Patent Application
20110086355
The present invention relates to the field of diagnostics and, in particular, to a method for selecting an individual neoadjuvant chemotherapy and for predicting the survival of breast cancer patients, based on the expression level of the BRCA1 gene in a sample from said patient.
Worldwide, breast cancer is the second most common type of cancer (10.4%; after lung cancer) and the fifth most common cause of cancer death (after lung cancer, stomach cancer, liver cancer, and colon cancer). Among women worldwide, breast cancer is the most common cause of cancer death. In 2005, breast cancer caused 502,000 deaths worldwide (7% of cancer deaths; almost 1% of all deaths). The number of cases worldwide has significantly increased since the 1970s, a phenomenon partly blamed on modern lifestyles in the Western world. North American women have the highest incidence of breast cancer in the world.
Because the breast is composed of identical tissues in males and females, breast cancer also occurs in males. Incidences of breast cancer in men are approximately 100 times less common than in women, but men with breast cancer are considered to have the same statistical survival rates as women.
Breast cancer is staged according to the TNM system. Prognosis is closely linked to results of staging, and staging is also used to allocate patients to treatments both in clinical trials and clinical practice. The information for staging is as follows:
The mainstay of breast cancer treatment is surgery when the tumor is localized, with possible adjuvant hormonal therapy (with tamoxifen or an aromatase inhibitor), chemotherapy, and/or radiotherapy. At present, the treatment recommendations after surgery (adjuvant therapy) follow a pattern. This pattern is subject to change, as every two years, a worldwide conference takes place in St. Gallen, Switzerland, to discuss the actual results of worldwide multi-center studies.
On the other hand, neoadjuvant chemotherapy, an adjunctive therapy given before a definitive treatment, is an essential component of modern multidisciplinary cancer therapy. Although neoadjuvant or induction therapy does not contribute the most to the treatment outcome, it may improve the result substantially. For example, neoadjuvant therapy allows patients with large breast cancer to undergo breast-conserving surgery. It enables patients with locally advanced laryngeal cancer to have their vocal function preserved. Many patients with rectal cancer can avoid permanent colostomy after undergoing this approach. In addition, in certain cancers, neoadjuvant therapy may improve long-term survival. Mouret-Reynier et al. (Clin Breast Cancer. 2004 October; 5 (4):303-7) investigated the efficacy of FEC as neoadjuvant chemotherapy in women with stage I-III primary operable breast cancer concluding that it was effective and well tolerated in patients with early-stage operable breast cancer.
During the past 30 years medical oncologists have focused to optimise the outcome of cancer patients and it is just now that the new technologies available are allowing to investigate polymorphisms, gene expression levels and gene mutations aimed to predict the impact of a given therapy in different groups of cancer patients to tailor chemotherapy. Representative examples include the relation between the TS mRNA expression and the response and the survival with antifolates (see EP 1 381 691), beta tubulin III mRNA levels and response to tubulin interacting agents, PTEN methylation and resistance to CPT-11 and STAT3 over expression and resistance to EGF interacting agents. PCR tests like Oncotype DX or microarray tests like MammaPrint can predict breast cancer recurrence risk based on gene expression. In February 2007, the MammaPrint test became the first breast cancer predictor to win formal approval from the Food and Drug Administration. This is a new gene test to help predict whether women with early-stage breast cancer will relapse in 5 or 10 years, this could help influence how aggressively the initial tumor is treated.
Breast Cancer 1 (BRCA1) plays a crucial role in DNA repair, and decreased BRCA1 mRNA expression has been observed in both sporadic and hereditary breast cancers (Kennedy R D, et al. (2002) Lancet, 360, 1007-1014). These patients can respond to DNA damage-based chemotherapy but not to antimicrotubule drugs. In addition, DNA damage-based chemotherapy confers a significant survival advantage to BRCA1 mutation carriers compared to non-mutation carriers. Also ovarian cancer patients with low levels of BRCA1 mRNA have improved survival following platinum-based chemotherapy compared to patients with high levels of BRCA1 mRNA (Quinn et al, Clin Cancer Res. 2007 Dec. 15; 13(24):7413-20).
