METHODS FOR ASSESSING THE TREATMENT RESPONSE OF TNBC PATIENTS TO NEO- ADJUVANT CHEMOTHERAPY BY ANALYSING CpG METHYLATION

FIELD OF THE INVENTION The present invention relates to the field of pharmacogenomics and in particular to assessing the response of a patient afflicted with triple-negative breast cancer (TNBC) to anthracycline-based neo-adjuvant chemotherapy (NAC) by analysing CpG methylation of the paired-like homeodomain 2 (ΡΓΓΧ2) gene. Depending on the result of the analysis, a decision can be made between anthracycline-based therapy and non-anthracycline -based treatment prior to NAC, thereby optimizing the treatment plan for each patient individually.

BACKGROUND OF THE INVENTION

Breast cancer is the most common malignancy in women with more than 464,000 new cases diagnosed in 2012 in Europe (source: Cancer Research UK) and with about 75,000 new cases per year in Germany (source: WHO IACR report 2014). Breast cancers are staged according to their size, location and occurrence of metastasis. Methods of treatment include the use of surgery, radiation therapy, chemotherapy and endocrine therapy, which are also used as adjuvant therapies to surgery.

Approximately 70,000 (15%) patients will suffer from so-called triple-negative breast cancer (TNBC). TNBC is characterized by the absence of estrogen-receptor, progesterone- receptor and HER2 overexpression/amplification. This specific breast cancer indication belongs to the high-risk group of patients according to the St. Gallen classification with very poor prognosis and clinical outcome. The poor outcome is also attributable to the fact that these patients cannot be treated by endocrine or HER2-targeted therapy.

In contrast to other types of breast cancer, TNBC is treated in the neo-adjuvant setting.

Neo-adjuvant treatment schedule is characterized by core biopsy taken from the breast to affirm the histological diagnosis followed by neo-adjuvant chemotherapy (NAC). Thereafter, patients will undergo breast surgery. Pathological examination of the whole resected breast tumor lesion will reveal whether invasive tumor is still present or not. If no invasive tumor is present, this result is classified as pathological complete remission (pCR). pCR has been acknowledged as a validated surrogate efficacy endpoint for overall survival (OS), yet, only ~ 30-50% of the TNBC patients will show pCR after neo-adjuvant chemotherapy. While TNBC patients with pCR have a good prognosis and do not need further systemic treatment, patients without pCR might benefit from further adjuvant systemic therapy. Since there is only one chance for a patient to undergo neo-adjuvant therapy, it is of utmost importance to choose the best care for the patient, which includes the therapy regimen with the highest probability of response in terms of pCR after neo-adjuvant treatment and to search for strategies for the prediction of chemotherapy response prior to NAC. In order to optimize the selection of treatment options, a rapid, specific and sensitive method for the assessment of a therapy response is of crucial importance.

The use of predictive biomarkers is becoming increasingly relevant in cancer therapy as it allows for better identification of patients who will respond positively to the therapy, and of patients not responding. In breast cancer, predictive markers can determine the benefits of chemotherapy, endocrine therapy, and other types of therapy, such as immunotherapy (Characiejus. Anticancer Res. 2011; 31:639; Duffy. Clin Chem. 2005; 51:494; van de Vijver. Virchows Arch. 2014; 464:283). There are many advantages to utilizing a predictive marker in cancer therapy. Predictive biomarkers can help to improve patient selection and can guide physicians to optimize the treatment plan for each patient individually including better patient management, minimizing unnecessary suffering from drug side effects, reducing loss of precious time whilst determining whether a therapy will provide any benefit, and a reduction in costs for both the patient and the health insurance systems.

Alterations of DNA-methylation in the promoter region of genes is an early and frequent change in cancer, including breast cancer.

More than 65 years ago Mandel and Metais described for the first time their observation of the presence of extracellular nucleic acids in humans (Mandel P, Metais P. Les acides nucleiques du plasma sanguin chez l'homme. C.R.Acad.Sci. Paris 142, 241-243. 1948) and more than four decades later it could be clearly demonstrated that tumor-associated genetic alterations can be found in cell-free nucleic acids isolated from plasma, serum and other body fluids (Fleischhacker M, Schmidt B. (2007) Circulating nucleic acids (CNAs) and cancer— a survey. Biochim Biophys Acta 1775: 181-232; Jung K, Fleischhacker M, Rabien A. (2010) Cell-free DNA in the blood as a solid tumor biomarker-a critical appraisal of the literature. Clin Chim Acta 411: 1611-1624). This includes epigenetic alterations observed in different forms of malignant tumors. A hallmark of mammalian chromatin is DNA methylation and it is known that cytosine methylation in the context of a CpG dinucleotide plays a role in the regulation of development and is important in basic biological processes like embryogenesis and cell differentiation (Smith ZD, Meissner A. (2013) DNA methylation: roles in mammalian development. Nat Rev Genet 14: 204-220; Gibney ER, Nolan CM. (2010) Epigenetics and gene expression. Heredity (Edinb) 105: 4-13). As such, methylation not only regulates gene transcription, but also plays a role in maintaining genome stability, imprinting and X- chromosome inactivation. Epigenetic alterations in oncogenes and tumor suppressor genes are of key importance in the development of cancer (Suva ML, Riggi N, Bernstein BE. (2013) Epigenetic reprogramming in cancer. Science 339: 1567-1570).The ubiquity of such epigenetic changes in cancer events through DNA-methylation has led to a variety of innovative diagnostic and therapeutic strategies; the most recent technical advances have shown the great potential of DNA-methylation markers as valuable tools for decision making in the treatment of cancer patients (Stefansson and Esteller. Am J Pathol. 2013; 183: 1052).

Although several genes altered by DNA-methylation have been associated with response to adjuvant therapy in breast cancer patients in small exploratory studies, currently, no predictive DNA-methylation test for breast cancer is commercially available. This is remarkable since, in contrast to RNA and proteins, DNA is a very stable biological material that can be extracted from the same clinical tissue samples that are subjected to analysis by the pathologist for clinical-routine malignancy diagnostics.

PITX2 has been identified as a result of a European transnational cooperation within the

FP6 framework program and the PathoBiology Group of the EORTC (European Organization for Research and Treatment of Cancer). The PITX2 (paired-like homeodomain 2) gene encodes a member of the RIEG/PITX homeobox family, which is in the bicoid class of homeodomain proteins. ΡΓΓΧ2 controls cell proliferation in a tissue- specific manner and is involved in morphogenesis. During embryonic development, it exerts a role in the expansion of muscle progenitors and plays a role in the proper localization of asymmetric organs such as the heart and stomach. As transcription factor, ΡΓΓΧ2 is strongly involved in developmental processes and functions as a main executor of transcriptional regulation of developmental target genes by interacting with other proteins like HDACl/3 and P300. ΡΓΓΧ2 itself mediates target gene activation/inactivation for many signaling pathways like Estrogen receptor pathway, WNT/β- catenin and TGF-beta pathway, regulating and being regulated by many second messengers of these pathways, which are distinctively active in different breast cancer subtypes.

Measurement of DNA-methylation in the promoter region of PITX2 was reported to estimate the potential response or failure of an individual breast cancer patient to systemic chemo- or endocrine therapy (Harbeck. J Clin Oncol. 2008; 26:5036; Nimmrich. Breast Cancer Res Treat. 2008; 111:429).

Methods for prognosis and/or for prediction of the outcome of estrogen-treatment in patients suffering from hormone receptor positive breast cancer by determining the expression level of ΡΓΓΧ2 or the genetic or epigenetic modifications of the genomic DNA associated with the gene PITX2 were also described in EP 1 561 821, EP 1 554 407, EP 1 561 821, and EP 2 157 191.

Recent data in non-metastasized breast cancer patients further suggested that ΡΓΓΧ2 DNA-methylation state may predict the response to adjuvant anthracycline-based chemotherapy. Specifically, in WO 2007/039128 the increase of methylation of PITX2 was used as a predictive marker for the outcome of an adjuvant anthracycline treatment in a variety of cell proliferative disorders including breast cancer.

Thus, at present, increased methylation of the PITX2 gen was described as potential marker for the prediction of the outcome of endocrine therapy in hormone receptor positive breast cancer and anthracycline-based therapy of various cell proliferative orders in the adjuvant setting, e.g. following surgery only.

The present inventors now for the first time found that determination of the expression of the PITX2 gene and/ or a genomic sequence thereof is also useful for predicting the outcome of chemotherapy of TNBC patients in the neo-adjuvant setting. The principle is to use the methylation state of tumor DNA from the breast cancer marker PITX2 that is obtained from biopsy-tissue obtained during routinely performed diagnostic biopsy as an indicator for the clinical outcome. While ΡΓΓΧ2 hypermethylation was reported to lead to worse prognosis in estrogen receptor positive breast cancer patients (Harbeck. J Clin Oncol. 2008; 26:5036; Maier. EJC. 2007; 43: 1679-1686), it was surprisingly found by the present inventors that in TNBC patients hypomethylation of the ΡΓΓΧ2 gene is indicative of a poor clinical outcome in terms of pathological complete remission (pCR).

Several neo-adjuvant chemotherapy (NAC)-regimens are currently being used for the treatment of TNBC.

While anthracycline/taxane combinations (herein referred to anthracycline-based chemotherapy) have shown pCR rates of 28-41% recent data also emphasize a benefit for treatment of TNBC patients with carboplatinum containing therapy regimens owing to their unique genetic properties.

Surprisingly, the present inventors found that DNA methylation state in the PITX2 gene, and, specifically, PITX2 hypomethylation could specifically predict a poor outcome of anthracycline-based neo-adjuvant chemotherapy in patients diagnosed with triple-negative breast cancer (TNBC). This is essential because there is only one chance for a patient to undergo neo-adjuvant therapy, and the prediction of response with respect to pCR will, for the first time, help to recommend anthracycline- versus non- anthracycline-based therapy. The present invention thus meets the long felt need to provide a rapid, specific and sensitive method for predicting the response of TNBC patients to anthracycline -based treatment prior to NAC, which allows to select those TNBC patients benefiting from such therapy. Since treatment regimens in neo-adjuvant chemotherapy not only differ with regard to efficacy, but often with regard to toxicity, said method will also encourage TNBC patients likely to respond to stay on a rationally selected therapy, despite possible toxic side effects.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to a method for predicting the outcome of neo-adjuvant chemotherapy of a subject afflicted with triple-negative breast cancer (TNBC) comprising the steps:

a) providing a biological sample from the subject,

b) determining the expression or methylation state of the gene and/or genomic sequence of PITX2 and/or regulatory sequences thereof within said sample; and

c) determining therefrom the outcome of a treatment of said subject.

