Introduction
Approximately 15% of breast cancers are classified as triple-negative breast cancer (TNBC), lacking expression of the estrogen receptor (ER) and the progesterone receptor (PR), and which is characterized by absence or low expression or no amplification on the gene level, of the human epidermal growth factor receptor 2 (HER2) (1,2). Thus, unfortunately, breast cancer patients suffering from TNBC do not benefit from cancer therapeutics targeting these receptors. Gene expression profiling revealed that TNBC shows overlapping characteristics with the basal-like breast cancer type, including various subtypes. Patients afflicted with this malignancy are known for early disease onset with high aggressiveness, poor clinical outcome and high nuclear grade (3–9). Often, TNBCs show BRCAness, characterized by clinicopathological features normally found with BRCA1-mutated tumors (10–14).
One has to admit that little progress has been made in the last decade regarding novel, suitable druggable targets and targeted drugs for TNBC patients (15), subsequently, chemotherapy remains the essential therapeutic tool in TNBC, both in the adjuvant and the neoadjuvant setting (3,13,15,16). Clinical data do suggest that the addition of platinum to anthracycline- and taxane-based chemotherapy regimens is an additional option in the treatment of both early-stage and advanced TNBC (4,17–25).
Breast cancer patients who are undergoing chemotherapy have an increased risk of developing cardiovascular complications, and anthracyclines (e.g. doxorubicin, daunorubicin, idarubicin and epirubicin), are some of the most frequently used agents. The administration of non-anthracycline agents, that also may cause cardiotoxicity, frequently results in synergistic toxicity when anthracyclines are given concurrently (26,27). Therefore, the identification of additional molecular biomarkers to predict response and/or potential cytotoxic side-effects to specific chemotherapeutics is still of high unmet medical need to further improve strategies to treat TNBC patients (3,7,19).
Epigenetic DNA-methylation plays an important role in controlling gene activity and nucleus architecture (28–31). DNA-methylation markers were shown to have prognostic and/or predictive value, thus, being considered valuable, additive tools for physicians to choose the appropriate therapy regimen for the cancer patient (32–34). The PITX2 gene (paired-like homeodomain transcription factor 2), a member of the paired-like homeodomain transcription factor family, which in the healthy organism is known to play an important role during embryogenesis and organogenesis, might serve as a prime example (35,36).
Recent data strongly suggest that methylation of certain CpG island promoters of the PITX2 gene may play an essential role in the very early stages of breast cancer pathogenesis and its methylation status being associated with response to adjuvant chemotherapy of a breast cancer patient (37–42). Thus, unexpectedly, in breast cancer patients, PITX2 emerged to be a key molecule in breast cancer pathophysiology, but not only associated with the course of the disease but also with response to adjuvant systemic endocrine or anthracycline-based chemotherapy (39–41).
The aim of this retrospective pilot study was to demonstrate that PITX2 DNA-methylation is a potential predictive breast cancer biomarker in the triple-negative breast cancer subgroup (TNBC), treated with adjuvant anthracycline-based chemotherapy regimens. Our present results, for the first time indicate that quantitative determination of the PITX2 DNA-methylation status in primary TNBC breast cancer tissues will allow selection of those TNBC patients who most probably will benefit from anthracycline-based chemotherapy or not. Consequently, TNBC patients who possibly will not respond should be spared the potentially toxic burden of such chemotherapy, but could be allocated to alternative treatment modalities (23,43–45).
Materials and methods
Materials
Unless otherwise stated, all the reagents applied in the present study were obtained from Qiagen (Hilden, Germany), Sigma-Aldrich (Taufkirchen, Germany), or Merck KGaA (Darmstadt, Germany).
