stage groupings according to the 7 th edition are provided in the Examples.
  • the inventors have also found that prognostic information in lung SCC patients is significantly improved when the Prognostic Index (PI) described above is further combined with a correction coefficient specific for the TNM stage of the particular SCC patient.
  • PI Prognostic Index
  • the likelihood ratio significantly increased after adding the molecular information of the Prognostic Index (PI) (P ⁇ 0.001 both for DFS and OS, Figure 17A). This improvement showed that the molecular model complements the TNM stage, adding very valuable prognostic information for the patients.
  • the prognosis method of the invention comprises further to step a):
  • step b) calculating a combined score from the protein expression levels of the markers determined in the biological sample as defined in step a) and the stage of TNM classification as defined in step Al);
  • the prognosis method of this particular embodiment is characterized by having corrected the combined score calculated on the basis of the levels of expression of the protein markers by the tumor TNM stage, i.e. the calculation of the combined score in step b) comprises correction of the protein marker based combined score with a TNM stage specific correction coefficient.
  • This improved combined score is herein referred to as Combined Prognostic Index (CPI).
  • said subject has resectable lung SCC (e.g., from stage I to IIIA).
  • the accuracy of the method of the invention can be further increased by determining the presence and/or quantification of other prognostic/predictive molecular markers (Coate LE et al, 2009; Ferte C et al 2010; Lin J and Beer DG, 2012; Zhu CQ and Tsao MS, 2014), and/or clinical signs or symptoms with reported prognostic/predictive value, such as morphological features of the tumor, histological subtypes, radiological traits of the imaging tests (e.g. size, shape, volume, radiological texture, morphological details or other features in a CT scan, X-Ray or SUV or alternative ways to analyze nuclear tracer levels in a PET imaging, etc); clinical characteristics of the patients (e.g. age, sex, race, respiratory function levels, performance status).
  • the potential additional molecular markers to be associated to the present invention can be found in the tumor specimen itself or other cells, body fluids or exhaled breath obtained from the same patient.
  • a further aspect of the invention refers to a computer implemented method, wherein the method is any of the methods disclosed herein or any combination thereof.
  • the invention relates to an in vitro method for determining the prognosis of a subject having lung squamous cell carcinoma (SCC), wherein said method comprises:
  • step b) calculating, using a computer, a combined score from the protein expression levels of the markers determined in the biological sample as defined in step a);
  • H-Score ⁇ intensity grade x % stained cells
  • This computer program is typically directly loadable into the internal memory of a digital computer, comprising software code portions for performing the steps of comparing the combined score (e.g., obtained from the level of one or more of the target markers as described in the invention), from the one or more biological samples of a subject with a reference value (e.g., reference combined value) and determining the prognosis of said subject or whether it would benefit from adjuvant therapy, when said product is run on a computer.
  • a combined score e.g., obtained from the level of one or more of the target markers as described in the invention
  • any device or apparatus comprising means for carrying out the steps of any of the methods of the present invention or any combination thereof, or carrying a computer program capable of, or for implementing any of the methods of the present invention or any combination thereof, is included as forming part of the present specification.
  • the methods of the invention may also comprise the storing of the method results in a data carrier, preferably wherein said data carrier is a computer readable medium.
  • the present invention further relates to a computer-readable storage medium having stored thereon a computer program of the invention or the results of any of the methods of the invention.
  • a computer readable medium can be any apparatus that may include, store, communicate, propagate, or transport the results of the determination of the method of the invention.
  • the medium can be an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system (or apparatus or device) or a propagation medium.
  • the present invention provides an in vitro method for selecting those subjects having lung SCC who are expected to benefit from an adjuvant treatment, wherein said method comprises:
  • adjuvant treatment refers to a treatment administered after surgical resection of a tumor, typically the primary tumor.
  • Said adjuvant treatment may comprise the administration of any cytotoxic or antiproliferative drug and includes chemotherapy and/or targeted therapies regimens.