BRCA1 is implicated in transcription-coupled nucleotide excision repair (TC-NER), and modulation of its expression leads to modification of TC-NER and hence to radio- and chemoresistance. Upregulation of BRCA1 expression led to increased cisplatin resistance in the SKOV-3 human ovarian cancer cell line (Husain A, et al. Cancer Res. 1998 Mar. 15; 58(6):1120-3) and restoration of BRCA1 in the BRCA1-negative HCC1937 human breast cancer cell line restored radioresistance. BRCA1 is also involved in homologous recombination repair (HRR) and non-homologous end joining in response to DNA damage. In addition, it is a component of a large DNA repair complex termed the BRCA1-associated genome surveillance complex, which contains a number of mismatch repair proteins, indicating a potential role for BRCA1 in mismatch repair. BRCA1 may also be a regulator of mitotic spindle assembly, as BRCA1 and 13-tubulin colocalize to the microtubules of the mitotic spindle and to the centrosomes. Finally, enhanced BRCA1 expression has been linked to apoptosis through the c-Jun N-terminal kinase pathway, which is activated by cisplatin-induced DNA damage; inhibition of this pathway increased cisplatin sensitivity in cell lines. Decreased BRCA1 mRNA expression in a breast cancer cell line, as determined by real-time quantitative polymerase chain reaction (RT-QPCR), led to greater sensitivity to cisplatin and etoposide but to greater resistance to the microtubule-interfering agents paclitaxel and vincristine (Lafarge S, et al. (2001) Oncogene, 20, 6597-6606). Recently, reconstitution of wild-type BRCA1 into the BRCA1-negative HCC1937 breast cancer cell line resulted in a 20-fold increase in cisplatin resistance and, in contrast, in a 1000-10,000-fold increase in sensitivity to antimicrotubule drugs (paclitaxel and vinorelbine).
Mouse models carrying conditional disruption of BRCA1 were highly sensitive to doxorubicin and gamma irradiation but resistant to tamoxifen, providing additional evidence for differential chemosensitivity linked to BRCA1 expression. When BRCA1 expression was examined by semi-quantitative PCR in women with sporadic breast cancer, lower BRCA1 mRNA levels (bottom quartile) were associated with a higher frequency of distant metastases (Seery L T, et al. (1999) Int. J. Cancer (Pred. Oncol.), 84, 258-262.
Despite the wealth of data in cell lines and mouse models, only one small study has examined the correlation of BRCA1 and BRCA2 mRNA expression with response to chemotherapy in the clinical setting (Egawa C., (2001) Int. J. Cancer (Pred. Oncol.), 95, 255-259). Among 25 women with docetaxel-treated locally advanced or metastatic breast cancer, only BRCA2 mRNA levels were significantly lower in responders than in non-responders, though a slight difference was also observed for BRCA1.
Martin-Richard et al (Oncology, 2004; 66: 388-94) describes the value of topoisomerase IIalpha (Topo II) in predicting the clinical response to anthracycline-based neoadjuvant chemotherapy in breast cancers and the potential changes in Topo II after chemotherapy. The results show that Topo II was overexpressed in 31% of tumors before treatment, and this overexpression was significantly associated with clinical response. Kandioler-Eckersberger D. et al (Clin Cancer Res. 2000; 6:50-6) describes the value of p53 to predict the cytotoxic effect of FEC (fluorouracil, epirubicin and cyclophosphamide) and microtubule stabilizing (paclitaxel) chemotherapies regimens in patients with advanced breast cancer. The results show that response to a combination of FEC was directly related to normal p53 and tumor cell apoptosis in breast cancer patients. Knoop (J Clin Oncol., 2005; 23:7483-90) describes that patients with TOP2A amplification had an increased recurrence-free and overall survival, respectively, if treated with CEF (cyclophosphamide, epirubicin, and fluorouracil) compared with CMF (cyclophosphamide, methotrexate, and fluorouracil) chemotherapies, and that patients with TOP2A deletions had an almost identical hazard ratio.
It is an object of the present invention to provide predictors of response to chemotherapy, in particular to neoadjuvant therapy, which can be a valuable clinical tool for use in the selection of optimal treatment modes, in particular for patients suffering from breast cancer.
The present invention provides a tool for use in predicting differential chemosensitivity and tailoring neoadjuvant chemotherapy in breast cancer.
