WO2009144155A1

WO2009144155A1 - Method for prediciting the clinical outcome of patients with non-small-cell lung cancer treated with an anti-metabolite plus an anti-microtubule agent

Info

Publication number: WO2009144155A1
Application number: PCT/EP2009/056048
Authority: WO
Inventors: Miguel Tarón Roca; Rafael Rosell Costa; Vassilis Georgoulias; Dimitris Mavroudis; John Souglakos
Original assignee: Pangaea Biotech, S.A.; University Of Crete
Priority date: 2008-05-30
Filing date: 2009-05-19
Publication date: 2009-12-03

Abstract

The invention relates to an in vitro method for predicting the clinical outcome of patients suffering from non-small-cell lung cancer who have been treated with an anti- metabolite plus an anti-microtubule agent, based on the expression of BRCA1 gene, RRM1 gene and/or RRM2 gene in a sample from said patient, as well as a method for designing an individual therapy for a subject suffering from NSCLC and a kit useful in the implementation of the methodology described herein.

Description

METHOD FOR PREDICITING THE CLINICAL OUTCOME OF PATIENTS WITH NON-SMALL-CELL LUNG CANCER TREATED WITH AN ANTIMETABOLITE PLUS AN ANTI-MICROTUBULE AGENT

FIELD OF THE INVENTION

The invention relates to the field of diagnostic and therapy, in particular to a method of predicting the clinical outcome of patients suffering from non-small-cell lung cancer based on the expression of certain biomarkers in a sample from said patients.

BACKGROUND

Non-small-cell lung cancer (NSCLC) accounts for approximately 80% of all lung cancers, with 1.2 million new cases worldwide each year. NSCLC resulted in more than one million deaths worldwide in 2001 and is the leading cause of cancer-related mortality in both men and women (31% and 25%, respectively). The prognosis of advanced NSCLC is dismal. A recent Eastern Cooperative Oncology Group trial of 1155 patients showed no differences among the chemotherapies used: cisplatin/p aclitaxel , cisp latin/gemcitabine , cisp latin/do cetaxel and carboplatin/paclitaxel.

Non-platinum-based combinations with gemcitabine plus docetaxel or paclitaxel have yielded a similar survival benefit with a more favourable toxicity profile. A feasibility study of customized treatment in NSCLC patients with high ERCCl and low ribonucleotide reductase subunit Ml (RRMl) mRNA expression found that gemcitabine plus docetaxel could be the optimal combination for this subgroup of patients (Simon G, et al. 2007, J Clin Oncol, vol. 25(19):2741-6).

RRMl and RRM2 are encoded by different genes on separate chromosomes and their mRNAs are differentially expressed throughout the cell cycle. In a study of metastatic lung adenocarcinoma patients treated with gemcitabine plus docetaxel, patients with low levels of both RRMl and RRM2 had a significantly higher response rate (60% vs 14.2%), time to progression (9.9 vs 2.3 months), and overall survival (15.4 vs 3.6 months) than patients with high levels of both genes (Souglakos J, et al. 2008, Br J Cancer, vol. 98(10): 1710-5). On the other hand, the patent application US2006094021 discloses a screening method for classifying patients and for selecting an effective chemotherapy for the treatment of a patient suffering from non-small-cell lung cancer (NSCLC), based on the use of his levels of BRCAl expression to predict the outcome of chemotherapy. Low levels of BRCAl also correlated with increased survival in NSCLC patients treated with gemcitabine plus cisplatin (Rosell R, et al. 2004, cited at supra).

A close correlation has been observed between expression levels of RRMl and BRCAl (Rosell R, et al. 2004, Clin Cancer Res, vol. 10(12 Pt 2):4215s-4219s; Taron M, et al. 2004, Hum MoI Genet, vol. 13(20):2443-9; Rosell R, et al 2007, PLoS ONE, vol. 2(1 l):el 129), and the loss of let-7 has been shown to upregulate BRCAl as well as RRMl and RRM2 (Johnson CD, et al. 2007, Cancer Res. vol. 67(16):7713-22).

Thus, there is a need in the art for further prognosis markers for predicting the clinical outcome of a patient suffering from NSCLC.

BRIEF DESCRIPTION OF THE INVENTION

In one aspect, the invention relates to an in vitro method for predicting the clinical outcome of a subject suffering from NSCLC who has been treated with an anti- metabolite plus an anti-microtubule agent comprising:

(i) determining the expression levels of a first gene and a second gene in a sample from said subject, wherein said first gene is BRCAl gene and the second gene is selected from RRMl gene, RRM2 gene and the combination thereof, and (ii) comparing said expression levels with reference values for each gene, wherein high expression levels of BRCAl gene and low expression levels of RRMl gene and/or RRM2 gene with respect to said reference values, are indicative of a positive clinical outcome of the subject.

In another aspect, the invention relates to an in vitro method for determining the risk of progression of a subject suffering from NSCLC who has been treated with an antimetabolite plus an anti-microtubule agent comprising (i) determining the expression levels of BRCAl and RRMl genes in a sample from said subject, (ii) comparing said expression levels with reference values for each gene, and (iii) categorizing said expression levels by terciles for each gene, wherein if

(a) the BRCAl expression levels are in the intermediate tercile and the RRMl expression levels are in the low tercile, or

(b) the BRCAl expression levels are in the high tercile and the RRMl expression levels are in the low tercile, or

(c) the BRCAl expression levels are in the high tercile and the RRMl expression levels are in the intermediate tercile, then the subject is in low risk of progression.

In yet another aspect, the invention relates to an in vitro method for designing an individual therapy for a subject suffering from NSCLC comprising

(i) determining the expression levels of a first and a second gene in a sample from said subject , wherein said first gene is BRCAl gene and the second gene is selected from RRMl gene, RRM2 gene and the combination thereof, and

(ii) comparing said expression levels genes with reference values for each gene, wherein low expression levels of BRCAl and high expression levels of RRMl and/or RRM2 with respect to said reference values, are indicative that the patient is a candidate for an anti-metabolite plus an anti-microtubule agent chemotherapy.

