The invention relates to the field of cancer prognosis and, more in particular, to methods for predicting the outcome of a cancer patient based on the expression levels of a gene signature as well as to methods for selecting a suitable therapy for a patient suffering from cancer, in particular, lung cancer and breast cancer.

BACKGROUND ART

Lung cancer is the leading cause of cancer death worldwide. In the United

States, its incidence rate is the second highest among men and women and is the most common cause of cancer death in both sexes. Similar data are found in Europe, where lung cancer is the third most common cancer and is the leading cause of cancer death. Lung cancer comprises two main histological subtypes: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). The latter accounts for 80-85% of all cases and includes the two most frequent lung cancer types: adenocarcinomas and squamous cell carcinomas.

Around 15% of people diagnosed with lung cancer survive five years after diagnosis. Survival depends mainly on stage. In early stage NSCLC (stages I to IIIA), curative surgery is the treatment of choice. However, even after complete surgical resection, patients are at substantial risk for recurrence. One third of patients with stage I, more than 50 percent with stage II, and more than 75% of those with stage IIIA eventually recur and die of their disease despite potentially curative surgery. For this reason, adjuvant cisplatin-based chemotherapy is recommended for routine use in patients with stages IIA, IIB, and IIIA disease. Adjuvant chemotherapy for patients with stage IA is not recommended. In stage IB, its use is controversial, as clinical trials have not clearly demonstrated a survival benefit.

It is generally thought that a fraction of patients with stage IB, II or IIIA would be receiving unnecessarily adjuvant chemotherapy, and, conversely, a subgroup of stage IA patients might benefit from adjuvant chemotherapy. Therefore, the ability to more accurately stratify patient may benefit health outcomes. In particular, molecular prognostic biomarkers may be used to better identify those patients with resectable NSCLC who are at high risk of recurrence and would benefit from adjuvant therapy. Prognostic biomarkers for relapse after local treatment are also very useful for therapy decision making in other tumor types, such as breast cancer. SUMMARY OF THE INVENTION

The present invention is based in the surprising finding that the expression levels of several genes involved in RNA metabolism are useful in prognosis of lung cancer patients. This method can be used to determine those patients with resectable NSCLC who are at a high risk of recurrence or progression. The identification of this subgroup of patients may guide the selection of therapies, improving financial and health outcomes. Moreover, the expression levels of these genes can also be used for the prognosis and therapy selection in other types of cancer, such as in breast cancer.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1. Kaplan-Meier curve and log rank statistics for overall survival in adenocarcinoma patients divided in high and low MARS mRNA expression.

FIG. 2. Kaplan-Meier curve and log rank statistics for overall survival in adenocarcinoma patients divided in high and low RAEl mRNA expression.

FIG. 3. Kaplan-Meier curve and log rank statistics for overall survival in adenocarcinoma patients divided in high and low SNRPB mRNA expression.

FIG. 4. Kaplan-Meier curve and log rank statistics for overall survival in adenocarcinoma patients divided in high and low SNRPE mRNA expression.

FIG. 5. Kaplan-Meier curve and log rank statistics for overall survival in adenocarcinoma patients divided in high and low AD ARB 1 mRNA expression.

FIG. 6. Kaplan-Meier curve and log rank statistics for overall survival in adenocarcinoma patients divided by the five-gene prognostic score.

FIG. 7. Kaplan-Meier curve and log rank statistics for disease-free survival in adenocarcinoma patients divided by the five-gene prognostic score.

FIG. 8. Kaplan-Meier curve and log rank statistics for overall survival in stage I adenocarcinoma patients divided by the five-gene prognostic score.

FIG. 9. Kaplan-Meier curve and log rank statistics for overall survival in stage II adenocarcinoma patients divided by the five-gene prognostic score. FIG. 10. Kaplan-Meier curve and log rank statistics for overall survival in stage III adenocarcinoma patients divided by the five-gene prognostic score.

FIG. 11. Kaplan-Meier curve and log rank statistics for overall survival in adenocarcinoma patients, divided by the five-gene prognostic score, in an independent validation series.

FIG. 12. Kaplan-Meier curve and log rank statistics for disease-free survival in adenocarcinoma patients, divided by the five-gene prognostic score, in an independent validation series.

FIG. 13. Kaplan-Meier curve and log rank statistics for distant metastasis- free survival in breast cancer patients divided in high and low MARS mRNA expression.

FIG. 14. Kaplan-Meier curve and log rank statistics for distant metastasis- free survival in breast cancer patients divided in high and low RAEl mRNA expression.

FIG. 15. Kaplan-Meier curve and log rank statistics for distant metastasis- free survival in breast cancer patients divided in high and low SNRPB mRNA expression.

FIG. 16. Kaplan-Meier curve and log rank statistics for distant metastasis- free survival in breast cancer patients divided in high and low SNRPE mRNA expression.

FIG. 17. Kaplan-Meier curve and log rank statistics for distant metastasis- free survival in breast cancer patients divided in high and low AD ARB 1 mRNA expression.

FIG. 18. Kaplan-Meier curve and log rank statistics for distant metastasis- free survival in breast cancer patients, divided by the five-gene prognostic score.

FIG. 19. Kaplan-Meier curve and log rank statistics for disease-specific survival in breast cancer patients, divided by the five-gene prognostic score, in an independent validation series.

FIG. 20. Kaplan-Meier curve and log rank statistics for recurrence in breast cancer patients, divided by the five-gene prognostic score, in an independent validation series.

DETAILED DESCRIPTION OF THE INVENTION Method for determining the prognosis of a patient suffering from cancer The authors of the present invention have identified several genes, the expression levels of which are a reliable marker for predicting the outcome of the cancer patient. Therefore, in a first aspect, the invention relates to a method (hereinafter first method of the invention) for determining the prognosis of a patient suffering from cancer which comprises the determination in a sample from said patient of the expression levels of at least one gene selected from the group consisting of genes MARS, SNRPB, ADARBl, RAEl and SNRPE,

wherein an increase in the expression of at least one gene selected from the group consisting of MARS, SNRPB, RAEl, SNRPE and/or a decrease in the expression of the ADARBl gene with respect to a reference value is indicative of a poor prognosis of the patient

or

wherein a decrease in the expression of at least one gene selected from the group consisting of MARS, SNRPB, RAEl and SNRPE and/or an increase in the expression of the ADARBl gene with respect to a reference value is indicative of a good prognosis of the patient.

In another aspect, the invention relates to the use of one or more genes selected from the group consisting of MARS, SNRPB, ADARBl, RAEl and SNRPE or of one or more of the polypeptides encoded by said genes from a sample obtained from the individual for the determination of the prognosis of a patient suffering from cancer.

The term "prognosis" refers to a prediction of medical outcome, for example, a poor or good outcome (e.g., likelihood of long-term survival, overall survival, disease- specific survival, progression-free survival or disease-free survival); a negative prognosis, or poor outcome, includes a prediction of relapse, disease progression (e.g., tumor growth or metastasis, or drug resistance), or mortality; a positive prognosis, or good outcome, includes a prediction of disease remission, (e.g., disease-free status), amelioration (e.g., tumor regression), or stabilization.

Any parameter which is widely accepted for determining prognosis of a patient can be used in the present invention including, without limitation:

· overall survival rate, as used herewith, relates to the percentage of people in a study or treatment group who are alive for a certain period of time after they were diagnosed with or treated for a disease, such as cancer. • disease-specific survival rate which is defined as the percentage of people in a study or treatment group who have not died from a specific disease in a defined period of time.

• disease-free survival (DFS), as used herewith, is understood as the length of time after treatment for a disease during which a subject survives with no sign of the disease.

• objective response which, as used in the present invention, describes the proportion of treated subjects in whom a complete or partial response is observed.

· tumor control which, as used in the present invention, relates to the proportion of treated subjects in whom complete response, partial response, minor response or stable disease > 6 months is observed.

• progression free survival which, as used herein, is defined as the time from start of treatment to the first measurement of cancer growth.

· time to progression (TTP), as used herein, relates to the time since a disease is treated until the disease starts to get worse. The term "progression" has been previously defined.

• six-month progression free survival or "PFS6" rate which, as used herein, relates to the percentage of subjects who are free of progression in the first six months after the initiation of the therapy and

• median survival which, as used herein, relates to the time at which half of the subjects enrolled in the study are still alive.

The term "patient" or "subject" refers to all 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. Preferably, the subject is a human man or woman of any age or race. In a preferred embodiment, the subject has not been treated with chemotherapy or radiotherapy prior to the determination of the expression levels of the gene or genes of interest. In yet another embodiment, the patient has undergone surgical resection of the tumor.

The terms "cancer" and "tumor" refer to the physiological condition in mammals characterized by unregulated cell growth. The methods of the present invention are useful in any cancer or tumor, such as, without limitation, breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head, neck, ovarian, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testicles, hepatobiliary and liver tumors. In particular, tumors whose clinical outcome may be predicted with the methods of the invention include adenoma, angiosarcoma, astrocytoma, epithelial carcinoma, germinoma, glioblastoma, glioma, hemangioendothelioma, hemangio sarcoma, hematoma, hepatoblastoma, leukaemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, hepatobiliary cancer, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma, and teratoma. In particular, the tumor/cancer is selected from the group of acrallentiginous melanoma, actinic keratosis adenocarcinoma, adenoid cystic carcinoma, adenomas, adenosarcoma, adenosquamous carcinoma, astro cytictumors, bartholin gland carcinoma, basal cell carcinoma, bronchial gland carcinoma, capillary carcinoid, carcinoma, carcinosarcoma, cholangiocarcinoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, ependymal sarcoma, Swing's sarcoma, focal nodular hyperplasia, germ cell tumors, glioblastoma, glucagonoma, hemangioblastoma, hemangioendothelioma, hemangioma, hepatic adenoma, hepatic adenomatosis, hepatocellular carcinoma, hepatobiliary cancer, insulinoma, intraepithelial neoplasia, interepithelial squamous cell neoplasia, invasive squamous cell carcinoma, large cell carcinoma, leiomyosarcoma, melanoma, malignant melanoma, malignant mesothelialtumor, medulloblastoma, medulloepithelioma, mucoepidermoid carcinoma, neuroblastoma, neuroepithelial adenocarcinoma, nodular melanoma, osteosarcoma, papillary serous adenocarcinoma, pituitary tumors, plasmacytoma, pseudosarcoma, pulmonary blastoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, small cell carcinoma, soft tissue carcinoma, somatostatin- secreting tumor, squamous carcinoma, squamous cell carcinoma, undifferentiated carcinoma, uveal melanoma, verrucous carcinoma, vipoma, Wilm's tumor. Even more preferably, the tumor/cancer include intracerebral cancer, head and neck cancer, rectal cancer, astrocytoma, glioblastoma, small cell cancer, and non-small cell cancer, preferably non-small cell lung cancer, metastatic melanoma, androgen-independent metastatic prostate cancer, androgen-dependent metastatic prostate cancer and breast cancer. In a preferred embodiment the cancer is selected from lung cancer, colon cancer, melanoma, pancreatic cancer, prostate cancer, glioma, bladder cancer, ovarian cancer, hepatobiliary cancer, breast cancer and lymphoma.

