Methods and devices for predicting anthracycline treatment efficacy

Abstract

The invention features methods, devices, and kits for predicting the responsiveness of a cancer patient (e.g., a breast cancer patient, such as a grade 1, 2, or 3 breast cancer patient) to anthracycline treatment by determining the expression levels of four chromosomal instability (CIN) genes including HDGF, KIAA0286, RFC4, and MSH6, collectively referred to as CIN4. Patients that have a low CIN4 score benefit from anthracycline treatment compared patients with a high CIN4 score.

Claims

1. A method of treating a cancer patient comprising a) obtaining the results of a test that determined the level of expression of biomarkers having at least 25 consecutive nucleotides of the sequences of each of SEQ ID NOs: 1-4 in a sample from the cancer patient, and compared: i) the levels of expression of the biomarkers in the sample from the cancer patient to the levels of expression of the same biomarkers in a sample from a first reference patient known to be responsive to anthracycline treatment, or ii) the levels of expression of the biomarkers in the sample from the cancer patient to the levels of expression of the same biomarkers in a sample from a second reference patient known to be non-responsive to anthracycline treatment, and wherein said results show that: iii) the levels of expression of the biomarkers in the sample from the cancer patient are similar to the levels of expression of the biomarkers in the sample from the first reference patient or iv) the levels of expression of the biomarkers in the sample from the cancer patient are dissimilar to the levels of expression of the biomarkers in the sample from the second reference patient; and (b) administering anthracycline treatment to the cancer patient; wherein the biomarkers are HDGF, KIAA0286, RFC4, and MSH6.

2. The method of claim 1, wherein the sample from the cancer patient is a tissue sample.

3. The method of claim 2, wherein the sample from the cancer patient is a tumor sample.

4. The method of claim 1, wherein the cancer is a breast cancer.

5. The method of claim 4, wherein the cancer is grade 1, 2, or 3.

6. The method of claim 1, wherein: (a) said obtaining occurs after said patient has received a first cancer treatment; (b) said obtaining occurs after said patient has received a second cancer treatment.

7. The method of claim 6, wherein: (a) said first cancer treatment comprises one or more of surgery, radiation therapy, and chemotherapy; (b) said first or second cancer treatment comprises one or more of surgery, radiation therapy, and chemotherapy; or (c) said second cancer treatment comprises one or more of surgery, radiation therapy, and chemotherapy.

8. The method of claim 1, wherein the level of expression of said biomarkers in said sample from the cancer patient, in said sample from said first reference patient, and in said sample from said second reference patient is determined by collecting nucleic acid molecules from said sample from the cancer patient.

9. The method of claim 1, wherein said anthracycline is selected from the group consisting of epirubicin, daunorubicin, doxorubicin, idarubicin, valrubicin, actinomycin-D, bleomycin, mitomycin-C, and mitoxantrone.

10. The method of claim 9, wherein said anthracycline is epirubicin.

11. The method of claim 1, further comprising treating said cancer patient with one or more chemotherapeutic agents listed in Table 2.

12. The method of claim 1, wherein said levels of expression of said biomarkers is determined using a microarray device.

13. The method of claim 1, wherein said method comprises determining the levels of expression of said biomarkers using a qRT-PCR.

14. A method of treating a cancer, said method comprising administering an anthracycline to a cancer patient that has been determined to have similar levels of expression of biomarkers having at least 25 consecutive nucleotides of the sequences of each of SEQ ID NOs: 1-4 relative to the levels of expression of the same biomarkers in a first reference patient known to be responsive to anthracycline treatment; wherein the biomarkers are HDGF, KIAA0286, RFC4, and MSH6.

15. The method of claim 14, wherein said anthracycline is selected from the group consisting of epirubicin, daunorubicin, doxorubicin, idarubicin, valrubicin, actinomycin-D, bleomycin, mitomycin-C, and mitoxantrone.

16. The method of claim 15, wherein said anthracycline is epirubicin.

17. The method of claim 14, wherein said cancer patient has been determined to have similar levels of expression of the biomarkers relative to the levels of expression of the same biomarkers in a first reference patient known to be responsive to anthracycline treatment by: a) determining the levels of expression of the biomarkers in a sample from the cancer patient, and b) i) comparing the levels of expression of the biomarkers in the sample from the cancer patient to the levels of expression of the same biomarkers in a sample from a first reference patient known to be responsive to anthracycline treatment, or ii) comparing the levels of expression of the biomarkers in the sample from the cancer patient to the levels of expression of the same biomarkers in a sample from a second reference patient known to be non-responsive to anthracycline treatment.

18. A method for treating a cancer patient comprising administering an anthracycline to a patient in which the levels of expression in a sample from the patient biomarkers having at least 25 consecutive nucleotides of the sequences of each of SEQ ID NOs: 1-4 has been determined to be similar to the levels of expression of said biomarkers in a reference patient known to be responsive to anthracycline treatment, wherein said levels of expression are determined using a device comprising at least four single-stranded nucleic acid molecules, each having at least 85% identity to a target nucleic acid molecule having a sequence that is complementary or identical to at least 5 consecutive nucleotides of said biomarkers respectively, wherein said at least four single-stranded nucleic acid molecules are sufficient for the detection of the levels of expression of said biomarkers and allow specific hybridization between said single stranded nucleic acid molecules and said target nucleic acid molecules, wherein the levels of expression of said biomarkers are predictive of responsiveness of said cancer patient to anthracycline treatment; wherein the biomarkers are HDGF, KIAA0286, RFC4, and MSH6.

19. The method of claim 18, wherein the target nucleic acid molecule has a sequence that is complementary or identical to at least 10 to 100, at least 20 to 100, at least 30 to 100, at least 40 to 100, at least 50 to 100, at least 60 to 100, at least 70 to 100, at least 80 to 100, or at least 90 to 100 consecutive nucleotides.

20. The method of claim 18, wherein said at least one single-stranded nucleic acid molecule has a length in the range of 10 to 100 nucleotides.

21. The method of claim 18, said device allowing, when contacted with a diverse population of nucleic acid molecules prepared from a sample under conditions allowing hybridization to occur, the determination of the levels of expression of said biomarkers.

22. The method of claim 18, wherein the device is a microarray device.

23. The method of claim 18, wherein the sample is a tissue sample.

24. The method of claim 23, wherein the sample is a tumor sample.

25. The method of claim 18, wherein the cancer is a breast cancer.

26. The method of claim 25, wherein the cancer is grade 1, 2, or 3.

27. The method of claim 18, wherein: (a) said administering occurs in said patient after said patient has received a first cancer treatment; or (b) said administering occurs in said patient after said patient has received a second cancer treatment.

28. The method of claim 27, wherein: (a) said first cancer treatment comprises any combination of one or more of surgery, radiation therapy, and chemotherapy; (b) said first cancer treatment comprises one or more of surgery, radiation therapy, and chemotherapy and combinations thereof; (c) said first or second cancer treatment comprises one or more of surgery, radiation therapy, and chemotherapy and combinations thereof; or (d) said second cancer treatment comprises one or more of surgery, radiation therapy, and chemotherapy and combinations thereof.

29. The method of claim 7, wherein said first or second cancer treatment is surgery.

30. The method of claim 8, further comprising detecting said nucleic acid molecules using one or more fluorescent probes or using a quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to amplify said nucleic acid molecules.