Inventors have surprisingly found that a neoadjuvant therapy based on a combination of an anti-metabolite, an intercalating agent and an alkylating agent improved survival in patients suffering from breast cancer with low expression levels of BRCA1.
Thus, in a first aspect, the present invention refers to a method for selecting an individual neoadjuvant therapy for a patient suffering from breast cancer which comprises determining the expression levels of BRCA1 gene in a sample from said patient, wherein if expression levels of BRCA1 gene are low when compared with reference values then, the patient is a good candidate for a neoadjuvant therapy based on a combination of an anti-metabolite, an intercalating agent and an alkylating agent.
In a second aspect, the invention refers to a method for determining the clinical response of a patient suffering from breast cancer to neoadjuvant therapy based on a combination of an anti-metabolite, an intercalating agent and an alkylating agent which comprises determining BRCA1 gene expression levels in a sample from said patient, wherein if the expression levels of BRCA1 gene are low when compared with reference values, then it is indicative of a good clinical response of said patient to said therapy.
In a further aspect, the invention relates to a method for evaluating the predisposition of a patient suffering from breast cancer to respond to a neoadjuvant therapy based on a combination of an anti-metabolite, an intercalating agent and an alkylating agent which comprises determining the expression levels of BRCA1 gene in a sample from said patient, wherein if the expression levels of BRCA1 gene are low, then it is indicative of favourable predisposition of said patient to respond to a neoadjuvant therapy based on a combination of an anti-metabolite, an intercalating agent and an alkylating agent.
In another aspect, the invention refers to a combination of an anti-metabolite, an intercalating agent and an alkylating agent as a neoadjuvant therapy for the treatment of breast cancer in a patient suffering from breast cancer wherein said patient presents low expression levels of the BRCA1 gene.
In another aspect, the invention relates to a method for classifying patients suffering from breast cancer comprising determining:
The authors of the present invention have surprisingly found that the clinical response of patients suffering from breast cancer being treated with a neoadjuvant therapy based on a combination of an anti-metabolite, an intercalating agent and an alkylating agent closely correlates with the expression levels of BRCA1.
This allows the physician to make an informed decision as to the therapeutic regimen most likely to improve survival according to the BRCA1 expression level with appropriate risk and benefit trade off to the patient. Based on these findings they have defined the method of the invention in its different embodiments that will be described now in detail.
Thus, in a first aspect, the invention provides a novel method for selecting an individual neoadjuvant therapy for a patient suffering from breast cancer which comprises determining the expression levels of BRCA1 gene in a sample from said patient, wherein if expression levels of BRCA1 gene are low when compared with reference values, then the patient is a good candidate for a neoadjuvant therapy based on a combination of an anti-metabolite, an intercalating agent and an alkylating agent.
The term “breast cancer” relates to a tumour of the breast and includes any histology subtype which typically appears in breast cancer such as ductal carcinoma, lobular carcinoma, haemangioma, sarcomas, etc. any clinical subtype such as superficial, muscle-invasive or metastatic disease cancer and any TMN stage including T is, T1, T2, T3 or T4 which depend on the presence or absence of invasive cancer, the dimensions of the invasive cancer, and the presence or absence of invasion outside of the breast, N0, N1, N2 or N3 which depend on the number, size and location of breast cancer cell deposits in lymph nodes and M0 or M1 which depend on the presence or absence of breast cancer cells in locations other than the breast and lymph nodes (so-called distant metastases, e.g. to bone, brain, lung).
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 tumour tissue sample or portion thereof. In a more particular embodiment, said tumor tissue sample is a breast tumor tissue sample from a patient suffering from breast cancer. 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.