In another aspect, the invention relates to an in vitro method for determining the risk of progression of a subject suffering from NSCLC who has received an anti-metabolite plus an anti-microtubule agent as first-line treatment and platin-based chemotherapy as second- line treatment comprising

(i) determining the expression levels of BRCAl gene in a sample from said subject and (ii) comparing said expression levels of BRCAl gene with reference values for said gene, wherein low expression levels of BRCAl gene with respect to said reference values are indicative of low risk of progression after second- line treatment.

In another aspect, the invention relates to a kit comprising a set of reagents, wherein said set consists of reagents for detecting BRCAl, RRMl, RRM2 genes and/or the combination thereof, or reagents for detecting BRCAl, RRMl, RRM2 proteins, variants thereof and/or the combination thereof and, optionally, a reagent for detecting a housekeeping gene or the protein encoded by said housekeeping gene.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 depicts a schematic diagram of the flow of patients through study. Figure 2 is a graph showing the time to progression according to risk groups. Figure 3 is a graph showing the time to progression after first-line treatment according to BRCAl terciles.

DETAILED DESCRIPTION OF THE INVENTION

The inventors of the present invention have discovered that the efficacy of the chemotherapy based on an anti-metabolite plus an anti-microtubule agent in subjects suffering from non-small-cell lung cancer (NSCLC), surprisingly, depends on the mRNA expression levels of BRCAl, RRMl and/or RRM2 genes. In this sense, high expression levels of BRCAl gene and low expression levels of RRMl gene and/or

RRM2 gene are correlated with a positive clinical outcome of the subject suffering from NSCLC who has been treated with an anti-metabolite plus an anti-microtubule agent.

Based on these findings the inventors have defined the method of the invention in its different embodiments that will be described now in detail.

Thus, in one aspect, the invention relates to an in vitro method for predicting the clinical outcome of a subject suffering from NSCLC who has been treated with an antimetabolite plus an anti-microtubule agent (hereinafter first method of the invention) comprising: (i) determining the expression levels of a first gene and a second gene in a sample from said subject, wherein said first gene is BRCAl gene and the second gene is selected from RRMl gene, RRM2 gene and the combination thereof, and (ii) comparing said expression levels with standard reference values for each gene, wherein high expression levels of BRCAl gene and low expression levels of RRMl gene and/or RRM2 gene with respect to said reference values, are indicative of a positive clinical outcome of the subject.

In the present invention "clinical outcome" is understood as the expected course of a disease. It denotes the doctor's prediction of how a subject's disease will progress, and whether there is chance of recovery or recurrence.

The prediction of the clinical outcome can be done by using any endpoint measurements used in oncology and known to the skilled practitioner. Useful endpoint parameters to describe the evolution of a disease include:

^■ disease-free progression which, as used herein, describes the proportion of subjects in complete remission who have had no recurrence of disease during the time period under study;

^■ objective response, which, as used in the present invention, describes the proportion of treated people in whom a complete or partial response is observed;

^■ tumour control, which, as used in the present invention, relates to the proportion of treated people 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.

^■ six-month progression free survival or "PFS6" rate which, as used herein, relates to the percentage of people wherein free of progression in the first six months after the initiation of the therapy.

^■ median survival which, as used herein, relates to the time at which half of the subjects enrolled in the study are still alive and ^■ time to progression, as used herein, relates to the time after a disease is diagnosed (or treated) until the disease starts to get worse

In a particular embodiment of the invention, the clinical outcome is measured as survival of the subject to the NSCLC, response to the treatment or time to progression.

The term "subject", as used herein, refers to all animals classified as mammals and includes, but is not restricted to, domestic and farm animals, primates and humans, e.g., human beings, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats, or rodents. Preferably, the subject is a male or female human of any age or race. In the context of the present invention, the subject is a subject suffering from NSCLC who has been treated with an anti-metabolite plus an anti-microtubule agent. In a particular embodiment, the NSCLC is selected from squamous cell carcinoma of the lung, large cell carcinoma of the lung, and adenocarcinoma of the lung. Furthermore, the present invention can also be applicable to a subject suffering from from any stage of NSCLC (stages 0, IA, IB, HA, HB, IIIA, IIIB or IV). However, in a particular embodiment, the stage of NSCLC is IIIB or IV.

In the context of the present invention, the term "subject" is a subject suffering from NSCLC who has received chemotherapy based on an anti-metabolite plus an anti- microtubule agent.

The term "anti-metabolite" refers to compounds which replace natural substances as building blocks in DNA molecules, thereby altering the function of enzymes required for cell metabolism and protein synthesis. Examples of anti-metabolite drugs which can be used according to the present invention include 5-fluorouracil, cytarabine, gemcitabine, aminopterin, methotrexate, pemetrexed, raltitrexed, cladribine, clofarabine, fludarabine, mercaptopurine, pentostatin, thioguanine, capecitabine, floxuridine, etc

The term "anti-microtubule agent" refers to compounds which promote the assembly of microtubules from tubulin dimmers and stabilizes microtubules by preventing depolymerisation. This stability results in the inhibition of the normal dynamic reorganization of the microtubule network that is essential for vital interphase and mitotic cellular functions. Examples of anti-micro tubules agents include vinca alkaloids such as vincristine, vinblastine, vinorelbine, etc.; Taxanes such as paclitaxel, docetaxel, etc.; colchicine, etc.

In a particular embodiment of the first method of the invention, the anti-metabolite is gemcitabine and the anti-microtubule agent is docetaxel.

In order to carry out the first method of the invention, a sample is obtained from the subject under study. The term "sample" as used herein, relates to any sample which can be obtained from the patient. The present method can be applied to any type of biological sample from a patient, such as a biopsy sample, tissue, cell or fluid (serum, saliva, semen, sputum, cerebral spinal fluid (CSF), tears, mucus, sweat, milk, brain extracts and the like). In a particular embodiment, said sample is a tissue sample or portion thereof, preferably tumour tissue sample or portion thereof. In a more particular embodiment, said tumour tissue sample is a lung tumour tissue sample from a subject suffering from NSCLC who has been treated with an anti-metabolite plus an anti- microtubule agent. Said sample can be obtained by conventional methods, e.g., biopsy, by using methods well known to those of ordinary skill in the related medical arts. Methods for obtaining the sample from the biopsy include gross apportioning of a mass, or microdissection or other art-known cell-separation methods. Tumour cells can additionally be obtained from fine needle aspiration cytology. In order to simplify conservation and handling of the samples, these can be formalin- fixed and paraffin- embedded or first frozen and then embedded in a cryosolidifiable medium, such as OCT-Compound, through immersion in a highly cryogenic medium that allows for rapid freeze.