In an embodiment of the first aspect, the method further comprises step d) wherein a suitable treatment regimen for the subject is determined.

In a further embodiment of the method according to the first aspect, the neo-adjuvant chemotherapy is anthracycline-based.

In a further embodiment of said method determining the expression or methylation state of the gene and/or genomic sequence of PITX2 and/or regulatory sequences thereof within said sample in step b) is accomplished by analysis of genomic DNA isolated from the biological sample. Said expression may be determined by determining the methylation state of one or more CpG sites within said gene and/or genomic sequence and/or regulatory regions thereof or by determining the amount of mRNA encoding PITX2 and/or the amount of PITX2 protein. The methylation state of one or more CpG sites may be determined by converting, in said genomic DNA, or a fragment thereof, cytosine unmethylated in the 5-position to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties (i.e. that does not hybridize to guanine), preferably by bisulfite conversion.

In a further embodiment of the method according to the first aspect, the sample in step a) is selected from the group consisting of body fluids such as nipple aspirate, blood, serum, plasma, cells, cell lines, blood cells, tissue, tissue biopsies, preferably breast tissue biopsies and all possible combinations thereof, preferably wherein said sample is provided in in a state selected from the group consisting of natural, frozen, lyophilized, preserved, embedded, paraffin embedded, and all possible combinations thereof, more preferably wherein the sample is a paraffin-embedded tissue sample or an anticoagulated blood sample.

In a further embodiment the method according to the first aspect comprises contacting genomic DNA isolated from a biological sample obtained from the subject with at least one reagent, or a series of reagents that distinguishes between methylated and non-methylated CpG dinucleotides within at least one target region of the genomic DNA, preferably wherein the target region comprises, or hybridizes under stringent conditions to a sequence of at least 16 contiguous nucleotides of the PITX2 gene and/or regulatory regions thereof, wherein said contiguous nucleotides comprise at least one CpG dinucleotide sequence, and whereby predicting the outcome of anthracycline treatment of cell proliferative disorders is at least in part, afforded.

In a further embodiment of said method, ΡΓΓΧ2 hypomethylation is indicative for a poor clinical outcome. Said hypomethylation may be a degree of methylation of the target DNA of up to 95%, up to 90%, up to 80%, up to 70%, up to 60%, up to 50%, up to 40%, up to 30%, up to 20%, or up to 10% lower than a degree of methylation of a control.

In a further embodiment of said method, said hypomethylation may be a percent methylation ratio (PMR) of the target DNA of less than 5% PMR, less than 4% PMR, less than 3% PMR, less than 2% PMR, or less than 1% PMR.

In a further embodiment of the method of the first aspect, step d) may comprise determining neoadjuvant chemotherapy as a suitable treatment regimen for a subject exhibiting a percent methylation ratio (PMR) value of >1% PMR, or a percent methylation ratio (PMR) value of >2% PMR. Preferably, said neo-adjuvant chemotherapy is anthracycline -based chemotherapy.

In a further embodiment of the method according to the first aspect, an endpoint of the clinical outcome is pathological complete remission (pCR).

In a second aspect, the present invention relates to a method for predicting the outcome of neo-adjuvant chemotherapy in a subject afflicted with triple-negative breast cancer (TNBC), comprising:

a) isolating genomic DNA from a biological sample of the subject;

b) converting, in said genomic DNA, or a fragment thereof, cytosine unmethylated in the 5-position to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties;

c) amplifying a region of the converted genomic DNA, or of the converted fragment thereof, using at least two primers, wherein said region comprises at least 16 contiguous nucleotides of the PITX2 gene and/or regulatory regions thereof, wherein said contiguous nucleotides comprise at least one CpG dinucleotide sequence;

d) detecting the presence or absence of, or the quantity of DNA amplified in step c); e) determining, based on the presence or absence of, or on the quantity of said amplificate, the methylation state of the gene and/or genomic sequence of PITX2; and

f) predicting from said methylation state the outcome of the neo-adjuvant treatment. In an embodiment of the second aspect, the method further comprises step g) wherein a suitable treatment regimen for the subject is determined.

In a further embodiment of the method according to the second aspect, the neo-adjuvant chemotherapy is anthracycline-based.

In an embodiment of the method according to the second aspect, at least two primers used in step c) each comprise a contiguous sequence of at least 16 nucleotides in length that is complementary to, or hybridizes under moderately stringent or stringent conditions to a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 30 and complements thereof.

In a further embodiment of said method, the neo-adjuvant treatment is an anthracycline- based chemotherapy.

In a yet further embodiment of said method, ΡΓΓΧ2 hypomethylation is indicative for a poor clinical outcome. Said hypomethylation may be a degree of methylation of the target DNA of up to 95%, up to 90%, up to 80%, up to 70%, up to 60%, up to 50%, up to 40%, up to 30%, up to 20%, or up to 10% lower than a degree of methylation of a control.

In a yet further embodiment of said method, said hypomethylation may be a percent methylation ratio (PMR) of the target DNA of less than 5% PMR, less than 4% PMR, less than 3% PMR, less than 2% PMR, or less than 1% PMR.

In a further embodiment of the method of the second aspect, step g) may comprise determining neoadjuvant chemotherapy as a suitable treatment regimen for a subject exhibiting a percent methylation ratio (PMR) value of >1% PMR, or a percent methylation ratio (PMR) value of >2% PMR. Preferably, said neo-adjuvant chemotherapy is anthracycline-based chemotherapy.

In a further embodiment of the method according to the second aspect, the sample in step a) is selected from the group consisting of body fluids such as nipple aspirate, blood, serum, plasma, cells, cell lines, blood cells, tissue, tissue biopsies, in particular breast tissue biopsies and all possible combinations thereof, preferably wherein said sample is provided in in a state selected from the group consisting of natural, frozen, lyophilized, preserved, embedded, paraffin embedded, and all possible combinations thereof, more preferably wherein the sample is a paraffin-embedded tissue sample or an anticoagulated blood sample.

In a further embodiment of the method according to the second aspect, an endpoint of the clinical outcome is pathological complete remission (pCR).

FIGURES

Figure 1: Schematic representation of the workflow established to assess the DNA- methylation status of the ΡΓΓΧ2 promoter gene extracted from formalin-fixed and paraffin- embedded (FFPE) breast tumor tissue, hereinafter referred to as "PITX2-Test" (see Example 3).

Figure 2: Schematic representation of the workflow of determining PMR value variations (heterogeneity) between consecutive tumor tissue sections and reproducibility of PMR values in 12 independent qPCR runs (see Example 4).

Figure 3: Schematic representation of the workflow to obtain macro-dissected tumor areas (see Example 5).

DESCRIPTION OF THE INVENTION

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, H.G.W, Nagel, B. and Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

In the following, the elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", are to be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents, unless the content clearly dictates otherwise. DETAILED DESCRIPTION OF THE INVENTION

Characterization of a cancer in terms of predicting treatment outcome enables the physician to make an informed decision as to a therapeutic regimen with appropriate risk and benefit tradeoffs to the patient.

Accordingly, the present invention provides methods for predicting the outcome of neo- adjuvant chemotherapy of a subject afflicted with triple-negative breast cancer (TNBC).

Subject

The term "subject" as used herein refers to an individual, such as a human, a non-human primate (e.g. chimpanzees and other apes and monkey species); farm animals, such as birds, fish, cattle, sheep, pigs, goats and horses; domestic mammals, such as dogs and cats; laboratory animals including rodents, such as mice, rats and guinea pigs. The term does not denote a particular age or sex. In a particular meaning, the subject is a mammal. In a preferred meaning, the subject is a human. Outcome

The term "predicting the outcome of neo-adjuvant chemotherapy" as used herein refers to the expected outcome of the cancer disease in response to said treatment and generally relates to the assessment of its state of development, progression, or of its regression, and/or the prognosis of the course of the cancer in the future. The prediction of the treatment effect can be done using any assessment criterion used in oncology and known to the person skilled in the art. Generally, the effect of the treatment can be assessed by determining the tumor size and/or the number of cancer cells. As will be understood by persons skilled in the art, such assessment normally may not be correct for 100% of the patients, although it preferably is correct. The term, however, requires that a correct prediction can be made for a statistically significant part of the subjects. Whether a part is statistically significant can be determined easily by the person skilled in the art using several well-known statistical evaluation tools, for example, determination of confidence intervals, determination of p values, Student's t-test, Mann- Whitney test, etc. Details are provided in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. The preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%. The p values are preferably 0.05, 0.01, or 0.005.

Pathological complete remission (pCR)

In a preferred embodiment of the present invention, an endpoint of the clinical outcome is pathological complete remission (pCR). Pathological examination of the whole resected breast tumor lesion will reveal whether invasive tumor is still present or not. More specifically, pCR is defined as absence of residual invasive cancer by histological evaluation (hematoxylin- eosin staining) of the complete resected breast specimen and all sampled regional lymph nodes, following completion of neo-adjuvant systemic therapy (i.e. ypTO/Tis ypNO in the current AJCC staging system). If no invasive tumor is present, this result is classified as pathological complete remission (pCR). pCR is an accepted endpoint by the EM A and FDA (Guidance for Industry, Pathological Complete Response in Neoadjuvant Treatment of High-Risk Early-Stage Breast Cancer: Use as an Endpoint to Support Accelerated Approval. U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER), October 2014 Clinical/Medical).