  • said adjuvant treatment may be selected from (but it is not limited to) any one or more of the group consisting of platinum anticancer agents (e.g., cisplatin, oxaliplatin, carboplatin, BBR3464, satraplatin, tetraplatin,ormiplatin, and iproplatin); antimetabolites (e.g., 5-fluorouracil, gemcitabine, cytarabine, capecitabine, decitabine, floxuridine, 6-mercaptopurine, methotrexate, fludarabine, aminopterin, pemetrexed, raltitrexed, cladribine, clofarabine, fludarabine, mercaptopurine, pentostatin, and thioguanine); mitotic inhibitors (e.g., paclitaxel, docetaxel, vinblastine, vincristine, vindesine, and vinorelbine); anthracycline antibiotics (e.
  • metabolic modulators e.g., mTOR inhibitors
  • epigenetic inhibitors e.g., DNMT inhibitors
  • immunotherapy agents e.g. Pembrolizumab, Nivolumab, Atezolizumab, Avelumab.
  • said adjuvant therapy comprises the administration of a platinum anticancer agent as single agent or in a combination therapy.
  • the platinum anticancer agent is cisplatin and/or carboplatin.
  • the present invention refers to an in vitro method for predicting the efficacy of an adjuvant treatment (e.g., adjuvant chemotherapy) in a subject having lung SCC, wherein said method comprises:
  • adjuvant treatment e.g. adjuvant chemotherapy
  • prognosis classification predicting the efficacy of said adjuvant treatment (e.g. adjuvant chemotherapy) according to the prognosis classification; preferably wherein classification of the subject as having poor prognosis is indicative of increased efficacy of the adjuvant treatment (e.g. adjuvant chemotherapy).
  • said adjuvant treatment comprises the administration of a platinum anticancer agent, and a poor prognosis is indicative of increased efficacy of the platinum anticancer agent.
  • the present invention refers to an in vitro method for selecting an adjuvant treatment for a subject having lung SCC, wherein said method comprises:
  • an adjuvant chemotherapy preferably, a platinum anticancer agent
  • the present invention provides a method for treating a subject having lung SCC comprising administering to said patient a therapeutically effective amount of a treatment, typically an adjuvant treatment, wherein said treatment is selected according to the classification of said patient according to the prognosis method of the invention.
  • a treatment typically an adjuvant treatment
  • the administered treatment comprises a platinum anticancer agent.
  • said lung SCC is resectable lung SCC which generally refers to stages I to III according to the TNM classification of lung tumors, preferably to stages I to Ilia.
  • said lung SCC is early stage lung SCC, which generally refers to stage I or II according to the TNM classification of lung tumors.
  • said lung SCC is stage I SCC according to the TNM classification of lung tumors.
  • TNM stages are defined according to 7th edition of the TNM classification of lung tumors (see Table 12 in the Examples). More specifically, these are defined as follows:
  • - stage IA is characterized as Tla-b, NO and MO;
  • stage IB is characterized as T2a, NO and MO;
  • - stage IIA is characterized as Tla-b, Nl and MO; or T2a, Nl and MO; or T2b, NO and MO; and
  • stage IIB is characterized as T2b, Nl and MO; or T3, NO and MO;
  • stage IIIA is characterized as T1/T2, N2 and MO; T3, Nl/2 and MO, or T4, N0/N1, MO; and
  • T4 N2 and MO
  • T3 Any T, N3 and MO.
  • T any N, and Mia
  • T any N, and Mlb.
  • Another aspect of the invention relates to prognostic and/or predictive biomarkers of lung squamous cell carcinoma (SCC) or combinations of a plurality thereof (signatures) as defined in any of the embodiments described herein above.
  • SCC lung squamous cell carcinoma
  • a preferred embodiment concerns a plurality of biomarkers for predicting disease progression or survival of a subject with lung squamous cell carcinoma (SCC), the plurality of biomarkers comprising STC1-C,_RAE1-C, RRM2-C, STC1-N, SRSF1-N, and SLC2A1-C.