In a more preferred embodiment the cancer is lung cancer. The term "lung cancer", as used herein, refers to any uncontrolled cell growth in tissues of the lung. In a preferred embodiment, the lung cancer is non-small cell lung cancer (NSCLC) or small cell lung cancer. The term non-small cell lung cancer (NSCLC), as used herein, refers to a group of heterogeneous diseases grouped together because their prognosis and management is roughly identical and includes, according to the histologic classification of the World Health Organization/International Association for the Study of Lung Cancer (Travis WD et al. Histological typing of lung and pleural tumors. 3 ^rd ed. Berlin: Springer- Verlag, 1999). Suitable NSCLC types, the prognosis of which can be determined with the method according to the present invention, include, without limitation, squamous cell carcinoma (SCC), lung adenocarcinoma, large cell carcinoma, adenosquamous carcinoma, carcinomas with pleomorphic, sarcomatoid or sarcomatous elements, carcinoid tumor, carcinomas of salivary gland and unclassified carcinomas of the lung. In a preferred embodiment, the NSCLC is selected from squamous cell carcinoma of the lung, large cell carcinoma of the lung or adenocarcinoma of the lung.

The predictive method according to the present invention allows the determination of the clinical outcome of patients having different stages of NSCLC, including patients having stage I NSCLC cancer, stage II NSCLC cancer, stage III NSCLC cancer or stage IV NSCLC cancer and, more in particular, stage IA NSCLC, stage IB NSCLC, stage IIA NSCLC, stage IIB NSCLC, stage IIIA NSCLC, stage IIIB and stage IV NSCLC. Stages I, II, III and IV in lung cancer are defined in Mountain CF. Revisions in the International System for Staging Lung Cancer. Chest. 1997; 11 1 : 1710-1717.

The predictive method according to the present invention allows the determination of the clinical outcome of patients having different stages of NSCLC, including patients in with stage TXN0M0 NSCLC (wherein X is an integer from 0 to 4), patients with T1N0M0 NSCLC, Stage T2M0N0 NSCLC, stage T1N1M0 NSCLC, stage T2N1M0 NSCLC, stage T3N0M0 NSCLC, stage T1N2M0 NSCLC, stage T2N2M0 NSCLC, stage T3N1M0 NSCLC, stage T3N2M0 NSCLC, stage T4N0M0, stage T4N1M0 NSCLC, stage T1N3M0 NSCLC, stage T2N3M0 NSCLC, stage T3N3M0 NSCLC, stage T4N2M0 NSCLC, stage T4N3M0 NSCLC or stage TXNYMl, wherein X is any value from 0 to 4 and Y is any value from 0 to 3, according to the TNM classification (AJCC Cancer Staging Manual, Lippincott, 5th edition, pp. 171-180, 1997).

In another preferred embodiment, the cancer is breast cancer. The terms breast cancer and malignant breast neoplasm are commonly used as the generic name for cancers originating from breast tissue, most commonly from the inner lining of milk ducts or the lobules that supply the ducts with milk. In a preferred embodiment, the first method of the present invention can be used for determining the prognosis of ER positive (ER+) breast cancer, ER negative (ER-) breast cancer, PR positive (PR+) breast cancer, PR negative (PR-) breast cancer, HER2 positive (HER2+) breast cancer (cancer over-expressing HER2), HER2 negative (HER2-) breast cancer (cancer expressing normal levels of HER2 or under-expressing HER2 or not expressing a detectable level of HER2), hormone receptor negative breast cancer, i.e. breast cancer with neither of estrogen nor progesterone receptors (abbreviated by ER-/PR- breast cancer); and triple negative breast cancer, i.e. breast cancer with neither of estrogen nor progesterone receptors and with normal expression/under-expression (or with the absence of detectable level of expression) of HER2 (abbreviated by ER-/PR-/HER2- breast cancer).

In another embodiment, the first method of the invention can be used for determining the prognosis of a patient suffering from luminal subtype A breast cancer, luminal subtype B breast cancer, normal-like breast cancer, HER2+ breast cancer and basal-like breast cancer.

The predictive method according to the present invention allows the prognosis of patients having different stages of breast cancer, including patients in with stage I breast cancer, stage II breast cancer, stage III breast cancer or stage IV breast cancer and, more in particular, stage IA breast cancer, stage IB breast cancer, stage IIA breast cancer, stage IIB breast cancer, stage IIIA breast cancer, stage IIIB and stage IV breast cancer. Stages I, II, III and IV in breast cancer are defined in Greene FL, Page DL, Fleming ID, et al, editors. AJCC cancer staging manual, 6th ed. New York: Springer- Verlag, 2002.

The predictive method according to the present invention allows the prognosis of patients having different stages of breast cancer, including patients with stage TXN0M0 breast cancer (wherein X is an integer from 0 to 4), stage T1N0M0 breast cancer, stage T2M0N0 breast cancer, stage T1N1M0 breast cancer, stage T2N1M0 breast cancer, stage T3N0M0 breast cancer, stage T1N2M0 breast cancer, stage T2N2M0 breast cancer, stage T3N1M0 breast cancer, stage T3N2M0 breast cancer, stage T4N0M0, stage T4N1M0 breast cancer, stage T1N3M0 breast cancer, stage T2N3M0 breast cancer, stage T3N3M0 breast cancer, stage T4N2M0 breast cancer, stage T4N3M0 breast cancer or stage TXNYM1 breast cancer, wherein X is any value from 0 to 4 and Y is any value from 0 to 3, according to the TNM classification (AJCC Cancer Staging Manual, Lippincott, 5th edition, pp. 171-180, 1997).

In another embodiment, the predictive method according to the present invention allows the prognosis of patients suffering breast cancer wherein the breast cancer is lymph-node negative breast cancer, i.e. a breast cancer that has not spread to the lymph node.

In a first step, the first method of the invention comprises the determination in a sample from a patient suffering from cancer of the expression levels of, at least, one gene selected from the group consisting of the MARS, SNRPB, AD ARB 1 , RAE1 and SNRPE genes.

As used herein, "sample" or "biological sample" means biological material isolated from a subject. The biological sample may contain any biological material suitable for determining the expression level of the MARS, SNRPB, AD ARB 1 , RAE1 and/or SNRPE genes. The sample can be isolated from any suitable biological tissue or fluid such as, for example, tumor tissue, blood, plasma, serum, sputum, bronchoalveolar lavage, urine or cerebral spinal fluid (CSF). In a preferred embodiment, the sample is a tumor tissue sample. The tumor tissue sample is understood as the tissue sample originating from the primary tumor or from a distant metastasis. Said sample can be obtained by conventional methods, for example biopsy, using methods well known by the persons skilled in related medical techniques. Alternatively, the tumor tissue sample may be a sample of a tumor which has been surgically resected. The methods for obtaining a biopsy sample include splitting a tumor into large pieces, or microdissection, or other cell separating methods known in the art. The tumor cells can additionally be obtained by means of cytology through aspiration with a small gauge needle. To simplify sample preservation and handling, samples can be fixed in formalin and soaked in paraffin or first frozen and then soaked in a tissue freezing medium such as OCT compound by means of immersion in a highly cryogenic medium which allows rapid freezing.

The term "expression level" of a gene as used herein refers to the measurable quantity of gene product produced by the gene in a sample of the subject, wherein the gene product can be a transcriptional product or a translational product. As understood by the person skilled in the art, the gene expression level can be quantified by measuring the messenger RNA levels of said gene or of the protein encoded by said gene. The expression levels of the at least one gene selected from the group consisting of MARS, SNRPB, AD ARB 1 , RAEl and SNRPE can be determined by measuring the levels of mRNA encoded by said gene, or by measuring the levels of the protein encoded by said gene, i.e. MARS, SNRPB, ADARB1, RAEl and SNRPE proteins, or of variants thereof.

In order to measure the mRNA levels of the one or more genes selected from the group consisting of MARS, SNRPB, AD ARB 1 , RAEl and SNRPE, the biological sample may be treated to physically, mechanically or chemically disrupt tissue or cell structure, to release intracellular components into an aqueous or organic solution to prepare nucleic acids for further analysis. The nucleic acids are extracted from the sample by procedures known to the skilled person and commercially available. RNA is then extracted from frozen or fresh samples by any of the methods typical in the art, for example, Sambrook, J., et al, 2001. Molecular cloning: A Laboratory Manual, 3 ^rd ed., Cold Spring Harbor Laboratory Press, N.Y., Vol. 1-3. Preferably, care is taken to avoid degradation of the RNA during the extraction process.

The expression level can be determined using mRNA obtained from a formalin- fixed, paraffin-embedded tissue sample. mRNA may be isolated from an archival pathological sample or biopsy sample which is first deparaffmized. An exemplary deparaffmization method involves washing the paraffinized sample with an organic solvent, such as xylene. Deparaffmized samples can be rehydrated with an aqueous solution of a lower alcohol. Suitable lower alcohols, for example, include methanol, ethanol, propanols and butanols. Deparaffmized samples may be rehydrated with successive washes with lower alcoholic solutions of decreasing concentration, for example. Alternatively, the sample is simultaneously deparaffmized and rehydrated. The sample is then lysed and RNA is extracted from the sample. Samples can be also obtained from fresh tumor tissue such as a resected tumor.

In a preferred embodiment samples can be obtained from fresh tumor tissue or from OCT embedded frozen tissue. In another preferred embodiment samples can be obtained by bronchoscopy and then paraffin-embedded.

Determination of the levels of mRNA of the one or more genes selected from the group consisting of MARS, SNRPB, AD ARB 1 , RAEl and SNRPE can be carried out by any method known in the art such as qPCR, northern blot, RNA dot blot, TaqMan, tag based methods such as serial analysis of gene expression (SAGE) including variants such as LongSAGE and SuperSAGE, microarrays. Determination of the levels of the above genes can also be carried out by Fluorescence In Situ Hybridization, including variants such as Flow-FISH, qFiSH and double fusion FISH (D-FISH) as described in WO2010030818, Femino et al. (Science, 1998, 280:585-590), Levsky et al. (Science, 2002, 297:836-840) or Raj et al. (PLoS Biology, 2006, 4:e309). The levels of the mRNA of the different genes can also be determined by nucleic acid sequence based amplification (NASBA) technology.

In a preferred embodiment, the gene mRNA expression levels are often determined by reverse transcription polymerase chain reaction (RT-PCR). The detection can be carried out in individual samples or in tissue microarrays.

Thus, in a particular embodiment, the mRNA expression levels of the one or more genes selected from the group consisting of genes MARS, SNRPB, ADARB1, RAEl and SNRPE are determined by quantitative PCR, preferably, Real-Time PCR. The detection can be carried out in individual samples or in tissue microarrays.

In order to normalize the values of mRNA expression among the different samples, it is possible to compare the expression levels of the mRNA of interest in the test samples with the expression of a control RNA. A "control RNA" as used herein, relates to RNA whose expression levels do not change or change only in limited amounts in tumor cells with respect to non-tumorigenic cells. Preferably, the control RNA is mRNA derived from housekeeping genes and which code for proteins which are constitutively expressed and carry out essential cellular functions. Preferred housekeeping genes for use in the present invention include β-2-microglobulin, ubiquitin, 18-S ribosomal protein, cyclophilin, IP08, HPRT, GAPDH, PSMB4, tubulin and β-actin. In a preferred embodiment, the control RNA is GAPDH, IP08, HPRT, β- actin, 18-S ribosomal protein or PSMB4 mRNA.