Pathological complete remission (pCR) may be classified into four different stages according to Sinn et al. (Sinn HP, Schmid H, Junkermann H, Huober J, Leppien G, Kaufmann M, Bastert G, Otto HF. Histologic regression of breast cancer after primary (neoadjuvant) chemotherapy. Geburtshilfe Frauenheilkd. 1994 Oct; 54(10):552-8), as depicted in Table 1 below:

Table 1: Classification of pCR according to Sinn et al. pCR Response to neoadjuvant chemotherapy:

according

to Sinn:

Sinn 0-1 no response

Sinn 2 largely tumor free

Sinn 3 tumor-free but in situ carcinoma present

Sinn 4 pathological complete remission (no invasive tumor present, no carcinoma in situ present and no invasive tumor cells present in axillary lymph nodes pCR has been acknowledged as a validated surrogate efficacy endpoint for overall survival (OS), yet, only ~ 30-50% of the TNBC patients will show pCR after neo-adjuvant chemotherapy (NAC). While TNBC patients with pCR have a good prognosis and do not need further systemic treatment, patients without pCR might benefit from further adjuvant systemic therapy.

Triple-negative breast cancer (TNBC)

The terms "triple-negative breast cancer" and "TNBC" as used herein refer to any breast cancer that does not express or overexpress the genes for estrogen receptor (ER), progesterone receptor (PR) or HER2 according to clinical guidelines stated in the American society of clinical oncologists for immunohistochemical assessment of ER/PR and HER2 expression state and clinical guidelines for TNBC (Oakman CI, Viale G, Di Leo A.; Management of triple negative breast cancer. Breast. 2010 Oct;19(5):312-21.).

The above-defined lack of gene expression or overexpression means that the growth of the cancer is not supported by the hormones estrogen and progesterone, nor by the presence of too many HER2 receptors. Therefore, triple-negative breast cancer does not respond to hormonal therapy (such as tamoxifen or aromatase inhibitors) or therapies that target HER2 receptors, such as Herceptin (chemical name: trastuzumab). Also for this reason, this specific breast cancer indication belongs to the high-risk group of patients according to the St. Gallen classification with very poor prognosis and clinical outcome. However, other medicines can be used to treat triple-negative breast cancer. In contrast to other types of breast cancer, TNBC is treated by neo-adjuvant chemotherapy. Neo-adjuvant chemotherapy (NAC) In the context of the present invention, the term "neo-adjuvant chemotherapy" or "NAC" refers to a treatment given as a first step to shrink a tumor before the main treatment, which is usually surgery, is given. Examples of neoadjuvant therapy include chemotherapy, radiation therapy, and hormone therapy. It is a type of induction therapy.

In the present context, said neo-adjuvant chemotherapy preferably is anthracycline - based. Anthracyclines are a large group of compounds synthesized by different Streptomyces species. They possess antibiotic activity and have cytotoxic effects on eukaryotic cells. All anthracyclines have a tetrahydronaphthacenedione ring structure attached by a glycosidic linkage to a sugar molecule, structural diversity of anthracyclines is generated by modifications of the backbone including a large number of different side chains. Anthracyclines have excellent antineoplastic activity in metastatic, neoadjuvant, and adjuvant settings and are used in the treatment of various haematopoietic and solid tumours. Although their mechanism of chemotherapeutic action is unclear it involves noncovalent DNA intercalation, formation of covalent DNA adducts, topoisomerase II (topo II) poisoning, and free radical effects on cellular membranes and DNA.

In the present context, anthracycline-based neo-adjuvant chemotherapy preferably comprises administering at least one anthracycline, which may be selected from the group consisting of mitoxantrone, doxorubicin, aclarubicin, daunorubicin, epirubicin, idarubicin and combinations thereof.

In an embodiment, anthracycline-based neo-adjuvant chemotherapy comprises administering to a subject at least one anthracycline selected from the above-mentioned group in combination with a platinum compound, which may be selected from cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin, nedaplatin, triplatin, lipoplatin, and combinations thereof. Typically, the at least one anthracycline and the platinum compound are administered to the subject in a sequential manner.

In a preferred embodiment, the subject receives anthracycline-based neo-adjuvant chemotherapy over a time period of 24 weeks. Within this time period the subject may receive four cycles of anthracycline-treatment followed by 12 weeks of treatment with carboplatin.

However, the clinical utility of anthracyclines are limited due to acute and chronic toxicities, particularly cardiotoxicity, myelosuppression, nausea and vomiting, and alopecia.

Heart failure following anthracycline therapy is a major clinical problem in cancer treatment. The establishment of predictors of the anthracycline treatment outcome would allow the identification and exclusion of individuals who would not benefit from said treatment, and thus to increase the safety of anthracycline treatment. Furthermore by determining which patients would benefit from anthracycline treatment, but wherein said predicted outcome is sub- optimal patients can be recommended for further chemotherapeutic or other treatments.

This is particularly essential in the treatment of TNBC because there is only one chance for a patient to undergo neo-adjuvant therapy. The prediction of response with respect to pCR after neo-adjuvant therapy would help to recommend anthracycline- versus non-anthracycline- based therapy.

Accordingly, there is a long felt need in the art for determining which patients afflicted with TNBC will benefit from anthracycline-based treatment in the neoadjuvant setting. Methods of the invention

This need has now been met by the methods according to the first and second aspect of the invention. Said method of the first aspect comprises: a) providing a biological sample from the subject, b) determining the expression or methylation state of the gene and/or genomic sequence of PITX2 and/or regulatory sequences thereof within said sample; and c) determining therefrom the outcome of a treatment of said subject.

The method of the second aspect comprises: a) isolating genomic DNA from a biological sample taken of a subject; b) converting, in said genomic DNA, or a fragment thereof, cytosine unmethylated in the 5-position to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties; c) amplifying a region of the converted genomic DNA, or of the converted fragment thereof, using at least two primers, wherein said region comprises at least 16 contiguous nucleotides of the PITX2 gene and/or regulatory regions thereof, wherein said contiguous nucleotides comprise at least one CpG dinucleotide sequence; d) detecting the presence or absence of, or the quantity of DNA amplified in step c); determining, based on the presence or absence of, or on the quantity of said amplificate, the methylation state of the gene and/or genomic sequence of PITX2; and e) predicting from said methylation state the outcome of the neo-adjuvant treatment.

Biological sample

The term "sample" or "biological sample" as used herein refers to biological material obtained from a subject and preferably comprises genomic DNA from all chromosomes, preferably genomic DNA covering the whole genome. The sample comprises, if a subject has cancer, cells of the cancer or free genomic DNA (including the target DNA) from cancer cells, preferably circulating genomic DNA from cancer cells. It can be derived from any suitable tissue or biological fluid such as nipple aspirate, blood, serum, plasma; cells, cell lines, blood cells, tissue, tissue biopsies and all possible combinations thereof. Preferably, said biological sample is provided in in a state selected from the group consisting of natural, frozen, lyophilized, preserved, embedded, paraffin embedded, and all possible combinations thereof. Methods for deriving samples from a subject are well known to those skilled in the art. In a preferred embodiment, the sample is a tumor biopsy or a liquid sample. The tumor biopsy is preferably a paraffin-embedded tissue sample and the liquid sample is preferably an anticoagulated blood sample.

Homeodomain transcription factor 2 (PITX2)

Within said biological sample, the expression or methylation state of the gene and/or genomic sequence of PITX2 and/or regulatory sequences thereof are determined. Determination of the expression may be through determination of the methylation state of one or more CpG. Alternatively, or additionally, determination of expression may be through determining the amount of mRNA encoding PITX2 or the amount of PITX2 protein. In a preferred embodiment, the said expression is determined by analysis of genomic DNA isolated from the biological sample.

Genomic DNA

The term "genomic DNA" as used herein refers to chromosomal DNA and is used to distinguish from coding DNA. As such, it includes exons, introns as well as regulatory sequences, in particular promoters, belonging to a gene. Genomic DNA may be isolated by any means standard in the art, including the use of commercially available kits. Briefly, wherein the DNA of interest is encapsulated in/by a cellular membrane the biological sample must be disrupted and lysed by enzymatic, chemical or mechanical means. In case DNA is extracted from formalin-fixed paraffin-embedded tissue deparaffinization and decros slinking steps are required. These steps may be performed as commonly known in the art, e.g. by using a kit from commercial providers according to the supplier protocols (e.g. QiaAmp DNA FFPE tissue kit by Qiagen GmbH, Hilden, Germany). The DNA solution may then be cleared of proteins and other contaminants e.g. by digestion with proteinase K. The genomic DNA is then recovered from the solution. This may be carried out by means of a variety of methods including salting out, organic extraction or binding of the DNA to a solid phase support. The choice of method will be affected by several factors including time, expense and required quantity of DNA. In the present context, the term "genomic DNA" preferably refers to genomic sequences of or within the PITX2 gene and treated variants thereof as displayed in Table 2 and Table 3 below. Methylation analysis

In a preferred embodiment, determining the expression of the gene and/or genomic sequence of PITX2 and/or regulatory sequences thereof comprises determining the methylation state of the gene and/or genomic sequence of ΡΓΓΧ2. It is particularly preferred that the methylation state of the CpG dinucleotides within the genomic sequence of said gene according to Table 2 (SEQ ID NO: 1) or in bisulfit converted derivatives thereof (SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13) are analyzed. More preferably, the methylation state of the CpG dinucleotides within the CpG rich regions of PITX2 gene with SEQ ID NO: 2 or in bisulfit converted derivatives thereof (SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 14), or within the CpG rich regions of PITX2 gene with SEQ ID NO: 3 or in bisulfit converted derivatives thereof (SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15), even more preferably within the CpG rich regions of ΡΓΓΧ2 gene with SEQ ID NO: 16 or in bisulfit converted derivatives thereof (SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28), and most preferably within the CpG rich regions of PITX2 gene with SEQ ID NO: 17 or in bisulfit converted derivatives thereof (SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29) or within the CpG rich regions of PITX2 gene with SEQ ID NO: 18 or in bisulfit converted derivatives thereof (SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30) are analysed (see Table 3).

In the present context, the genomic sequences with SEQ ID NO: 1 to 3 and 16 to 18 or the bisulfite converted sequences with SEQ ID NO: 4 to 15 and 19 to 30 corresponding thereto will also be referred to as "target DNA".