  • SCC lung squamous cell carcinoma
  • Another preferred embodiment relates to a plurality of biomarkers for predicting disease progression or survival of a subject with lung squamous cell carcinoma (SCC), the plurality of biomarkers comprising STC1-C, RAE1-C, RRM2-C, STC1-N, LIG1-N, and QKI-N.
  • it refers to a plurality of biomarkers for predicting disease progression or survival of a subject with lung squamous cell carcinoma (SCC), the plurality of biomarkers comprising STC1-C, RAE1-C, RRM2-C, STC1-N, LIG1-N, QKI-N, RAD51-C, and SRSF1-N.
  • it pertains to a plurality of biomarkers for predicting disease progression or survival of a subject with lung squamous cell carcinoma (SCC), the plurality of biomarkers comprising STC1-C, RAE1-C, RRM2-C, LIG1-N, and QKI-N.
  • SCC lung squamous cell carcinoma
  • it relates to a plurality of biomarkers for predicting disease progression or survival of a subject with lung squamous cell carcinoma (SCC), the plurality of biomarkers comprising STC1-C, RAE1-C, STC1-N, QKI-N, RAD51-C, and SIRT2- N.
  • the present invention provides a method for determining target protein expression levels by immunohistochemistry, said method being suitable for carrying out the determination of the protein expression levels of the markers in the biological sample as defined in step a) of the prognosis method of the invention.
  • said method for determining target protein expression levels comprises incubating the biological sample isolated from said subject with an affinity reagent for STC1, an affinity reagent for RAEl, and optionally, an affinity reagent for each of the at least one, two, three, four, five, six or the seven additional proteins of the additional protein markers defined in step a) of the method of prognosis of the invention.
  • said sample is a fixed biopsy sample, typically a formalin fixed-paraffin embedded sample.
  • one or more lung cancer cells are used as low and/or high expression controls as has been described herein.
  • said cells or tissues are fixed, typically formalin- fixed and paraffin embedded.
  • low expression controls may be used lung cancer cells or tissues known to have low protein marker expression
  • cells or tissues known to have high protein marker expression can be used as high expression controls.
  • said lung cancer cell lines may be selected from (but are not limited to) any one or more of the group consisting of NCI-H1395, NCI-H23, NCI-H441, A549, NCI-H358, NCI- H1299, NCI-H460, Calu-1, NCI-H1869, NCI-H520 y HCC15.
  • the recited cell lines may be used as high or low expression controls:
  • - STC1 HCC15 and/or NCI-358 as high expression controls and NCI-H520 and/or NCI- H1299 as low expression controls
  • RAE1 NCI-H520 and/or NCI-H520 as high expression controls and HCC15 and/or NCI- HI 299 as low expression controls
  • NCI-H520 and/or HCC15 as high expression controls and NCI-H441 and/or NCI-H358 as low expression controls
  • NCI-441 and/or NCI-H460 as high expression controls and HCC15 and/or Calu-1 as low expression controls
  • NCI-H520 and/or NCI-H1299 as high expression controls NCI-H23 and/or A549 as low expression controls
  • NCI-H1395 and/or NCI-H1299 as high expression controls NCI-H23 and/or NCI-H441 as low expression controls
  • NCI-H520 and/or NCI-H1395 as high expression controls NCI-H1299 and/or Calu-1 as low expression controls
  • NCI-H1395 and/or NCI-H1299 as high expression controls NCI-H460 and/or A549 as low expression controls
  • sample and/or control cells Prior to incubation with the affinity reagent as defined herein above said sample and/or control cells may be submitted to a deparaffinization and to an antigen retrieval process.
  • the antigen retrieval process is conducted at a temperature of 90°C to 100°C during 10 to 30 minutes, preferably at 95°C during 20 minutes.
  • the antigen retrieval process comprises the treatment with a citrate buffer when the antigen to be retrieved is selected from the group consisting of STC1, RAEl, RRM2, SIRT2, and SLC2A1; and comprises the treatment with a EDTA buffer when the antigen to be retrieved is selected from the group consisting of LIG1, QKI, RAD51, and SRSF1.