As the person skilled in the art understands, the expression levels of the BRCAl gene, RRMl gene and RRM2 gene can be determined by measuring the levels of mRNA encoded by said genes or by measuring both the levels of proteins encoded by said genes, i.e. BRCAl protein, RRMl protein and RRM2 protein, and the levels of variants thereof. Thus, in a particular embodiment of the invention, the expression levels of the BRCAl gen, RRMl gene and/or RRM2 gene are determined by measuring the levels of mRNA encoded by the BRCAl gene, RRMl gene and/or RRM2 gene. For this purpose, the biological sample may be treated to physically or mechanically 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, 3rd 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.

In a particular embodiment, the expression level is 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 deparaffinized. An exemplary deparaffinization method involves washing the paraffinized sample with an organic solvent, such as xylene. Deparaffinized samples can be rehydrated with an aqueous solution of a lower alcohol. Suitable lower alcohols, for example include, methanol, ethanol, propanols, and butanols. Deparaffinized samples may be rehydrated with successive washes with lower alcoholic solutions of decreasing concentration, for example. Alternatively, the sample is simultaneously deparaffinized and rehydrated. The sample is then lysed and RNA is extracted from the sample.

While all techniques of gene expression profiling (RT-PCR, SAGE, or TaqMan) are suitable for use in performing the foregoing aspects of the invention, the gene mRNA expression levels are often determined by reverse transcription polymerase chain reaction (RT-PCR). In a particular embodiment, the mRNA expression levels of

BRCAl gene, RRMl gene and/or RRM2 gene 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 a RNA whose expression levels do not change or change only in limited amounts in tumour cells with respect to non-tumourigenic 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, GAPDH and actin. In a preferred embodiment, the control RNA is β-actin mRNA. In one embodiment relative gene expression quantification is calculated according to the comparative Ct method using β- actin as an endogenous control and commercial RNA controls as calibrators. Final results, are determined according to the formula 2-(ΔCt sample-ΔCt calibrator), where ΔCT values of the calibrator and sample are determined by subtracting the CT value of the target gene from the value of the β-actin gene.

The determination of the expression levels of the BRCAl gene, RRMl gene and RRM2 gene needs to be correlated with reference values which correspond to the median value of expression levels of BRCAl gene, RRMl gene and RRM2 gene measured in a collection of tumour tissue in biopsy samples from subjects suffering from NSCLC who have been treated with an anti-metabolite plus an anti-microtubule agent. Once this median value is established, the level of this marker expressed in tumour tissues from the subject can be compared with this median value, and thus be assigned a level of "low," "normal" or "high" expression. The collection of samples from which the reference level is derived will preferably be constituted from subjects suffering from the same type of cancer, i.e. NSCLC, who have been treated with the same chemotherapeutic treatment, i.e. an anti-metabolite plus an anti-microtubule agent.

For example, the one described in the examples which is statistically representative was constituted with 96 samples from NSCLC patients treated with gemcitabine (an antimetabolite) plus docetaxel (an anti-microtubule agent). In any case it can contain a different number of samples. The use of a reference value used for determining whether the expression of a gene sample is "increased" or "decreased" corresponds to the median value of expression levels of said gene measured in a RNA sample obtained by pooling equal amounts of RNA from each of the tumour samples obtained by biopsy from NSCLC patients treated with gemcitabine plus docetaxel. Once this median value is established, the level of this marker expressed in tumours tissues from patients can be compared with this median value, and thus be assigned a level of "increased" or "decreased". Due to inter-subject variability (e.g. aspects relating to age, race, etc.) it is very difficult (if not practically impossible) to establish absolute reference values for BRCAl gene, RRMl gene or RRM2 gene. Thus, in a particular embodiment, the reference values for "increased" or "decreased" BRCAl gene expression, RRMl gene expression or RRM2 gene expression are determined by calculating percentiles by conventional means involving the testing of a group of samples isolated from normal subjects (i.e. people with no diagnosis of NSCLC) for the expression levels of the BRCAl gene, RRMl gene or RRM2 gene. The "increased" levels can then be assigned, preferably, to samples wherein expression levels for the BRCAl gene, RRMl gene or RRM2 gene are equal to or in excess of percentile 50 in the normal population, including, for example, expression levels equal to or in excess to percentile 60 in the normal population, equal to or in excess to percentile 70 in the normal population, equal to or in excess to percentile 80 in the normal population, equal to or in excess to percentile 90 in the normal population, and equal to or in excess to percentile 95 in the normal population.

In a preferred embodiment, the BRCAl gene, RRMl gene and RRM2 gene expression values are divided into terciles. The term "terciles " refers to the percentiles that divide the distribution of data into 3 equal parts. In the contex of the present invention, the distribution of data are divided into 3 equal part according to the expression levels of BRCAl , RRMl or RRM2. As an example, real-time quantitative PCR was used to determine BRCAl, RRMl or RRM2 mRNA levels in 96 tumour biopsies from NSCLC patients treated with gemcitabine (an anti-metabolite) plus docetaxel (an anti- microtubule agent), and divided the gene expression values into terciles. When results were correlated with the risk of progression, it was observed that the risk of progression was greater for patients in the intermediate (T2) and lowest tercile (Tl) of BRCAl than those in the highest tercile (T3) [Table 3].

Alternatively, in another particular embodiment, the expression levels of the BRCAl gene, RRMl gene and/or RRM2 gene can be determined by measuring both the levels of proteins encoded by said genes, i.e. BRCAl protein, RRMl protein and RRM2 protein, and the levels of variants thereof.