Table 2: Genomic sequence of the PITX2 gene and bisulfite converted variants thereof

Table 3: Preferred CpG rich regions of PITX2 and bisulfite converted variants thereof Gene Genomic Pretreated Pretreated Pretreated Pretreated

SEQ ID methylated methylated unmethylated unmethylated

NO: sequence sequence sequence sequence

(sense) SEQ ID (antisense) (sense) SEQ (antisense)

NO: SEQ ID NO: ID NO: SEQ ID NO:

ΡΓΓΧ2 2 5 8 11 14

ΡΓΓΧ2 3 6 9 12 15

ΡΓΓΧ2 16 19 22 25 28

ΡΓΓΧ2 17 20 23 26 29

ΡΓΓΧ2 18 21 24 27 30

DNA methylation

The term "methylation" or "DNA methylation" as used herein refers to a biochemical process involving the addition of a methyl group to the cytosine or adenine DNA nucleotides. DNA methylation at the 5 position of cytosine, especially in promoter regions, can have the effect of reducing gene expression and has been found in every vertebrate examined. In adult non-gamete cells, DNA methylation typically occurs in a CpG site.

CpG dinucleotides

The term "CpG site" or "CpG dinucleotide", as used herein, refers to regions of DNA where a cytosine nucleotide occurs next to a guanine nucleotide in the linear sequence of bases along its length. "CpG" is shorthand for "C-phosphate-G", that is, cytosine and guanine separated by only one phosphate; phosphate links any two nucleosides together in DNA. The "CpG" notation is used to distinguish this linear sequence from the CG base-pairing of cytosine and guanine. Cytosines in CpG dinucleotides can be methylated to form 5-methylcytosine. The term "CpG site" or "CpG site of genomic DNA" is also used with respect to the site of a former (unmethylated) CpG site in DNA in which the unmethylated C of the CpG site was converted to another as described herein (e.g. by bisulfite to uracil). The application provides in Table 2 and Table 3 the genomic sequence of each relevant DNA region as well as the bisulfite converted sequences of each converted strand. Herein, bisulfite converted sequences are also referred to as "treated sequences" or "pretreated sequences". Table 3 specifically provides CpG rich regions of the ΡΓΓΧ2 gene and treated variants thereof. CpG sites referred to are always the CpG sites of the genomic sequence, even if the converted sequence does no longer contain these CpG sites due to the conversion.

Suitable methods for quantifying CpG methylation in genomic DNA are known in the art. In the context of the present invention, methylation within the ΡΓΓΧ2 gene and/or regulatory or promoter regions thereof may be analyzed by the any of the methods as described in WO 2007/03128 and in United States Patent No. 6,265,171 to Herman, the disclosure of which is herein incorporated by reference.

DNA conversion

Briefly, determining CpG methylation state within the PITX2 gene requires in a first step, converting, in genomic DNA, or a fragment thereof, cytosine unmethylated in the 5- position to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties.

The term "hybridization", when used with respect to an oligonucleotide, is to be understood as a bond of an oligonucleotide to a complementary sequence along the lines of the Watson-Crick base pairings in the sample DNA, forming a duplex structure, under moderate or stringent hybridization conditions. When it is used with respect to a single nucleotide or base, it refers to the binding according to Watson-Crick base pairings, e.g. C-G, A-T and A-U. Stringent hybridization conditions involve hybridizing at 68°C in 5x SSC/5x Denhardt's solution/1.0% SDS, and washing in 0.2x SSC/0.1% SDS at room temperature, or involve the art-recognized equivalent thereof (e.g., conditions in which a hybridization is carried out at 60°C in 2.5 x SSC buffer, followed by several washing steps at 37°C in a low buffer concentration, and remains stable). Moderate conditions involve washing in 3x SSC at 42°C, or the art-recognized equivalent thereof. The parameters of salt concentration and temperature can be varied to achieve the optimal level of identity between the probe and the target nucleic acid. Guidance regarding such conditions is available in the art, for example, by Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; and Ausubel et al. (eds.), 1995, Current Protocols in Molecular Biology, (John Wiley & Sons, N.Y.) at Unit 2.10.

The phrase "converting, in said genomic DNA, or a fragment thereof, cytosine unmethylated in the 5-position to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties" as used herein refers to a process of chemically treating the DNA in such a way that all or substantially all of the unmethylated cytosine bases are converted to uracil bases, or another base which is dissimilar to cytosine in terms of base pairing behaviour, i.e. that does not hybridize to guanine, while the 5-methylcytosine bases remain unchanged. The conversion of unmethylated, but not methylated, cytosine bases within the DNA sample is conducted with a converting agent. The term "converting agent" as used herein relates to a reagent capable of converting an unmethylated cytosine to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties. The converting agent is preferably a bisulfite such as disulfite, or hydrogen sulfite. The reaction is performed according to standard procedures (Frommer et al., 1992, Proc Natl Acad Sci USA 89: 1827-31; Olek, 1996, Nucleic Acids Res 24:5064-6; EP 1394172). It is also possible to conduct the conversion enzymatically, e.g. by use of methylation specific cytidine deaminases. Most preferably, the converting agent is sodium bisulfite or bisulfite.

The disclosed invention provides treated nucleic acid sequences, derived from genomic SEQ ID NO: 1 to SEQ ID NO: 3 and SEQ ID NO: 16 to SEQ ID NO: 18, wherein the treatment is suitable to convert at least one unmethylated cytosine base of the genomic DNA sequence to uracil or another base that is detectably dissimilar to cytosine in terms of hybridization. The genomic sequences in question may comprise one, or more, consecutive or random methylated CpG positions.

DNA amplification

In the next step, a region of the converted genomic DNA, or a region of a converted fragment thereof, is amplified, preferably in a methylation dependent manner.

The term "amplifying" or "generating an amplicon" as used herein refers to an increase in the number of copies of the target nucleic acid and its complementary sequence, or particularly a region thereof. The amplification may be performed by using any method known in the art. The amplification of nucleic acid includes methods that require multiple cycles during the amplification process or method that are performed at a single temperature. Cycling techniques are exemplified by methods requiring thermo -cycling. The methods requiring thermo-cycling include polymerase chain reaction (PCR), which is well known in the art. The PCR includes denaturing a double-stranded DNA into single stranded DNAs by thermal denaturation, annealing a primer to the single stranded DNAs; and synthesizing a complementary strand from the primer. Isothermal amplification is an amplification performed at a single temperature or where the major aspect of the amplification process is performed at a single temperature. In the PCR process, the product of the reaction is heated to separate the two strands such that another primer may bind to the template. Conversely, the isothermal techniques rely on a strand displacing polymerase in order to separate the two strands of a double strand and re-copy the template. Isothermal techniques may be classified into methods that rely on the replacement of a primer to initiate a reiterative template copying and those that rely on continued re -use or new synthesis of a single primer molecule. The methods that rely on the replacement of the primer include helicase dependant amplification (HDA), exonuclease dependent amplification, recombinase polymerase amplification (RPA), and loop mediated amplification (LAMP). The methods that rely on continued re -use or new synthesis of a single primer molecule include strand displacement amplification (SDA) or nucleic acid based amplification (NASBA and TMA). Methylation-specific PCR

The amplification is preferably performed by methylation-specific PCR by use of methylation- specific primer oligonucleotides, or, in an alternative embodiment, by use of primer oligonucleotides which are methylation-unspecific, but specific to bisulfite-converted DNA (i.e. hybridize only to converted DNA by covering at least one converted C). The latter method has been described in WO 2007/03128, the disclosure of which is herein incorporated by reference.

Primer oligonucleotides

The term "primer oligonucleotide" as used herein refers to a single-stranded oligonucleotide sequence substantially complementary to a nucleic acid sequence sought to be copied (the template) and serves as a starting point for synthesis of a primer extension product. "Substantially complementary" means that a primer oligonucleotide does not need to reflect the exact sequence of the template and can comprise mismatches and/or spacers, as long as it is still capable of annealing and serving as a starting point for extension under the chosen annealing and extension conditions (e.g. of a PCR cycle).

The term "mismatch" as used herein refers to base-pair mismatch in DNA, more specifically a base-pair that is unable to form normal base -pairing interactions (i.e., other than "A" with "T" or "U", or "G" with "C").

The term "spacer" as used herein refers to a non-nucleotide spacer molecule, which increases, when joining two nucleotides, the distance between the two nucleotides to about the distance of one nucleotide (i.e. the distance the two nucleotides would be apart if they were joined by a third nucleotide). Non-limiting examples for spacers are Inosine, d-Uracil, halogenated bases, Amino-dT, C3, C12, Spacer 9, Spacer 18, and dSpacer). Methylation-specific primer oligonucleotides

In an embodiment of the method, the methylation state of preselected CpG positions within one or more of the nucleic acid sequences selected from the group comprising SEQ ID NO: 1 to SEQ ID NO: 3 and SEQ ID NO: 16 to SEQ ID NO: 18 , may be detected by use of methylation- specific primer oligonucleotides. This technique (MSP) has been described in United States Patent No. 6,265,171 to Herman.

The use of methylation state specific primers for the amplification of bisulfite treated DNA allows the differentiation between methylated and unmethylated nucleic acids. MSP primers pairs contain at least one primer which hybridizes to a bisulfite treated CpG dinucleotide. Therefore, the sequence of said primers comprises at least one CpG , TpG or CpA dinucleotide. MSP primers specific for non-methylated DNA contain a "T' at the 3' position of the C position in the CpG. Preferably, therefore, the base sequence of said primers is required to comprise a sequence having a length of at least 18 nucleotides which hybridizes to a pretreated nucleic acid sequence according to SEQ ID NO: 4 to SEQ ID NO:9 or SEQ ID NO: 19 to SEQ ID NO: 24 and sequences complementary thereto, or to a pretreated nucleic acid sequence according to SEQ ID NO: 10 to SEQ ID NO: 15 or SEQ ID NO: 25 to SEQ ID NO: 30 and sequences complementary thereto, wherein the base sequence of said oligomers comprises at least one CpG, tpG or Cpa dinucleotide. In this embodiment of the method according to the invention it is particularly preferred that the MSP primers comprise between 2 and 4 CpG , tpG or Cpa dinucleotides. It is further preferred that said dinucleotides are located within the 3' half of the primer e.g. wherein a primer is 18 bases in length the specified dinucleotides are located within the first 9 bases form the 3 'end of the molecule. In addition to the CpG, tpG or Cpa dinucleotides it is further preferred that said primers should further comprise several bisulfite converted bases (i.e. cytosine converted to thymine, or on the hybridizing strand, guanine converted to adenosine). In a further preferred embodiment said primers are designed so as to comprise no more than 2 cytosine or guanine bases.