  • citrate buffer refers to a buffer system comprising citric acid and/or a salt thereof (i.e., comprising the corresponding conjugate base, namely citrate ion).
  • a citrate buffer are sodium citrate buffer or potassium citrate buffer.
  • the pH of the citrate buffer may be from pH 5.0 to pH 7.0, preferably from pH 5.5 to pH 6.5, more preferably about pH 6.0.
  • EDTA buffer refers to a buffer system comprising Ethylenediaminetetraacetic acid (EDTA) and/or salt thereof (i.e., comprising the corresponding conjugate base, namely ethylenediaminetetraacetate).
  • EDTA buffer examples include disodium EDTA buffer and calcium disodium EDTA buffer.
  • the pH of the EDTA buffer may be from pH 8.0 to pH 10.0, preferably from pH 7.5 to pH 9.5, more preferably about pH 9.0.
  • the present invention refers to a kit for determining the levels of one or more of the target markers as described herein in a biological sample (preferably a tumor biopsy sample) isolated from a subject.
  • the kit may also contain instructions indicating how the materials within the kit may be used.
  • kit or "testing kit” denotes combinations of reagents and adjuvants required for an analysis. Although a test kit consists in most cases of several units, one-piece analysis elements are also available, which must likewise be regarded as testing kits.
  • said kit is suitable for determining the levels of at least STC1 and RAEl in a biological sample (preferably a tumor biopsy) and comprises:
  • step a) of the prognosis method of the invention optionally, further comprising an affinity reagent for each of the one, two, three, four, five, six or the seven additional proteins of the additional protein markers defined in step a) of the prognosis method of the invention;
  • cancer cells to be used as low and/or high expression controls optionally, further comprising cancer cells to be used as low and/or high expression controls
  • said kit is suitable for determining the levels of at least STCl, PvAEl and one or more of the additional protein markers defined in step a) of the prognosis method of the invention in a biological sample (preferably a tumor biopsy) and comprises:
  • cancer cells to be used as low and/or high expression controls optionally, further comprising cancer cells to be used as low and/or high expression controls
  • an affinity reagent for refers to an affinity reagent capable of specifically binding to the recited target protein.
  • the various affinity reagents may be labelled with the same or different tags. Preferably, these will be labelled with different tags for multiplex analysis.
  • said kit comprises an affinity reagent for each of the proteins in the set of markers selected from the group consisting of:
  • said kit comprises an affinity reagent for each of the proteins in the set of markers selected from the group consisting of:
  • said affinity reagent is an antibody, preferably a monoclonal antibody.
  • the affinity reagent may bind to any linear or conformational region (e.g. epitope) specific for the target protein.
  • the affinity reagents are the antibodies defined in Table 3 and antibodies binding to the same antigenic region and/or epitope in the marker protein (see Table I below):
  • said kit comprises reagents to perform an immunohistochemistry (ICH) assay.
  • ICH immunohistochemistry
  • it may contain inter alia: an enzyme-conjugated secondary antibody (e.g. conjugated to horseradish peroxidase or alkaline phosphatase), an enzyme substrate, and a counterstain such as hematoxylin.
  • Kits for ICH are well known in the art and commercially available (http://www.sigmaaldrich.com/life-science/cell-biology/antibodies/antibodies- application/protocols/immunohistochemistry.html#reagents_equipment).
  • ICH assay kits for use in a method of the invention also contain reagents for quantification of the targ protein markers, as described herein above.
  • kits in a method for the prognosis of a subject having lung SCC according to the invention, wherein said kit comprises:
  • step a) optionally, further comprising a reagent for each of the one, two, three, four, five, six or the seven additional proteins of theadditional protein markers defined in step a) for determining the protein expression levels thereof;
  • cancer cells to be used as low and/or high expression controls optionally, further comprising cancer cells to be used as low and/or high expression controls;
  • reagents may be useful for determining the expression levels of the target protein marker(s) using any suitable method for determining the expression of the target marker(s) in a given subcellular localization as described herein above.