In one embodiment relative gene expression quantification is calculated according to the comparative threshold cycle (Ct ) method using GAPDH, IP08, HPRT, β-actin or PSMB4 as an endogenous control and commercial RNA controls as calibrators. Final results are determined according to the formula 2-(ACt sample-ACt calibrator), where ACT values of the calibrator and sample are determined by subtracting the Ct value of the target gene from the value of the control gene.

Alternatively, in another embodiment of the first method of the invention, the expression levels of the one or more genes selected from the group consisting of MARS, SNRPB, ADARB1, RAE1 and SNRPE are determined by measuring the expression of the polypeptides encoded by said genes or of variants thereof. In a preferred embodiment the expression levels of the proteins or of variants thereof are determined by Western blot, ELISA or by immunohistochemistry.

The expression levels of the protein encoded by the one or more genes selected from the group consisting of MARS, SNRPB, AD ARB 1 , RAE1 and SNRPE can be quantified by means of conventional methods, for example, using antibodies with a capacity to specifically bind to the proteins encoded by said genes (or to fragments thereof containing antigenic determinants) and subsequent quantification of the resulting antibody-antigen complexes.

The antibodies to be employed in these assays can be, for example, polyclonal sera, hybridoma supernatants or monoclonal antibodies, antibody fragments, Fv, Fab, Fab' and F(ab')2, ScFv, diabodies, triabodies, tetrabodies and humanized antibodies. At the same time, the antibodies can be labeled or not. Illustrative, but non-exclusive examples of markers which can be used include radioactive isotopes, enzymes, fluorophores, chemiluminescent reagents, enzymatic substrates or cofactors, enzymatic inhibitors, particles, colorants, etc. There are a wide variety of well-known assays that can be used in the present invention, which use non-labeled antibodies (primary antibody) and labeled antibodies (secondary antibodies); among these techniques are included Western blot or Western transfer, ELISA (enzyme linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (enzymatic immunoassay), DAS- ELISA (double antibody sandwich ELISA), immunocytochemical and immunohistochemical techniques, techniques based on the use of biochips or protein microarrays including specific antibodies or assays based on colloidal precipitation in formats such as dipsticks. Other ways of detecting and quantifying the levels of the protein of interest include techniques of affinity chromatography, binding- ligand assays, etc.

On the other hand, the determination of the levels of protein encoded by the one or more genes selected from the group consisting of MARS, SNRPB, ADARB1, RAEl and SNRPE can be carried out by constructing a tissue microarray (TMA) containing the subject samples assembled, and determining the expression levels of each corresponding protein by immunohistochemistry techniques. Immunostaining intensity can be evaluated by two or more different pathologists and scored using uniform and clear cut-off criteria, in order to maintain the reproducibility of the method. Discrepancies can be resolved by simultaneous re-evaluation. Briefly, the result of immunostaining can be recorded as negative expression (0) versus positive expression, and low expression (1+) versus moderate (2+) and high (3+) expression, taking into account the expression in tumor cells and the specific cut-off for each marker. As a general criterion, the cut-offs are selected in order to facilitate reproducibility, and when possible, to translate biological events. Alternatively, the immunostaining intensity can be evaluated by using imaging techniques and automated methods such as those disclosed in Rojo, M.G. et al. (Folia Histochem. Cytobiol. 2009; 47: 349-54) or Mulrane, L. et al. (Expert Rev. Mol. Diagn. 2008; 8: 707-25).

Alternatively, in another particular embodiment, the levels of the protein encoded by the one or more genes selected from the group consisting of MARS, SNRPB, ADARBl, RAEl and SNRPE or of the variants thereof are determined by Western blot. Western blot is based on the detection of proteins previously resolved by gel electrophoreses under denaturing conditions and immobilized on a membrane, generally nitrocellulose, by the incubation with an antibody specific and a developing system (e.g. chemoluminiscent).

The term "MARS", also known as MRS, MetRS, cytosolic methionyl-tRNA synthetase, methionine tRNA ligase 1, cytoplasmic METRS, methionine-tRNA ligase, or cytoplasmic MTRNS, as used herein, refers to a gene encoding a methionyl-tRNA synthetase. The human gene is shown in the Ensembl database with accession number ENSG00000166986. The mRNA encoded by the human gene is shown in the NCBI nucleotide database with accession number NM 004990 and the corresponding polypeptide is shown with accession number SYMC HUMAN or P56192 in the UniProtKB/SwissProt database.

The term "SNRPB", also known as Sm protein B/B', COD1, B polypeptide of

Sm protein, SNRPB 1, sm-B/Sm-B', Sm-B/B', small nuclear ribonucleoprotein polypeptide B, snRNP-B, small nuclear ribonucleoprotein polypeptides B and B', smB/SmB', small nuclear ribonucleoprotein-associated proteins B and B' and SmB/B', as used herein, refers to a gene encoding the small nuclear ribonucleoprotein polypeptides B and Bl . The human gene is shown in the Ensembl database under accession number ENSG00000125835. The human gene produces two alternative transcripts which are shown in the NCBI nucleotide database with accession numbers NP 003082.1 and NP 937859.1 which produce three different isoforms known as isoform SM-B' (identifier: P14678-1 in the UniProtKB/SwissProt database), isoform SM-B (identifier: P14678-2 in the UniProtKB/SwissProt database) and isoform SM-B1 (identifier: PI 4678-3 in the UniProtKB/SwissProt database).

The term "AD ARB 1", also known as ADAR2, DRADA2 or REDl, as used herein, refers to a gene encoding an adenosine deaminase, RNA- specific, Bl . The human gene is shown in the Ensembl database with accession number ENSG00000197381. The mRNA produced by the human gene is shown in the GenEMBL nucleotide database with accession number U82120.1. The polypeptide appears as five different isoforms, namely, isoform 1 (identifier: P78563-1), also known as REDl-L or DRADA2B; Isoform 2 (identifier: P78563-2), also known as REDl-S or DRADA2A; Isoform 3 (identifier: P78563-3), also known as DRADA2C; Isoform 4 (identifier: P78563-4); and Isoform 5 (identifier: P78563-5).

The term "RAE1", also known as Rael protein homolog mRNA-associated protein mrnp 41, as used herein, refers to a gene which encodes the RNA export 1 homolog polypeptide. The human gene is shown in the Ensembl database with accession number ENSG00000101146. The mRNA produced by the human gene is shown in the GenEMBL database with accession number U84720. The corresponding polypeptide is shown with accession number and the corresponding polypeptide is shown with accession number P78406 or RAE1L HUMAN in the UniProtKB/SwissProt database.

The term "SNRPE", also known as Sm-E, SME, Sm protein E, small nuclear ribonucleoprotein E2, snRNP-E and SmE, refers to a gene encoding a small nuclear ribonucleoprotein polypeptide E. The human gene is shown in the Ensembl database under accession number ENSGOOOOO 182004. The mRNA produced by the human gene is shown in the GenEMBL database with accession number M37716. The corresponding polypeptide is shown with accession number and the corresponding polypeptide is shown with accession number P62304 or RUXE HUMAN in the UniProtKB/SwissProt database.

The term "at least a gene selected from the group consisting of the MARS, SNRPB, ADARBl, RAEl and SNRPE", as used herein, means that the method may involve the determination of the expression levels of 1, 2, 3, 4 or 5 genes. In a preferred embodiment, the method comprises the determination of the expression levels of the MARS gene, of the SNRPB gene, of the ADARBl gene, of the RAEl gene or of the SNRPE gene. In another embodiment, the first method of the invention comprises determining the expression levels of two of the above genes. Suitable combinations of two genes which can be measured include, MARS and SNRPB, MARS and ADARBl, MARS and RAEl, MARS and SNRPE, SNRPB and ADARBl, SNRPB and RAEl, SNRPB and SNRPE, ADARBl and RAEl, ADARBl and SNRPE, RAEl and SNRPE. In another embodiment, the method of the invention comprises the determination of the expression levels of three of the above genes. Suitable combinations of three genes include MARS, SNRPB and ADARBl; MARS, SNRPB and RAEl; MARS, SNRPB and SNRPE; MARS, ADARBl and RAEl; MARS, ADARBl and SNRPE; MARS, RAEl and SNRPE; SNRPB, ADARBl and RAEl; SNRPB, ADARBl and SNRPE; SNRPB, RAEl and SNRPE; and ADARBl, RAEl and SNRPE. In another embodiment, the method of the invention comprises the determination of the expression levels of four of the above genes. Suitable combinations of four genes include MARS, SNRPB, ADARBl and RAEl; MARS, SNRPB, ADARBl and SNRPE; MARS, SNRPB, RAEl, SNRPE; MARS, ADARBl, RAEl and SNRPE; and SNRPB, ADARBl, RAEl and SNRPE. In yet another embodiment, the method of the present invention comprises the determination of the expression levels of the MARS, SNRPB, ADARB1, RAEl and the SNRPE genes.

In a preferred embodiment, the first method of the invention further comprises the determination of the expression levels of one or more cancer marker genes and/or the determination of one or more clinical parameters which are also indicative of the prognosis of the cancer.

Such indicators include the presence or levels of known cancer markers, or can be clinical or pathological indicators (for example, age, tumor size, tumor histology, differentiation grade, clinical stage, family history and the like).

In the particular case of lung tumors, suitable clinical markers that can be combined with the first method of the invention include, without limitation, tumor size, cell type (histology), differentiation grade, stage, ECOG performance status. Suitable marker molecules include, without limitation, ERCC1, RRM1, TP53, KRAS, HRAS, NRAS, CDK 1B, CDK 2A, BRCA1, EGFR, CCND1, CCND3, CCNE, TUBB3, HER2, DUSP6, MMD, STAT1, ERBB3, LCK, EML4, ALK, BRAF, RET, PIK3CA, CT NB1, ROS1, PDGFRA, DDR1, DDR2, FGFR1, PTEN, VEGF; VEGFR1, VEGFR2, VEGFR3, CEA, IL6, IL8, IL10, OPN, MCP1, NSE, SCC, CYFRA 21-1, TK, KLKB1, SMRP, CA 125.

In the particular case of breast cancer, suitable clinical markers include, without limitation, location of the tumor (carcinoma in situ vs. lymph node vs. metastasis), tumor size and shape, rate of cell division. Suitable marker molecules which may be combined with the method of the invention include, without limitation, hormone receptors wherein presence of hormone receptors (progesterone receptor and estrogen receptor) is associated with a better prognosis, HER2, wherein HER2 positive cancer is associated with a worse prognosis, CA 15-3, CA 27.29, CEA, ER, PgR, uPA, PAI-1, the marker profile used in the Oncotype DX™ comprising the BAG1, BCL2, CCNB1, CD68, SCUBE2, CTSL2, ESR1, GRB7, GSTM1, ERBB2, MKI67, MYBL2, PGR, AURKA, MMPl l and BIRC5 markers or any of the 70 genes which form part of the Mammaprint™ profiling test.