Patients
Inclusion criteria for the retrospective study were breast cancer patients with histologically confirmed invasive triple-negative breast cancer (n=56), no signs of distant metastasis at time of diagnosis, availability of frozen tumor tissue specimens for DNA extraction, follow-up data and signed informed patient consent. All patients were treated between 1991 and 2006 at the Department of Obstetrics and Gynecology, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany. Study approval was obtained from the Ethics Committee of the Medical Faculty of the Technical University of Munich. Clinical and histomorphological patient-related data are summarized in Table I. Histopathologic tumor grade was determined according to the Nottingham modification of the Scarff-Bloom-Richardson grading scheme. Absence of estrogen receptor (ER) and/or progesterone receptor (PR) protein expression was confirmed either by the dextran-coated charcoal method, by enzyme immunoassay, or immunohistochemistry, whereby positive staining of either receptor denoted receptor positivity. Presence of human epidermal growth factor receptor 2 (HER2) expression/amplification was demonstrated by immunohistochemistry using a semi-quantitative scoring system or by fluorescent in situ hybridization analysis (40). Twenty patients were younger than 50 years at the time of diagnosis. Median time of follow-up was 74 months (range, 8–179). Fifteen patients were treated with breast conserving therapy, 41 patients with mastectomy and 51 patients received radiotherapy. TNBC patients were allocated to various types of adjuvant anthracycline-based polychemotherapy regimens including FEC (n=19), EC (n=13); EC+CMF (n=3), anthracycline plus taxane (n=20), or idarubicin-based therapeutics (n=1).
Immunohistochemistry
2–4 µm thick sections were cut from FFPE blocks of TNBC breast cancer patients and mounted on microscope slides (R. Langenbrinck GmbH, Emmendingen, Germany). Sections were deparaffinized by xylene (2 × 10 min) and then rehydrated in a series of graded ethanol, followed by washing in TBS, pH 7.6, 5 min, as previously described (46). All steps were performed at room temperature. Antigen retrieval was accomplished by exposing the slides to 4 min of pressure cooking (WMF, Geislingen an der Steige, Germany). Since a peroxidase-dependent antibody-binding system was applied (Dako EnVision + Dual Link System; Dako Deutschland GmbH, Hamburg, Germany), endogenous peroxidase activities were blocked by use of the peroxidase/alkaline phosphatase blocking reagent (Dako Deutschland GmbH). For immunohistochemical staining of PITX2 protein expressed in tumor tissues, the polyclonal rabbit antibody PITX2-484 to the human PITX2 molecule was added (1:50) in antibody diluent (Dako Deutschland GmbH). Sections were stored overnight at 4°C to allow solid interaction of antibody PITX2-484 with its target molecule PITX2 expressed in the tumor sections. After washing with TBS, the secondary horseradish peroxidase-conjugated polymer antibody to the Fc-region of rabbit immunoglobulin G was added according to the manufacturer's recommendation (30 min, room temperature). After another washing step with TBS, the peroxidase detection solution (3,3′-diaminobenzidine; Dako Deutschland GmbH) was added (8 min, room temperature). The sections were then washed with TBS, nuclei of the tissue sections counterstained, and then sealed with Pertex embedding medium as previously described (46). Stained sections were scanned and digitized using the NanoZoomer Digital Pathology RS (NDP) scanner (Hamamatsu Photonics Deutschland GmbH, Herrsching am Ammersee, Germany), utilizing the NDP scan 2.2 software. A selection of TNBC primary tumors ± hematoxylin counterstain plus a prostate cancer specimen for comparison are shown in Fig. 1.
Generation of PITX2-directed antibody PITX2-484
Polyclonal antibody PITX2-484 was produced by Pineda Antibody Service (Berlin, Germany), after two PITX2-peptides were selected common for all known three PITX2 variants, by generation in rabbits after combined immunization with the PITX2-derived peptides Y (aa 154-170: NGFGPQFNGLMQPYDDM) and Z (aa 243-260: NNLNNLSSPSLNSAVPTP). Peptides Y and Z relate to PITX2-B, which is referred to by UniProt as canonical sequence of PITX2. These peptides were synthesized plus an additional N-terminal Cys-residue which was used for S-S-based linkage to the carrier protein KLH. Sera of the immunized rabbits were purified by affinity chromatography on vIDR-pHis (aa 153-261 of PITX2B).