The determination of the expression levels of the proteins can be carried out by immunological techniques such as ELISA, Western Blot or immunofluorescence. 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 analysis by immunofluorescence requires the use of an antibody specific for the target protein for the analysis of the expression. ELISA is based on the use of antigens or antibodies labelled with enzymes so that the conjugates formed between the target antigen and the labelled antibody results in the formation of enzymatically-active complexes. Since one of the components (the antigen or the labelled antibody) are immobilised on a support, the antibody-antigen complexes are immobilised on the support and thus, it can be detected by the addition of a substrate which is converted by the enzyme to a product which is detectable by, e.g. spectrophotometry or fluorometry.

When an immunological method is used, any antibody or reagent known to bind with high affinity to the target proteins can be used for detecting the amount of target proteins. It is preferred nevertheless the use of antibody, for example polyclonal sera, hybridoma supernatants or monoclonal antibodies, antibody fragments, Fv, Fab, Fab' y F(ab')2, ScFv, diabodies, triabodies, tetrabodies and humanised antibodies.

On the other hand, the determination of the protein expression levels can be carried out by constructing a tissue microarray (TMA) containing the subject samples assembled, and determining the expression levels of the proteins by immunohistochemistry techniques well known in the state of the art.

As previously cited, the expression levels of the BRCAl gene, RRMl gene and RRM2 gene can be determined by measuring both the levels of protein encoded by said genes, i.e. BRCAl protein, RRMl protein and RRM2 protein, and the levels of variants thereof, such as fragments, analogues and/or derivatives. Variants of the BRCAl protein, RRMl protein and RRM2 protein may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non- conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, (ii) one in which there are one or more modified amino acid residues, e.g., residues that are modified by the attachment of substituent groups, (iii) one in which the protein is an alternative splice variant of the proteins of the present invention and/or (iv) fragments of the proteins. The fragments include proteins generated via proteolytic cleavage (including multi-site proteolysis) of an original sequence. Variants are deemed to be within the scope of those skilled in the art from the teaching herein.

As known in the art the "similarity" between two proteins is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one protein to a sequence of a second protein. Variants according to the present invention includes amino acid sequences that are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90%, or 95% similar or identical to the original amino acid sequence. The degree of identity between two proteins is determined using computer algorithms and methods that are widely known for the persons skilled in the art. The identity between two amino acid sequences is preferably determined by using the BLASTP algorithm [BLASTManual, Altschul, S., et al, NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. MoI. Biol. 215: 403-410 (1990)].

The proteins can be post-translationally modified. For example, post-translational modifications that fall within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis myristoylation, protein folding and proteolytic processing, etc. Additionally, the proteins may include unnatural amino acids formed by post-translational modification or by introducing unnatural amino acids during translation.

The findings of the inventors allow the physician to determine the risk of progression of a subject suffering from NSCLC who has been treated with an anti-metabolite plus an anti-micro tubule agent based on the interaction observed between BRCAl and RRMl, as well as to design an individual therapy for a subject suffering from NSCLC based on the interaction observed between BRCAl, RRMl and RRM2.

Thus, in another aspect, the invention relates to an in vitro method for determining the risk of progression of a subject suffering from NSCLC who has been treated with an anti-metabolite plus an anti-microtubule agent (hereinafter second method of the invention) comprising

(i) determining the expression levels of BRCAl and RRMl genes in a sample from said subject, (ii) comparing said expression levels with standard reference values for each gene, and

(iii) categorizing said expression levels by terciles for each gene, wherein if

In another aspect, the invention relates to an in vitro method for designing an individual therapy for a subject suffering from NSCLC (hereinafter third method of the invention) comprising (i) determining the expression levels of a first and a second gene in a sample from said subject , wherein said first gene is BRCAl and the second gene is selected from RRMl gene, RRM2 gene and the combination thereof, and

(ii) comparing said expression levels genes with standard reference values for each gene, wherein low expression levels of BRCAl and high expression levels of RRMl and/or RRM2 are indicative that the subject is a candidate for anti-metabolite plus an anti- microtubule agent chemotherapy. In the context of the third method of the invention, the term "subject" is understood as a subject suffering from NSCLC who has not received or is not receiving chemotherapy based on an anti-metabolite plus an anti-microtubule agent.

The skilled person will appreciate that the particular embodiments developed in the first method of the invention are also applicable to the second and third methods of the invention, such as (i) the anti-metabolite and the anti-microtubule agent used in the chemotherapy (gemcitabine and docetaxel respectively), (ii) the kind of sample obtained from the subject (tissue sample, preferably tumour tissue sample, more preferably lung tumour tissue sample), (iii) the different procedures for determining the expression levels of BRCAl, RRMl and RRM2 genes (measuring the levels of mRNA or proteins encoded by said genes), or (iv) the stage of the NSCLC (preferably, stage IIIB or IV), etc. Moreover, the skilled person will also understand that all method and techniques previously cited for determining the protein and mRNA expression levels can also be used in the second and third methods of the invention.

On the other hand, the invention also contemplates the use of the expression levels of BRCAl gene for determining the risk of progression of a subject suffering from NSCLC who has received a second-line cisp latin-based treatment after a first-line gemcitabine plus docetaxel. The inventors have discovered that patients suffering from NSCLC with low BRCAl mRNA expression have poor response and time to progression to first-line gemcitabine plus docetaxel; in contrast, they obtained the maximum benefits from second-line cisplatin-based treatment.

Thus, in another aspect, the invention relates to an in vitro method for determining the risk of progression of a subject suffering from NSCLC who has received an antimetabolite plus an anti-microtubule agent as first-line treatment and platinum-based chemotherapy as second- line treatment (hereinafter fourth method of the invention) comprising

(i) determining the expression levels of BRCAl gene in a sample from said subject and (ii) comparing said expression levels of BRCAl gene with standard reference values for said gene, wherein low expression levels of BRCAl gene with respect to said reference values are indicative of low risk of progression after second- line treatment.

In a particular embodiment of the fourth method of the invention, the anti-metabolite is gemcitabine and the anti-microtubule agent is docetaxel.

In another particular embodiment, the sample is a tissue sample, preferably a tumour sample, more preferably a lung sample.

In another particular embodiment, the expression levels of the BRCAl, RRMl and/or RRM2 genes are determined by measuring the levels of mRNA encoded by the BRCAl, RRMl and/or RRM2 genes or the level of BRCAl, RRMl and/or RRM2 proteins.