31. The method of claim 1, wherein the biomarker comprises all or a portion of the sequences of each of SEQ ID NOs: 1-4.

32. The method of claim 1, wherein the biomarker consists of all or a portion of the sequences of each of SEQ ID NOs: 1-4.

33. The method of claim 14, wherein the biomarker comprises all or a portion of the sequences of each of SEQ ID NOs: 1-4.

34. The method of claim 14, wherein the biomarker consists of all or a portion of the sequences of each of SEQ ID NOs: 1-4.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows graphs of Kaplan-Meier survival curves for low CIN25 score (lower line) and high CIN25 score (higher line) for overall survival (A) and distant relapse free survival (B).

(2) FIG. 2 shows graphs of Kaplan-Meier survival curves for epirubicin plus cyclophosphamide, methotrexate and fluorouracil (E-CMF) (higher line) and CMF (lower line) treated low CIN25 score (A, C) or high CIN25 score (B, D) for overall survival (A, B) and distant relapse free survival (C, D).

(3) FIG. 3 shows a graph of Kaplan-Meier survival curve for low CIN4 score treated with epirubicin plus cyclophosphamide, methotrexate and fluorouracil (E-CMF) (Line 2), high CIN4 score treated with E-CMF (Line 3), low CIN4 score treated with CMF (Line 1) and high CIN4 score treated with CMF (Line 4) for distant relapse free survival.

DETAILED DESCRIPTION OF THE INVENTION

(4) CIN4 Signature is a Predictive Marker of Anthracycline Benefit

(5) We have discovered that a four-gene-based signature (CIN4) is associated with a cancer patient's response to anthracycline treatment. The CIN4 signature includes the expression levels of one or more, or all of the genes HDGF, KIAA0286, RFC4, and MSH6. In multivariate regression analysis, the CIN4 signature conferred predictive responsiveness to anthracycline treatment. Interestingly, three of the four genes in our CIN4 signature are involved in DNA repair/DNA-binding activity. Anthracyclines are thought to exert their actions by intercalation with DNA, generation of free radicals, and crosslinking DNA to proteins. Therefore, dysregulation of genes involved in DNA repair leads to anthracycline sensitivity.

(6) We discovered that grade 3 breast cancer patients with a high level of CIN benefited from anthracycline therapy. It is possible that low grade tumors are more susceptible to a taxane treatment while higher grade tumors are sensitive to anthracycline treatment. A previous study has demonstrated a high level of CIN70 gene expression is associated with paclitaxel resistance (Swanton et al., Proc Natl Acad Sci. 106:8671-8676, 2009).

(7) In some embodiments, the CIN4 signature is an independent predictor of anthracycline sensitivity. In other embodiments, the combination of chromosomal instability and high tumor grade may predict anthracycline sensitivity and taxane resistance.

(8) Correlation of CIN25 or CIN70 and Clinicopathological Parameters

(9) We successfully analysed 282 of 321 (87.9%) and 421 of 440 (95.7%) tumors from BR9601 and MA.5, respectively. High CIN70 and CIN25 scores were defined as above the median as previously described (Carter et al., Nat Genet 38:1043-1048, 2006). High CIN70 and CIN25 scores were associated with age (p<0.0001), grade (p<0.0001), PgR negativity (p<0.0001), and ER negativity (p<0.0001), but not with tumor size, nodal status, or HER2 status.

(10) CIN Signature as a Prognostic Marker for Overall Survival (OS) and Distant Recurrence-Free Survival (DRFS)

(11) The prognostic significance of CIN25 and CIN70 was tested on the entire patient cohort, irrespective of allocated adjuvant chemotherapy. No significant association between CIN70 expression and OS or DRFS was evident. Tumors with high CIN25 scores were associated with reduced OS (HR: 0.69, 95% Cl 0.54-0.88, p=0.003, FIG. 1A) and DRFS (HR: 0.70, 95% Cl 0.60-0.90, p=0.004, FIG. 1B). After multivariate analysis and adjustment for nodal status, grade, size, age, HER2, ER, and PgR status, high CIN25 score was not an independent predictor for OS or DRFS.

(12) CIN Signature as a Biological Marker for Responsiveness to Anthracycline Treatment

(13) We analysed the differential effects of the CIN signatures on OS and DRFS between patients receiving anthracycline treatment (E-CMF) and those given CMF alone by assessing hazard ratios. No significant differential benefit from E-CMF treatment was demonstrated between patients whose tumors had high or low CIN70 expression (Table 1)

(14) In univariate analysis, patients whose tumors had high CIN25 gene expression scores had a decreased risk of distant relapse (HR: 0.74, 95% Cl 0.54-0.99, p=0.046) when treated with E-CMF compared with patients treated with CMF alone (Table 1). There was no apparent benefit of E-CMF vs CMF noted in patients with low CIN25 scores for DRFS (HR: 0.87, 95% Cl 0.61-1.21, p=0.374). In a multivariate analysis with adjustment for size, nodal status, ER, pathological grade, HER2, CIN25, treatment and CIN25*treatment (a marker by treatment interaction test; e.g., McShane, BMC Medicine 10:52, 2012, and Mandrekar et al., Journal of clinical Oncology 27:4027, 2009) showed only pathological grade, nodal status, tumor size, and polysomy to be significant predictors of outcome. No significant differential benefit from E-CMF treatment was demonstrated between patients whose tumors had high or low CIN25 expression for OS (Table 1). The hazard ratio for treatment marker effect of CIN25 was 0.86 (95% CI 0.53-1.40, p=0.549) for OS and 0.86 (95% CI 0.54-1.36, p=0.519) (Table 1).

(15) CIN Signature as a Biological Marker for Responsiveness to Anthracycline Treatment in Grade 3 Patients

(16) Previous research identified a significant association between CIN gene expression and grade 3 tumors (Carter et al., Nat Genet 38:1043-1048, 2006). Therefore, an exploratory analysis was performed on patients that were pathological grade 3 only. We analysed the differential effects of the CIN signatures on OS and DRFS between patients receiving anthracycline treatment (E-CMF) and those given CMF alone by assessing hazard ratios.

(17) In univariate analysis, patients whose tumors had high CIN25 gene expression scores had a reduced risk of distant relapse (HR: 0.66, 96% Cl 0.46-0.94, p=0.021) and increased OS (HR: 0.73, 95% Cl 0.57-0.95, p=0.05) when treated with E-CMF compared with patients treated with CMF alone (FIG. 2, Table 1). No significant benefit from E-CMF treatment versus CMF treatment was demonstrated in patients with tumors exhibiting low CIN25 gene expression (Table 1). In patients with grade 1 and 2 tumors, no significant benefit from E-CMF treatment versus CMF treatment was demonstrated with either high or low CIN25 gene expression scores (Table 1). The hazard ratio for treatment marker effect of CIN25 in grade 3 tumors was 0.78 (95% CI 0.42-1.43, p=0.413) for OS and 0.81 (95% CI 0.45-1.46, p=0.479) (Table 1).