DNA extraction
Immediately after excision of the primary breast tumor tissue at the Department of Obstetrics and Gynecology, Klinikum rechts der Isar, Technical University of Munich, Germany, the removed tissues were placed on ice and transported to the university's nearby pathologist to examine the removed tissue for the presence of malignant cells. Approved malignant tissue was snap-frozen and stored in the liquid nitrogen tumor bank of the Klinikum rechts der Isar of the Technical University of Munich until further use. On demand, tissue was removed from the liquid nitrogen storage container and the still-frozen tumor tissue pulverized by use of the Mikro-Dismembrator S (Sartorius Stedim Biotech, Göttingen, Germany), the powder was then suspended in Tris-buffered saline (0.02 M Tris-HCl/0.125 M NaCl, pH 8.5) containing 0.1% of the non-ionic detergent Triton X-100, to be centrifuged at 100,000 × g (60 min, 4°C) (47). The supernatant and the cellular debris, containing the DNA-containing nuclei, were aliquoted separately and stored in liquid nitrogen until further use. Aliquots of cellular debris representing ~30 mg of breast cancer tissue were used for DNA extraction by following the QIAamp DNA Mini and Blood Mini Handbook protocol, employing the semi-automated QIAcube system (Qiagen, Hilden, Germany). Extracted genomic DNA was aliquoted and stored at −80°C until further use. DNA concentration was determined by use of the NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). Breast cancer cell lines MCF-7 and MDA-MB-231 (CLS Cell Lines Service GmbH, Eppelheim, Germany), genomic unconverted DNA, and water as no template control, as well as fully methylated bisulfite-converted DNA (EpiTect PCR Control DNA Set; Qiagen) served as controls for PITX2 DNA-methylation status.
PITX2-probe and primer system specifications (according to patent EP1561821)
Entrez gene ID: 5308. Amplicon length 144. Reference sequence (RefSeq) ID: NT_016354.18. Detected CpG in RefSeq: 3 CpG in 36106573 - 36106600 (39).
DNA-methylation-specific quantitative real-time PCR
For PITX2 DNA-methylation status determination, for each specimen, 310 ng of DNA was applied in the subsequent bisulfite conversion step which was performed following the EpiTect Bisulfite Handbook protocol (Qiagen) employing an ABI PCR Cycler (Applied Biosystems, Darmstadt, Germany). Program details: 1st: 5 min at 99°C. 2nd: 25 min at 60°C. 3rd: 5 min at 99°C. 4th: 85 min at 60°C. 5th: 5 min at 99°C. 6th: 175 min at 60°C. Clean-up of the bisulfite-converted DNA was carried out following the EpiTect Bisulfite kit protocol. Primers and probes for the methylated and unmethylated PITX2 DNA-methylation status were applied in a duplex probe system combined in a 10× ready to use primer and probe Master Mix; qPCR was performed according to the provider protocol (EpiTect MethyLight Assay Hs_PITX2; Qiagen) using the ABI 7000 Taqman system (Applied Biosystems). Run details: 1st: 15 min at 95°C. 2nd: 48 cycles comprising of each 15 sec at 95°C and of 1 min at 60°C, including 2 µl primer and probe Master Mix, 2 µl bisulfite converted DNA (7.5 ng) and 10 µl QuantiTect 2× QPCR Master Mix (Qiagen), supplemented with water to a final volume of 20 µl. Each specimen was assessed in triplicates. A total of 5 ng fully methylated bisulfite-converted human control DNA (Qiagen) and 7.75 ng MCF-7 bisulfite-converted DNA served as positive controls, RNAse-free water as the negative control.