Platinum-based chemotherapy, as used herein, refers to chemotherapy using compounds which contain a platinum atom and which are capable of binding DNA inducing the activation of the DNA repair and subsequent cell death. Platinum-based compounds which are suitable for use in chemotherapy include, without limitation, cisplatin, carboplatin, iproplatin, tetraplatin, JMl 18, JM149, JM216, JM335, transplatin, cis, trans, CW-Pt(NH₃)(C₆HIiNH₂)(OOCC₃Hv)₂Cl, nedap latin, malanato-1,2- diaminocyclohexaneplatinum(II), 5-Sulfosalicylato-£ra/?s-(l ,2 diaminocyclohexane)Platinum(II) (SSP), Poly-[(traτw-l,2- diaminocyclohexane)platinum]-carboxyamylose (POLY-PLAT) and 4-Hydroxy- sulfonylphenylacetato(£ra/?s- 1 ,2-diaminocyclohexane)platinum(II) (SAP).

In another particular embodiment, the NSCLC is in stage IIIB or IV.

As the skilled person understands, all method and techniques previously cited for determining the protein and gene expression levels can also be used in the fourth method of the invention. In another aspect, the invention relates to a kit useful in the implementation of the methodology described herein.

Thus, in another aspect, the invention relates to a kit comprising a set of reagents, wherein said set consists of reagents for detecting BRCAl, RRMl, RRM2 genes and/or the combination thereof, or reagents for detecting BRCAl, RRMl, RRM2 proteins, variants thereof and/or the combination thereof, and optionally, a reagent for detecting a housekeeping gene or the protein encoded by said housekeeping gene.

In a particular embodiment of the kit of the invention, the set of reagents is selected from the group of: a) A set of nucleic acids capable of specifically hybridising with the BRCAl, RRMl and/or RRM2 genes, and/or b) A set of antibodies, or fragments thereof capable of specifically binding to BRCAl , RRM 1 and/or RRM2 proteins or to variants thereof.

Nucleic acids capable of specifically hybridizing with the BRCAl, RRMl and/or RRM2 genes are, for example, one or more pairs of primer oligonucleotides for the specific amplification of fragments of the mRNAs (or of their corresponding cDNAs) of said genes and/or one or more probes for the identification of one or more genes selected from said genes.

Antibodies, or a fragment thereof, capable of detecting an antigen, capable of specifically binding to BRCAl, RRMl and/or RRM2 proteins or to variants thereof are, for example, monoclonal and polyclonal antibodies, antibody fragments, Fv, Fab, Fab' y F(ab')2, ScFv, diabodies, triabodies, tetrabodies and humanised antibodies.

Said reagents, specifically, the probes and the antibodies, may be fixed onto a solid support, such as a membrane, a plastic or a glass, optionally treated in order to facilitate fixation of said probes or antibodies onto the support. Said solid support, which comprises, at least, a set of antibodies capable of specifically binding to BRCAl, RRMl and/or RRM2 proteins or to variants thereof, and/or probes specifically hybridized with the BRCAl, RRMl and/or RRM2 genes, may be used for the detection of the expression levels by means of array technology.

The kits of the invention optionally comprise additional reagents for detecting a polypeptide encoded by a housekeeping gene or the mRNA encoded by said housekeeping genes. The availability of said additional reagent allows the normalization of measurements taken in different samples (e.g. the test sample and the control sample) to exclude that the differences in expression of the different bio markers are due to a different amount of total protein in the sample rather than to real differences in relative expression levels. Housekeeping genes, as used herein, relates to genes 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, GAPDH and actin.

The following examples are provided as merely illustrative and are not to be construed as limiting the scope of the invention.

EXAMPLES

I. MATERIAL AND METHODS Patients

Tumour samples were collected from patients with histologically confirmed inoperable stage IIIB and IV NSCLC, who were included in the experimental arm of a HORG randomised trial. Eligibility criteria have been previously reported. The study was approved by the Ethics Committees of the participating hospitals, and all patients gave their signed informed consent prior to study entry. Patients received first-line gemcitabine (Gemzar^®; Eli Lilly, Indianapolis, IN, USA) 1000 mg/m² on days 1 and 8 and docetaxel (Taxotere^®; Sanofi-Aventis, Collegeville, NJ, USA) 100 mg/m² on day 8, with human granulocyte colony- stimulating factor support every 3 weeks. Patient evaluation was performed at baseline and after every three cycles of chemotherapy.

Study design

The present study was a retrospective analysis of the prognostic value of BRCAl,

RRMl and RRM2 mRNA expression in NSCLC patients treated with first-line gemcitabine plus docetaxel. All available tumour biopsies of the primary tumour with more than 100 cells per section were included in the analysis. All efficacy results were assessed for all enrolled patients on an intent-to-treat basis.

Gene expression analysis

All paraffin-embedded tumours were reviewed by two independent pathologists in order to ensure the validity of the sample and define the most appropriate tumour area for microdissection. Malignant cells were procured using an Eppendorf piezoelectric microdissector (Eppendorf, Hamburg, Germany).

The pellet of microdissected cells was resuspended in 200 μl RNA lyses buffer and incubated at 60⁰C for 16 hours until the tissue was completely solubilized. RNA was purified by trizol LS (Invitrogen, Carlsbad, CA, USA) extractions followed by isopropanol precipitation and DNase (DNase Free, Ambion, Austin, TX, USA) treatment in order to avoid genomic DNA contamination of the samples. cDNA synthesis was performed in a final volume of 22 μl using Superscript III reverse transcriptase according to the manufacturer's protocol (Invitrogen, Carlsbad, CA, USA). The QPCR reaction was performed using 2.5 μl of template cDNA to 6.25 μl Taqman Universal Master Mix (Applied Biosystems, Foster City, CA, USA) with the addition of specific primers and probe for each gene and adjusted with DEPC water to a final volume of 12.5 μl per reaction. The primers and the probe were as follows: BRCAl : forward 5 'GGC TAT CCT CTC AGA GTG ACA TTT TA 3', reverse 5 ' GCT TTA TCA GGT TAT GTT GCA TGG T 3 ', probe 5 ' CCA CTC AGC AGA GGG 3 '; RRMl : forward 5'-ACT AAG CAC CCT GAC TAT GCT ATC C-3', reverse 5'-CTT CCA TCA CAT CAC TGA ACA CTT T-3', probe 5'-CAG CCA GGA TCG CTG TCT CTA ACT TGC A-3'; and RRM2: forward 5'-CCT GGC CAG CAA GAC CG-3', reverse 5'-TAG TTT TCG GCT CCG TGG G-3', probe 5'-CGA GGA GGA TCT TCC AGG A-3'. Quantification of gene expression was performed using the ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA). All primers and probe sets were designed to spread into an exon-exon junction.