  • the determination of the levels of said protein marker(s) may be carried out by a mass spectrometry (MS)-based method, and said kit may comprise said marker unlabelled and/or said marker stably labelled for detection by a mass spectrometry (MS)-based method, preferably wherein the marker is labelled with a tag which comprises one or more stable isotope.
  • Isotopic atoms which may be incorporated into the tag are heavy atoms for example 13 C, 15 N, 17 0 and/or 34 S, which can be distinguished by MS.
  • these reagents are affinity reagents as described herein for use in immunoassay methods as described herein.
  • said immunoassay is an immunohistochemistry assay.
  • said kit is as defined in the above aspect of the invention or in any of its preferred embodiments. It is contemplated that any features described herein can optionally be combined with any of the embodiments of any method, kit, use of a kit, or computer program of the invention; and any embodiment discussed in this specification can be implemented with respect to any of these. It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
  • the term “comprises” also encompasses and expressly discloses the terms “consists of and “consists essentially of.
  • A, B, C, or combinations thereof refers to all permutations and combinations of the listed items preceding the term.
  • A, B, C, or combinations thereof is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.
  • expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth.
  • BB BB
  • AAA AAA
  • AB BBC
  • AAABCCCCCC CBBAAA
  • CABABB CABABB
  • words of approximation such as, without limitation, "about”, “around”, “approximately” refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skilled in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature.
  • a numerical value herein that is modified by a word of approximation such as “about” may vary from the stated value by ⁇ 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10%. Accordingly, the term “about” may mean the indicated value ⁇ 5% of its value, preferably the indicated value ⁇ 2% of its value, most preferably the term "about” means exactly the indicated value ( ⁇ 0%).
  • Example 1 generation of a protein-based signature that correlates with the clinical outcome of the patient
  • the protein expression levels of 11 genes were analyzed in tumor samples from early stage lung cancer patients by IHC and semiquantitative analysis.
  • the prognostic signature was generated performing a manual regression Cox analysis by steps.
  • Tumor samples (from 239 ADCs and 117 SCC) were collected from consecutive population cohorts surgically treated at MD Anderson Cancer Center (MDA). Inclusion criteria were as follows: patients with NSCLC, complete resection of the primary tumor, no treatment with radiation or chemotherapy before surgery and absence of cancer within the 5 years before surgery. Lung tumors were classified according to the WHO 2004 classification (Travis WD, 2004) and 7th TNM edition was used for the tumors stratification (Goldstraw P, 2009). The study protocol was approved by local ethics committees of each center. Written informed consent was obtained from each patient. Reported recommendations for tumor marker prognostic studies (REMARK) criteria were followed throughout the study (Altman DG, 2012). For survival analysis the follow-up period was restricted to 60 months in all cohorts.
  • MDA MD Anderson Cancer Center
  • the PI was significantly prognostic of the five-year outcome for both DFS (Table 12-16, P ⁇ 0.05) and OS (Table 17-21, P ⁇ 0.05) in a univariate Cox proportional hazards analysis.
  • stage prognosis was highly significant (P ⁇ 0.05 for DFS and OS).
  • the PI score after adjustment for the clinical parameters was evaluated in a multivariate Cox regression analysis.
  • the molecular prognostic model and the stage remained the most significant predictors of five-year outcome for DFS and OS (all the models P ⁇ 0.05). All the results from the univariate and multivariate Cox proportional hazards analysis are summarized in Tables 12-21.
  • the obtained data confirmed the usefulness of the selected histotype-specific protein-based prognostic signatures to stratify the five-year risk of lung cancer recurrence or/and death in patients with either lung ADC or SCC.
  • Example 2 independent validation of the prognostic signature
  • Example 1 The protein expression levels of all the genes comprised in both signatures selected as the best model developed in Example 1 were analyzed in tumor samples from an independent cohort of early stage lung cancer patients (CUN-CIBERES cohort) and the models were validated.