(18) TABLE-US-00001 TABLE 1 Hazard ratios for overall survival and distant relapse free survival comparing epirubicin plus cyclophosphamide, methotrexate and fluorouracil (E-CMF) with CMF alone by biomarker status. Treatment*Marker Low Biomarker High Biomarker Test for HR 95% CI HR 95% CI HR Interaction P Overall Survival (OS) CIN70 0.82 0.57-1.17 0.82 0.59-1.14 0.99 0.977 CIN25 0.87 0.61-1.29 0.76 0.56-1.05 0.86 0.549 CIN25 in 0.91 0.55-1.48 0.70 0.41-1.36 0.78 0.413 grade 3 CIN25 in 0.74 0.41-1.36 1.27 0.58-2.80 1.76 0.266 grade 1&2 Distant Relapse Free Survival (DRFS) CIN70 0.79 0.57-1.10 0.79 0.58-1.08 0.97 0.904 CIN25 0.85 0.61-1.21 0.74 0.54-0.99 0.86 0.519 CIN25 in 0.81 0.51-1.30 0.66 0.46-0.94 0.81 0.479 grade 3 CIN25 in 0.85 0.50-1.43 1.12 0.58-2.12 1.30 0.541 grade 1&2
CIN4 Signature Predicts Responsiveness to Anthracycline Treatment

(19) In order to select a more limited set of genes that reflects CIN, we used the merged clinical cohort (containing both BR9601 and MA.5). The cohort was split into anthracycline treated and CMF treated cohorts. The patients were clustered using the expression profile of the 70 genes, which led to nine clusters. A multivariate Cox model was fit for each gene, adjusting for clinical variables including HER2, ER, PgR, tumor size, grade, and nodal status. The top genes from each expression cluster, with the most significant p-value in the anthracycline treated cohort and a non-significant CMF cohort, were selected to make a list of 20 genes.

(20) From this list of 20 genes, all possible combinations of 2, 3, 4, and 5 gene signatures were examined (210, 1330, 5985 and 20349 combinations, respectively) and bootstrapped 100 times, with the median area under the curve (AUC) noted. In each bootstrap, the treatment cohort was split into 60% training and 40% test sets. The AUC was calculated from the test sets and the gene signature selected had the greatest AUC and had four genes, HDGF, KIAA0286, RFC4, and MSH6, termed the CIN4 signature. A multivariate Cox regression was fit using the 4 genes, adjusting for the same clinical variables mentioned above. A CIN4 score was generated using the expression values of the 4 genes, weighted by their regression coefficients. Patients that have a low CIN4 score benefit from anthracycline treatment compared to high CIN4 score (HR 2.72, 95% Cl 1.48-5.02, p=0.001) (FIGS. 2 and 3). No significant benefit with CMF treatment was observed in either low or high CIN4 score. The hazard ratio for treatment marker effect of CIN4 was 0.35 (95% CI 0.15-0.79, p=0.01) for DRFS.

(21) Methods of Determining Gene Expression and CIN4 Signature Score

(22) In some embodiments, formalin-fixed paraffin embedded (FFPE) tissue samples may be used. In other embodiments, cell or tissue samples (e.g., tissue samples from tumors) taken from patients (e.g., cancer patients, such as breast cancer patients (e.g., grade 1, 2, or 3 breast cancer patients)) may be snap frozen in liquid nitrogen until processing or by other methods known in the art. Total RNA may be extracted from cell or tissue samples using one of the commercially available kits, e.g., preferably RecoverAll Total Nucleic Acid Isolation kit (Life Technologies), or using agents well known in the art, e.g., Trizol Reagent.

(23) The expression levels of the various biomarkers, e.g., one or more (e.g., all) of the four genes in the CIN4 signature (HDGF, KIAA0286, RFC4, and MSH6), using, e.g., isolated RNA, may be determined using procedures, such as a microarray or other known device or platform and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), that can be used to measure the level of biomarkers expression in a sample. In some embodiments, microarray and qRT-PCR may be performed in combination. Alternatively, the method may determine the expression levels of one or more of the four genes (HDGF, KIAA0286, RFC4, and MSH6) in the CIN4 signature using isolated proteins corresponding to the product of these genes.

(24) A microarray of the invention may include one or more oligonucleotide probes that have nucleotide sequences that are identical to or complementary to, e.g., at least 5, 8, 12, 20, 25, 30, 40, 60, 80, 100, 150, or 200 consecutive nucleotides (or nucleotide analogues) of the biomarkers, e.g., HDGF, KIAA0286, RFC4, and MSH6. The oligonucleotide probes may be, e.g., 5-20, 25, 5-50, 50-100, or over 100 nucleotides long. The oligonucleotide probes may be deoxyribonucleic acids (DNA) or ribonucleic acids (RNA). Additionally, probes employed on microarrays of the invention may also include proteins, peptides, or antibodies that selectively bind any of the oligonucleotide sequences or their complementary sequences of a polypeptide encoded by the gene or mRNA of HDGF, KIAA0286, RFC4, or MSH6.

(25) Procedures for performing qRT-PCR are described in, e.g., U.S. Pat. No. 7,101,663 and U.S. Patent Application Nos. 2006/0177837 and 2006/0088856, each of which is incorporated herein by reference in its entireties.

(26) The resulting gene or protein expression measurements are further processed and statistically analyzed as described further herein. A CIN4 signature score is calculated using statistical analyses described in Example 4.

(27) Identifying a Subpopulation of Patients Sensitive to a Treatment for Cancer

(28) The methods of the invention may be used to identify a subpopulation of cancer patients, e.g., breast cancer patients, such as grade 1, 2, or 3 breast cancer patients, responsive to a treatment, e.g., anthracycline treatment, or other medical treatment. To this end, the level of expression of one or more (e.g., all) of the biomarkers in the CIN4 signature (HDGF, KIAA0286, RFC4, and MSH6) correlating to responsiveness to anthracycline treatment, may be determined so that patients responsive to anthracycline treatment may be identified.

(29) Alternatively, genes may be identified as biomarkers (e.g., biomarkers in the CIN4 signature (HDGF, KIAA0286, RFC4, and MSH6)) according to their ability to discriminate patients known to be responsive to a treatment (e.g., anthracycline treatment) from those known to be resistant. The significance of the differences in gene (HDGF, KIAA0286, RFC4, and/or MSH6) expression between the responsive and resistant patients may be measured using, e.g., t-tests.

(30) The patient populations (e.g., cancer patients, such as breast cancer patients (e.g., grade 1, 2, or 3 breast cancer patients)) considered may be further divided into patients predicted to survive without treatment (e.g., anthracycline treatment), patients predicted to die without treatment (e.g., anthracycline treatment), and patients predicted to have symptoms without treatment (e.g., anthracycline treatment). The above methodology may be similarly applied to any of these further defined patient subpopulations to identify biomarkers (e.g., biomarkers in the CIN4 signature (HDGF, KIAA0286, RFC4, and MSH6)) that are able to predict a patient's responsiveness to treatments, e.g., anthracycline treatment, or other treatments for cancer, e.g., breast cancer, such grade 1, 2, or 3 breast cancer.

(31) Anthracycline and Non-Anthracycline Treatments

(32) Patients with CIN4 signature scores that identify them as responsive to anthracycline treatment may be administered anthracycline treatment, which includes, but is not limited to, treatment with one or more of the following chemotherapeutic agents and their derivatives: epirubicin, daunorubicin, doxorubicin, idarubicin, valrubicin, actinomycin-D, bleomycin, mitomycin-C, and mitoxantrone.

(33) Cancer patients with CIN4 signature scores identifying them as likely to be non-responsive to anthracycline treatment may be administered one or more chemotherapeutic agents other than an anthracycline. Some examples of non-anthracycline chemotherapeutic agents are listed in Table 2.