Relative gene expression quantification was performed according to the comparative Ct method using β-actin as an endogenous control and commercial RNA controls (Stratagene, La Jolla, CA, USA) as calibrators. In all experiments, only triplicates with a standard deviation of the Ct value <0.25 were accepted. In addition, genomic DNA contamination was excluded by non-reverse transcript RNA for each sample analysed.

Statistical analyses

Besides analyzing the expression levels of each gene as a continuous variable, gene expression was also categorized in terciles in order to explore the risk trend of the gene variables and in order to easily identify groups of gene expression levels with different risk. Responses were recoded according to the RECIST criteria. Median time to tumour progression and overall survival were calculated from the start of treatment to the first documented disease progression or death, respectively.

The potential association between baseline characteristics, response and gene expression levels were compared with either the two-sided Fisher's exact test or the Chi-square test for categorical variables and the Kruskal-Wallis test for continuous variables. The normality of continuous variables was verified with a Kolmogorov- Smirnov test. Pearson's exact test was used to evaluate the correlation between BRCAl, RRMl and RRM2 mRNA expression. All potential risk factors for response were evaluated in a univariate analysis, and a multivariate logistic regression analysis, with adjusted odd ratios and their 95% confidence intervals (CI), was used to evaluate which of the factors had a significant influence on response. The Hosmer-Lemeshow likelihood test was used to assess the goodness of fit.

The association of risk factors with time-to-event endpoints was analyzed with the log- rank test, and the Kaplan-Meier method was used to plot the corresponding time-to- progression and survival curves. A univariate Cox regression analysis, with hazard ratios and 95% CIs, was used to assess the association between each potential prognostic factor and survival and time to progression. These factors were then included in a multivariate Cox proportional hazards regression model with a stepwise procedure (both forward and backward) to evaluate the independent significance of different variables on survival and time to progression. The likelihood ratio test was used to assess the goodness of fit, and the Wald's test was used to assess the coefficient significance. In the case of potential multiple comparisons, the p-values were corrected with the Bonferroni correction.

All statistical calculations were performed with the SPSS (SPSS, Inc., Chicago, IL, USA). Two-sided p-values of less than 0.05 were considered significant.

II- RESULTS

Patient characteristics and clinical outcome In the original randomised trial (Georgoulias V, et al. 2005. J Clin Oncol; vol. 23(13): 2937-45), 209 NSCLC patients were treated with gemcitabine plus docetaxel; 107 were not included in the present study due to lack of tumour tissue (Figure 1). Clinical data and samples from primary tumours were available for 102 patients, who were included in the present study. Amplification of BRCAl, RRMl and RRM2 was successful in 96 samples. Eighty-one were adenocarcinomas, ten squamous cell carcinomas, and five large-cell carcinomas. Patient characteristics are shown in Table 1. Table 1

Table 1 (continuation)

In the original trial, the response rate was 30%, time to progression 4 months, and median survival 9 months (Georgoulias V, et al. 2005 cited at supra). Outcome for the 96 patients assessed in the present study was similar: response rate 30.5%, time to progression 4.2 months, and median survival 10.5 months.

BRCAl, RRMl and RRM2 mRNA expression levels

Median mRNA expression levels were 3.64 (range 0-34.37) for BRCAl, 0.82 (range 0- 325.23) for RRMl and 27.16 (range 0.97-256.84) for RRM2 (Table 1). There was no correlation between age, gender, PS, or disease stage and BRCAl, RRMl or RRM2 mRNA levels. Significant correlations were observed overall between BRCAl and

RRMl (p²= 0.23; p=0.02) and between RRMl and RRM2 (p²=0.23; p=0.02) mRNA levels. There was also a significant inverse correlation between BRCAl and RRM2 mRNA levels (p²⁼ -0.33; p=0.001). Table 1 also shows the mRNA expression levels of the three genes according to terciles.

Gene expression and response to treatment

In order to predict response to treatment, a logistic regression model was fitted for the expression of each gene as a continuous variable. As BRCAl levels increased, the probability of response increased significantly (Odds Ratio [OR] = 1.09; 95% CI, 1.02- 1.16; p=0.01). In contrast, as RRM2 levels increased, the probability of response decreased (OR = 0.94; 95% CI, 0.91-0.97; p<0.0001). A similar but non-significant trend was observed for RRMl levels (OR = 0.97; 95% CI, 0.77-1.23; p=0.82).

When responders were classified according to their gene expression levels by terciles, the majority of responders had high BRCAl expression and low RRM2 expression: 58.6% in the highest tercile of BRCAl expression (p=0.002) and 72.4% in the lowest tercile of RRM2 expression (p<0.001) (Table 2).

Table 2

Table 2 (continuation)

CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease; OR, odds ratio; T, terciles; PS, performance status The univariate logistic regression analysis revealed that low RRM2 expression, ECOG PS 0, and high BRCAl expression were significantly associated with a higher probability of response (Table 2). In the multivariate logistic regression analysis of these variables together with RRMl and disease stage, only low RRM2 expression emerged as an independent predictive factor for response (Table 2).