In a second step, the first method of the invention comprises comparing the expression levels of the genes with a reference value. "Reference value", as used herein, refers to a laboratory value used as a reference for values/data obtained by laboratory examinations of subjects or samples collected from subjects. The reference value or reference level can be an absolute value; a relative value; a value that has an upper and/or lower limit; a range of values; an average value; a median value, a mean value, or a value as compared to a particular control or baseline value. A reference value can be based on an individual sample value, such as for example, a value obtained from a sample from the subject being tested, but at an earlier point in time or from a non-cancerous tissue. The reference value can be based on a large number of samples, such as from population of subjects of the chronological age matched group, or based on a pool of samples including or excluding the sample to be tested. Various considerations are taken into account when determining the reference value of the marker. Among such considerations are the age, weight, sex, general physical condition of the patient and the like. For example, equal amounts of a group of at least 2, at least 10, at least 100 to preferably more than 1000 subjects, preferably classified according to the foregoing considerations, for example according to various age categories, are taken as the reference group.

In a preferred embodiment, the reference value is the expression level of the gene of interest in a non-cancerous tissue. In case that the method is used for determining the prognosis of a lung cancer patient, the reference value is the expression level of the gene of interest in non-cancerous lung tissue. In case that the method is used for determining the prognosis of a breast cancer patient, the reference value is the expression level of the gene of interest in non-cancerous breast cancer tissue. In another embodiment, the reference value can correspond to an average value obtained from a pool of non-cancerous tissues. Said reference sample is typically obtained by combining equal amounts of samples from a subject population.

In another embodiment, the reference value is the expression levels of the gene or genes of interest in a pool obtained from tumor tissues obtained from patients having the same type of cancer. This pool will include patients with good prognosis and patients with bad prognosis and therefore, the expression levels would be an average value of the values found in the different types of patients.

In another embodiment, the reference value is the expression levels of the gene or genes of interest in a tumor tissue obtained from a patient or patients identified as patients having a good prognosis. In another embodiment, the reference value is the expression levels of the gene or genes of interest in a tumor tissue obtained from a patient or patients identified as patients having a bad prognosis.

In case that the reference value is the expression level of the gene or genes of interest obtained from tumor tissues from patients having a good prognosis, then patients can be identified as having poor prognosis if the expression levels are lower than the reference value. In case that the reference value is the expression level of the gene or genes of interest obtained from tumor tissues from patients having a poor prognosis, then patients can be identified as having good prognosis if the expression levels are higher than the reference value.

The sample collection from which the reference level is derived will preferably be formed by subjects suffering from the same type of cancer as the patient object of the study. Moreover, a reference value has to be established for each gene to be measured. In another embodiment, the quantity of any one or more biomarkers in a sample from a tested subject may be determined directly relative to the reference value (e.g., in terms of increase or decrease, or fold-increase or fold-decrease). Advantageously, this may allow to compare the quantity of any one or more biomarkers in the sample from the subject with the reference value (in other words to measure the relative quantity of any one or more biomarkers in the sample from the subject vis-a-vis the reference value) without the need to first determine the respective absolute quantities of said one or more biomarkers.

In another embodiment, the expression levels are normalized expression levels. The term "normalized" expression level as used herein refers to an expression level of a gene relative to the expression level of a single reference gene, or a particular set of reference genes. Suitable reference genes include, without limitation, β-2- microglobulin, ubiquitin, ribosomal protein 18S, cyclophilin A, transferrin receptor, actin, GAPDH, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (YWHAZ), HPRT, and IP08.

Once this reference value is established, the level of this marker expressed in tumor tissues from subjects can be compared with this reference value, and thus be assigned a level of "increased" or "decreased". For example, an increase in expression levels above the reference value of at least 1.1-fold, 1.5-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or even more compared with the reference value is considered as "increased" expression level. On the other hand, a decrease in expression levels below the reference value of at least 0.9-fold, 0.75-fold, 0.2-fold, 0.1 -fold, 0.05-fold, 0.025-fold, 0.02-fold, 0.01 -fold, 0.005-fold or even less compared with reference value is considered as "decreased" expression level.

In a third step, the comparison of the expression levels of the gene or genes of interest with the reference value for each gene or genes of interest allows to determine whether the patient will show a good or poor prognosis. In particular, an increase in the expression of at least one gene selected from the group consisting of MARS, SNRPB, RAEl, SNRPE and/or a decrease in the expression of the AD ARB 1 gene with respect to a reference value is indicative of a poor prognosis of the patient or a decrease in the expression of at least one gene selected from the group consisting of MARS, SNRPB, RAEl and SNRPE and/or an increase in the expression of the AD ARB 1 gene with respect to a reference value is indicative of a good prognosis of the patient.

As used herein, "good prognosis" indicates that the subject is expected (e.g. predicted) to survive and/or have no, or is at low risk of having, recurrence or distant metastases within a set time period. The term "low" is a relative term and, in the context of this application, refers to the risk of the "low" expression group with respect to a clinical outcome (recurrence, distant metastases, etc.). A "low" risk can be considered as a risk lower than the average risk for a heterogeneous cancer patient population. In the study of Paik et al. (2004), an overall "low" risk of recurrence was considered to be lower than 15 percent. The risk will also vary in function of the time period. The time period can be, for example, five years, ten years, fifteen years or even twenty years after initial diagnosis of cancer or after the prognosis is made.

As used herein, "poor prognosis" indicates that the subject is expected, i.e. predicted, to not survive and/or to have, or is at high risk of having, recurrence or distant metastases within a set time period. The term "high" is a relative term and, in the context of this application, refers to the risk of the "high" expression group with respect to a clinical outcome (recurrence, distant metastases, etc.). A "high" risk can be considered as a risk higher than the average risk for a heterogeneous cancer patient population. The risk will also vary in function of the time period. The time period can be, for example, five years, ten years, fifteen years or even twenty years of initial diagnosis of cancer or after the prognosis was made.

The person skilled in the art will understand that the determination of the prognosis is not needed to be correct for all the subjects (i.e., for 100% of the subjects). Nevertheless, the term requires enabling the identification of a statistically significant part of the subjects (for example, a cohort in a cohort study). Whether a part is statistically significant can be determined in a simple manner by the person skilled in the art using various well known statistical evaluation tools, for example, the determination of confidence intervals, determination of p values, Student's T test, Mann- Whitney test, etc. Details are provided in Dowdy and Wearden, Statistics for Research, John Wiley and Sons, New York 1983. The preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99%. The p values are preferably 0.1, 0.05, 0.01, 0.005 or 0.0001. More preferably, at least 60%, at least 70%, at least 80%> or at least 90%> of the subjects of a population can be suitably identified by the method of the present invention.

In a preferred embodiment, the method according to the invention further comprises determining a risk value based on the expression levels of the gene or genes which have been determined.

The term "risk value" or "score", as used herein, refer to a value assigned to a given combination of factors and which reflects the degree to which said combination of factors influences the probability of an outcome, such as the clinical outcome of a patient.

In a preferred embodiment, the risk value or score is calculated from the weighted expression levels of the genes assayed, where the weighted expression levels are obtained by multiplying the expression level of each gene by a weighting factor or "weight", to arrive at weighted expression levels for each of the one or more genes. In a preferred embodiment, the weight is the same for every gene. In this case, the risk value can be determined by adding a value of 1 for each gene within the group consisting of MARS, SNRPB, RAE1, SNRPE which is/are up-regulated with respect to their reference values and/or a value of 1 if the expression level of AD ARB 1 is down- regulated with respect to the reference value. This leads to a risk value which can take values from 0 to 5 wherein a risk value of 0 indicates good prognosis, a risk value of 1-3 indicates a medium prognosis and a risk value of 4 or 5 indicates a poor prognosis. It will be appreciated that the risk value can be determined based on the expression of any number of genes within the 5 genes which can be determined in the present invention. Thus, if only one is determined, the risk value can take the value of 0 or 1. If two genes are determined, the risk value can take the value of 0, 1 or 2. If three genes are determined, the risk value can take the value of 0, 1, 2 or 3. If four genes are determined, the risk value can take the value of 0, 1, 2, 3 or 4. If five genes are determined, the risk value can take the value of 0, 1, 2, 3, 4 and 5. Method for the identification of a patient suffering from cancer which requires adjuvant therapy

Around 15% of people diagnosed with lung cancer survive five years after diagnosis. Survival depends mainly on stage. In early stage NSCLC (stages I to IIIA), curative surgery is the treatment of choice. However, even after complete surgical resection, patients are at substantial risk for recurrence. Some patients might benefit from adjuvant therapy, but others would be treated unnecessarily. Thus, the method according to the present invention, by providing a reliable prognosis of patients, may also be used for deciding whether a patient would benefit from adjuvant therapy. In particular, patients with poor prognosis determined according to the first method of the invention would be candidates for adjuvant therapy (even if they are in stage IA for which adjuvant chemotherapy is usually not recommended). Conversely, patients with a good prognosis determined according to the method of the invention would not require adjuvant therapy (even if they are in stage IIA, IIB or IIIA, for which adjuvant therapy is usually recommended).

Therefore, in another aspect, the invention relates to a method (hereinafter second method of the invention) for the identification of a patient suffering from cancer which requires adjuvant therapy which comprises the determination in a sample from said patient of the expression levels of at least one gene selected from the group consisting of MARS, SNRPB, AD ARB 1 , RAE 1 and SNRPE,

wherein an increase in the expression of at least one gene selected from the group consisting of MARS, RAEl, SNRPB, SNRPE and/or a decrease in the expression of the AD ARB 1 gene with respect to a reference value is indicative that the patient requires adjuvant therapy

or

wherein a decrease in the expression of at least one gene selected from the group consisting of MARS, RAEl, SNRPB, SNRPE and/or an increase in the expression of the AD ARB 1 gene with respect to a reference value is indicative that the patient does not require adjuvant therapy.

In another embodiment, the invention relates to the use of one or more genes selected from the group consisting of MARS, SNRPB, AD ARB 1 , RAEl and SNRPE or of one or more of the polypeptides encoded by said genes from a sample obtained from the individual for the identification of a patient suffering from cancer which requires adjuvant therapy.

The term "cancer" has been defined in the context of the first method of the invention.

In a preferred embodiment, the cancer is lung cancer. In a more preferred embodiment, the lung cancer is non-small cell lung cancer (NSCLC) or small cell lung cancer. In another embodiment, the cancer is stage I lung cancer, stage II lung cancer, stage III lung cancer or stage IV lung cancer. In another embodiment, the NSCLC is stage IA NSCLC, stage IB NSCLC, stage IIA NSCLC, stage IIB NSCLC, stage IIIA NSCLC, stage IIIB and stage IV NSCLC. Stages I, II, III and IV in lung cancer are defined in Mountain CF. Revisions in the International System for Staging Lung Cancer. Chest. 1997;111 : 1710-1717.