(34) TABLE-US-00002 TABLE 2 Therapeutic Class Exemplary, Non-Limiting Agents Alkylating Nitrogen mustards: such as mechlorethamine Agents (nitrogen mustard), chlorambucil, cyclophosphamide (Cytoxan ®), ifosfamide, and melphalan Nitrosoureas: which include streptozocin, carmustine (BCNU), and lomustine Alkyl sulfonates: busulfan Triazines: dacarbazine (DTIC) and temozolomide (Temodar ®) Ethylenimines: thiotepa and altretamine (hexamethyl- melamine) Antimetabolites 5-fluorouracil (5-FU) 6-mercaptopurine (6-MP) Capecitabine (Xeloda ®) Cladribine Clofarabine Cytarabine (Ara-C ®) Floxuridine Fludarabine Gemcitabine (Gemzar ®) Hydroxyurea Methotrexate Pemetrexed (Alimta ®) Pentostatin Thioguanine Topoisomerase topoisomerase I inhibitors inhibitors topotecan irinotecan (CPT-11). topoisomerase II inhibitors etoposide (VP-16) teniposide Mitotic Taxanes such as paclitaxel (Taxol ®) and docetaxel inhibitors (Taxotere ®) Epothilones: ixabepilone (Ixempra ®) Vinca alkaloids such as vinblastine (Velban ®), vincristine (Oncovin ®), and vinorelbine (Navelbine ®) Estramustine (Emcyt ®) Corticosteroids Examples include prednisone, methylprednisolone (Solumedrol ®), and dexamethasone (Decadron ®). Miscellaneous L-asparaginase Chemo- bortezomib (Velcade ®) therapeutics imatinib (Gleevec ®) gefitinib (Iressa ®) sunitinib (Sutent ®) Differentiating retinoids agents tretinoin (ATRA or Atralin ®) bexarotene (Targretin ®) arsenic trioxide (Arsenox ®). Hormone The anti-estrogens: fulvestrant (Faslodex ®), therapy tamoxifen, and toremifene (Fareston ®) Aromatase inhibitors: anastrozole (Arimidex ®), exemestane (Aromasin ®), and letrozole (Femara ®) Progestins: megestrol acetate (Megace ®) Estrogens Anti-androgens: bicalutamide (Casodex ®), flutamide (Eulexin ®), and nilutamde (Nilandron ®) Gonadotropin-releasing hormone (GnRH), also known as luteinizing hormone-releasing hormone (LHRH) agonists or analogs: leuprolide (Lupron ®) and goserelin (Zoladex ®) Immunotherapy Monoclonal antibody therapy (passive immuno- therapies), such as rituximab (Rituxan ®) and alemtuzumab (Campath ®) Non-specific immunotherapies and adjuvants (other substances or cells that boost the immune response), such as BCG, interleukin-2 (IL-2), and interferon-alfa Immunomodulating drugs, for instance, thalidomide and lenalidomide (Revlimid ®) Cancer vaccines (active specific immunotherapies)

(35) One or more of the above-mentioned chemotherapeutic agents may be administered to the appropriate patient populations identified based on their CIN4 scores using any methods known in the art, including but not limited to, oral, topical, transdermal, parenteral, subcutaneous, intranasal, intramuscular and intravenous routes, including both local and systemic applications. The chemotherapeutic agents, and pharmaceutical compositions thereof, may be administered to a patient in need thereof, for example, one or more times (e.g., 1-10 times or more) daily, weekly, monthly, biannually, annually, or as medically necessary. Dosages may be provided in either a single or multiple dosage regimens. Methods of administering chemotherapeutic agents are known in the art. See, for example, U.S. Pat. Nos. 7,811,998, 6,201,554, and 8,497,274, and U.S. Patent Application Publication Nos. US20090048301, WO2013025882, US20040063705, the disclosures of which are incorporated by reference in their entireties.

EXAMPLES

Example 1—BR9601 Clinical Trial

(36) The BR9601 trial recruited 374 pre- and post-menopausal women with completely excised, histologically confirmed breast cancer and a clear indication for adjuvant chemotherapy. Patients were randomized between 8 cycles of CMF (i.v. cyclophosphamide 750 mg/m.sup.2, methotrexate 50 mg/m.sup.2, and 5-fluorouracil 600 mg/m.sup.2) every 21 days, and E-CMF (4 cycles of epirubicin 100 mg/m.sup.2 every 21 days followed by 4 cycles of the same CMF regimen). Patient characteristics are shown in Table 3. The protocol was approved by central and local ethics committees, and each patient provided written informed consent prior to randomization. For the current analysis, tissue blocks were retrieved and RNA was extracted.

Example 2—MA.5 Clinical Trial

(37) The MA.5 trial randomized 716 premenopausal women with node-positive breast cancer to receive either adjuvant CEF or CMF. The CEF regimen consisted of 6 cycles of epirubicin 60 mg/m.sup.2 and 5-fluorouracil (5-FU) 500 mg/m.sup.2, both delivered intravenously on days 1 and 8, and oral cyclophosphamide 75 mg/m.sup.2 daily on days 1 through 14. Patients randomized to the CEF regimen also received antibiotic prophylaxis throughout. The CMF regimen consisted of 6 cycles of methotrexate 40 mg/m.sup.2 and 5-FU 600 mg/m.sup.2, both delivered intravenously on days 1 and 8, and oral cyclophosphamide 100 mg/m.sup.2 daily on days 1 through 14. Patient characteristics are shown in Table 3. The MA.5 protocol was approved by the institutional review board at each participating center and registered as NCI-V90-0027 on cancer.gov. Written informed consent was obtained from each woman.

(38) TABLE-US-00003 TABLE 3 Baseline characteristics for patients from BR9601 and MA.5 clinical trials Clinical trial CIN analysis BR9601 MA.5 BR9601 MA.5 Number 374  710  282  421  Age   50.6   43.9   50.6   43.9 Mean (22.7-76.0) (23.4-57.2) (26.2-76.0) (23.4-57.2) (range) Treatment E-CMF 183 (48.9%) 350 (49.3%) 138 (48.9) 208 (49.4%) CMF 191 (51.1%) 360 (50.7%) 144 (51.1%) 213 (50.6%) Size <2.0 cm 123 (32.9%) 265 (37.9%) 94 (33.3%) 150 (36.1%) >2.0 cm 251 (67.1%) 435 (62.1%) 188 (66.7) 265 (63.9%) Missing 10 Nodes 0  48 (12.8%)  0 1-3 214 (57.3%) 433 (61.0%) ≥4 112 (29.9%) 277 (39.0%) Grade 1 22 (6.1%)  77 (12.4%) 19 (6.8%)  42 (10.2%) 2 126 (35.2%) 204 (32.9%) 96 (34.3%) 120 (29.1%) 3 210 (58.7%) 340 (54.8%) 165 (58.9%) 250 (60.7%) Unknown 16 89  2  9 ER Status Positive 202 (62.9%) 424 (59.7%) 155 (62.8%) 253 (67.3%) Negative 119 (37.1%) 200 (32.1%) 92 (37.2%) 123 (32.7%) Unknown 53 86 35 45

Example 3—RNA Purification and Gene Expression Analysis

(39) Total RNA from formalin-fixed paraffin embedded (FFPE) tissue samples (2×10 μM sections) were extracted using the RecoverAll Total Nucleic Acid Isolation kit (Life Technologies) according to the manufacturers protocol and concentrations were determined using the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies).