Gene expression and time to progression

The univariate analysis for time to progression revealed that the only clinical variable associated with time to progression was PS (Hazard Ratio [HR] for PS 1-2, 1.55; 95% CI, 0.99-2.41 ; p=0.05) (Table 3). The univariate analysis for time to progression according to gene expression levels as continuous variables showed that as RRMl and RRM2 values increased, the risk of progression increased significantly: RRMl (HR, 1.02; 95% CI, 1.01-1.02; p=0.001); RRM2 (HR, 1.005; 95% CI, 1.001-1.008; p=0.01). However, as BRCAl levels increased, the risk of progression decreased (HR, 0.99; 95% CI, 0.95-1.02; p=0.36).

Table 3

Table 3 (continuation)

TTP, time to progression; HR, hazard ratio; T, tercile; PS, performance status

When gene expression levels were categorized by terciles, the risk of progression was greater for patients in the intermediate and highest terciles of RRMl and RRM2 than for those in the lowest tercile: RRMl intermediate tercile (HR, 1.20; 95% CI, 0.72-1.97; p=0.49); RRMl highest tercile (HR, 1.51; 95% CI, 0.91-2.51; p=0.11); RRM2 intermediate terciles (HR, 1.28; 95% CI, 0.77-2.13; p=0.35); RRM2 highest terciles (HR, 1.93; 95% CI, 1.16-3.22; p=0.01) (Table 3). The risk of progression was greater for patients in the intermediate and lowest tercile of BRCAl than for those in the highest tercile: BRCAl intermediate tercile (HR, 1.33; 95% CI, 0.80-2.22; p=0.28); BRCAl lowest tercile (HR, 1.51; 95% CI, 0.91-2.49; p=0.11) (Table 3). Time to progression according to gene expression by terciles is shown in Table 3.

A multivariate model was fitted with the variables examined in the univariate setting. When interaction terms were examined to check whether they significantly improved the fit, none was significant except for BRCA* RRMl, which gave a significance of p=0.02 to the model without the interaction term (Supplementary Table 1). The multivariate model was then stratified by RRMl (Supplementary Table 2) and without disease stage. In this model, patients in the lowest tercile of RRM2 continued to have the lowest risk of progression, independently of RRMl levels.

Supplementary Table 1. Interactions for time to progression

Patients were classified in three groups according to risk of progression, based on the interaction observed between RRMl and BRCAl. Twenty- four patients were in the low-risk group (intermediate BRCAl + low RRMl; high BRCAl + low RRMl; high BRCAl + intermediate RRMl); 42 patients were in the intermediate-risk group (low BRCAl + low RRMl ; intermediate BRCAl + high RRMl ; high BRCAl + high RRMl); and 30 patients were in the high-risk group (low BRCAl + intermediate RRMl; intermediate BRCAl + intermediate RRMl; low BRCAl + high RRMl).

The median time to progression was 10.13 months (95% CI, 7.65-12.62) for patients in the low-risk group, 4.17 months (95% CI, 72.90-5.44) for patients in the intermediate- risk group, and 2.30 months (95% CI, 1.76-2.84) for patients in the high-risk group (p=0.001) (Supplementary Table 3; Figure 2).

Supplementary Table 2. Multivariate analysis of time to progression stratify by RRMl

K)

Supplementary Table 3. Median time to progression stratified by RRMl

Gene expression and survival

In the univariate analysis of survival, the only significant clinical variable was PS (HR for PS 1-2, 1.94; 95% CI, 1.21-3.12; p=0.005) (Table 4). As RRMl and RRM2 values increased, the risk of death increased: RRMl (HR, 1.01; 95% CI, 1.00-1.02; p=0.005); RRM2 (HR, 1.004; 95% CI, 1.00-1.008; p=0.06). However, as BRCAl levels increased, the risk of death decreased (HR, 0.99; 95% CI, 0.96-1.03; p=0.60). When gene expression levels were categorized in terciles, the same pattern of increased risk of death was observed for higher levels of both RRMl and RRM2 and lower levels of

BRCAl (Table 4). In the multivariate model including all the variables from the univariate analysis, only PS emerged as a significant factor for survival (Table 4).

Table 4: Median survival according to gene expression, PS and disease stage

MS, median survival; T, tercile. Table 4 (continuation)

Univariate Cox Multivariate Cox

HR (95% CI) P ] HR (95% CI) P

BRCAl

Tl 1 .28 (0.74-2 21) 0 .37 1 .39 (0.75-2 56) 0 .29

T2 1 .51 (0.85-2 88) 0 .16 1 .48 (0.79-2 79) 0 .22

T3 1 1

RRMl

Tl 1 1

T2 1 .17 (0.68-2 02) 0 .57 1 .37 (0.76-2 46) 0 .29

T3 1 .47 (0.85-2 56) 0 .17 1 .73 (0.94-3 18) 0 .08

RRM2

Tl 1 1

T2 1 .15 (0.66-2 OD 0 .62 0 .74 (0.39-1 40) 0 .35

T3 1 .40 (0.81-2 42) 0 .23 0 .91 (0.49-1 71) 0 .77

PS

0 1 1

1-2 1 .94 (1.21-3 12) 0. 006 2 .07 (1.25-3 43) 0. 005

STAGE

IHB 1 1

IV 1 .57 (0.92-2 63) 0 .10 1 .45 (0.85-2 47) 0 .18

HR, hazard ratio; T, tercile; PS, performance status

Gene expression and second-line treatment Second-line therapy was administered in 31 patients, 90.3% of whom received cisplatin- based chemotherapy. Gene expression levels were not related to whether patients received second- line therapy or not. Time to progression for all 31 patients calculated from the start of second- line therapy was 3.40 months (95% CI, 2.73-4.07). In contrast to the pattern observed with first-line therapy, low levels of BRCAl were significantly associated with the lowest risk of progression to second-line therapy. Median time to progression was 6.60 months for patients in the lowest tercile, 2 months for those in the intermediate tercile, and 2.40 months for those in the highest tercile of BRCAl expression (p=0.004) (Table 5, Figure 3). BRCAl mRNA expression emerged as the only significant factor in both the univariate and multivariate analyses of time to progression in the 31 patients receiving second- line therapy (Table 6). Table 5. Time to progression after first-line treatment according to gene expression levels in 31 patients receiving second- line therapy

TTP, time to progression; T, tercile

Table 6. Univariate and multivariate analyses of time to progression after first-line therapy for 31 patients receiving second- line treatment.