The method of the invention requires determining the expression levels of the BRCA1 gene. In a preferred embodiment, the determination of the expression levels of the BRCA1 gene can be carried out by measuring the expression levels of the mRNA encoded by the BRCA1 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, Fischer and Maniatis, Molecular Cloning, a laboratory manual, (2nd ed.), Cold Spring Harbor Laboratory Press, New York, (1989). 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, for example. 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). 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 tumor cells with respect to non-tumorigenic cells. Preferably, the control RNA is mRNA derived from housekeeping genes and which code for proteins which are constitutively expressed and carry out essential cellular functions. Preferred housekeeping genes for use in the present invention include β-2-microglobulin, ubiquitin, 18-S ribosomal protein, cyclophilin, GAPDH and actin. In a preferred embodiment, the control RNA is beta-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 level of expression of the BRCA1 gene needs to be correlated with the reference values which correspond to the median value of expression levels of BRCA1 measured in a collection of tumor tissue in biopsy samples from cancer patients, previous to the neoadjuvant chemotherapeutic treatment. Once this median value is established, the level of this marker expressed in tumor tissues from patients can be compared with this median value, and thus be assigned a level of “low,” “normal” or “high”. The collection of samples from which the reference level is derived will preferably be constituted from patient suffering from the same type of cancer. For example, the one described in the examples which is statistically representative was constituted with 41 samples from breast cancer patients. 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 BRCA1 measured in a RNA sample obtained by pooling equal amounts of RNA from each of the tumour samples obtained by biopsy from cancer patients previous to the neoadjuvant chemotherapeutic treatment. 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 BRCA1. Thus, in a particular embodiment, the reference values for “increased” or “decreased” BRCA1 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 breast cancer) for the expression levels of the BRCA1 gene. The “increased” levels can then be assigned, preferably, to samples wherein expression levels for the BRCA1 genes are equal to or in excels 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 BRCA1 expression values are divided into terciles. As an example, real-time quantitative PCR was used to determine BRCA1 mRNA levels in 41 tumor biopsies from breast cancer patients who had received neoadjuvant FEC chemotherapy, and divided the gene expression values into terciles. When results were correlated with outcome (DFS and MS), it was observed that patients with BRCA1 levels in the bottom tercile (tercile 1) had a significantly decreased risk of relapse (DFS) and a significantly better survival (MS) when compared to those in the top and middle terciles (see
In another embodiment, the expression levels of the BRCA1 gene are determined by measuring the expression of the BRCA1 protein. The determination of the expression levels of the BRCA1 protein can be carried out by immunological techniques such as e.g. 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 and subcellular localization by microscopy. Generally, the cells under study are previously fixed with paraformaldehyde and permeabilised with a non-ionic detergent. 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. This technique does not allow the exact localisation of the target protein or the determination of its molecular weight but allows a very specific and highly sensitive detection of the target protein in a variety of biological samples (serum, plasma, tissue homogenates, postnuclear supernatants, ascites and the like). In a preferred embodiment, the BRCA1 protein is detected by immunohistochemistry (IHC) analysis using thin sections of the biological sample immobilised on coated slides. The sections are then deparaffinised, if derived from a paraffinised tissue sample, and treated so as to retrieve the antigen. The detection can be carried out in individual samples or in tissue microarrays.
Any antibody or reagent known to bind with high affinity to the target protein can be used for detecting the amount of target protein. 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.
In yet another embodiment, the determination of BRCA1 protein expression levels can be carried out by constructing a tissue microarray (TMA) containing the patient samples assembled, and determining the expression levels of BRCA1 protein by immunohistochemistry techniques. Immunostaining intensity can be evaluated by two different pathologists and scored using uniform and clear cut-off criteria, in order to maintain the reproducibility of the method. Discrepancies can be resolved by simultaneous re-evaluation. Briefly, the result of immunostaining can be recorded as negative expression (0) versus positive expression, and low expression (1+) versus moderate (2+) and high (3+) expression, taking into account the expression in tumoral cells and the specific cut-off for each marker. As a general criterion, the cut-offs were selected in order to facilitate reproducibility, and when possible, to translate biological events.
