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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
  • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/118—Prognosis of disease development
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers

Definitions

• stage I NSCLC
• stage IIA stage IIA
• stage IIIA Advanced Chemsor-based chemotherapy
• 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.
• stage IB its use is controversial, as clinical trials have not clearly demonstrated a survival benefit.
• 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.
• 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.
• FIG. 3 Kaplan-Meier curve and log rank statistics for overall survival in adenocarcinoma patients divided in high and low SNRPB 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. 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. 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. 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.
• 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,
• 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.
• 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.
• ⁇ overall survival rate 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.
• DFS disease-free survival
• ⁇ 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.
• PFS6 six-month progression free survival
• 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.
• 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, germ
• 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.
• 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.
• Suitable NSCLC types 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.
• SCC squamous cell carcinoma
• 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.
• 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 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
• 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 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, Lip
• 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.
• 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.
• 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.
• 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.
• the levels of the mRNA of the different genes can also be determined by nucleic acid sequence based amplification (NASBA) technology.
• NASBA nucleic acid sequence based amplification
• control RNA relates to RNA whose expression levels do not change or change only in limited amounts in tumor cells with respect to non-tumorigenic cells.
• the control RNA is mRNA derived from housekeeping genes and which code for proteins which are constitutively expressed and carry out essential cellular functions.
• housekeeping genes for use in the present invention include ⁇ -2-microglobulin, ubiquitin, 18-S ribosomal protein, cyclophilin, IP08, HPRT, GAPDH, PSMB4, tubulin and ⁇ -actin.
• the control RNA is GAPDH, IP08, HPRT, ⁇ - actin, 18-S ribosomal protein or PSMB4 mRNA.
• 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.
• the expression levels of the proteins or of variants thereof are determined by Western blot, ELISA or by immunohistochemistry.
• 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.
• the cut-offs are selected in order to facilitate reproducibility, and when possible, to translate biological events.
• 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).
• 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 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).
• 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).
• isoform 1 (identifier: P78563-1), also known as REDl-L or DRADA2B
• Isoform 2 identifier: P78563-2
• DRADA2A REDl-S or DRADA2A
• Isoform 3 identifier: P78563-3
• Isoform 4 identifier: P78563-4
• Isoform 5 identifier: P78563-5.
• 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.
• 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.
• the first method of the invention comprises determining the expression levels of two 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.
• the method of the present invention comprises the determination of the expression levels of the MARS, SNRPB, ADARB1, RAEl and the SNRPE genes.
• the first method of the invention comprises comparing the expression levels of the genes with a reference value.
• Reference value 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.
• 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.
• 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.
• 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.
• 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).
• 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.
• 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, ⁇ -2- microglobulin, ubiquitin, ribosomal protein 18S, cyclophilin A, transferrin receptor, actin, GAPDH, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (YWHAZ), HPRT, and IP08.
• 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.
• 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.
• 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.
• 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.
• risk value refers 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.
• 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.
• the weight is the same for every gene.
• 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.
• 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.
• 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).
• 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).
• 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,
• 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
• 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.
• cancer has been defined in the context of the first method of the invention.
• the cancer is lung cancer.
• the lung cancer is non-small cell lung cancer (NSCLC) or small cell lung cancer.
• the cancer is stage I lung cancer, stage II lung cancer, stage III lung cancer or stage IV lung cancer.
• 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.
• the cancer is breast cancer.
• 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).
• 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 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).
• the breast cancer is lymph-node negative breast cancer, i.e. a breast cancer that has not spread to the lymph node.
• 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.
• the patient has undergone surgical resection of the tumor.
• adjuvant chemotherapy 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.
• 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.
• FEC epirubicin/ cyclophosphamide
• CMF 5-fluorouracil
• 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.
• 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.
• 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.
• 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.
• the second method of the invention comprises the determination of the expression levels of four of the above genes.
• 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.
• 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.
• the second method of the invention comprises correlating the expression levels of the genes with a reference value.
• the reference value has been described in the context of the first method of the invention.
• the reference value is the expression level of the gene of interest in a non-cancerous tissue.
• the reference value corresponds to the expression levels of the gene of interest in a healthy lung tissue.
• the reference value corresponds to the expression levels of the gene of interest in a healthy breast tissue.
• 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".
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• treatment refers to any type of therapy, which is aimed at terminating, preventing, ameliorating or reducing the susceptibility to a clinical condition as described herein.
• 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.
• prophylactic treatment i.e. a therapy to reduce the susceptibility to a clinical condition
• treatment refers to obtaining a desired pharmacologic or physiologic effect, covering any treatment of a pathological condition or disorder in a mammal, including a human.
• 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.
• a parameter such as inflammation, pain, or immune deficiency
• 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.
• the cancer is lung cancer.
• the lung cancer is non-small cell lung cancer (NSCLC) or small cell lung cancer.
• NSCLC non-small cell lung cancer
• 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.
• 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,
• the cancer is breast cancer.
• 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).
• 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.
• 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.
• 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
• the breast cancer is lymph-node negative breast cancer, i.e. a breast cancer that has not spread to the lymph node.
• 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.
• the patient has undergone surgical resection of the tumor.
• the adjuvant therapy is chemotherapy, radiotherapy or a combination thereof. Suitable chemotherapeutic treatments are indicated in the second method of the invention.
• 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.
• the invention in another embodiment, 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.
• kits 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.
• 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 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.).
• 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.
• kits or assay devices 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.
• 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.
• 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.
• 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.
• 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.
• the reagents specific for said gene i.e.
• 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.
• the first component of the kit of the invention comprises probes which can specifically hybridize to the genes mentioned above.
• “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 ⁇ / ⁇ 1), 0.1% SDS, and 10% dextran sulfate at 42°C
• Modely 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.
• washing solution and hybridization conditions e.g., temperature, ionic strength and % SDS
• An example of moderately stringent conditions is overnight incubation at 37°C.
• the hybridization pattern is detected, which provides information on the genetic profile of the sample analyzed.
• 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).
• PNA peptide nucleic acids
• 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.
• Probes suitable for use as expression controls correspond to genes expressed constitutively, such as genes encoding proteins which exert essential cell functions such as ⁇ -2-microglobulin, ubiquitin, ribosomal protein 18S, cyclophilin A, transferrin receptor, actin, GAPDH, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (YWHAZ), HPRT, and IP08.
• proteins which exert essential cell functions such as ⁇ -2-microglobulin, ubiquitin, ribosomal protein 18S, cyclophilin A, transferrin receptor, actin, GAPDH, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (YWHAZ), HPRT, and IP08.
• 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.
• the arrays are low density arrays or LDA containing less than 10000 probes per square centimeter.
• 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.
• kits according to the present invention comprise reagents which are capable of specifically binding to said polypeptide or polypeptides.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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:
• 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.
• 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.
• 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.
• 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 prognostic performance of the combined expression of the five genes was also evaluated in breast cancer.
• 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.
• 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).

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