(40) RNA (400 ng) was used for the analysis with the nCounter system, according to the manufacturer's protocol (Nanostring® Technologies, Seattle, Wash., USA). In brief, 5 μl of RNA was hybridized at 96° C. overnight with the Nanostring Codeset.

(41) Probes for the analysis of 70 genes and controls were synthesized by Nanostring technologies, including probes for the 70 genes of interest and 6 normalising genes. All 76 genes and controls were assayed simultaneously in multiplexed reactions (gene list, Table 4). After probe hybridizations and Nanostring nCounter digital reading, counts for each RNA species were extracted and analyzed. The nCounter CodeSet contains two types of built-in controls: positive controls (spiked RNA at various concentrations to assess the overall assay performance) and negative controls (alien probes for background calculation). To account for slight differences in hybridization and purification efficiency, the raw data were normalized to the standard curve generated via the nCounter system spike-in controls present in all reactions.

(42) TABLE-US-00004 TABLE 4 Housekeeping CIN70 genes genes DHCR7 CCNB2 ch-TOG RNASEH2A AURKB ATM GUSB GPIandMGC13096 FEN1 MSH6 RRM1 CDC2 ATR PUM1 CKS2 FLJ10036 PCNA RRM2 MAD2L1 CDC25B SF3A1 BRRN1 H2AFX RAD21 TGIF2 PRC1 CDC25C TBP CNAP1 H2AFZ RFC4 ATAD2 TPX2 CHEK1 TFRC MCM10 HDGF UNG NDUFAB1 TTK CHEK2 TMED10 CDC20 KIF4A CDC45L KIAA0286 UBE2C MDM2 ESPL1 PTTG1 CDC6 KIF20A ZWINT P53 FOXM1 AURKA CDCA8 CDC3A CMAS CDKN1A MTB MELK CEP55 ACTL6A DKC1 NEK2 RAD51AP1 CTPS LSM4 TRIP13 OIP5 TOPK ECT2 SFRS2 CCT5 TOP2A EZH2 MCM2 ELAV1 MTCH2 CCNB1 ASF1B MCM7 NXT1 NUP205

Example 4—Statistical Analysis

(43) The SPSS (v20) statistical package was used for statistical analysis. Kaplan-Meier estimates of survival were used for analysis of relapse free survival (RFS) and overall survival (OS). The Cox's proportional hazard model was used to obtain hazard ratios for relapse or death. When comparing outcomes between the treatment arms within the groups of patients identified by biomarker expression, formal p-values were not calculated for sub-groups to avoid multiple testing and bias where one group was much smaller than the other. The Cox model was instead used to identify statistically significant interactions (p<0.05) between biomarkers and outcome on the different treatments (treatment by marker effect), in models that also included biomarker status (marker effect) and treatment, as covariates.

Example 5—Generation of CIN4 Score

(44) The combined cohort was split to two groups according to the randomzied treatment. Using Affinity propagation clustering (R package apcluster.sup.1), the 70 genes were clustered into 9 groups according to their expression profiles. A multivariate Cox model was fit for each gene, adjusting for clinical variables including HER2, ER, PgR, tumor size, grade, and nodal status. The top genes from each expression cluster, with the most significant p-value in the anthracycline treated cohort and a non-significant CMF cohort, were selected to make a list of 21 genes. From this list, all possible combinations of 2,3,4, and 5 genes signatures were examined (210,1330,5985, and 20,349 combinations, respectively) and bootstrapped 100 times, with the median area under the curve (AUC) noted. In each bootstrap, the treatment cohort was split into 60% training and 40% test sets. The AUC was calculated from the test sets (R package survivalROC.sup.2). The gene signature selected had the greatest AUC and had four genes, termed the CIN4 signature. A multivariate Cox regression was fit using the four genes, adjusting for the same clinical variables mentioned above. A CIN4 score was generated using the expression values of the four genes, weighted by their regression coefficients.

Other Embodiments

(45) All publications, patents, and patent applications mentioned in the above specification are hereby incorporated by reference. Various modifications and variations of the described methods and uses of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention.