Methylation-specific PCR (MSP) is a methylation assay well-known in the art and was described by Herman et al. Proc. Natl. Acad. Sei. USA 93:9821-9826, 1996. In a most preferred embodiment, the step of amplifying comprises a real-time PCR as disclosed in EP 1 561 821 Bl, in particular MethyLight™. In the context of the present invention, the term "MethyLight™" refers to a methylation assay comprising four oligonucleotides, i.e. two methylation un-specific primer oligonucleotides and two oligonucleotide probes that competitively hybridize to the binding site. The two methylation un-specific primers are used to amplify a segment of the treated genomic DNA containing a methylation variable oligonucleotide probe binding site.

In the context of the present disclosure, the term "variable oligonucleotide probe binding site" refers to to the binding site of two differential fluorescent labelled oligonucleotide probes, detecting the fully methylated or fully unmethylated state of one or more CpG-motifs covered by the sequence of the respective probes.

Preferably, said two methylation un-specific PCR primers are oligonucleotides with SEQ-ID NO: 31 and SEQ-ID NO: 32.

Following the non-methylation specific amplification of the target region the PITX2 methylation state is determined by methylation specific detection using two different oligonucleotide probes that competitively hybridize to the binding site.

In the context of the present invention the term "methylation state" refers to the degree of methylation present in a nucleic acid of interest. This may be expressed in absolute or relative terms i.e. as a percentage or other numerical value or by comparison to another tissue and may be described as hypermethylated, hypomethylated or as having significantly similar or identical methylation state.

The two oligonucleotide probes used in the present methylation assay competitively hybridize to the binding site, one specific for the methylated version of the binding site, the other specific to the unmethlyated version of the binding site. Accordingly, one of the probes comprises a CpG at the methylation variable position (i.e. anneals to methylated bisulphite treated sites) and the other comprises a TpG at said position (i.e. anneals to unmethylated bisulphite treated sites). Each species of probe is labeled with a 5' fluorescent reporter dye and a 3' quencher dye wherein the CpG and TpG oligonucleotides are labeled with different dyes.

In a preferred embodiment, said oligonucleotide probes are oligonucleotides with SEQ- ID NO: 33 and SEQ-ID NO: 34. Said 5' fluorescent reporter dye may be any 5' fluorescent reporter dye known in the art and is particularly selected from 6-carboxyfluorescein (FAM). The 3' quencher dye may be any 3' quencher dye known in the art and is particularly selected from 5-Carboxytetramethylrhodamine (TAMRA).

In a particularly preferred embodiment, the PITX2 methylation state is determined by the art-recognized fluorescence-based real-time PCR technique described by Eads et al., Cancer Res. 59:2302-2306, 1999.

In a preferred embodiment of the invention, the amplified region of the PITX2 gene or fragment thereof is at least 16 contiguous nucleotide bases in length of a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3 and SEQ ID NO: 16 to SEQ ID NO: 18, wherein said sequence comprises at least one CpG dinucleotide and sequences complementary thereto, for which the amount of methylation is to be determined. The sequences of SEQ ID NO: 4 to SEQ ID NO: 15 and SEQ ID NO: 19 to SEQ ID NO: 30 provide non-naturally occurring modified versions of the nucleic acid according to SEQ ID NO: 1 to SEQ ID NO: 3 and SEQ ID NO: 16 to SEQ ID NO: 18, wherein the modification of each genomic sequence results in the synthesis of a nucleic acid having a sequence that is unique and distinct from said genomic sequence as follows. For each sense strand genomic DNA, e.g., SEQ ID NO: 1, two converted versions are disclosed. A first version wherein "C" to "T," but"CpG" remains "CpG" (i.e., corresponds to case where, for the genomic sequence, all "C" residues of CpG dinucleotide sequences are methylated and are thus not converted); a second version discloses the complement of the disclosed genomic DNA sequence (i.e. antisense strand), wherein "C" to "T," but "CpG" remains "CpG" (i.e., corresponds to case where, for all "C" residues of CpG dinucleotide sequences are methylated and are thus not converted). The 'unmethylated' converted sequences of SEQ ID NO: 1 to SEQ ID NO: 3 and SEQ ID NO: 16 to SEQ ID NO: 18 correspond to SEQ ID NO: 10 to SEQ ID NO: 15 and SEQ ID NO: 25 to SEQ ID NO: 30, wherein "C" is converted to "T" for all "C" residues, including those of "CpG" dinucleotide sequences (i.e., corresponds to case where, for the genomic sequences, all "C" residues of CpG dinucleotide sequences are unmethylated). The complementary strands are in silico built up from chemically converted, methylated and unmethylated DNA sequences (SEQ ID NO: 1 to SEQ ID NO: 3 and SEQ ID NO: 16 to SEQ ID NO: 18), representing complemented PCR products after the first amplification step (i.e. complementary sequence of fully methylated chemically converted sense strand). The 'complementary' converted sequences of SEQ ID NO: 1 to SEQ ID NO: 3 and SEQ ID NO: 16 to SEQ ID NO: 18 correspond to SEQ ID NO: 7 to SEQ ID NO: 9; SEQ ID NO: 13 to SEQ ID NO: 15; SEQ ID NO: 22 to SEQ ID NO: 24 and SEQ ID NO: 28 to SEQ ID NO: 30.

Particularly suitable methylation- specific primers for use in determining the ΡΓΓΧ2 methylation state are given in Table 4 below.

Table 4: Methylation-specific primers and amplificates for use in ΡΓΓΧ2 methylation analysis

The primer and probe oligonucleotdides according to SEQ ID NO: 31 to SEQ ID NO: 34 particularly anneal to nucleotide positions 10990 to 11011 (SEQ ID NO: 31), 10874 to 10887 (SEQ ID NO: 32), 10951 to 10970 (SEQ ID NO: 33) and 10944 to 10970 (SEQ ID NO: 34) of SEQ ID NO: 1, respectively. More particularly, three CpG site in the nucleotide range of from 10952 to 10967 of SEQ ID NO: 1 may be detected by use of the primers and probes referred to in Table 4.

Methylation unspecific primer oligonucleotides

In an alternative embodiment, the methylation state of preselected CpG positions within one or more of the nucleic acid sequences selected from the group comprising SEQ ID NO: 1 to SEQ ID NO: 3 and SEQ ID NO: 16 to SEQ ID NO: 18 is analysed using primers which are methylation-unspecific, but specific to bisulfite-converted DNA (i.e. hybridize only to converted DNA by covering at least one converted C). In this case, methylation-specificity is achieved by using methylation- specific blocker oligonucleotides, which hybridize specifically to converted or non-converted CpG sites and thereby terminate the PCR polymerization. In this alternative embodiment, the step of amplifying comprises a real-time PCR as disclosed in WO 2007/03128, in particular HeavyMethyl™. In the context of the present invention, the term "HeavyMethyl™" refers to a methylation assay comprising methylation specific blocking probes covering CpG positions between the amplification primers.

Specifically, a "blocker oligonucleotide" or "blocking probe" may be a blocker that prevents the extension of the primer located upstream of the blocker oligonucleotide. It comprises nucleosides/nucleotides having a backbone resistant to the 5' nuclease activity of the polymerase. This may be achieved, for example, by comprising peptide nucleic acid (PNA), locked nucleic acid (LNA), Morpholino, glycol nucleic acid (GNA), threose nucleic acid (TNA), bridged nucleic acids (BNA), N3'-P5' phosphoramidate (NP) oligomers, minor groove binder-linked-oligonucleotides (MGB-linked oligonucleotides), phosphorothioate (PS) oligomers, CrC4alkylphosphonate oligomers, phosphoramidates, β-phosphodiester oligonucleotides, a-phosphodiester oligonucleotides or a combination thereof. Alternatively, it may be a non-extendable oligonucleotide with a binding site on the DNA single-strand that overlaps with the binding site of a primer oligonucleotide. When the blocker is bound, the primer cannot bind and therefore the amplicon is not generated. When the blocker is not bound, the primer-binding site is accessible and the amplicon is generated. For such an overlapping blocker, it is preferable that the affinity of the blocker is higher than the affinity of the primer for the DNA. Also, a blocker oligonucleotide cannot by itself act as a primer (i.e. cannot be extended by a polymerase) due to a non-extensible 3' end. In a preferred embodiment, a set of at least two primer oligonucleotides is used for amplifying DNA sequences of one of SEQ ID NO: 1 to SEQ ID NO: 30 and sequences complementary thereto, or segments thereof.

In a particularly preferred embodiment, said at least two primers each comprise a contiguous sequence of at least 18 nucleotides in length that is complementary to, or hybridizes under moderately stringent or stringent conditions to a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 30 and sequences complementary thereto, or segments thereof.

Particular methylation un- specific primers for use in determining the ΡΓΓΧ2 methylation state are listed in Table 5.

Table 5: Methylation un-specific primers and amplificates for use in ΡΓΓΧ2 methylation analysis

The resulting amplification product is isolated and used as a template for determining the methylation state of at least one CpG dinucleotide.

Accordingly, in a next step, the methylation state of the target DNA is determined, based on the presence or absence of, or on the quantity of said amplificate.

In the context of the present invention the term "methylation state" refers to the degree of methylation present in a nucleic acid of interest. This may be expressed in absolute or relative terms i.e. as a percentage or other numerical value or by comparison to another tissue and may be described as hypermethylated, hypomethylated or as having significantly similar or identical methylation state.