The authors of the present invention have further shown that survival of patients suffering from breast cancer who have been treated with a neoadjuvant therapy based on a combination of an anti-metabolite, an intercalating agent and an alkylating agent also correlates with the expression levels of the progesterone receptor. Thus, measurement of both, BRCA1 expression and progesterone receptor expression, can be used as predictive markers of the clinical outcome of patients suffering from breast cancer who have been treated with a neoadjuvant therapy based on based on a combination of an anti-metabolite, an intercalating agent and an alkylating agent. Therefore, in a particular embodiment of the invention and in order to further improve the survival rate in patients with breast cancer and in order to provide more effective therapeutic options according to the invention, the method further comprises determining progesterone receptor expression, wherein if the progesterone receptor expression is positive when compared with reference values, then the patient is a good candidate for a neoadjuvant therapy based on a combination of an anti-metabolite, an intercalating agent and an alkylating agent. In a particular embodiment, the expression levels of the progesterone receptor are determined by measuring the expression of the progesterone receptor protein. The determination of the expression levels of the progesterone receptor protein can be carried out by any immunological means as described before. In a more particular embodiment, the determination of progesterone receptor protein expression levels is carried out by tissue microarray (TMA) determining the expression levels of progesterone receptor protein by immunohistochemistry techniques. Thus, as an illustrative, non limitative example of determining PR expression, immunohistochemical expression of PR is quantified by immunohistochemistry techniques by means of quantifying the number of PR-positive nuclei in a sample as described, for example, by Mohsin et al. (Modern Pathology; 2004 17, 1545-1554) wherein a tumor sample showing 10% or more PR-positive nuclei is considered as PR positive.
The authors of the present invention have also found that the degree of lymph node involvement, i.e. lymphatic invasion, can be used as a predictive marker of the clinical outcome of patients suffering from breast cancer who have been treated with a neoadjuvant therapy based on based on a combination of an anti-metabolite, an intercalating agent and an alkylating agent. Therefore, in another embodiment the method of the invention further comprises measuring lymph node involvement, wherein if lymph node involvement is negative then, the patient is a good candidate for a neoadjuvant therapy based on a combination of an anti-metabolite, an intercalating agent and an alkylating agent.
The expression “lymph node involvement” as used herein, is understood as the spread of the tumor cells to the lymph nodes and blood vessels located in the vicinity of the tissue which contains the tumor.
The chemotherapy neoadjuvant agents to be used in the method of this invention will be administered in doses commonly employed clinically. Such doses will be calculated in the normal fashion, for example on body surface area.
Examples of antimetabolite 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.
Examples of alkylating agents include chlorambucil, chlormethine, cyclophosphamide, ifosphamide, melphalan, carmustine, fotemustine, lomustine, streptozocin, carboplatin, cisplatin, oxaliplatin, satraplatin, busulfan, dacarbazine, procarbazine, temozolomide, thioTEPA, treosulfan, and uramustine.
Examples of intercalating agents include daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, pixantrone, valrubicin.
In a particular embodiment of the invention, the neoadjuvant chemotherapy administered to said breast cancer patient comprises the administration of the anti-metabolite fluorouracil.
5-FU (fluorouracil) acts in several ways, but principally as a thymidylate synthase inhibitor. Interrupting the action of this enzyme blocks synthesis of the pyrimidine thymidine, which is a nucleotide required for DNA replication. Thymidylate synthase methylates deoxyuracilmonophoshate (dUMP) into deoxythyminemonophosphate (dTMP).
Like many anti-cancer drugs, 5-FU's effects are felt system-wide but fall most heavily upon rapidly dividing cells that make heavy use of their nucleotide synthesis machinery. As a pyrimidine analogue, it is transformed inside the cell into different cytotoxic metabolites which are then incorporated into DNA and RNA, finally inducing cell cycle arrest and apoptosis by inhibiting the cell's ability to synthesize DNA. It is an S-phase specific drug and only active during certain cell cycles. In addition to being incorporated in DNA and RNA, the drug has been shown to inhibit the activity of the exosome complex, an exoribonuclease complex of which the activity is essential for cell survival.
In another particular embodiment of the invention, said intercalating agent is epirubicin.
Epirubicin acts by intercalating DNA strands. Intercalation results in complex formation which inhibits DNA and RNA synthesis. It also triggers DNA cleavage by topoisomerase II, resulting in mechanisms that lead to cell death. Binding to cell membranes and plasma proteins may be involved in the compound's cytotoxic effects. Epirubicin also generates free radicals that cause cell and DNA damage. Epirubicin is favoured over doxorubicin, the most popular anthracycline, in some chemotherapy regimens as it appears to cause fewer side-effects. Epirubicin has a different spatial orientation of the hydroxyl group at the 4′ carbon of the sugar, which may account for its faster elimination and reduced toxicity. Epirubicin is primarily used against breast and ovarian cancer, gastric cancer, lung cancer, and lymphomas.