In another embodiment, the NSCLC is stage TXNOMO (wherein X is an integer from 0 to 4), stage T1N0M0 NSCLC, Stage T2M0N0 NSCLC, stage T1N1M0 NSCLC, stage T2N1M0 NSCLC, stage T3N0M0 NSCLC, stage T1N2M0 NSCLC, stage T2N2M0 NSCLC, stage T3N1M0 NSCLC, stage T3N2M0 NSCLC, stage T4N0M0, stage T4N1M0 NSCLC, stage T1N3M0 NSCLC, stage T2N3M0 NSCLC, stage T3N3M0 NSCLC, stage T4N2M0 NSCLC, stage T4N3M0 NSCLC or stage TXNYM1, wherein X is any value from 0 to 4 and Y is any value from 0 to 3, according to the TNM classification (AJCC Cancer Staging Manual, Lippincott, 5th edition, pp. 171- 180, 1997). In another preferred embodiment, the cancer is breast cancer. In a preferred embodiment, the breast cancer is ER positive (ER+) breast cancer, ER negative (ER-) breast cancer, PR positive (PR+) breast cancer, PR negative (PR-) breast cancer, HER2 positive (HER2+) breast cancer (cancer over-expressing HER2), HER2 negative (HER2-) breast cancer (cancer expressing normal levels of HER2 or under-expressing HER2 or not expressing a detectable level of HER2), hormone receptor negative breast cancer, i.e. breast cancer with neither of estrogen nor progesterone receptors (abbreviated by ER-/PR- breast cancer); and triple negative breast cancer, i.e. breast cancer with neither of estrogen nor progesterone receptors and with normal expression/under-expression (or with the absence of detectable level of expression) of HER2 (abbreviated by ER-/PR-/HER2- breast cancer).

In another embodiment, the first method of the invention can be used for determining the prognosis of a patient suffering from luminal subtype A breast cancer, luminal subtype B breast cancer, normal-like breast cancer, HER2+ breast cancer and basal- like breast cancer.

In another embodiment, the breast cancer is stage I breast cancer, stage II breast cancer, stage III breast cancer or stage IV breast cancer. In another embodiment, the cancer is stage IA breast cancer, Stage IB breast cancer, stage IIA breast cancer, stage IIB breast cancer, stage IIIA breast cancer, stage IIIB and stage IV breast cancer. In another embodiment, the breast cancer is stage TXN0M0 breast cancer (wherein X is an integer from 0 to 4), stage T1N0M0 breast cancer, stage T2M0N0 breast cancer, stage T1N1M0 breast cancer, stage T2N1M0 breast cancer, stage T3N0M0 breast cancer, stage T1N2M0 breast cancer, stage T2N2M0 breast cancer, stage T3N1M0 breast cancer, stage T3N2M0 breast cancer, stage T4N0M0, stage T4N1M0 breast cancer, stage T1N3M0 breast cancer, stage T2N3M0 breast cancer, stage T3N3M0 breast cancer, stage T4N2M0 breast cancer, stage T4N3M0 breast cancer or stage TXNYMl breast cancer, wherein X is any value from 0 to 4 and Y is any value from 0 to 3, according to the TNM classification (AJCC Cancer Staging Manual, Lippincott, 5 ^th edition, pp. 171-180, 1997).

In another embodiment, the breast cancer is lymph-node negative breast cancer, i.e. a breast cancer that has not spread to the lymph node. In a preferred embodiment, the patient has not been treated with adjuvant therapy, such as chemotherapy or radiotherapy prior to the determination of the expression levels of the gene or genes of interest. In another preferred embodiment, the patient has undergone surgical resection of the tumor.

The term "adjuvant chemotherapy" as used herein means treatment of cancer with standard chemotherapeutic agents after surgery where all detectable disease has been removed, but where there still remains a risk of small amounts of remaining cancer. Adjuvant therapy can include chemotherapy or radiotherapy.

The term "chemotherapy" refers to the use of drugs to destroy cancer cells. The drugs are generally administered through oral or intravenous route. Sometimes, chemotherapy is used together with radiation treatment.

Suitable chemotherapeutic treatments for breast cancer include, without limitation, anthracyclines (doxorubicin, epirubicin, pegylated liposomal doxorubicin), Taxanes (paclitaxel, docetaxel, albumin nano-particle bound paclitaxel), 5-fluorouracil (continuous infusion 5-FU, capecitabine), Vinca alkaloids (vinorelbine, vinblastine), Gemcitabine, Platinum salts (cisplatin, carboplatin), cyclophosphamide, Etoposide and combinations of one or more of the above such as Cyclophosphamide/anthracycline +/- 5-fluorouracil regimens (such as doxorubicin/ cyclophosphamide (AC), epirubicin/cyclophosphamide, (EC) cyclophosphamide/epirubicin/5-fluorouracil (CEF), cyclophosphamide/doxorubicin/5-fluorouracil (CAF), 5-fluorouracil

/ epirubicin/ cyclophosphamide (FEC)), cyclophosphamide/metothrexate/ 5-fluorouracil (CMF), anthracyclines/taxanes (such as doxorubicin/paclitaxel or doxorubicin/docetaxel), Docetaxel/capecitabine, Gemcitabine/paclitaxel,

Taxane/platinum regimens (such as paclitaxel/carboplatin or docetaxel/carboplatin).

Suitable chemotherapeutic treatments for lung cancer include, without limitation, platinum-based drugs (either cisplatin or carboplatin), etoposide, gemcitabine, paclitaxel, docetaxel, cisplatin or carboplatin, in combination with gemcitabine, paclitaxel, docetaxel, etoposide, or vinorelbine, pemetrexed.

The term "radiotherapy" or "radiotherapeutic treatment" is a term commonly used in the art to refer to multiple types of radiation therapy including internal and external radiation therapies or radio immunotherapy, and the use of various types of radiations including X-rays, gamma rays, alpha particles, beta particles, photons, electrons, neutrons, radioisotopes, and other forms of ionizing radiations.

In a first step, the second method of the invention comprises the determination in a sample from said patient of the expression levels of at least one gene selected from the group consisting of the MARS, SNRPB, ADARBl, RAEl and SNRPE genes.

The terms "MARS", "SNRPB", "ADARBl", "RAEl" and "SNRPE" have been described in detail in the context of the first method of the invention.

The term "at least a gene selected from the group consisting of the MARS, SNRPB, ADARBl, RAEl and SNRPE", as used herein, means that the method may involve the determination of the expression levels of 1, 2, 3, 4 or 5 genes. In a preferred embodiment, the second method comprises the determination of the expression levels of the MARS gene, of the SNRPB gene, of the ADARBl gene, of the RAEl gene or of the SNRPE gene. In another embodiment, the second method of the invention comprises determining the expression levels of two of the above genes. Suitable combinations of two genes which can be measured include, MARS and SNRPB, MARS and ADARBl, MARS and RAEl, MARS and SNRPE, SNRPB and ADARBl, SNRPB and RAEl, SNRPB and SNRPE, ADARBl and RAEl, ADARBl and SNRPE, RAEl and SNRPE. In another embodiment, the second method of the invention comprises the determination of the expression levels of three of the above genes. Suitable combinations of three genes include MARS, SNRPB and ADARBl; MARS, SNRPB and RAEl; MARS, SNRPB and SNRPE; MARS, ADARBl and RAEl; MARS, ADARBl and SNRPE; MARS, RAEl and SNRPE; SNRPB, ADARBl and RAEl; SNRPB, ADARBl and SNRPE; SNRPB, RAEl and SNRPE; and ADARBl, RAEl and SNRPE. In another embodiment, the second method of the invention comprises the determination of the expression levels of four of the above genes. Suitable combinations of four genes include MARS, SNRPB, ADARBl and RAEl; MARS, SNRPB, ADARBl and SNRPE; MARS, SNRPB, RAEl, SNRPE; MARS, ADARBl, RAEl and SNRPE; and SNRPB, ADARBl, RAEl and SNRPE. In yet another embodiment, the second method of the present invention comprises the determination of the expression levels of the MARS, SNRPB, AD ARB 1 , RAE 1 and the SNRPE genes. In a preferred embodiment, the sample is a tumor tissue sample, which may be either a biopsy of the tumor or a fragment of the tumor obtained after the tumor has been surgically resected.

In a preferred embodiment, determination of the expression levels of the gene or genes of interest is carried out by determination of the levels of the corresponding mRNAs. In another embodiment, determination of the expression levels of the gene or genes of interest is carried out by determination of the levels of the corresponding polypeptides encoded by said genes.

In a preferred embodiment, the second method of the invention further comprises the determination of the expression levels of one or more cancer marker genes and/or the determination of one or more clinical parameters which are also indicative of the prognosis of the cancer. Such indicators include the presence or levels of known cancer markers, or can be clinical or pathological indicators (for example, age, tumor size, tumor histology, differentiation grade, clinical stage, family history and the like). Suitable clinical and molecular markers for lung and breast cancer have been defined in the context of the first method of the invention.

In a second step, the second method of the invention comprises correlating the expression levels of the genes with a reference value.

The term "reference value" has been described in the context of the first method of the invention. In a preferred embodiment, the reference value is the expression level of the gene of interest in a non-cancerous tissue. Thus, in the case of lung cancer, the reference value corresponds to the expression levels of the gene of interest in a healthy lung tissue. In the case of breast cancer, the reference value corresponds to the expression levels of the gene of interest in a healthy breast tissue.

In another embodiment, the reference value is the expression levels of the gene or genes of interest in a pool obtained from tumor tissues obtained from patients having the same type of cancer. In another embodiment, the reference value is the expression levels of the gene or genes of interest in a tumor tissue obtained from a patient or patients identified as patients having a good prognosis. In another embodiment, the reference value is the expression levels of the gene or genes of interest in a tumor tissue obtained from a patient or patients identified as patients having a bad prognosis. Once this reference value is established, the level of this marker expressed in tumor tissues from subjects can be compared with this reference value, and thus be assigned a level of "increased" or "decreased". For example, an increase in expression levels above the reference value of at least 1.1-fold, 1.5-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or even more compared with the reference value is considered as "increased" expression level. On the other hand, a decrease in expression levels below the reference value of at least 0.9-fold, 0.75-fold, 0.2-fold, 0.1 -fold, 0.05-fold, 0.025-fold, 0.02-fold, 0.01 -fold, 0.005-fold or even less compared with reference value is considered as "decreased" expression level In a third step, the second method of the invention comprising selecting a patient for treatment with adjuvant therapy depending on the comparison of the expression levels of the gene or genes of interest with the reference value for each gene of interest. In particular, an increase in the expression of at least one gene selected from the group consisting of MARS, SNRPB, RAE1, SNRPE and/or a decrease in the expression of the AD ARB 1 gene with respect to a reference value is indicative that the patient requires adjuvant therapy or a decrease in the expression of at least one gene selected from the group consisting of MARS, SNRPB, RAEl and SNRPE and/or an increase in the expression of the AD ARB 1 gene with respect to a reference value is indicative that the patient does not require adjuvant therapy.