The methylation state of the target DNA may be determined by means of one or more methods taken from the group consisting oligonucleotide hybridization analysis, Ms-SnuPE, sequencing, Real Time detection probes and oligonucleotide array analysis. These methods may be performed as commonly know in the art and/or as disclosed in WO 2007/03128. In a particularly preferred embodiment, PITX2 methylation state is determined by quantitative real-time PCR (QM-PCR) as described by Harbeck et al. 2008; J Clin Oncol; 26:5036-5042 or as in patent EP1561821 Bl.

In a preferred embodiment, the methylation state is determined using oligonucleotides detecting the cytosine methylation state within genomic or pre-treated DNA, according to SEQ ID NO: 1 to SEQ ID NO: 30. It is particularly preferred that said oligonucleotides comprise a nucleic acid sequence having a length of at least nine nucleotides which hybridizes, under moderately stringent or stringent conditions (as defined herein above), to a treated nucleic acid sequence according to SEQ ID NO: 4 to SEQ ID NO: 15 and SEQ ID NO: 19 to SEQ ID NO: 30 and/or sequences complementary thereto, or to a genomic sequence according to SEQ ID NO: 1 to SEQ ID NO: 3 or SEQ ID NO: 16 to SEQ ID NO: 18 and/or sequences complementary thereto. Most preferably, detection oligonucleotides for determining the methylation state of ΡΓΓΧ2 are selected from SEQ ID NO: 52 to SEQ ID NO: 71. Predictive value of PITX2 hypomethylation

From the methylation state determined by any of the herein described or referred to methods, the outcome of neo-adjuvant treatment of an TNBC patient may be predicted. Specifically, it was found that ΡΓΓΧ2 hypomethylation is indicative for a poor clinical outcome of anthracycline -based neo-adjuvant chemotherapy.

The term "poor clinical outcome" as used herein means the absence of pathological complete remission (pCR), i.e. the presence of residual invasive cancer after completion of neoadjuvant systemic therapy. While TNBC patients with pCR have a good prognosis and do not need further systemic treatment, patients without pCR might benefit from further adjuvant systemic therapy.

Preferably, the term "poor clinical outcome" as used herein refers to pCR 0-1 according to Sinn et al. (see Table 1).

The term "hypomethylation" as used herein refers to an aberrant methylation pattern or state (i.e. the presence or absence of methylation of one or more nucleotides), wherein one or more nucleotides, preferably C(s) of a CpG site(s), are un-methylated compared to a control.

In an embodiment, said control may be the same genomic DNA from a non-cancer cell of the patient or a subject not suffering or having suffered from the cancer the patient is treated for, preferably any cancer (healthy control). In particular, it may refer to a decreased presence of 5-mCyt at one or a plurality of CpG dinucleotides within a DNA sequence comprised in a biological sample, relative to the amount of 5-mCyt found at corresponding CpG dinucleotides within a healthy control DNA sample.

In an alternative embodiment, the term "control" as used herein may refer to a reference sample comprising control DNA with known DNA concentration and known target methylation state. In this alternative embodiment, the control DNA is preferably, but not necessarily, human DNA that is artificially methylated, preferably substantially fully methylated. Preferably, said artificial methylation is achieved by using DNA-Methyltransferases. The DNA itself can be, for example, cell line DNA, plasmid DNA, artificial DNA, or combinations/mixtures thereof. Substantially fully methylated genomic DNA preferably is DNA, particularly genomic DNA, which has all or substantially all CpG sites methylated. "Substantially all" in this respect means at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9%. It is preferred that the methylation of all or substantially all CpG sites is achieved by treating the DNA with a CpG methyltransferase in a manner that provides for the methylation of all or substantially all CpG sites.

In a preferred embodiment, the term "hypomethylation" as used herein refers to a degree of methylation of the target DNA of up to 95%, up to 90%, up to 80%, up to 70%, up to 60%, up to 50%, up to 40%, up to 30%, up to 20%, or up to 10% lower than a degree of methylation of a control as defined herein.

In a further preferred embodiment, the term "hypomethylation" as used herein refers to a percent methylation ratio (PMR) of the target DNA of less than 5% PMR, less than 4% PMR, or less than 3% PMR, and, preferably, of less than 2% PMR, or less than 1 % PMR. Said percent methylation ratio (PMR) may be determined by data transformation of CT values obtained from quantitative methylation-specific PCR and a modified 2exp ^ACT method for a duplex probe system, with internal calibration and standardization according to the following formula:

PMR (sample well): l/(l+2 ^ex P(CT _met h. - CT _un meth.)) .

MV PMR (sample duplicate): (PMR (sample well 1) + PMR (sample well 2))/2

Error (absolute): STDEV MV PMR (sample duplicate). meth. = methylated signal

unmeth. = unmethylated signal

PMR = Percent Methylation Ratio

MV PMR (sample duplicate): Mean value of PMR values calculated for each separate well in the technical assay duplicate of one sample.

CT = Cycle Threshold. PCR cycle number, at which the reporter dye fluorescent signal reaches an assay specific threshold. For further analysis the mean value and STDEV of technical replicates (n=2) was established.

STDEV = Standard deviation of the Mean value PMR for the technical replicates.

Included control references plasmid mixtures (2500 copies input per well: 1: 1 mixture in case of REF50) containing the bisulfite-converted fully methylated and fully unmethylated sequence to be amplified by the assay can be used for absolute quantification of methylated and unmethylated copies of the PITX2 gene promoter in a sample according to the following formula:

Copy number methylated (sample): 1250/(2 ^exp (CT _m eth sample - CTmeth REFSO)) .

Copy number unmethylated (sample): 1250/(2 ^exp (CT _un meth sample - CT _un meth REFSO)).

CTmeth REFSO: CT value of methylated signal of reference 50 sample (1250 methylated and 1250 unmethylated copies input per assay well).

CTunmeth REFSO: CT value of unmethylated signal of reference 50 sample (1250 methylated and 1250 unmethylated copies input per assay well).

The term "PITX2 hypomethylation" as used herein refers to hypomethylation of the gene and/or genomic sequence of homeodomain transcription factor 2 (PITX2) and/or regulatory sequences thereof and, preferably, to hypomethylation of one or more CpG sites within said gene and/or genomic sequence and/or regulatory regions, preferably within the genomic region with SEQ ID NO: 1, more preferably within the genomic region with SEQ ID NO: 2 and SEQ ID NO: 3 and most preferably within the genomic region with SEQ ID NO: 2.

Selection of therapy

Depending on the methylation state determined by any of the herein described or referred to methods, a suitable treatment regimen for a patient suffering from triple-negative breast cancer (TNBC) may be determined. While anthracycline-based neoadjuvant chemotherapy is recommended in the absence PITX2 hypomethylation, an alternative, non- anthracycline-based therapy should be selected for TNBC patients showing said hypomethylation. In a preferred embodiment, anthracycline-based neoadjuvant chemotherapy is selected for patients exhibiting a percent methylation ratio (PMR) value of >1 PMR and, preferably, for patients exhibiting a percent methylation ratio (PMR) value of >2 PMR.

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

EXAMPLE 1

A retrospective analysis of 120 tumor specimens from triple-negative breast cancer patients treated in the neo-adjuvant setting with anthracycline-based therapy was conducted and the optimal cut-off value of the methylation state of PITX2 was determined to predict pCR in TNBC patients.

EXAMPLE 2 In addition, a prospective clinical trial was conducted to support the retrospective analysis results. This study has prospectively determined diagnostic efficacy of the "PITX2- Test" to predict pCR for anthracycline-based combination therapy. The prospective study was outlined as follows: Title

Prospective trial to predict the efficacy of platinum-based neo-adjuvant chemotherapy by BRCAness and PITX2 in triple-negative breast cancer patients (P ⁴ -trial))

Study design

Except for some biopsy-tissue obtained during routinely performed diagnostic biopsy and three blood samples taken, TNBC patients were treated as per standard of care. Standard of care for these patients consists of 24 weeks NAC (4 cycles of EC, followed by 12 weeks of carboplatin), followed by primary surgery. Afterwards, patients with no pCR received further adjuvant treatment with 12 x paclitaxel weekly while patients with pCR were submitted to active surveillance. Primary surgery was considered end of the active study part, while follow- up was conducted for 3 years to determine disease-free survival (DFS) and overall survival (OS). Tissue and blood sampling

Tissue: Pre-treatment core-biopsies were collected, formalin-fixed and paraffin- embedded (FFPE). FFPE tumor tissue samples were provided for evaluation by the PITX2- and MLPA tests. Detailed instructions were supplied to all sites to allow for consistent sampling and processing of the tissues.

Blood: Anticoagulated EDTA -blood samples were taken at three time points to analyze free circulating DNA for ΡΓΓΧ2 methylation state.

Diagnostic PITX2-Test

The methylation state of the promoter region of the PITX2 gene was determined as described by Harbeck et al. 2008; J Clin Oncol; 26:5036-5042. For this, FFPE tissue was sampled and sent to a central laboratory to assess the DNA-methylation state. The central laboratory performed DNA extraction, bisulfite-conversion and quantitative methylation- specific real-time PCR. ΡΓΓΧ2 methylation scores ("PITX2-Test") were calculated and categorized as PITX2-high or PITX2-low according to a pre-defined cut-off. pCR definition and assessment

pCR was defined as absence of residual invasive cancer by histological evaluation (hematoxylin-eosin staining) of the complete resected breast specimen and all sampled regional lymph nodes, following completion of neo-adjuvant systemic therapy (i.e. ypTO/Tis ypNO in the current AJCC staging system). Histological sections of FFPE tumor blocks were evaluated for pathological response by local pathologists and a central pathologist.