In another particular embodiment of the invention, said alkylating agent is cyclophosphamide.
Cyclophosphamide, also known as cytophosphane, is a nitrogen mustard alkylating agent, from the oxazophorines group. It is a “prodrug”; it is converted in the liver to active forms that have chemotherapeutic activity. Cyclophosphamide is converted by mixed function oxidase enzymes in the liver to active metabolites. The main active metabolite is 4-hydroxycyclophosphamide. 4-hydroxycyclophosphamide exists in equilibrium with its tautomer, aldophosphamide. Most of the aldophosphamide is oxidised by the enzyme aldehyde dehydrogenase (ALDH) to make carboxyphosphamide. A small proportion of aldophosphamide is converted into phosphoramide mustard and acrolein. Acrolein is toxic to the bladder epithelium and can lead to hemorrhagic cystitis. This can be prevented through the use of aggressive hydration and/or Mesna.
Recent clinical studies have shown that cyclophosphamide induce beneficial immunomodulatory effects in the context of adoptive immunotherapy. The main effect of cyclophosphamide is due to its metabolite phosphoramide mustard. This metabolite is only formed in cells which have low levels of ALDH. Phosphoramide mustard forms DNA crosslinks between (interstrand crosslinkages) and within (intrastrand crosslinkages) DNA strands at guanine N-7 positions. This leads to cell death. Cyclophosphamide has relatively little typical chemotherapy toxicity as ALDHs are present in relatively large concentrations in bone marrow stem cells, liver and intestinal epithelium. ALDHs protect these actively proliferating tissues against toxic effects phosphoramide mustard and acrolein by converting aldophosphamide to carboxyphosphamide that does not give rise to the toxic metabolites (phosphoramide mustard and acrolein).
Conventional FEC regimen consists of 5-Fluorouracil 600 mg/m2, Epirubicin 60 mg/m2, Cyclophosphamide 600 mg/m2. However, it is feasible to vary said dose according to patients requirements. For example, the dose of epirubicin can be increased by more than 50 percent with increased dose intensity between 25 and 70 percent. Additionally, the dose of cyclophosphamide can be increased by more than 100 percent without severe increase in toxicity for the patient.
The authors of the present invention have found that BRCA1 expression can be used as a good predictive marker of survival in patients suffering from breast cancer. Thus, in another aspect, the present invention refers to a method for determining the clinical response of a patient suffering from breast cancer to neoadjuvant therapy based on a combination of an anti-metabolite, an intercalating agent and an alkylating agent which comprises determining BRCA1 gene expression levels in a sample from said patient, wherein if expression levels of BRCA1 gene are low when compared with reference values then, it is indicative of a good clinical response of said patient to said therapy.
The prediction of the clinical response 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:
In a particular embodiment, prediction of the clinical response is carried out by measuring disease free survival and median survival.
The term “sample” has been previously defined and can be applied to any type of biological sample from a patient. In a particular embodiment, said sample is a tumour tissue sample or portion thereof. In a more particular embodiment, said tumor tissue sample is a breast tumor tissue sample from a patient suffering from breast cancer or a formalin embedded breast tissue sample. In a preferred embodiment, the sample is a tumor biopsy.
In a particular embodiment, the determination of the expression levels of the BRCA1 gene is carried out by measuring the expression levels of the mRNA encoded by the BRCA1 gene or by measuring the expression levels of the BRCA1 gene product using any of the procedures previously mentioned.
As explained before, determining progesterone receptor expression besides BRCA1 expression can be used as a good predictive marker of DFS and MS. Thus, in a particular embodiment, the method also comprises measuring progesterone receptor expression, wherein if the progesterone receptor expression is positive when compared with reference values then, it is indicative of a good clinical response of said patient to said therapy.
In a particular embodiment, the expression levels of the progesterone receptor are determined by measuring the expression of the progesterone receptor protein. The determination of the expression levels of the progesterone receptor protein can be carried out by any immunological means as described before.
In a more particular embodiment, the determination of progesterone receptor protein expression levels is carried out by tissue microarray (TMA) determining the expression levels of progesterone receptor protein by immunohistochemistry techniques.