III. DISCUSSION

The present invention shows an association correlation between RRM2 mRNA expression and response to gemcitabine plus docetaxel in advanced NSCLC patients. Patients with low RRM2 mRNA expression attained a significantly higher response rate and time to progression than those with high RRM2 expression. In addition, RRM2 mRNA expression was revealed as an independent predictive factor for response. These results confirm earlier findings in a small cohort of lung adenocarcinomas treated with the same regimen (Souglakos J, et al. 2008. Br J Cancer, vol. 98(10): 1710-5). Intriguingly, transgenic mice developed lung adenocarcinoma but not other tumours in the presence of RRM2 overexpression. In earlier retrospective studies (Rosell R, et al. 2003. Oncogene; vol. 22(23): 3548-53; Rosell R, et al. 2004. Clin Cancer Res; vol. 10(4): 1318-25), it was found that high levels of RRMl predicted longer survival in stage IV NSCLC patients treated with gemcitabine plus cisplatin but not in those treated with cisp latin-based regimens without gemcitabine.

The significant correlation between the top tercile of BRCAl mRNA expression and improved response observed in the present invention adds to the growing body of evidence that BRCAl is a crucial mediator of DNA damage response. Low BRCAl expression confers increased sensitivity to cisplatin and etoposide and resistance to antimicrotubule drugs, such as paclitaxel, docetaxel and vinorelbine, while high BRCAl expression leads to resistance to cisplatin and etoposide and sensitivity to paclitaxel, docetaxel and vinorelbine. In the present invention, patients with low BRCAl mRNA expression had poor response and time to progression to first-line gemcitabine plus docetaxel; in contrast, they obtained the maximum benefit from second-line cisplatin- based treatment, attaining a median time to progression of 6.6 months. BRCAl downregulation has been reported to mediate paclitaxel resistance through premature inactivation of spindle checkpoint in MCF-7 breast cancer cells. An analysis of the SV40 T/t-antigen signature revealed that BRCAl is overexpressed in a subset of lung cancers. Although the authors of the present invention do not want to be attached to any theory, several layers of evidence indicate that loss of let-7 miRNA leads to upregulation of BRCAl as well as of RRMl and RRM2. Therefore, the authors of the present invention can speculate that a subgroup of NSCLCs overexpressing BRCAl can benefit from a non-platinum regimen containing docetaxel. Importantly, a meaningful number of patients - whose tumours have high BRCAl and low RRMl expression - will obtain the maximum benefit from gemcitabine plus docetaxel.

In summary, the findings showed herein indicate that the efficacy of gemcitabine plus docetaxel depends on the mRNA expression of BRCAl, RRMl and RRM2.

Claims

1. An in vitro method for predicting the clinical outcome of a subject suffering from NSCLC who has been treated with an anti-metabolite plus an anti-microtubule agent comprising: (i) determining the expression levels of a first gene and a second gene in a sample from said subject, wherein said first gene is BRCAl gene and the second gene is selected from RRMl gene, RRM2 gene and the combination thereof, and

(ii) comparing said expression levels with reference values for each gene, wherein high expression levels of BRCAl gene and low expression levels of RRMl gene and/or RRM2 gene with respect to said reference values, are indicative of a positive clinical outcome of the subject.

2. Method according to claim 1, wherein the clinical outcome is measured as survival of the subject to the NSCLC, response to the treatment or time to progression.

3. An in vitro method for determining the risk of progression of a subject suffering from NSCLC who has been treated with an anti-metabolite plus an anti-microtubule agent comprising

(i) determining the expression levels of BRCAl and RRMl genes in a sample from said subject, (ii) comparing said expression levels with reference values for each gene, and (iii) categorizing said expression levels by terciles for each gene, wherein if

4. An in vitro method for designing an individual therapy for a subject suffering from NSCLC comprising

(i) determining the expression levels of a first and a second gene in a sample from said subject , wherein said first gene is BRCAl gene and the second gene is selected from RRMl gene, RRM2 gene and the combination thereof, and (ii) comparing said expression levels genes with reference values for each gene, wherein low expression levels of BRCAl and high expression levels of RRMl and/or RRM2 with respect to said reference values, are indicative that the patient is a candidate for an anti-metabolite plus an anti-microtubule agent chemotherapy.

5. An in vitro method for determining the risk of progression of a subject suffering from NSCLC who has received an anti-metabolite plus an anti-microtubule agent as first- line treatment and platinum-based chemotherapy as second-line treatment comprising

(i) determining the expression levels of BRCAl gene in a sample from said subject and

(ii) comparing said expression levels of BRCAl gene with reference values for said gene, wherein low expression levels of BRCAl gene with respect to said reference values are indicative of low risk of progression after second- line treatment.

6. Method according to anyone of claims 1 to 5, wherein the anti-metabolite is gemcitabine and the anti-microtubule agent is docetaxel.

7. Method according to anyone of claims 1 to 6, wherein the sample is a tissue sample, preferably a tumour tissue sample, more preferably a lung tumour tissue sample.

8. Method according to anyone of claims 1 to 7, wherein the expression levels of the BRCAl gene, RRMl gene and/or RRM2 gene are determined by measuring the levels of mRNA encoded by said genes or the levels of BRCAl protein, RRMl protein and/or RRM2 protein or of variants thereof.

9. Method according to claim 8, wherein the mRNA expression levels are determined by quantitative PCR, preferably, Real-Time PCR.

10. Method according to claim 1 to 9, wherein the NSCLC is in stage IIIB or IV.

11. A kit comprising a set of reagents, wherein said set consists of reagents for detecting BRCAl, RRMl, RRM2 genes and/or the combination thereof or reagents for detecting BRCAl, RRMl, RRM2 proteins, variants thereof and/or the combination thereof and, optionally, a reagent for detecting a housekeeping gene or the protein encoded by said housekeeping gene.

12. Kit according to claim 11, wherein the set of reagents is selected from the group of: a) A set of nucleic acids capable of specifically hybridising with the BRCAl, RRMl and/or RRM2 genes, and/or b) A set of antibodies, or fragments thereof capable of specifically binding to BRCAl, RRMl and/or RRM2 proteins or to variants thereof.