Objectives

Primary objective

• Employ PITX2-Test as biomarker test for prediction of pCR in TNBC patients following

NAC EC/carboplatin therapy (pCR rate of PITX2-low is <20% compared to PITX2-high is >50%)

Secondary objectives

· Determine BRCAness in FFPE tumor specimens as a predictive marker for pCR in TNBC patients following EC/carboplatin therapy (pCR rate of BRCAness-positive patients is >20% higher compared to BRCAness-negative patients) • Determine diagnostic efficacy (sensitivity, specificity, accuracy, PPV and NPV) of PITX2- Test score determined in FFPE tumor specimens to predict pCR in patients with TNBC following NAC

• Determine PITX2-Test score in FFPE tumor specimens to predict patient outcome (DFS/OS)

• Determine correlation of clinico-pathological factors (stage, nuclear grade, nodal state, pathological remission grade according to Sinn, age, therapy schedule) with PrfX2-Test score obtained by the PITX2-Test

• Determine correlation of clinico-pathological factors (stage, nuclear grade, nodal state, pathological remission grade according to Sinn, age, therapy schedule) with BRCAness as the predictive test

Exploratory objectives

• Determine correlation of PITX2 quantified in FFPE tumor specimens with PITX2 quantified from circulating DNA in blood.

Patient population

Major inclusion criteria

1. Female

2. >18 years old at time of written informed consent

3. Written informed consent to provide biomarker samples, clinico-pathological data and to comply with 60 months follow-up

4. Patients with histologically confirmed, non-metastatic TNBC (HER2-negative by FISH or IHC staining 0 or 1+, 2+ (however, only if FISH is negative), ER- and PR-negative per local immunohistochemistry assessment, <10% reactive cells)

5. Indication for primary, systemic, neo-adjuvant chemotherapy

Statistical methodology

Sample size calculation

The primary study objective was to determine the diagnostic efficacy by comparing the pCR rate in PrfX2-low patients of <20% to the pCR rate in PrfX2-high patients of >50%. It was further assumed that the prevalence of PITX2-low patients will be approximately 33%. Based on these assumptions using a power of 90% and a significance level of 5% (two-sided), the total sample size was determined to be about 200 patients.

Statistical analyses: Primary objectives: The primary study objective was to determine the diagnostic efficacy by comparing the pCR rate in PITX2-low patients to the pCR rate in PITX2-high patients. This comparison will be performed with the Chi-square test. In addition, logistic regression analysis was applied in order to identify variables which are also associated with the pCR-rate. Computations are performed using the PASSU software.

Secondary objectives: For determination of BRCAness as a predictive marker for pCR in TNBC patients following EC/carboplatin therapy (pCR rate of BRCAness-positive patients is >20% higher compared to BRCAness-negative patients), a sample size of about 200 patients is required using a power of 90% and a significance level of 5% (two-sided). The predictive potential of the PITX2-Test score was tested by Kaplan Meier analyses displaying OS and DFS for PTIX2-high and PTIX2-low patients. Clinico-pathological factors (stage, nuclear grade, nodal state, pathological remission grade according to Sinn, age, therapy schedule) was included as covariates to test whether PITX2 DNA-methylation adds predictive and/or prognostic statistically independent information. For this, multivariable Cox proportional models were applied to calculate hazard ratios and their 95% confidence intervals.

EXAMPLE 3

An assay was established to assess the DNA-methylation status of the PITX2 promoter gene extracted from formalin-fixed and paraffin-embedded (FFPE) breast tumor tissue (herein referred to as "PITX2-Test").

The PITX2-Test predicts response to therapy in triple negative breast cancer patients (TNBC) treated with systemic cancer therapy in the (neo-)adjuvant setting. In short, it comprises the extraction of DNA from formalin fixed, paraffin embedded core biopsy sections (5μιη with an overall surface area of 30-600mm ² , 1-2 sections, QIAamp DSP ^CE DNA FFPE Kit, Qiagen, Hilden), concentration determination by OD measurement (QiaXpert, Qiagen Hilden), bisulfite conversion (Epitect Fast Bisulfite Kit, Qiagen, Hilden, optional with semi- automated clean up step on the QIAcube, Qiagen, Hilden) and semi-quantitative analysis of the DNA-Methylation status in the promoter region of the PITX2 gene by quantitative real-time PCR with dual labelled Taqman probes specific for the methylated and unmethylated status of 3 CpGs in the ΡΓΓΧ2 promoter gene on the Rotorgene Q 5-plex HRM (Qiagen, Hilden) platform using the Rotorgene Q software for data analysis. An overview of the workflow of the PITX2- Test is given in Figure 1. Specifically, this test was performed as follows:

DNA isolation from formalin-fixed paraffin-embedded (FFPE) breast cancer tissue 1-2 x 5 μηι sections with an overall surface area of 30-600mm2 were cut from FFPE- tumor tissue blocks according to manufacturer instructions (QIAamp DSP ^CE DNA FFPE Kit). Optionally, a macro-dissection of the paraffin-embedded tumor samples has been performed for enrichment of tumor cell content in the extraction sample. FFPE-material was transferred to a nuclease-free 1.5 ml reaction tube and deparaffinization using xylene and ethanol was performed. Subsequently, a proteinase K digestion step was performed overnight, formaline- crosslinks were de-modified by a de-crosslinking step at 90°C for 1 hour and DNA was purified according to manufacturer's instructions. OD determination of extracted DNA

DNA concentration was determined and DNA quality was controlled by spectrophotometric analysis of OD 260/280 and OD 230/260 ratios using a QIAxpert UV/VIS spectrophotometer. Bisulfite conversion

For quantitative methylation specific analysis bisulfite conversion of extracted DNA was performed using the Epitect Fast Bisulfite Kit according to manufacturer's instructions. Specifically, 120-1000 ng (400 ng recommended input amount) DNA solution was added to a 200 μΐ PCR-reaction tube (e.g. Eppendorf PCR-Tube) and adjusted to a final volume of 40 μΐ with nuclease-free water.

85 μΐ bisulfite solution immediately followed by 15 μΐ DNA-Protect-Buffer (green color) were added, the cap was immediately closed and subsequently vortexed until a homogeneously blue coloured reaction mix was obtained. Bisulfite conversion of the DNA was mediated by the following thermal program in a standard Thermocycler (e.g. Eppendorf PCR Cycler):

• 5 min, 95 °C (1. Denaturing step)

• 10 min, 60 °C (Incubation)

• 5 min, 95 °C (2. Denaturing step)

• 10 min, 60 °C (Incubation)

· infinite, 20 °C (storage)

Sample clean up followed the manual instructions or was performed semi- automated on the QIAcube platform.

Quantitative methylation-specific PCR (qMS-PCR, Methylight) For the PITX2 marker a Methylight™ reaction system on the basis of fluorescent labelled hydrolysis probes was established (for details it is referred to European Patent EP 1 561 821). The Methylight™ system contains two differently fluorescent labelled probes (FAM- and HEX fluorescent dyes plus Quencher e.g. with TAMRA or BHQ1), specific for methylated (FAM-label) and unmethylated (HEX-label) status of clinical response predictive CpG-sites in the PITX2 gene. As reference control 2500 copies of plasmids containing the bisulfite- converted fully methylated and fully unmethylated sequence to be amplified by the assay were included as positive controls (Reference 50 with a ratio meth:unmeth 1 : 1 and Reference 10 with a ration meth:unmeth 1 :9) as well as genomic DNA as negative control and no template controls were used. Assessment of each sample was performed in technical duplicates or single reactions in a reaction volume of 20 μΐ, as followed: lx Reaction mix: 10 μΐ Quantinova Probe PCR mastermix (2x, Qiagen, Hilden); 2 μΐ ΙΟχ Primer and probe mastermix containing methylated and unmethylated probe (2μΜ) and both primers (6μΜ); 5 μΐ bisDNA (up to 30-250 ng); 3 μΐ H ₂ 0 ad 20 μΐ end volume. Final reaction concentrations of primers and probes were 600nM and 200nM. 15 μΐ of the mastermix (containing primers and probes, qPCR Mastermix and water) were pipetted in 4-cap-strips for analysis on the Rotorgene qPCR platform (Qiagen, Hilden) and 5μ1 of bisulfite-converted DNA or control samples were added, the strips sealed and qPCR performed according to the following protocol:

A standard qPCR protocol template (qPCR ΡΓΓΧ2 template) with 2 Reporter-Dyes (green channel (FAM) and yellow channel (HEX)) was used with the Rotor Gene Q Software 2.3.1. No internal reference dye was used. Auto-gain compensation on sample Pos 1 was performed before begin of 1 ^st fluorescence signal acquisition in qPCR cycle 1 to optimize gain setting for both reporter dyes. Detection of the respective methylation status was performed in channel green- (methylated Status; 516 nm) and channel yellow- filter modus (unmethylated Status; 555 nm). Thermal PCR program contained a polymerase activation step for 2 min at 95 °C, followed by 40 cycles with 5 sec, 95 °C (denaturing step) and 5 sec, 60 °C (annealing and elongation step). Fluorescence signal readout follows at each end of the cycle.

Data analysis

Cycle threshold values (CT) were determined automatically for each marker separately by the Rotor Gene Q Software2.3.1. (dynamic well compensation, adaptive baseline correction for cycles 2-10, Threshold- setting for channel green: 0.058 and for channel yellow: 0.015) by the course of the fluorescent signal readout during the PCR cycle program and including adaptive baseline correction. Data transformation of CT values in percent methylation ratio (PMR) values is facilitated by a modified 2exp ^A method for a duplex probe system, with internal calibration and standardization according to the following formula:

PMR (sample well): l/(l+2 ^ex P(CT _met h. - CT _un meth.)) .

MV PMR (sample duplicate): (PMR (sample well 1) + PMR (sample well 2))/2

Error (absolute): STDEV MV PMR (sample duplicate). meth. = methylated signal

unmeth. = unmethylated signal

PMR = Percent Methylation Ratio

MV PMR (sample duplicate): Mean value of PMR values calculated for each separate well in the technical assay duplicate of one sample .

CT = Cycle Threshold. PCR cycle number, at which the reporter dye fluorescent signal reaches an assay specific threshold. For further analysis the mean value and STDEV of technical replicates (n=2) was established.

STDEV = Standard deviation of the Mean value PMR for the technical replicates.

Assay performance quality control: The CT value of at least one probe signal of the duplex system had to be <31.5.