In another particular embodiment, the method for predicting the clinical response further comprises measuring lymph node involvement, wherein if lymph node involvement is negative then, it is indicative of a good clinical response of said patient to said therapy.
The chemotherapy neoadjuvant agents to be used in the method of this invention will be administered in doses commonly employed clinically. In a particular embodiment of the invention, the neoadjuvant chemotherapy administered to said breast cancer patient comprises the administration of the anti-metabolite fluorouracil. In another particular embodiment, said intercalating agent is epirubicin. In another particular embodiment said alkylating agent is cyclophosphamide.
The findings of the inventors allow the development of personalised therapies for patients suffering from breast cancer wherein the expression of BRCA1 correlates with the possibility that the patient will respond to a neoadjuvant chemotherapy based on a combination of an anti-metabolite, an intercalating agent and an alkylating agent. Thus, in another aspect, the invention relates to a method for evaluating the predisposition of a patient suffering from breast cancer to respond to a neoadjuvant therapy based on a combination of an anti-metabolite, an intercalating agent and an alkylating agent which comprises determining the expression levels of BRCA1 gene in a sample from said patient, wherein if expression levels of BRCA1 gene are low, then it is indicative of favourable predisposition of said patient to respond to a neoadjuvant therapy based on a combination of an anti-metabolite, an intercalating agent and an alkylating agent.
In a particular embodiment of the invention, such sample is a biopsy sample.
In another particular embodiment, the determination of the expression levels of the BRCA1 gene is carried out by measuring the expression levels of the mRNA encoded by the BRCA1 gene or by measuring the expression levels of the BRCA1 gene product using any of the procedures previously mentioned.
The inventors have shown that positive progesterone receptor expression besides low BRCA1 expression correlates with the possibility that the patient will respond to a neoadjuvant chemotherapy based on a combination of an anti-metabolite, an intercalating agent and an alkylating agent. Thus, in a particular embodiment, the method also comprises measuring progesterone receptor expression as explained before, wherein if the progesterone receptor expression is positive when compared with reference values then, it is indicative of favourable predisposition of said patient to respond to a neoadjuvant therapy based on a combination of an anti-metabolite, an intercalating agent and an alkylating agent.
The progesterone receptor (PR) also known as NR3C3 (nuclear receptor subfamily 3, group C, member 3), is an intracellular steroid receptor that specifically binds progesterone.
In a particular embodiment, the expression levels of the progesterone receptor are determined by measuring the expression of the progesterone receptor protein. The determination of the expression levels of the progesterone receptor protein can be carried out by any immunological means as described before.
In a more particular embodiment, the determination of progesterone receptor protein expression levels is carried out by tissue microarray (TMA) determining the expression levels of progesterone receptor protein by immunohistochemistry techniques.
In another particular embodiment, the method for evaluating the predisposition of a patient suffering from breast cancer to respond to a neoadjuvant therapy based on a combination of an anti-metabolite, an intercalating agent and an alkylating agent further comprises measuring lymph node involvement, wherein if lymph node involvement is negative then, it is indicative of favourable predisposition of said patient to respond to a neoadjuvant therapy based on a combination of an anti-metabolite, an intercalating agent and an alkylating agent.
The chemotherapy neoadjuvant agents to be used in the method of this invention will be administered in doses commonly employed clinically. In a particular embodiment of the invention, the neoadjuvant chemotherapy administered to said breast cancer patient comprises the administration of the anti-metabolite fluorouracil. In another particular embodiment, said intercalating agent to be administered is epirubicin. In another particular embodiment said alkylating agent is cyclophosphamide.
In another aspect, the invention refers to a combination of an anti-metabolite, an intercalating agent and an alkylating agent as a neoadjuvant therapy for the treatment of breast cancer in a patient suffering from breast cancer wherein said patient presents low expression levels of the BRCA1 gene.
For the same reasons as explained above, in a particular embodiment of the invention, said patient further presents progesterone receptor positive expression and in another particular embodiment, said patient further presents negative lymph node involvement.
In another aspect, the invention further refers to a method for classifying patients suffering from breast cancer comprising determining:
The following examples are provided as merely illustrative and are not to be construed as limiting the scope of the invention.