In a preferred embodiment, the second method of the invention further comprises determining a risk value based on the expression levels of the gene or genes which have been determined. In another preferred embodiment, the risk value is calculated by multiplying values of the expression levels of each gene to a weighting factor or "weight" specific for each gene, to arrive at weighted expression levels for each of the one or more genes. In a preferred embodiment, the weight is the same for every gene. In yet another embodiment, the weighting factors for all genes is the same. In this case, the risk value can be determined by adding a value of 1 for each gene within the group consisting of MARS, SNRPB, RAEl, SNRPE which is/are up-regulated with respect to the reference value and/or a value of 1 if the expression level of AD ARB 1 is down- regulated with respect to a reference value. This leads to a risk value which can take values from 0 to 5 wherein a risk value of 0 indicates that the patient does not require adjuvant therapy and a risk value of 4 or 5 indicates that the patient requires adjuvant therapy. It will be appreciated that the risk value can be determined based on the expression of any number of genes within the 5 genes which can be determined in the present invention. Thus, if only one is determined, the risk value can take the value of 0 or 1. If two genes are determined, the risk value can take the value of 0, 1 or 2. If three genes are determined, the risk value can take the value of 0, 1 , 2 or 3. If four genes are determined, the risk value can take the value of 0, 1, 2, 3 or 4. If five genes are determined, the risk value can take the value of 0, 1, 2, 3, 4 and 5.

Method for personalized therapy

In another aspect, the invention allows applying personalized therapy to patients based on the identification of the patient as a patient who might benefit from the adjuvant therapy according to the second method of the invention. Thus, in another aspect, the invention relates to a method for the treatment of patients suffering from cancer with adjuvant therapy which comprises the administration to said patient of said adjuvant therapy, wherein the patient has been selected using a method according to the second method of the invention.

In another aspect, the invention relates to an adjuvant therapy for use in the treatment or prevention of cancer in a subject wherein the subject has been selected using a method according to the second method of the invention.

The term "treatment", as used herein, refers to any type of therapy, which is aimed at terminating, preventing, ameliorating or reducing the susceptibility to a clinical condition as described herein. In a preferred embodiment, the term treatment relates to prophylactic treatment (i.e. a therapy to reduce the susceptibility to a clinical condition), of a disorder or a condition as defined herein. Thus, "treatment," "treating," and their equivalent terms refer to obtaining a desired pharmacologic or physiologic effect, covering any treatment of a pathological condition or disorder in a mammal, including a human. The effect may be prophylactic in terms of completely or partially preventing a disorder or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disorder and/or adverse effect attributable to the disorder. That is, "treatment" includes (1) preventing the disorder from occurring or recurring in a subject, (2) inhibiting the disorder, such as arresting its development, (3) stopping or terminating the disorder or, at least, symptoms associated therewith, so that the host no longer suffers from the disorder or its symptoms, such as causing regression of the disorder or its symptoms, for example, by restoring or repairing a lost, missing or defective function, or stimulating an inefficient process, or (4) relieving, alleviating, or ameliorating the disorder, or symptoms associated therewith, where ameliorating is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, such as inflammation, pain, or immune deficiency.

When the cancer has metastasized, systemic treatments including but not limited to chemotherapy, hormone treatment, immunotherapy, or a combination thereof are used. Additionally, radiotherapy and/or surgery can be used. The choice of treatment generally depends on the type of primary cancer, the size, the location of the metastasis, the age, the general health of the patient and the types of treatments used previously.

In a preferred embodiment, the cancer is lung cancer. In a more preferred embodiment, the lung cancer is non-small cell lung cancer (NSCLC) or small cell lung cancer. In another embodiment, the NSCLC is stage IA NSCLC, stage IB NSCLC, stage IIA NSCLC, stage IIB NSCLC, stage IIIA NSCLC, stage IIIB and stage IV NSCLC. Stages I, II, III and IV in lung cancer are defined in Mountain CF. Revisions in the International System for Staging Lung Cancer. Chest. 1997;111 : 1710-1717.

In another embodiment, the NSCLC is stage TXN0M0 (wherein X is an integer from 0 to 4), stage T1N0M0 NSCLC, Stage T2M0N0 NSCLC, stage T1N1M0 NSCLC, stage T2N1M0 NSCLC, stage T3N0M0 NSCLC, stage T1N2M0 NSCLC, stage T2N2M0 NSCLC, stage T3N1M0 NSCLC, stage T3N2M0 NSCLC, stage T4N0M0, stage T4N1M0 NSCLC, stage T1N3M0 NSCLC, stage T2N3M0 NSCLC, stage T3N3M0 NSCLC, stage T4N2M0 NSCLC, stage T4N3M0 NSCLC or stage TXNYMl, wherein X is any value from 0 to 4 and Y is any value from 0 to 3, according to the TNM classification (AJCC Cancer Staging Manual, Lippincott, 5th edition, pp. 171- 180, 1997).

In another preferred embodiment, the cancer is breast cancer. In a preferred embodiment, the breast cancer is ER positive (ER+) breast cancer, ER negative (ER-) breast cancer, PR positive (PR+) breast cancer, PR negative (PR-) breast cancer, HER2 positive (HER2+) breast cancer (cancer over-expressing HER2), HER2 negative (HER2-) breast cancer (cancer expressing normal levels of HER2 or under-expressing HER2 or not expressing a detectable level of HER2), hormone receptor negative breast cancer, i.e. breast cancer with neither of estrogen nor progesterone receptors (abbreviated by ER-/PR- breast cancer); and triple negative breast cancer, i.e. breast cancer with neither of estrogen nor progesterone receptors and with normal expression/under-expression (or with the absence of detectable level of expression) of HER2 (abbreviated by ER-/PR-/HER2- breast cancer).

In another embodiment, the third method of the invention can be used for determining the prognosis of a patient suffering from luminal subtype A breast cancer, luminal subtype B breast cancer, normal-like breast cancer, HER2+ breast cancer and basal- like breast cancer.

In another embodiment, the breast cancer is Stage IA breast cancer, Stage IB breast cancer, stage IIA breast cancer, stage IIB breast cancer, stage IIIA breast cancer, stage IIIB and stage IV breast cancer. In another embodiment, the breast cancer is stage TXN0M0 breast cancer (wherein X is an integer from 0 to 4), stage T1N0M0 breast cancer, stage T2M0N0 breast cancer, stage T1N1M0 breast cancer, stage T2N1M0 breast cancer, stage T3N0M0 breast cancer, stage T1N2M0 breast cancer, stage T2N2M0 breast cancer, stage T3N1M0 breast cancer, stage T3N2M0 breast cancer, stage T4N0M0, stage T4N1M0 breast cancer, stage T1N3M0 breast cancer, stage T2N3M0 breast cancer, stage T3N3M0 breast cancer, stage T4N2M0 breast cancer, stage T4N3M0 breast cancer or stage TXNYM1 breast cancer, wherein X is any value from 0 to 4 and Y is any value from 0 to 3, according to the TNM classification (AJCC Cancer Staging Manual, Lippincott, 5 ^th edition, pp. 171-180, 1997).

In another embodiment, the breast cancer is lymph-node negative breast cancer, i.e. a breast cancer that has not spread to the lymph node.

In a preferred embodiment, the patient has not been treated with chemotherapy or radiotherapy prior to the determination of the expression levels of the gene or genes of interest. In another preferred embodiment, the patient has undergone surgical resection of the tumor.

The different preferred embodiments of the third method of the invention apply equally to the method of personalized therapy according to the invention. In another embodiment, the adjuvant therapy is chemotherapy, radiotherapy or a combination thereof. Suitable chemotherapeutic treatments are indicated in the second method of the invention.

In a preferred embodiment, the patients who show an increase in the expression of at least one gene selected from the group consisting of MARS, SNRPB, RAE1, SNRPE and/or a decrease in the expression of the AD ARB 1 gene with respect to a reference value are treated with adjuvant therapy. In a preferred embodiment, the patients are treated with adjuvant therapy based on a risk value which is determined based on the expression levels of the gene or genes which have been determined. In another preferred embodiment, the risk value is calculated by multiplying values of the expression levels of each gene to a weighting factor or "weight" specific for each gene, to arrive at weighted expression levels for each of the one or more genes. In a preferred embodiment, the weight is the same for every gene. In yet another embodiment, the weighting factors for all genes is the same. In this case, the risk value can be determined by adding a value of 1 for each gene within the group consisting of MARS, SNRPB, RAEl, SNRPE which is/are up-regulated with respect to the reference value and/or a value of 1 if the expression level of AD ARB 1 is down-regulated with respect to a reference value. This leads to a risk value which can take values from 0 to 5 wherein a risk value of 0 indicates that the patient does not require adjuvant therapy and a risk value of 4 or 5 indicates that the patient requires adjuvant therapy. It will be appreciated that the risk value can be determined based on the expression of any number of genes within the 5 genes which can be determined in the present invention. Thus, if only one is determined, the risk value can take the value of 0 or 1. If two genes are determined, the risk value can take the value of 0, 1 or 2. If three genes are determined, the risk value can take the value of 0, 1 , 2 or 3. If four genes are determined, the risk value can take the value of 0, 1 , 2, 3 or 4. If five genes are determined, the risk value can take the value of 0, 1, 2, 3, 4 and 5.

Kits and assay devices

In another embodiment, the invention relates to a kit or assay device comprising reagents adequate for the determination of the expression levels of at least two genes selected from the group consisting of MARS, SNRPB, AD ARB 1 , RAE1 and SNRPE wherein said reagents comprise at least 10% of the reagents present in the kit.

In the context of the present invention, "kit" or "assay device" is understood as a product or device containing the different reagents necessary for carrying out the methods of the invention packed so as to allow their transport and storage. Materials suitable for packing the components of the kit include crystal, plastic (polyethylene, polypropylene, polycarbonate and the like), bottles, vials, paper, envelopes and the like. Additionally, the kits of the invention can contain instructions for the simultaneous, sequential or separate use of the different components which are in the kit. Said instructions can be in the form of printed material or in the form of an electronic support capable of storing instructions such that they can be read by a subject, such as electronic storage media (magnetic disks, tapes and the like), optical media (CD-ROM, DVD) and the like. Additionally or alternatively, the media can contain Internet addresses that provide said instructions.

The expression "reagent which allows determining the expression level of a gene" means a compound or set of compounds that allows determining the expression level of a gene both by means of the determination of the level of mRNA or by means of the determination of the level of protein. Thus, reagents of the first type include probes capable of specifically hybridizing with the mRNAs encoded by said genes. Reagents of the second type include compounds that bind specifically with the proteins encoded by the marker genes and preferably include antibodies, although they can be specific aptamers.

The reagents for use in the first or second methods of the invention or in the method for personalized therapy according to the invention may be formulated as a "kit" and thus, may be combined with one or more other types of elements or components (e.g., other types of biochemical reagents, containers, packages such as packaging intended for commercial sale, substrates to which the reagents are attached, electronic hardware components, etc.).

