Clinical Validation of PITX2 DNA Methylation to Predict Outcome in High-Risk Breast Cancer Patients Treated with Anthracycline-Based Chemotherapy

Abstract

Background: Breast cancer patients at high risk for recurrence are treated with anthracycline-based chemotherapy, but not all patients do equally benefit from such a regimen. To further improve therapy decision-making, biomarkers predicting outcome are of high unmet medical need. Methods: The percent DNA methylation ratio (PMR) of the promoter gene coding for the Paired-like homeodomain transcription factor 2 (PITX2) was determined by a validated methylation-specific real-time polymerase chain reaction (PCR) test. The multicenter study was conducted in routinely collected archived formalin-fixed paraffin-embedded (FFPE) tissue from 205 lymph node-positive breast cancer patients treated with adjuvant anthracycline-based chemotherapy. Results: The cut-off for the PITX2 methylation status (PMR = 12) was confirmed in a randomly selected cohort (n = 60) and validated (n = 145) prospectively with disease-free survival (DFS) at the 10-year follow-up. DFS was significantly different between the PMR ≤ 12 versus the PMR > 12 group with a hazard ratio (HR) of 2.74 (p < 0.001) in the validation cohort and also for the patient subgroup treated additionally with endocrine therapy (HR 2.47; p = 0.001). Conclusions: Early-stage lymph node-positive breast cancer patients with low PITX2 methylation do benefit from adjuvant anthracycline-based chemotherapy. Patients with a high PITX2 DNA methylation ratio, approximately 30%, show poor outcome and should thus be considered for alternative chemotherapy regimens.

Current status of development of methylation biomarkers for in vitro diagnostic IVD applications

“…The clinical validation of the test was initially performed in the study that included 205 archival FFPE breast cancer samples of lymph node-positive (LN + ), estrogen receptor-positive (ER + ), HER2-negative patients (HER2 − ), treated with adjuvant anthracycline-based chemotherapy. This study reported that the test allowed to stratify patients undergoing adjuvant anthracyclinebased chemotherapy regarding disease-free survival, with HR 2.74 (95% CI 1.65-3.54; p < 0.001) [90].…”

Epigenetic modulators combination with chemotherapy in breast cancer cells

“…DNA methylation‐mediated gene inactivation was found in various cellular signalling pathways which influence transcription of tumour suppressor genes involved in DNA repair, hormonal signal transduction, cell adhesion, drug degradation and apoptosis 6,44 . Moreover, it has been shown, that epigenetic markers can be of clinical relevance in breast cancer 45‐50 …”

Establishment of a RESEARCH USE ONLY Condensed-Efficient-Fast (CEF) PITX2 workflow for analysis of PITX2 DNA methylation in small tumor tissue samples&nbsp;

“…If the PMR obtained is equal to or lower than 12, the patient is likely to benefit from anthracycline-based chemotherapy. In contrast, if the PMR obtained is higher than 12, an alternative treatment may be proposed, because the patient has a lower probability of responding to anthracycline-based chemotherapy 20 .…”

“…DNA methylation patterns such as methylation status of the pituitary homeobox gene 2 (PITX2) play a key role in several cancer indications [6][7][8][9][10][11][12] including breast cancer [13][14][15][16][17] and published data of a Research Use Only (RUO) PITX2 DNA methylation assay using fresh-frozen tissue showed that PITX2 DNA methylation can be a predictive marker for response to adjuvant anthracycline-based chemotherapy in breast cancer 18 . The CE-marked therascreen ® PITX2 RGQ PCR Kit by QIAGEN was entering the market in 2018 as clinically validated biomarker test for breast cancer [19][20][21] .…”

“…The clinical validity of the pre-defined PMR cut-off of 12 to predict patient outcome to anthracyclinebased chemotherapy with or without endocrine therapy was shown by using 145 patient samples with 10-year follow-up data and disease-free survival as the primary end point. PITX2 hypermethylation (PMR > 12) indicates a poor outcome to ANT-based chemotherapy whereas PITX2 hypomethylation (PMR ≤ 12) indicates a good outcome to ANT-based chemotherapy 20 . The CE-marked therascreen ® PITX2 RGQ PCR assay is able to guide the clinician to select the appropriate chemotherapy regimen for high-risk BC patient group.…”