(46) TABLE-US-00005 APPENDIX Sequences SEQ ID NO: 1 is the nucleic acid sequence of HDGF (GenBank: NM_004494.2). SEQ ID NO: 1 1 gagggaggag gaggagtggg gaccgggcgg ggggtggagg aagaggcctc gcgcagagga   61 gggagcaatt gaatttcaaa cacaaacaac tgcacgagcg cgcacccacc gcgccggagc  121 cttgccccga tccgcgcccg ccccgtccgt gcggcgcgcg ggcggagacg ccgtggccgc  181 gccggagctc gggccggggg ccaccatcga ggcgggggcc gcgcgagggc cggagcggag  241 cggcgccgcc accgccgcac gcgcaaactt gggctcgcgc ttcccggccc ggcgcggagc  301 ccggggcgcc cggagccccg ccatgtcgcg atccaaccgg cagaaggagt acaaatgcgg  361 ggacctggtg ttcgccaaga tgaagggcta cccacactgg ccggcccgga ttgacgagat  421 gcctgaggct gccgtgaaat caacagccaa caaataccaa gtcttttttt tcgggaccca  481 cgagacggca ttcctgggcc ccaaagacct cttcccttac gaggaatcca aggagaagtt  541 tggcaagccc aacaagagga aagggttcag cgaggggctg tgggagatcg agaacaaccc  601 tactgtcaag gcttccggct atcagtcctc ccagaaaaag agctgtgtgg aagagcctga  661 accagagccc gaagctgcag agggtgacgg tgataagaag gggaatgcag agggcagcag  721 cgacgaggaa gggaagctgg tcattgatga gccagccaag gagaagaacg agaaaggagc  781 gttgaagagg agagcagggg acttgctgga ggactctcct aaacgtccca aggaggcaga  841 aaaccctgaa ggagaggaga aggaggcagc caccttggag gttgagaggc cccttcctat  901 ggaggtggaa aagaatagca ccccctctga gcccggctct ggccgggggc ctccccaaga  961 ggaagaagaa gaggaggatg aagaggaaga ggctaccaag gaagatgctg aggccccagg 1021 catcagagat catgagagcc tgtagccacc aatgtttcaa gaggagcccc caccctgttc 1081 ctgctgctgt ctgggtgcta ctggggaaac tggccatggc ctgcaaactg ggaacccctt 1141 tcccacccca acctgctctc ctcttctact cacttttccc actccaagcc cagcccatgg 1201 agattgacct ggatggggca ggccacctgg ctctcacctc taggtcccca tactcctatg 1261 atctgagtca gagccatgtc ttctccctgg aatgagttga ggccactgtg ttccttccgc 1321 ttggagctat tttccaggct tctgctgggg cctgggacaa ctgctcccac ctcctgacac 1381 ccttctccca ctctcctagg cattctggac ctctgggttg ggatcagggg taggaatgga 1441 aaggatggag catcaacagc agggtgggct tgtggggcct gggaggggca atcctcaaat 1501 gcggggtggg ggcagcacag gagggcggcc tccttctgag ctcctgtccc ctgctacacc 1561 tattatccca gctgcctaga ttcagggaaa gtgggacagc ttgtagggga ggggctcctt 1621 tccataaatc cttgatgatt gacaacaccc atttttcctt ttgccgaccc caagagtttt 1681 gggagttgta gttaatcatc aagagaattt ggggcttcca agttgttcgg gccaaggacc 1741 tgagacctga agggttgact ttacccattt gggtgggagt gttgagcatc tgtccccctt 1801 tagatctctg aagccacaaa taggatgctt gggaagactc ctagctgtcc tttttcctct 1861 ccacacagtg ctcaaggcca gcttatagtc atatatatca cccagacata aaggaaaaga 1921 cacatttttt aggaaatgtt tttaataaaa gaaaattaca aaaaaaaatt ttaaagaccc 1981 ctaacccttt gtgtgctctc cattctgctc cttccccatc gttgccccca tttctgaggt 2041 gcactgggag gctccccttc tatttggggc ttgatgactt tctttttgta gctggggctt 2101 tgatgttcct tccagtgtca tttctcatcc acataccctg acctggcccc ctcagtgttg 2161 tcaccagatc tgatttgtaa cccactgaga ggacagagag aaataagtgc cctctcccac 2221 cctcttccta ctggtctctc tatgcctctc tacagtctcg tctcttttac cctggcccct 2281 ctcccttggg ctctgatgaa aaattgctga ctgtagcttt ggaagtttag ctctgagaac 2341 cgtagatgat ttcagttcta ggaaaataaa acccgttgat tactataaaa aaaaaaa SEQ ID NO: 2 is the nucleic acid sequence of KIAA0286 (GenBank: NM_015257.2). SEQ ID NO: 2 1 agttgctggg gtaaggcacg tgaggaggag gtggcttgag gcaaccatgg cgggaggaat   61 gaaagtggcg gtctcgccgg cagttggtcc cgggccctgg ggctcgggag tcgggggcgg  121 tgggacagtg cggctactct tgatcctctc cggctgcttg gtctacggca cagctgaaac  181 tgatgtaaat gtggtcatgc ttcaggaatc ccaagtttgt gaaaagcgtg ccagccaaca  241 attctgttac acaaatgtgc ttatcccaaa atggcatgat atatggacac ggatacagat  301 ccgagtaaat agttccagat tggttcgagt cacccaggtg gagaatgagg agaaactgaa  361 ggagctagag caaagtcaaa ttttctacta ctctactggg atgactgtgg gaattgtggc  421 ctctctgcta atcatcattt ttatactatc taagtttatg cctaagaaaa gtcccattta  481 cgtcatcctg gtgggaggct ggtctttttc tctgtacctc attcaactag tttttaaaaa  541 tttacaagag atctggaggt gttactggca gtatctttta agttatgtcc tcacagttgg  601 attcatgagt tttgcagtat gttacaagta tgggcccttg gagaatgaac gaagtatcaa  661 cctgctgacc tggaccttgc agctgatggg cctgtgtttc atgtattctg gcatccagat  721 accacatatt gcccttgcca ttatcatcat tgctctttgt actaagaacc tggaacaccc  781 tattcagtgg ctgtacatca cctgcagaaa ggtgtgtaag ggagcagaaa agcctgttcc  841 ccctcgtctc ctgacagaag aagaatatcg gatacaagga gaggtagaaa cccgaaaggc  901 tttagaggag ctccgagaat tttgtaacag tccagactgc tctgcttgga agactgtttc  961 tcgaatccag tctccaaaaa gatttgctga ctttgtggaa ggctcttccc acctcacgcc 1021 aaatgaagtt tctgtccatg agcaggagta tggattaggg agcattattg cccaggatga 1081 aatctatgag gaagcatcct ctgaggagga ggactcatat tctcggtgtc ctgctatcac 1141 acagaacaac tttctaacct aggtagtggt cagttatctt tacgtggact ggcttggtgc 1201 cttggtccat gttgcatgtg ttgtgcaatt gctttcaacc ctttgaaaca gagtgagata 1261 gatagggtag aaattctcct actgaaataa gaggcctaaa aaggcctccc tttggaaatg 1321 ggaggtctct atgggatccc tgaggaagga gagtggataa agtagtgaat gctgggtagt 1381 tcacttccca ttggttaagc taacagccca cttttatgtt tccagagaaa ttggatggcc 1441 acagctagca tggcattcta gctccttctt gaaagttgat tcaatcatgg catttctgtc 1501 actggctggc tctccaaagt aagaactgtt gttaagtgca ggaatgcttt tagactatag 1561 gctgcaactt ccagagagaa atccacaaat ctgagcctcc ttcactccag cttttatttc 1621 agtgacttta gaataattat tgatttaact gttttgggag gaaaatagat ttttattgtt 1681 ttgtttttta aatgaatgtc ttttaaaaaa cataacaaac tcatgttcca gaaccagcaa 1741 gtgctccaga gtgacacacc ccctaggccc ctacatattt attaatatgg attatccatt 1801 aaagccccag gagctgttgt tttaagcttt gatttagttc tcatacatat gatagaaagt 1861 cctatttgcc tttaggaaca tgcctgtagg ctcttctgca ggtgagatgt actgggcttt 1921 ttattatatt caactttcaa ttccatctta aaaaacattt gtattcttct cttcccattc 1981 ttccttaccc tgcctttgcc ctttcaggaa gggtcagttc ccttacctgt gaactatgta 2041 tgttcagagt agcattattc ctgctagcta