EXAMPLE 4

In addition, PMR value variations (heterogeneity) between consecutive tumour tissue sections were determined and reproducibility of PMR values were assessed in 12 independent qPCR runs.

Therefore, five tissue sections of 5μιη thickness each were obtained from an FFPE- tumor block of a breast cancer patient (TEC3). DNA was extracted from these tissue sections, bisulfite-converted and PMR ( ) determined in 12 independent qPCR runs performed in duplicates according to the assay described in Example 3 above (see Figure 1). An overview of this workflow is provided in Figure 2.

Results

The mean values of the 24 PMR analyses for each of the 5 tissue sections are shown in the graph below: Sing le PM R value d istribution

ver all 1 2 Ru ns ( n = 24)

REF50P/10P: Reference Plasmid mixtures (meth: unmeth 1 : 1 and 1 :9)

The mean values and standard deviations of the 12 qPCR runs (design identical to the graph above) as well as the maximum and minimum values (REF50P and REFIOP are references in the assay) are given in Table 6 below:

Table 6

iSample MV all STDEV all CV all Maximum Minimum

IREF50P 62.42 2.91 4.66% 67.13 57.57

IREFIOP 9.61 1.27 13.26% 11.89 7.57

[TEC3.1 77.86 3.54 4.55% 83.98 69.97

TEC3.2 79.37 3.21 4.04% 86.98 74.54

1TEC3.3 76.20 4.22 5.53% 83.12 69.82

1 1 ( 3.4 75.25 4.65 6.18% 84.35 64.95

ITEC3.5 74.51 5.06 6.79% 84.16 62.71

IMV TEC3.1-5 76.64 1.98 2.58%

The experiment was repeated with 7 other breast cancer FFPE tumor blocks. Regarding the tumor tissue heterogeneity of the gene promoter PITX2 DNA-methylation status, comparable results were obtained as shown above.

Conclusion Tissue heterogeneity of the gene promoter ΡΓΓΧ2 DNA-methylation status (PMR %) is very low as assessed in 5 consecutive sections. Reproducibility is very solid with a coefficient of variation (CV) of 2.58%.

EXAMPLE 5

In addition, the gene promoter DNA-methylation status of PITX2 was assessed in full- face breast cancer FFPE-tissue sections with either low or high tumor cell content and compared to macro-dissected tissue enriched for tumor cell content from the same FFPE tumor blocks.

Therefore, macrodissections were performed first by inspection of a corresponding hematoxyline and eosine stained section for tumor area with high tumor content by an experienced pathologist, marking of the corresponding tumor area on an unstained and unprocessed FFPE section by optical overlay with a permanent marker, scratching of associated low tumor containing tissue areas with a scalpel blade and transfer of tumor enriched areas as well as low tumor containing areas in separate Eppendorf tubes (corresponding to samples TECXXXT and TECXXXN) and processed according to the same workflow as in Example 3. Full face sections were prepared from whole sections (TECXXXff) by the same workflow as in Example 3 (see Figure 3). DNA was extracted from these tissue sections, bisulfite-converted and PMR (%) determined according to the assay described in Example 3 above (see Figure 1).

Results

The mean values of the PMR analyses for macro-dissected tissue areas with high (T) or low (N) tumor cell content as compared to full-face FFPE tumor tissue sections (ff) are shown in the graph below:

Conclusion

For macro-dissected breast cancer with high tumor cell content (T) and full-face sections (ff) of the same tumor block similar results were obtained for the PITX2 DNA-methylation status. Macrodissected FFPE tumor sections containing low tumor cell content (N) displayed lower PMR values compared to non-dissected full-face specimens.

EXAMPLE 6

Further, the PITX2 DNA-methylation status was determined in 21 FFPE core biopsies taken from patients afflicted with triple-negative breast cancer (TNBC) treated with neoadjuvant anthracycline -based chemotherapy and correlated to pathological complete remission data (pCR).

Patient selection

Only TNBC patients were included in the study, who were estrogen-receptor (ER) and progesterone -receptor (PR) negative with <1 ER/PR-positive tumor cells and with negative HER2 status (immunoreactive score 0 or 1 if FISH HER2 amplification test was negative).

PITX2-methylation assay and pCR correlation

DNA extraction from FFPE core biopsy sections, bisulfite conversion and semi- quantitative analysis of the DNA-methylation status in the promoter region of the PITX2 gene was performed in accordance with the PITX2-methylation assay described in Example 3 above (see Figure 1). The PITX2 DNA methylation status (PMR ) in TNBC FFPE tumor tissue sections was correlated with pathological complete remission (pCR) according to Sinn et al. (see Table 1).

Experimental outline

Two independent Experiments 1 and 2 were performed and the results correlated with each other. Experiment 1:

Two independent experimental runs were performed and mean values for both experiments were taken. TNBC patients included pCR 2-4 according to Sinn et al. The raw data and correlation results of the two independent experimental runs of Experiment 1 are given in in Table 7 and the graph below: pCR according to Sinn and PITX2 PMR,

HER2 IRS 0+1

Table 7: Raw data of Experiment 1

pCR Sample MV cv according to 1st run both runs both runs Sinn

¹ Tneol5 0.01 0.00

¹ Tneo31 0.13 0.04

¹ Tneol4 0.13 0.04

0-1 Tneo48 0.17 0.09

¹ Tneo42 0.20 0.03

¹ TneolO 0.23 0.21

¹ Tneo43 1.58 1.79

¹ Tneo49 1.91 1.35

4 Tneo04 0.02 0.01

4 Tneo32 0.03 0.01

4 Tneol8 0.04 0.02

4 Tneol2 0.04 0.01

4 Tneo47 0.04 0.01

4 Tneo03 0.06 0.02

2 Tneo28 0.13 0.14

4 Tneol l 0.30 0.23

4 Tneo40 2.67 1.73

3 Tneol9 4.10 0.54

4 Tneo22 6.04 0.78

Results

• PMR (%) mean values of TNBC FFPE tumor tissues samples for patients with no response (Sinn 0-1) were 0.54 %, patients with pCR (Sinn 2-4) had a mean value of 3.24 %, i.e. a 6-fold increase of PMR (%).

• Applying a cut-off value of 2% PMR leaves 8 out of 8 patient tumor samples with no identification of therapy response (Sinn 0-1).

• Applying the same cut-off value of 2% PMR, 4 out of 12 patient tumor samples with pCR (Sinn 2-4) had PMRs above 2%.

• If PITX2 is methylated >2 PMR, 4 out of 4 PITX2-methylated samples were pCR (Sinn 2-4) corresponding to a positive predictive value* of 100%.

• If the PITX2-DNA is methylated <2% PMR, 8 out of 16 tumor samples were defined no-responders, corresponding to a negative -predictive* value of 50%.

*Positive predictive value is referring to the number of correctly predicted responders divided by the total number of patients with a positive biomarker result, whereas negative predictive value is referring to the number of correctly predicted non-responders divided by the total number of patients with a negative biomarker result.

Correlation of experimental results

Further, the results of the two independent qPCR runs (using independent bisulfite conversion batches) from Experiment 1 were correlated with each other as demonstrated in the graph below. A high statistical correlation for the two independent qPCR runs was obtained with a correlation coefficient of R = 0.983, proving that sufficient DNA is extracted from breast cancer FFPE core biopsies to obtain reproducible ΡΓΓΧ2 PMR values.

-1

30 35

PITX2 PMR 2nd run

HER2: Human epidermal growth factor receptor 2

IHC: Immunoreactive score (IRS) 0-3. 0 = no protein expression; 1 = low protein expression; 2 = moderate protein expression; 3 = high protein expression. Score 2 only qualifies for targeted therapy if FISH HER2 gene amplification test is positive.

Experiment 2:

A single experimental run was performed, wherein TNBC patients showing 3 and 4 according to Sinn et al. were included in the analysis. The raw data from Experiment 2 is given in Table 8 and the correlation results of PITX2 DNA-methylation status and pathological complete remission (pCR) are given in Table 9 below:

Table 8: Raw data of Experiment 2

pCR according Sample PMR STDEV

to Sinn 1st run duplicate duplicate

¹ Tneol5 0.01 0.00

¹ Tneo23 0.02 0.00

¹ TneolO 0.08 0.01

¹ Tneo31 0.16 0.01

¹ Tneol4 0.16 0.04

¹ Tneo42 0.22 0.01

0-1 Tneo48 0.23 0.24

¹ Tneo43 0.31 0.29

¹ Tneo49 0.96 0.96

¹ Tneo24 12.95 0.88 4 Tneo32 0.03 0.00

4 Tneo04 0.03 0.00

4 Tneol2 0.03 0.00

4 Tneo47 0.05 0.00

4 Tneol8 0.05 0.01

4 Tneo03 0.08 0.01

4 Tneol l 0.46 0.18

4 Tneo40 1.45 0.01

4 Tneo25 2.15 0.50

3 Tneol9 4.48 1.87

4 Tneo22 6.59 1.11

Table 9: Correlation results of Experiment 2

Results

• Applying a cut-off value of 1% PMR, 9 out of 16 patient tumor samples are with no response (Sinn 0-1), with PMRs < 1%.

• Applying the same cut-off value of 1% PMR, 4 out of 5 patient tumor samples with pCR (Sinn 3-4) showed PMRs > 1%.

• If PITX2-DNA is methylated > 1% PMR, 4 out of 5 ΡΓΓΧ2 DNA-methylated samples were pCR Sinn 3-4, corresponding to a positive predictive value* of 80%.

• If PITX2-DNA is methylated < 1% PMR, 9 out of 16 tumor samples were no- responders, corresponding to a negative predictive* value of 56%.

*Positive predictive value is referring to the number of correctly predicted responders divided by the total number of patients with a positive biomarker result, whereas negative predictive value is referring to the number of correctly predicted non-responders divided by the total number of patients with a negative biomarker result. Conclusions

In the above experiments a cut-off value of 2% PMR was determined for patients whose tumor had responded to neoadjuvant anthracycline -based chemotherapy according to Sinn 2 to 4 (Experiment 1), and a cut-off value of 1% PMR was determined for responses according to Sinn 3 and 4 (Experiment 2).