Statistics
Reporting of this study was carried out respecting the REMARK criteria (48,49). For calculation of the PITX2 DNA-methylation status, the modified ΔΔCT-method as described by Harbeck et al (40) was employed. Mean values of triplicates were calculated for the methylated and the unmethylated PITX2 DNA-methylation status, respectively, which were then used for calculation of the individual PITX2 DNA-methylation scores. CT-values (methylated or unmethylated) obtained with >38 cycles were disregarded. Mean values, standard deviation, and coefficient of variation of the different qPCR runs were calculated. Only values with a coefficient of variation <0.3 were considered for the statistical evaluation. The relationship between PITX2 DNA-methylation score and established clinical factors to the primary endpoints disease-free (DFS) and overall survival (OS) was calculated applying univariate and multivariate Cox proportional hazard models. The date of surgery was considered as the follow-up index date. In order to discriminate between low- and high-risk patients with regards to DFS and OS, optimized cut-off values were calculated with the 'maximum-selected log-rank statistic' using the maxstat.test function as implemented from the program library 'maxstat' of the program 'R' (R Development Core Team 2012) (50,51). Death before incidence of distant recurrence was considered censoring event. Survival curves were calculated according to the Kaplan-Meier method (40). The log-rank test was used for calculating the respective P-values. Cox regression models were employed for univariate risk estimation (hazard ratios, HR) for DFS and OS. Due to the limited numbers of events (disease recurrence, deaths), multivariate analyses were carried out in an exploratory fashion. For this, covariates (tumor size, tumor grading and age) were added stepwise to the variable PITX2 DNA-methylation (high vs. low) and the according hazard ratios and 95% confidence intervals depicted in forest plot diagrams in order to test whether the PITX2 DNA-methylation status adds statistically independent additional information to DFS and OS.
Discussion
During embryonic development, expression of PITX2, a bicoid-like developmental transcription factor, determines the left-right symmetry of the body and tightly controls the correct placement of various internal organs (36,52–54). In breast cancer, the methylation status of the PITX2 gene has been reported to be both a prognostic and a predictive biomarker for response of patients to endocrine therapy or anthracycline-based chemotherapy (39–41). In the present study, a test system was applied which determines the PITX2 DNA-methylation status in FFPE-breast cancer tumor tissues, first described by Harbeck et al (40), resulting from a multicenter trans-European cooperation of European academic and commercial partners supported by the European Union Framework Program FP6. It is a real-time quantitative methylation-specific PCR-based (qMSP) assay; the sample type is bisDNA, i.e. bisulfite-converted gDNA. For this, gDNA is extracted from FFPE breast cancer tumor tissues, then exposed to bisulfite treatment to distinguish between the methylated and unmethylated PITX2 status (39,40).
DNA methylation changes in routinely available FFPE tissues or fresh-frozen tissues not only can serve as biomarkers for the detection of malignant disease and for the assessment of the clinical course of cancer disease but also as specific biomarkers to predict whether a cancer patient will respond to systemic drug therapy or not (39–41,55–61). Tests to assess promoter methylation patterns of some genes (e.g. MGMT, GSTP1, SHOX2, SEPT9, ASTN1 and ZNF671), in malignancies other than breast cancer, have already been transformed into commercially available clinical assays (e.g. by MDxHealth, Epigenomics and Oncgnostics).
Increasing evidence suggests that aberrant PITX2 DNA methylation is not only prominent in breast cancer but is also associated with other malignant diseases, e.g. cancers of the urogenital and gastrointestinal tract, that of the thyroid and of head and neck, and of leukemia (62–71). Yet, the regulatory role that PITX2 DNA methylation plays in these diseases is still not fully explored. Further clinical studies are warranted to determine whether the results available are transferable to larger patient cohorts.
In a recent Clinical Practice Guideline published on behalf of the American Society of Clinical Oncology (ASCO), evidenced-based recommendations were published on the appropriate use of breast tumor biomarker assay results to guide decisions on adjuvant systemic therapy for women with early-stage invasive breast cancer with known ER/PR/HER2 status (72). One focal point of the guideline was to provide recommendations to physicians and patients on the potential ability of predictive biomarkers to indicate benefit of a certain chemotherapeutic for a respective class of ER/PR/HER2-positive and negative breast cancer patients. In summary, in view of the guideline panel, sufficient clinical evidence was provided in the scientific literature to recommend the use of multiplex biomarker assays to manage treatment of ER/PR-positive breast cancer patients. Such recommendation could not be given for the group of TNBC patients, which means that there is still a fundamental lack to guide decision on adjuvant systemic therapy for this group of patients (72).