ggagaagtca tcttgtttag gggatttgga 2101 tgctttttat acgttctcca ttttcctgtc attgggtcat gttatctttg agttgctatg 2161 aaatcaggaa actgtctcct tttcctttcc cttcctttgt ctacatgctc tgtccattcc 2221 tttcagcctt ttctcaccac ccatactccc ccaaatctgg gtaattttta agccttgaaa 2281 ctatgtagtt tcttgataca caatttgtag ttatgcagca gccacaattt gcattgccag 2341 gaaataggct ccaggttatc ttcatgcctc tgggtgctca ttcagctgtc aagtttccat 2401 gaacttacac ttatttatga ttgcgtttct gacctgagat gtatgctgcc tgttattgca 2461 gtagcattag tttcagattc ttttgccatt gcaaagtacc ccttataaac cagcaatgtc 2521 atctgtgagg aagcaaattc tcaagtgtct gtcatttact tggttctttt tctttgtggt 2581 cttcaccctt ataccctgga aaagtctgta attaccttag ccaggaagat agatggtcat 2641 ggcaagcgca cagcaccaga cttactggct caccaagatg atggaaaaag gcagatgatt 2701 ttttaaaaag ccgtaatgac tcctttagac cagccattta gcgtggtaat tttgaaaggc 2761 ctagctccat tgcagacttc caaagggtca gctctgagac tgccctccag gtgggcagtt 2821 gattatttcc accagtgttt tccagagcct taaactgtcc taagtgacaa ctacctcagt 2881 tggcaggaaa gagacatata gtagaaagtg aaaaatgagc agtatttggg cagatgctat 2941 gggttacagt tgaagggtaa aaggaacttt acattgggaa acctttatac ccttgtgaat 3001 tatgtacatg gtaaaatgtt ctctctctac aaagaactat taaaacttct gaaatatact 3061 attttttacc ttatttatag aaattgagac ctagcatatt taagcataag tttattttaa 3121 aaaataattc aactcgtgca agtggtctca ggattctctg gagattttgg tgcctcccct 3181 acttagggag gtgatagctt gcctataagg gtgacttttc ctgatcatgt ctttatttca 3241 atgagaaagc actgtgaaat tgtgaaagat tctcctcttt ctctgtttaa taaaccccca 3301 tgaaatatag tttccatctc tagaccagtt ttttttccac cgtgtttaga cttgaggtga 3361 ataaaatcaa actgtttttt actccctatc tggtagttgg agacctgagc tgtaggcagt 3421 ggagatggca attggttctg cagcctgaga gttgctctca cacagtgaag gacggtgctg 3481 ctctggtgtg ctgtgtgtcc ttgccctgcc tgcctgtggc tctgcccaga tgcttcagat 3541 cctctgtgtt ccggagattg cttgacttca accttcttta ggagctgctc ttgtctccct 3601 cttggccact tagtttgctg gctcagtcac tacttgaaga ccccatttaa tttttctctg 3661 gcagttatag ctcttgtgat ttcagtacag tctcatctct cagaccaatc tcatcaagaa 3721 ggattgaagg gataactatg aggtaagctg gacattggag ccgtgtttgc tgccacgtca 3781 gcgtcttgct gggtgaatgt caagccataa atgggctcca gggctctgga tctcatcagc 3841 attggaaatc tattgcctct catcagtctg accaaattat gtagagcatt aatgtagaga 3901 ctcccattaa tgggaataca agaggcagct ggcataaaac atttctttca ctttcctttc 3961 ccactcagat tgcttcaaga gaccaacaga acacagggat caaaaacaag gaaaatttag 4021 caacttcatt accttctaat aagtaattcc tgttagccac tgcatcccac caaaactagt 4081 ttatttttcc cctcaaattc atgattttta cgtctgttac aaagggaatt ttgctgatag 4141 ctctttgggt cccactgttc cattttatgc taatagattc cattctaggg cccagccgtc 4201 tcttgactga tggtgttccc tttaaccctt ggcatgtata atagaatttt ggtgaatgaa 4261 agaacccaaa taggccagat agtcccccca ggccctgata tccataaaag gcttgggaat 4321 gcattatgta attgtcctta gtctttttgt tgttttagaa aaaaaaaaca agatgggctc 4381 agatggatgc ctacgtaaaa atggttccta gctgtgtact cataactttt ctttgaattg 4441 agtagtgaaa ggaaggagga ggaaaggaaa ttaaatgtcc ttctagtatt ctctggactc 4501 aagtctgaca tatgagataa taacctatat tgaaatgcca agaattgtat ctgaaacaag 4561 agaacagttt gacacattta tcatgccttc atattacata ttaactgaaa ccaattaata 4621 aacatatgaa atatccattg cacaaggcaa aggcacctaa accttttgtt tctttttcta 4681 catagcagaa attgattttt tttttatttt tttaggggaa cctatataat tatgacccag 4741 tgatgtcttt tggtgactta agcttatgaa ttcaggttac aattgagttg attctagatg 4801 gttactacct tgaaaaggat gttggtgcct tatgtgacac gagccagagc ctgctgggaa 4861 taaacaaagc agattcatgc caacaccaac tcgtagcttt agtggcagat gggagtggtc 4921 acagactccc aaaatgtggg gctttggatt tccacaccat cccacgtgtg tgtcatcttc 4981 ctctttcaca ctcttgatga taatttgaaa atggtgaaat cacctctgaa tttgcctata 5041 gcatgagcac attcttatga caacataaca aatagttcat aatgtgaata ttagaaactg 5101 ttacagcctg cagttaccat aattttccat gtttgtggaa ttgatattga aatagcaggg 5161 ctaaggaatt actggcaagt tttagcctgt gggtaatacc ttagggttat ttaaatattt 5221 gtaattttat ttaaatgttc atgaatgttt gaaaggaaca aaattatcag ggatggctct 5281 ttgccatggg tcttattttc accctctttt ctgtaagaaa aaagaacaat gtcttaatgt 5341 atttttaaag tttttggtat agtttctaat tccaatttta ataaaagttt tatagataaa 5401 aaaaaaaaaa aaa SEQ ID NO: 3 is the nucleic acid sequence of RFC4 (GenBank: NM_181573.2). SEQ ID NO: 3 1 cgcgctcacg tctgaagtgg gagcaatgca ccgggacagg gacacctcct aggccatgcc   61 tgttccagtc cagttctgcc tgaaagtccg gctggctcat cacctgccta aataaaaccg  121 tatacgggca aactccctcc gcaagcagcg cgccccagca ccggaagtga cgcgttacgt  181 gcccgcgtat tcctaccggc gtattcccgc cctgcttttc gcccgccgtt ccgtggcggg  241 aactgaggcg actgtgggga catcagtgat cggtgaagta ccatgcaagc atttcttaaa  301 ggtacatcca tcagtactaa acccccgctg accaaggatc gaggagtagc tgccagtgcg  361 ggaagtagcg gagagaacaa gaaagccaaa cccgttccct gggtggaaaa atatcgccca  421 aaatgtgtgg atgaagttgc tttccaggaa gaagtggttg cagtgctgaa aaaatcttta  481 gaaggagcag atcttcctaa tctcttgttt tacggaccac ctggaactgg aaaaacatcc  541 actattttgg cagcagctag agaactcttt gggcctgaac ttttccgatt aagagttctt  601 gagttaaatg catctgatga acgtggaata caagtagttc gagagaaagt gaaaaatttt  661 gctcaattaa ctgtgtcagg aagtcgctca gatgggaagc cgtgtccgcc ttttaagatt  721 gtgattctgg atgaagcaga ttctatgacc tcagctgctc aggcagcttt aagacgtacc  781 atggagaagg agtcgaaaac cacccgattc tgtcttatct gtaactatgt cagtcgaata  841 attgaacccc tgacctctag atgttcaaaa ttccgcttca agcctctgtc agataaaatt  901 caacagcagc gattactaga cattgccaag aaggaaaatg tcaaaattag tgatgaggga  961 atagcttatc ttgttaaagt gtcagaagga gacttaagaa aagccattac atttcttcaa 1021 agcgctactc gattaacagg tggaaaggag atcacagaga aagtgattac agacattgcc 1081 ggggtaatac cagctgagaa aattgatgga gtatttgctg cctgtcagag tggctctttt 1141 gacaaactag aagctgtggt caaggattta atagatgagg gtcatgcagc aactcagctc 1201 gtcaatcaac tccatgatgt ggttgtagaa