  • Tumor samples (from 116 ADCs and 106 SCC) were collected from consecutive population cohorts surgically treated at Clinica Universidad de Navarra (CUN) and CIBERES network. Inclusion criteria were the same as in example 1.
  • the clinical and pathological data are summarized in Tables 1 and 2.
  • the expression of the seven proteins (BRCA1, QKI, SLC2A1, RAE1, RRM2, SRSF1, y STC1) was evaluated using IHC and semiquantitative method (H- score determination) by two experienced observers. Later, for each patient, the immunostaining scores were applied to the prognostic formulas, previously generated in example 1. We calculated the CH and the survival curves with Kaplan-Meier method, which differences were compared using log-rank test as previously described.
  • example 1 the two histotype specific protein-based signatures identified in example 1 were validated in an independent cohort of ADC or SCC patients, respectively.
  • Example 3 risk stratification in stage I-II patients
  • T primary tumor
  • N lymph nodes
  • M metastasis
  • the T component is defined by tumor size, tumor location the involved structures or the effects of the tumor growth and has seven categories.
  • the N component is defined by the absence or presence and location of the involved nodes and has five categories and the M component has two different categories defined by the absence or presence and location of the metastasis (Table 26).
  • TNM subsets of similar prognosis are grouped in 7 different stages.
  • the likelihood ratio significantly increased after adding the molecular information (PI) (P ⁇ 0.001 both for DFS and OS). This improvement showed that the molecular model complements the stage, adding very valuable prognostic information for the patients.
  • PI molecular information
  • prognostic capability improved in all cases after addition of the Pis variables. All the prognostic models were complementary to the stage and were able to add additional prognostic information to the patients.
  • the likelihood ratio significantly increased after adding the molecular information (PI) (P ⁇ 0.001 both for DFS and OS).
  • PI molecular information
  • This improvement showed that the molecular model complements the stage, adding very valuable prognostic information for the patients.
  • CPI combined prognostic index
  • CPI combined model
  • Example 6 Predictive value of protein-based models for the benefit of the adjuvant therapy
  • stage I is the most controversial subgroup in terms of adjuvant treatment effectiveness.
  • our models are able to select those stage I-II patients who could obtain a benefit from an adjuvant therapy, in particular from platinum-based adjuvant chemotherapy treatment, in both histological subtypes.
  • Ferte C, Andre F, Soria JC Molecular circuits of solid tumors: prognostic and predictive tools for bedside use. Nat Rev Clin Oncol 2010 Jul;7(7):367-380.

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Abstract

L'invention concerne le domaine du pronostic du cancer et, plus particulièrement, des méthodes de prédiction du résultat d'un patient atteint d'un carcinome squameux pulmonaire (SCC) sur la base des niveaux d'expression de plusieurs protéines et de signatures et d'algorithmes de pronostic spécifiques, ainsi que des méthodes de sélection de patients qui bénéficieraient d'un traitement adjuvant après une résection tumorale, ainsi que des kits pour la mise en œuvre de ces méthodes. Plus particulièrement, ladite signature pronostique/prédictive est basée sur les niveaux d'expression de protéines de STC1 cytoplasmique (STC1-C) et de RAE1 cytoplasmique (RAE1-C) dans un échantillon biologique d'un sujet atteint de SCC pulmonaire.
PCT/EP2018/075641 2017-09-21 2018-09-21 Méthode et kits pour le pronostic du carcinome squameux pulmonaire (scc) WO2019057913A1 (fr)

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CN116718784A (zh) * 2023-06-19 2023-09-08 十堰市太和医院(湖北医药学院附属医院) Stc1作为胶质瘤标记物的应用

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111793691A (zh) * 2020-08-04 2020-10-20 中国科学院昆明动物研究所 一种特征mRNA表达谱组合及肺鳞癌早期预测方法
CN116718784A (zh) * 2023-06-19 2023-09-08 十堰市太和医院(湖北医药学院附属医院) Stc1作为胶质瘤标记物的应用

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