This assessment supports the idea that the PITX2 assay should be considered as a novel, valuable additive clinically useful test, different from the established multiplex gene signatures as depicted by Harris et al (72). The PITX2 assay may assist the physician to guide decisions on adjuvant systemic therapy, both for the ER/PR-positive and negative (TNBC) breast cancer subsets (39–42).
The present clinical investigation centered on the question whether breast cancer patients afflicted with the aggressive TNBC subtype would benefit from anthracycline-containing adjuvant chemotherapy by grouping the respective TNBC patients to their low or high PITX2 gene methylation status. Different from that, previously published investigations described the relation of PITX2 DNA-methylation status with estrogen/progesterone receptor-positive breast cancer disease, not including the TNBC phenotype (39–42).
PITX2 DNA-methylation profiles in TNBC patients' tumor tissues apply to the methylation status of particular CpG sites in this gene; such changes can be used clinically as a prognostic and/or predictive marker for this kind of malignant disease (39–42,55). Thus, the aim of the present study was to explore a possible statistical correlation between methylation in the promoter region of the PITX2 gene and clinical outcome of TNBC patients treated with anthracycline-based chemotherapy.
In addition, we also analyzed PITX2 protein expression in a limited number of randomly selected TNBC primary breast cancer tumor tissue specimens by immunohistochemistry, by employing the proprietary antibody PITX2-484, but did not observe any lack of PITX2-expression in the breast cancer tumor tissue specimens looked at, demonstrating that with respect to PITX2 protein expression, partial methylation of CpG islands is not associated with complete gene silencing.
In our TNBC cases, the antibody applied (PITX2-484 to two PITX2-peptides located on canonical PITX2-variant PITX2-B) reacted predominantly with cytoplasmic PITX2 protein but occasionally nuclear and perinuclear staining was observed as well. Since PITX2 is a transcription factor, nuclear localization of PITX2 was expected. Yet, in the immunohistochemical assessment of PITX2 in breast cancer tissues presented by Wan Abdul Rahman et al (73), the authors claimed that in invasive ductal breast cancer, including TNBC, PITX2 protein expression is preferentially associated with the cytoplasm of the tumor cells. Other but single clinically relevant studies assessed cytoplasmic PITX2 protein expression in malignant diseases as well, e.g. in odontogenic tumors, thyroid and esophageal cancer (69,74,75). Another study was related to PITX2-protein expression in ovarian cancer. The authors observed both cytoplasmic and nuclear localization of PITX2, depending on the malignant stage (62).
TNBC is characterized by large-scale transcriptional, mutational, and copy number heterogeneity; thus, in the past, most targeted chemotherapeutic agents have demonstrated low overall activity in unselected TNBC patients since various biological TNBC subgroups are overlapping and so far cannot be combined into a 'one-fits-all' model of TNBC biology (61,76–78). Oppositely, this molecular heterogeneity has allowed to categorize TNBC for different novel targeted therapeutic interventions, having led to ongoing innovative clinical strategies for early-stage and advanced TNBC, including immunotherapy and modified chemotherapy (76,79). Besides that, TNBC is typically treated with various combinations of chemotherapy; a sequential anthracycline-taxane combination is the standard of care for TNBC (3). Yet, systemic chemotoxic treatment of TNBC patients needs to be personalized for a specific patient to suit her best, preferentially depending on the molecular characteristics of her disease, since at least ten different molecular TNBC subtypes have been identified using gene copy number and expression analyses (3,77).
Many clinical studies have demonstrated that TNBC is sensitive to anthracycline-containing adjuvant chemotherapy regimens (3,80,81); anthracyclines are considered to be among the most active drugs for the treatment of breast cancer by destabilizing the DNA through intercalation (3,80,81). Otherwise, anthracyclines may cause severe side-effects, including cardiac toxicity, which can lead to heart failure and which therefore may hamper their optimal use in treatment of TNBC (82). The risk of TNBC patients to experience severe side-effects caused by systemic cancer treatment can be higher when other treatments, e.g. taxanes or platinum-based therapeutics are used in combination with an anthracycline (3,83).