aataacttat ctgataaaca gaagtctatt 1261 atcacagaaa aacttgccga agttgacaaa tgcctagcag atggtgctga tgaacatttg 1321 caactcatca gcctttgtgc aactgtgatg cagcagttat ctcagaattg ttaacgtgaa 1381 tatatctgga tggggggttt tgtaaataat gaagttgtaa taaaaataaa atgaccaaaa 1441 gcacctttaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa SEQ ID NO: 4 is the nucleic acid sequence of MSH6 (GenBank NM_000179.1). SEQ ID NO: 4 1 atttcccgcc agcaggagcc gcgcggtaga tgcggtgctt ttaggagctc cgtccgacag   61 aacggttggg ccttgccggc tgtcggtatg tcgcgacaga gcaccctgta cagcttcttc  121 cccaagtctc cggcgctgag tgatgccaac aaggcctcgg ccagggcctc acgcgaaggc  181 ggccgtgccg ccgctgcccc cggggcctct ccttccccag gcggggatgc ggcctggagc  241 gaggctgggc ctgggcccag gcccttggcg cgatccgcgt caccgcccaa ggcgaagaac  301 ctcaacggag ggctgcggag atcggtagcg cctgctgccc ccaccagttg tgacttctca  361 ccaggagatt tggtttgggc caagatggag ggttacccct ggtggccttg tctggtttac  421 aaccacccct ttgatggaac attcatccgc gagaaaggga aatcagtccg tgttcatgta  481 cagttttttg atgacagccc aacaaggggc tgggttagca aaaggctttt aaagccatat  541 acaggttcaa aatcaaagga agcccagaag ggaggtcatt tttacagtgc aaagcctgaa  601 atactgagag caatgcaacg tgcagatgaa gccttaaata aagacaagat taagaggctt  661 gaattggcag tttgtgatga gccctcagag ccagaagagg aagaagagat ggaggtaggc  721 acaacttacg taacagataa gagtgaagaa gataatgaaa ttgagagtga agaggaagta  781 cagcctaaga cacaaggatc taggcgaagt agccgccaaa taaaaaaacg aagggtcata  841 tcagattctg agagtgacat tggtggctct gatgtggaat ttaagccaga cactaaggag  901 gaaggaagca gtgatgaaat aagcagtgga gtgggggata gtgagagtga aggcctgaac  961 agccctgtca aagttgctcg aaagcggaag agaatggtga ctggaaatgg ctctcttaaa 1021 aggaaaagct ctaggaagga aacgccctca gccaccaaac aagcaactag catttcatca 1081 gaaaccaaga atactttgag agctttctct gcccctcaaa attctgaatc ccaagcccac 1141 gttagtggag gtggtgatga cagtagtcgc cctactgttt ggtatcatga aactttagaa 1201 tggcttaagg aggaaaagag aagagatgag cacaggagga ggcctgatca ccccgatttt 1261 gatgcatcta cactctatgt gcctgaggat ttcctcaatt cttgtactcc tgggatgagg 1321 aagtggtggc agattaagtc tcagaacttt gatcttgtca tctgttacaa ggtggggaaa 1381 ttttatgagc tgtaccacat ggatgctctt attggagtca gtgaactggg gctggtattc 1441 atgaaaggca actgggccca ttctggcttt cctgaaattg catttggccg ttattcagat 1501 tccctggtgc agaagggcta taaagtagca cgagtggaac agactgagac tccagaaatg 1561 atggaggcac gatgtagaaa gatggcacat atatccaagt atgatagagt ggtgaggagg 1621 gagatctgta ggatcattac caagggtaca cagacttaca gtgtgctgga aggtgatccc 1681 tctgagaact acagtaagta tcttcttagc ctcaaagaaa aagaggaaga ttcttctggc 1741 catactcgtg catatggtgt gtgctttgtt gatacttcac tgggaaagtt tttcataggt 1801 cagttttcag atgatcgcca ttgttcgaga tttaggactc tagtggcaca ctatccccca 1861 gtacaagttt tatttgaaaa aggaaatctc tcaaaggaaa ctaaaacaat tctaaagagt 1921 tcattgtcct gttctcttca ggaaggtctg atacccggct cccagttttg ggatgcatcc 1981 aaaactttga gaactctcct tgaggaagaa tattttaggg aaaagctaag tgatggcatt 2041 ggggtgatgt taccccaggt gcttaaaggt atgacttcag agtctgattc cattgggttg 2101 acaccaggag agaaaagtga attggccctc tctgctctag gtggttgtgt cttctacctc 2161 aaaaaatgcc ttattgatca ggagctttta tcaatggcta attttgaaga atatattccc 2221 ttggattctg acacagtcag cactacaaga tctggtgcta tcttcaccaa agcctatcaa 2281 cgaatggtgc tagatgcagt gacattaaac aacttggaga tttttctgaa tggaacaaat 2341 ggttctactg aaggaaccct actagagagg gttgatactt gccatactcc ttttggtaag 2401 cggctcctaa agcaatggct ttgtgcccca ctctgtaacc attatgctat taatgatcgt 2461 ctagatgcca tagaagacct catggttgtg cctgacaaaa tctccgaagt tgtagagctt 2521 ctaaagaagc ttccagatct tgagaggcta ctcagtaaaa ttcataatgt tgggtctccc 2581 ctgaagagtc agaaccaccc agacagcagg gctataatgt atgaagaaac tacatacagc 2641 aagaagaaga ttattgattt tctttctgct ctggaaggat tcaaagtaat gtgtaaaatt 2701 atagggatca tggaagaagt tgctgatggt tttaagtcta aaatccttaa gcaggtcatc 2761 tctctgcaga caaaaaatcc tgaaggtcgt tttcctgatt tgactgtaga attgaaccga 2821 tgggatacag cctttgacca tgaaaaggct cgaaagactg gacttattac tcccaaagca 2881 ggctttgact ctgattatga ccaagctctt gctgacataa gagaaaatga acagagcctc 2941 ctggaatacc tagagaaaca gcgcaacaga attggctgta ggaccatagt ctattggggg 3001 attggtagga accgttacca gctggaaatt cctgagaatt tcaccactcg caatttgcca 3061 gaagaatacg agttgaaatc taccaagaag ggctgtaaac gatactggac caaaactatt 3121 gaaaagaagt tggctaatct cataaatgct gaagaacgga gggatgtatc attgaaggac 3181 tgcatgcggc gactgttcta taactttgat aaaaattaca aggactggca gtctgctgta 3241 gagtgtatcg cagtgttgga tgttttactg tgcctggcta actatagtcg agggggtgat 3301 ggtcctatgt gtcgcccagt aattctgttg ccggaagata cccccccctt cttagagctt 3361 aaaggatcac gccatccttg cattacgaag actttttttg gagatgattt tattcctaat 3421 gacattctaa taggctgtga ggaagaggag caggaaaatg gcaaagccta ttgtgtgctt 3481 gttactggac caaatatggg gggcaagtct acgcttatga gacaggctgg cttattagct 3541 gtaatggccc agatgggttg ttacgtccct gctgaagtgt gcaggctcac accaattgat 3601 agagtgttta ctagacttgg tgcctcagac agaataatgt caggtgaaag tacatttttt 3661 gttgaattaa gtgaaactgc cagcatactc atgcatgcaa cagcacattc tctggtgctt 3721 gtggatgaat taggaagagg tactgcaaca tttgatggga cggcaatagc aaatgcagtt 3781 gttaaagaac ttgctgagac tataaaatgt cgtacattat tttcaactca ctaccattca 3841 ttagtagaag attattctca aaatgttgct gtgcgcctag gacatatggc atgcatggta 3901 gaaaatgaat gtgaagaccc cagccaggag actattacgt tcctctataa attcattaag 3961 ggagcttgtc ctaaaagcta tggctttaat gcagcaaggc ttgctaatct cccagaggaa 4021 gttattcaaa agggacatag aaaagcaaga gaatttgaga agatgaatca gtcactacga 4081 ttatttcggg aagtttgcct ggctagtgaa aggtcaactg tagatgctga agctgtccat 4141 aaattgctga ctttgattaa ggaattatag actgactaca ttggaagctt tgagttgact 4201 tctgaccaaa ggtggtaaat tcagacaaca ttatgatcta ataaacttta ttttttaaaa 4261 atga

(47) Other embodiments are within the following claims.