Tumor biopsies were obtained from 86 patients with locally advanced breast cancer who were treated with four cycles of neoadjuvant chemotherapy fluorouracil, epirubicin and cyclophosphamide (FEC).
Estrogen receptor(ER), progesterone receptor (PR), HER2, cytokeratin 6/7, vimentin, Huntingtin interacting protein 1 (HIP1) and BRCA1 expression were examined by tissue microarray. HER2 was also assessed by chromogenic in situ hybridization (CISH), and BRCA1 mRNA was analyzed in samples of 41 patients by quantitative PCR.
The BRCA1 gene expression was measured as previously described by Specht K, et al. (2001) (Am. J. Pathol., 158, 419-429 and Krafft A E, et al. (1997) Mol. Diagn. 3, 217-230. After standard tissue sample deparaffinization using xylene and alcohols, samples were lysed in a Tris-chloride, EDTA, sodium dodecyl sulphate (SDS) and proteinase K containing buffer. RNA was then extracted with phenol-chloroform-isoamyl alcohol followed by precipitation with isopropanol in the presence of glycogen and sodium acetate. RNA was resuspended in RNA storage solution (Ambion Inc; Austin Tex., USA) and treated with DNAse I to avoid DNA contamination. cDNA was synthesized using M-MLV retrotranscriptase enzyme. Template cDNA was added to Taqman Universal Master Mix (AB; Applied Biosystems, Foster City, Calif., USA) in a 12.5-μl reaction with specific primers and probe for each gene. The primer and probe sets were designed using Primer Express 2.0 Software (AB). Quantification of gene expression was performed using the ABI Prism 7900HT Sequence Detection System (AB). Primers and probe for BRCA1 mRNA expression analysis were designed according to the Ref Seq NM—007294 (http://www.ncbi.nlm.nih.gov/LocusLink). Forward primer is located in exon 8 (position 4292 bp to 4317 bp), reverse primer in exon 9 (position 4336 bp to 4360 bp), and probe in the exon 8/9 junction (position 4313 bp to 4333 bp). The PCR product size generated with these primers was 69 bp. The primers and 5′ labeled fluorescent reporter dye (6FAM) probe were as follows: β-actin: forward 5′ TGA GCG CGG CTA CAG CTT 3′ (SEQ ID NO: 1), reverse 5′ TCC TTA ATG TCA CGC ACG ATT T 3′ (SEQ ID NO: 2), probe 5′ ACC ACC ACG GCC GAG CGG 3′ (SEQ ID NO: 3); BRCA1: forward 5′GGC TAT CCT CTC AGA GTG ACA TTT TA 3′ (SEQ ID NO: 4), reverse 5′ GCT TTA TCA GGT TAT GTT GCA TGG T 3′ (SEQ ID NO: 5), probe 5′ CCA CTC AGC AGA GGG 3′ (SEQ ID NO: 6). Relative gene expression quantification was calculated according to the comparative Ct method using β-actin as an endogenous control and commercial RNA controls (Stratagene, La Jolla, Calif.) as calibrators. Final results, were determined as follows: 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. In all experiments, only triplicates with a standard deviation (SD) of the Ct value <0.20 were accepted. In addition, for each sample analyzed, a retrotranscriptase minus control was run in the same plate to assure lack of genomic DNA contamination.
Pathological response was attained in 57% of patients. Median disease-free survival (DFS) was 30 months (m) and median survival (MS) was 41 months.
Table 3 shows the relationship between BRCA1 expression by terciles (T1, T2 and T3) and the clinical characteristics of the patients.
On the other hand, as it is shown in
In the multivariate analysis for DFS and MS (Table 5), it is shown that low levels of BRCA1 mRNA, together with positive PR and negative lymph node involvement predicted significantly lower risk of relapse (DFS), while low levels of BRCA1 mRNA and positive PR were the only variables associated with significantly better survival.
The inventors have provided evidences to support a major role for BRCA1 gene expression as a predictive marker of DFS and MS in breast cancer. These findings can be useful for customizing chemotherapy.
| Number | Date | Country | Kind |
|---|---|---|---|
| 08380053.2 | Feb 2008 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2009/052027 | 2/20/2009 | WO | 00 | 11/18/2010 |