The term "at least two genes selected from the group consisting of MARS, SNRPB, AD ARB 1 , RAE1 and SNRPE", as used herein, means that the kit or assay device may contain reagents suitable for the determination of the expression levels of at least 2, 3, 4 or 5 of the above genes. Suitable combinations of two genes which can be measured include, MARS and SNRPB, MARS and ADARBl, MARS and RAEl, MARS and SNRPE, SNRPB and ADARBl, SNRPB and RAEl, SNRPB and SNRPE, ADARBl and RAEl, ADARBl and SNRPE, RAEl and SNRPE. In another embodiment, the kit or assay device of the invention comprises the reagents suitable for the determination of the expression levels of three of the above genes. Suitable combinations of three genes include MARS, SNRPB and ADARBl; MARS, SNRPB and RAEl; MARS, SNRPB and SNRPE; MARS, ADARBl and RAEl; MARS, ADARBl and SNRPE; MARS, RAEl and SNRPE; SNRPB, ADARBl and RAEl; SNRPB, ADARBl and SNRPE; SNRPB, RAEl and SNRPE; and ADARBl, RAEl and SNRPE. In another embodiment, the kit or assay device of the invention comprises reagents for the determination of the expression levels of four of the above genes. Suitable combinations of four genes include MARS, SNRPB, ADARBl and RAEl; MARS, SNRPB, ADARBl and SNRPE; MARS, SNRPB, RAEl, SNRPE; MARS, ADARBl, RAEl and SNRPE; and SNRPB, ADARBl, RAEl and SNRPE. In yet another embodiment, the kit or assay device of the invention comprises reagents adequate for the determination of the expression levels of the MARS, SNRPB, ADARBl, RAEl and the SNRPE genes.

In a preferred embodiment, the reagents adequate for the determination of the expression levels of one or more genes comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% of the total amount of reagents adequate for the determination of the expression levels of genes forming the kit. Thus, in the particular case of kits comprising reagents for the determination of the expression levels of the MARS, SNRPB, ADARBl, RAEl and SNRPE genes, the reagents specific for said gene (i.e. probes which are capable of hybridizing under stringent conditions to the MARS, SNRPB, ADARBl, RAEl and SNRPE genes) comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% of the probes present in the kit. In further embodiments, the reagents adequate for the determination of the expression levels of one or more genes comprise at least 55% at least 60%>, at least 65%, at least 70%>, at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the total amount of reagents forming the kit. In a particular embodiment of the kit of the invention, the reagents of the kit are nucleic acids which are capable of specifically detecting the mR A level of the genes mentioned above and/or the level of proteins encoded by one or more of the genes mentioned above. Nucleic acids capable of specifically hybridizing with the genes mentioned above can be one or more pairs of primer oligonucleotides for the specific amplification of fragments of the mRNAs (or of their corresponding cDNAs) of said genes.

In a preferred embodiment, the first component of the kit of the invention comprises probes which can specifically hybridize to the genes mentioned above.

The term "specifically hybridizing", as used herein, refers to conditions which allow hybridizing of two polynucleotides under high stringent conditions or moderately stringent conditions.

"Stringency" of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and the hybridizable sequence, the higher the relative temperature which can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al, Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

"Stringent conditions" or "high stringency conditions", as defined herein, typically: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Fico 11/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) employ 50%> formamide, 5xSSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50 μ§/ι 1), 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2xSSC (sodium chloride/sodium citrate) and 50%> formamide, followed by a high- stringency wash consisting of O. lxSSC containing EDTA at 55 °C.

"Moderately stringent conditions" may be identified as described by Sambrook et al, Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and % SDS) less stringent that those described above. An example of moderately stringent conditions is overnight incubation at 37°C. in a solution comprising: 20% formamide, 5xSSC (150 mMNaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10%> dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in lxSSC at about 37-50°C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.

In the event that the expression levels of several of the genes identified in the present invention are to be simultaneously determined, it is useful to include probes for all the genes the expression of which is to be determined in a microarray hybridization.

The microarrays comprise a plurality of nucleic acids that are spatially distributed and stably associated to a support (for example, a bio chip). The nucleic acids have a sequence complementary to particular subsequences of genes the expression of which is to be detected, therefore are capable of hybridizing with said nucleic acids. In the methods of the invention, a microarray comprising an array of nucleic acids is put into contact with a preparation of nucleic acids isolated from the patient object of the study. The incubation of the microarray with the preparation of nucleic acids is carried out in conditions suitable for the hybridization. Subsequently, after the elimination of the nucleic acids which have not been retained in the support, the hybridization pattern is detected, which provides information on the genetic profile of the sample analyzed. Although the microarrays are capable of providing both qualitative and quantitative information of the nucleic acids present in a sample, the invention requires the use of arrays and methodologies capable of providing quantitative information.

The invention contemplates a variety of arrays with regard to the type of probes and with regard to the type of support used. The probes included in the arrays that are capable of hybridizing with the nucleic acids can be nucleic acids or analogs thereof which maintain the hybridization capacity such as for example, nucleic acids in which the phosphodiester bond has been substituted with a phosphorothioate, methylimine, methylphosphonate, phosphoramidate, guanidine bond and the like, nucleic acids in which the ribose of the nucleotides is substituted with another hexose, peptide nucleic acids (PNA). The length of the probes can of 5 to 50 nucleotides and, preferably, of 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 100 nucleotides and vary in the range of 10 to 1000 nucleotides, preferably in the range of 15 to 150 nucleotides, more preferably in the range of 15 to 100 nucleotides and can be single-stranded or double- stranded nucleic acids. The array can contain all the specific probes of a certain mR A of a certain length or can contain probes selected from different regions of an mRNA. Each probe is assayed in parallel with a probe with a changed base, preferably in a central position of the probe. The array is put into contact with a sample containing nucleic acids with sequences complementary to the probes of the array and the signal of hybridization with each of the probes and with the corresponding hybridization controls is determined. Those probes in which a higher difference is observed between the signal of hybridization with the probe and its hybridization control are selected. The optimization process can include a second round of optimization in which the hybridization array is hybridized with a sample that does not contain sequences complementary to the probes of the array. After the second round of selection, those probes having signals of hybridization lower than a threshold level will be selected. Thus, probes which pass both controls, i.e., which show a minimum level of unspecific hybridization and a maximum level of specific hybridization with the target nucleic acid are selected.

The microarrays of the invention contain not only specific probes for the polynucleotides indicating a determined pathophysiological situation, but also containing a series of control probes, which can be of three types: normalization controls, expression level controls and hybridization controls.

Probes suitable for use as expression controls correspond to genes expressed constitutively, such as genes encoding proteins which exert essential cell functions such as β-2-microglobulin, ubiquitin, ribosomal protein 18S, cyclophilin A, transferrin receptor, actin, GAPDH, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (YWHAZ), HPRT, and IP08.

Once a set of probes showing the suitable specificity and a set of control probes are provided, the latter are arranged in the array in a known position such that, after the steps of hybridization and of detection, it is possible to establish a correlation between a positive signal of hybridization and the particular gene from the coordinates of the array in which the positive signal of hybridization is detected.

The microarrays can be high density arrays with thousands of oligonucleotides by means of photolithographic in situ synthesis methods (Fodor et al, 1991, Science, 767-773). This type of probe is usually redundant, i.e., they include several probes for each mR A which is to be detected. In a preferred embodiment, the arrays are low density arrays or LDA containing less than 10000 probes per square centimeter. In said low density arrays, the different probes are manually applied with the aid of a pipette in different locations of a solid support (for example, a crystal surface, a membrane). The supports used to fix the probes can be obtained from a large variety of materials, including plastic, ceramics, metals, gels, membranes, crystals and the like. The microarrays can be obtained using any methodology known for the person skilled in the art.

In the event that the expression levels of the genes according to the present invention is determined by measuring the levels of the polypeptide or polypeptides encoded by said gene or genes, the kits according to the present invention comprise reagents which are capable of specifically binding to said polypeptide or polypeptides. For this purpose, the arrays of antibodies such as those described by De Wildt et al. (2000) Nat. Biotechnol. 18:989-994; Lueking et al. (1999) Anal. Biochem. 270: 103- 111; Ge et al. (2000) Nucleic Acids Res. 28, e3, 1- VII; MacBeath and Schreiber (2000) Science 289: 1760-1763; WO 01/40803 and WO 99/51773A1 are useful. The antibodies of the array include any immunological agent capable of binding to a ligand with high affinity, including IgG, IgM, IgA, IgD and IgE, as well as molecules similar to antibodies which have an antigen binding site, such as Fab', Fab, F(ab')2, single domain antibodies or DABS, Fv, scFv and the like. The techniques for preparing said antibodies are very well known for the person skilled in the art and include the methods described by Ausubel et al. (Current Protocols in Molecular Biology, eds. Ausubel et al, John Wiley & Sons (1992)). The antibodies of the array can be applied at high speed, for example, using commercially available robotic systems (for example, those produced by Genetic Microsystems or Biorobotics). The substrate of the array can be nitrocellulose, plastic, crystal or can be of a porous material as for example, acrylamide, agarose or another polymer. In another embodiment, it is possible to use cells producing the specific antibodies for detecting the proteins of the invention by means of their culture in array filters. After the induction of the expression of the antibodies, the latter are immobilized in the filter in the position of the array where the producing cell was located. An array of antibodies can be put into contact with a labeled target and the binding level of the target to the immobilized antibodies can be determined. If the target is not labeled, a sandwich type assay can be used in which a second labeled antibody specific for the polypeptide which binds to the polypeptide which is immobilized in the support is used. The quantification of the amount of polypeptide present in the sample in each point of the array can be stored in a database as an expression profile. The array of antibodies can be produced in duplicate and can be used to compare the binding profiles of two different samples.

In another aspect, the invention relates to the use of a kit of the invention for determination of the prognosis of a patient suffering from cancer or for the identification of a patient suffering from cancer which requires adjuvant therapy. In a preferred embodiment the cancer is lung or breast cancer.

Computer systems and programs

In another aspect, the invention relates to a computer system that is provided with means for implementing the methods according to the first, second or third method of the invention. In another aspect, the invention relates to a computer program comprising a programming code to execute the steps of the methods according to the invention if carried out in a computer. In yet another aspect, the invention relates to a computer-readable data medium comprising a computer program according to the invention in the form of a computer-readable programming code.

The term "computer-readable medium" may refer to any storage device used for storing data accessible by a computer, as well as any other means for providing access to data by a computer. Examples of a storage device-type computer-readable medium include: a magnetic hard disk; a floppy disk; an optical disk, such as a CD-ROM and a DVD; a magnetic tape; a memory chip.

The term "software" is used interchangeably herein with "program" and refers to prescribed rules to operate a computer. Examples of software include: software; code segments; instructions; computer programs; and programmed logic.

The term a "computer system" may refer to a system having a computer, where the computer comprises a computer-readable medium embodying software to operate the computer.

In another aspect, the invention relates to a computer system that is provided with means for implementing the first or second according to the invention. The computer system can include:

(a) at least one memory containing at least one computer program adapted to control the operation of the computer system to implement a method that includes: (i) receiving gene expression data e.g., the expression data of a tumor biopsy sample (mRNA levels or protein levels) and the expression data of the same genes in a reference sample, (ii) comparing the expression levels of the different genes in the tumor biopsy and the reference sample.

(b) at least one processor for executing the computer program.

Another aspect of the present invention relates to a computer program for controlling a computer system to execute the steps according to the first or second method of the invention.

The following invention is hereby described by way of the following examples, which are to be construed as merely illustrative and not limitative of the scope of the invention.