There is general consensus that for TNBC systemic adjuvant therapy anthracycline-containing regimens are the standard approach for patients after primary surgery. This is also the opinion of the American Society of Clinical Oncology (ASCO), it recommends chemotherapy treatment for TNBC patients based on the combination of an anthracycline with a taxane but does not currently recommend tailoring therapy for TNBC patients by stratifying treatment by implementing non-validated results for TNBC biomarkers, such as tumor cell surface receptors (EGFR, IGFBP, C-kit and PD-L1) which potentially could serve as novel target molecules to block tumor cell proliferation and dissemination (16). If comorbidities forbid the use of anthracyclines, treatment with taxane-based regimens and cyclophosphamide are recommended as alternative adjuvant treatment; treatments using paclitaxel or CMF (cyclophosphamide, methotrexate and 5-fluorouracil) should be considered as well (45,84–87).
Only scarce data are available in the scientific literature naming biomarkers predictive for response of TNBC patients to anthracycline-based adjuvant chemotherapy. For example, Mori et al (88) speculated on the predictive value of BRCAness as a predictive factor for the effectiveness of anthracycline-based adjuvant chemotherapy for patients with TNBCs. Different from that analysis, Bouchalova et al (89) demonstrated the usefulness of the biomarker BCL2 to predict the level of recurrence-free and overall survival in TNBC patients treated with anthracycline-based adjuvant chemotherapy.
More information is available for non-breast cancer patients. For these patients, especially concerning association of PITX2 DNA-methylation status with other biomarkers, several epigenetic studies were published, demonstrating co-expression of hypo- or hypermethylated biomarkers in conjunction with PITX2 DNA-methylation profiles (37,38,90–92). None of these studies, however, specifically addressed the issue of the potential benefit of adjuvant anthracycline treatment with regard to PITX2 expression or its DNA-methylation status, except the work performed by Hartmann et al (41). Obviously, there is still a strong need to address the question of anthracycline efficacy in TNBC and associated biomarkers to predict sensitivity or resistance to adjuvant anthracycline-based chemotherapy and for new cellular targets for individualized TNBC patients.
On this line, accumulated substantial evidence was presented in the past by a European EU-FP6-framework consortium that among all genes analyzed, PITX2 DNA-methylation analysis performed on routinely available FFPE-tumor tissue specimens holds promise as a novel practical assay for routine clinical use to predict outcome of these patients, treated with endocrine or anthracycline-based adjuvant therapy providing a potential link between PITX2 expression and breast cancer progression (39–42).
Otherwise, these studies on steroid hormone receptor-positive breast cancer were different from the present investigation which exclusively focusses on the TNBC subgroup of breast cancer patients, lacking both ER/PR-steroid hormone receptor and oncoprotein HER2 expression (3). To the best of our knowledge, this is the first clinical study examining whether determination of the methylation status of the promoter region of the PITX2 gene in primary tumor tissues can serve as a biomarker to predict response of TNBC patients to adjuvant anthracycline-based chemotherapy.
Collectively, we show by statistical analyses of TNBC patients treated with anthracycline-based adjuvant chemotherapy that low PITX2 DNA-methylation is associated with poor clinical outcome, demonstrating its statistically independent nature by univariate and multivariate statistics. Notably, the PITX2-TNBC response data indicate a reverse relationship between PITX2 DNA-methylation and response to anthracy-cline-based TNBC when compared to the non-TNBC breast cancer studies (39–42), supporting the notion that TNBC reflects a selected kind of breast cancer disease, different in many phenotypic and genomic aspects from endocrine receptor-positive breast cancer (3). Remarkably, our results also demonstrated that the sequential addition of taxanes to adjuvant systemic anthracycline-based chemotherapy did not alter the predictive value of PITX2.
Since searching for optimised cut-off values could potentially lead to overfitting, it is important to note that this study does not claim that the optimized cut-off value defined in this anthracycline-receiving TNBC subgroup is the optimal one for future application. The pilot character of the present study is reflected by its retrospective design and the non-homogeneous therapy regimens applied but also by a small number of cases and number of events. Therefore, the current results of this first-time observation study should rather serve as a clue towards validation of the data which have to be evaluated in future clinically relevant studies.