Example 1. Expression of RNA metabolism-related genes and lung cancer clinical outcome

The association of the expression of different genes with clinical outcomes in patients with lung cancer was investigated. Kaplan-Meier plots were used to illustrate differences in progression according to the mRNA levels of the selected genes. Significant differences in survival were analyzed using the log-rank test. mRNA expression data were obtained from an extensive study of lung adenocarcinomas (Shedden et al, 2008, Nat Med 14: 822-827). Patients with adjuvant chemo- or radiotherapy were excluded. After this selection, information about survival and gene expression was available from 213 patients. Clinicopathological features of these patients are shown in Table 1. Patients were divided according to high and low mRNA expression levels using the median as the cutoff point. Overall survival (censored at 60 months) was used as the outcome variable.

Table 1. Demographic and clinical characteristics of the 213 patients selected from the cohort of adenocarcinoma patients (Shedden et al, 2008, Nat Med 14: 822-827)

Age - years

Median 65

SD 10

Gender - n (%)

Female 116 (54%)

Male 97 (46%)

Smoking status - n (%)

Nonsmoker 27 (13%)

Current 19 (9%)

Former 160 (78%)

NA 7

Stage - n (%)

I 157 (74%)

II 35 (17%)

III 19 (9%)

IV 0 (0%)

NA 2

NA: not available

The expression of the five deregulated genes correlated with prognosis: high expression of MARS, RAEl, SNRPB, and SNRPE was significantly associated with reduced overall survival (Figures 1-4); and high expression of AD ARB 1 was significantly associated with a better outcome (Figure 5). Probesets analyzed for these genes were: 201475_s_at (MARS), 211318_s_at (RAEl), 208821_s_at (SNRPB), 203316_s_at (SNRPE); and 203865_s_at (ADARB1). Example 2. Generation of a gene signature that correlates with the clinical outcome of lung cancer patients For a combined analysis of the five prognostic genes, a score was assigned based on the number of deregulating events in the tumor. A deregulating event was defined by a high expression of an up-regulated gene (MARS, RAEl, SNRPB or SNRPE) or a low expression of the down-regulated one (ADARBl). Therefore, scores ranged between 0 and 5. Patients were divided into three groups: patients with score 0, patients with score 1-3, and patients with score 4-5. Kaplan-Meier plots were used to illustrate differences in progression according to the three groups. Significant differences in survival were analyzed using the log-rank test. Overall survival and disease-free survival (censored at 60 months) were used as the outcome variable.

The combined score of the five genes was a strong prognostic marker for both overall survival (Figure 6) and disease-free survival (Figure 7). Thus, patients with no deregulating events exhibited very good prognosis. In contrast, patients with deregulation in four or five of the genes exhibited the worst prognosis. Interestingly, the combined score was able to predict survival when the patients were divided by stages I (Figure 8), II (Figure 9) and III (Figure 10).

The Cox proportional hazards model was also used, in both univariate and multivariate analyses. Results clearly showed that the five-gene signature is an independent prognostic marker for both overall survival (Table 2) and disease-free survival (Table 3).

Table 2. Cox proportional-hazards models for association of the five-gene signature and overall survival of patients with lung adenocarcinoma (Shedden et al, 2008, Nat Med 14: 822-827)

Hazard ratio (95% CI) p

Univariate analysis

Age

<70

>70 1.363 (0.831-2.236) 0.220

Gender

Female

Male 1.221 (0.756-1.972) 0.413

Smoking status 0.435

Never

Former 1.273 (0.578-2.808) 0.549

Current 1.904 (0.690-5.252) 0.214

Stage <0.001

I

II 3.073 (1.737-5.437) <0.001

III 5.742 (3.013-10.943) <0.001

Prognostic score <0.001

0

1-3 7.487 (1.022-54.850) 0.048

4-5 16.028 (2.195-117.025) 0.006

Multivariate analysis

Prognostic score* <0.001

0

1-3 8.490 (1.154-62.443) 0.036

4-5 22.703 (3.062-168.343) 0.002

^Adjusted by stage

Table 3. Cox proportional- hazards models for association of the five-gene signature and disease-free survival of patients with lung adenocarcinoma (Shedden et al, 2008, Nat Med 14: 822-827) Hazard ratio (95% CI) p

Univariate analysis

Gender

Female

Male 1.214 (0.735-2.005) 0.450

Age

<70

>70 0.841 (0.469-1.506) 0.560

Smoking status 0.401

Never

Former 1.621 (0.694-3.790) 0.265

Current 2.090 (0.701-6.224) 0.186

Stage <0.001

I

II 3.115 (1.743-5.568) <0.001

III 2.041 (0.858-4.859) 0.107

Prognostic score 0.038

0

1-3 2.200 (0.775-6.248) 0.139

4-5 3.435 (1.201-9.829) 0.021

Multivariate analysis

Prognostic score* 0.032

0

1-3 2.072 (0.728-5.901) 0.172

4-5 3.452 (1.195-9.975) 0.022

* Adjusted by stage

Example 3. Independent validation of the prognostic signature

The prognostic performance of the combined expression of the five genes was validated using mRNA expression data from an independent series of lung adenocarcinomas (Okayama et al, 2012, Cancer Res. 72: 100-11). Patients with incomplete resection or adjuvant therapy were excluded. After the selection, information about survival and gene expression was available from 204 patients. Clinicopatho logical features of these patients are shown in Table 4. Overall survival and disease-free survival were used as the outcome variable. Patients were divided according to the prognostic score in three groups: 0, 1-3, and 4-5. Table 4. Demographic and clinical characteristics of the 213 patients selected from the cohort of lung adenocarcinoma patients (Okayama et al, 2012, Cancer Res. 72: 100-11)

Age - years

Median 60

SD 8

Gender - n (%)

Female 109 (53%)

Male 95 (47%)

Smoking status - n (%)

Never-smoker 105 (52%)

Ever-smoker 99 (48%)

Stage - n (%)

IA 109 (53%)

IB 53 (26%)

II 42 (21%)

The combined score of the five genes was a strong prognostic marker for both overall survival (Figure 11) and disease-free survival (Figure 12). The Cox proportional hazards also showed that the combined expression of the five genes is an independent prognostic marker for overall survival (Table 5) and disease-free survival (Table 6). These results fully validate the utility, in lung adenocarcinoma patients, of the prognostic signature based on the mRNA expression of MARS, RAEl, SNRPB, SNRPE, and AD ARB 1.

Table 5. Cox proportional- hazards models for association of the five-gene signature and overall survival of patients with lung adenocarcinoma (Okayama et al, 2012, Cancer Res. 72: 100-11)

Hazard ratio (95% CI) p

Univariate analysis

Gender

Female

Male 1.686 (0.818-3.476) 0.157

Age

<70

>70 4.563 (1.380-15.090) 0.013

Smoking status

Never

Ever 1.908 (0.918-3.966) 0.084

Stage <0.001

IA

IB 2.098 (0.787-5.597) 0.139

II 5.831 (2.438-13.942) <0.001

Prognostic score 0.011

0

1-3 2.845 (0.370-21.886) 0.048

4-5 7.493 (0.977-56.331) 0.050

Multivariate analysis

Prognostic score* 0.054

0

1-3 2.037 (0.258-16.087) 0.500

4-5 4.752 (0.616-36.675) 0.135

* Adjusted by stage and age

Table 6. Cox proportional-hazards models for association of the five-gene signature and disease-free survival of patients with lung adenocarcinoma (Okayama et al, 2012, Cancer Res. 72: 100-11)

Hazard ratio (95% CI) p ^~

Univariate analysis

Gender

Female

Male 1.397 (0.819-2.384) 0.220

Age

<70

>70 2.363 (0.736-7.584) 0.148

Smoking status 0.401

Never

Ever 1.430 (0.837-2.443) 0.190

Stage <0.001

IA

IB 2.970 (1.508-5.849) 0.002

II 5.486 (2.801-10.741) <0.001

Prognostic score <0.001

0

1-3 5.959 (0.805-44.128) 0.139

4-5 15.633 (2.131-114.690) 0.021

Multivariate analysis*

Prognostic score 0.003

0

1-3 5.981 (0.804-44.467) 0.081

4-5 12.519 (1.687-92.914) 0.013

* Adjusted by stage

Example 4. Application of the signature to other cancers

The prognostic performance of the combined expression of the five genes was also evaluated in breast cancer. First, a cohort of 200 cases was studied (Schmidt et al, 2008, Cancer Res 68: 5405-5413). The cohort consisted of lymph node-negative breast cancer patients treated with surgery and without any systemic therapy in the adjuvant setting. Data from mRNA expression and distant metastasis- free survival were available. Clinicopathological features of these patients are shown in Table 7. Distant metastasis- free survival (censored at 120 months) was used as the outcome variable. The expression of the five deregulated genes correlated with prognosis: high expression of MARS, RAE1, SNRPB, and SNRPE was associated with reduced overall survival (Figures 13-16); and high expression of AD ARB 1 was associated with a better outcome (Figure 17). According to the prognostic score, in this case, patients were divided in three groups: 0, 1-2, and 3-5. The score was a strong prognostic marker for metastasis- free survival (Figure 18). Table 7. Demographic and clinical characteristics of the 200 patients selected from a cohort of breast cancer patients (Schmidt et al., 2008, Cancer Res 68: 5405-5413)

Tumor size - mm

Median 20

SD 10

Grade - n (%)

1 24 (14%)

2 136 (68%)

3 35 (18%)

A second cohort of patients from a series of 251 primary breast cancers was used to validate the results in breast cancer (Miller et al, 2005, Proc Natl Acad Sci USA. 102: 13550-13555). Information about survival was available from 236 patients. Clinicopatho logical features of these patients are shown in Table 8. Disease-specific survival was used as the outcome variable (censored at 120 months). Patients were divided according to the prognostic score in two groups: 0-3, and 4-5. The score was a significant prognostic marker for disease survival (Figure 19).

Table 8. Demographic and clinical characteristics of the 236 patients selected from a cohort of breast cancer patients (Miller et al., 2005, Proc. Natl. Acad. Sci USA., 102: 13550-13555)

Age - years

Median 65

SD 14

Tumor size - mm

Median 20

SD 10

Lymph node - n (%)

Negative 149 (63%)

Positive 78 (33%)

NA 9 (4%)

Grade - n (%)

1 62 (26%)

2 121 (51%)

3 51 (22%)

NA 2 (1%)

ER - n (%)

Negative 31 (13%)

Positive 201 (85%)

NA 4 (2%)

PgR - n (%)

Negative 57 (24%)

Positive 179 (76%)

NA: not available

A third cohort of patients was also used (Chanrion et al., 2008, Clin Cancer Res 14: 1744-1833). This series contains 155 primary tumors from breast cancer patients who received adjuvant tamoxifen. Clinicopathological features of these patients are shown in Table 9. Recurrence was used as the outcome variable (censored at 120 months). Patients were divided according to the prognostic score in two groups: 0-3, and 4-5. The score was again a significant prognostic marker for disease survival (Figure 20).

Table 9. Demographic and clinical characteristics of breast cancer patients (Chanrion et al, 2008, Clin Cancer Res 14: 1744-1833)

Age - years

Median 67

SD 10

Tumor size - mm

Median 20

SD 8

Radiotherapy- n (%)

No 34 (22%)

Yes 121 (78%)

Grade - n (%)

1 21 (13%)

2 94 (61%)

3 33 (21%)

NA 7 (5%)

NA: not available