Abstract P3-08-65: PITX2 DNA methylation: A prognostic/predictive biomarker for anthracycline-based chemotherapy
Authors: Kiechle Marion , Schricker Gabriele , Napieralski Rudolf , Aubele Michaela , Auer Gert , Ulm Kurt , Perkins Jonathan , Paepke Stefan , Hamann Moritz , Wilhelm Olaf G. 
Published: March 2020

Abstract
Background: Published data on the basis of a research-use-only PITX2 methylation assay using fresh-frozen tissue showed that the PITX2 DNA methylation may be a predictive marker for response to (neo) adjuvant anthracycline-based chemotherapy in breast cancer patients, including high-risk lymph node positive, estrogen receptor-positive, HER2-negative breast cancer and triple-negative breast cancer (TNBC). A retrospective study showed that high-risk ER-positive patients with a high PITX2 methylation ratio (PMR&gt;12) had a poor outcome after anthracycline-based chemotherapy [HR 2,28; 95%-Cl 1,49 - 3,49; p&lt;0.001, median DFS 71 months] compared to patients with low PITX2 methylation ratios [PMR≤12, median DFS 105 months]. Nevertheless, the question regarding the prognostic versus predictive value of PITX2 methylation for anthracycline therapy remained unanswered so far. Within an exploratory study using archived tissue specimen, this question was addressed by comparing the impact of PITX2 methylation on outcome in an untreated versus treated TNBC population. Methods: Patient samples and clinical data from 144 TNBC patients of 2 cohorts were analyzed: 1) 66 untreated patients from the Karolinska Institute in Sweden (primary diagnosis 1971-1976) and 2) 78 patients treated with anthracycline-based chemotherapy from the Comprehensive Cancer Center TUM in Germany (primary diagnosis 1996 to 2014). The main eligibility criteria were as follows: ER-negative, PR-negative, HER2-negative breast cancer (TNBC); no endocrine therapy; untreated collective: no further therapy besides surgery and radiation therapy; treated collective: surgery followed by anthracycline based chemotherapy. Samples were assessed for PITX2 methylation using a CE marked PITX2 RGQ PCR Test. The study was designed to determine the PITX2 PMR cut-off value in the untreated population (n=66, prognostic value) and to subsequently determine if this cut-off provides statistical significance in the treated population (n=78, prognostic & predictive value) as well. If no statistically significant cut-off value can be determined in the untreated population, cut-off determination was performed in the treated population and the cut-off was applied in the untreated population. The primary clinical endpoint for all cut-offs was 10-years DFS, secondary clinical endpoints were DFS censored at 5-years and OS censored at 5- and 10-years. Results: The untreated TNBC study population demonstrated a higher event rate at 10-years DFS/OS compared to the anthracycline-treated population [36 DFS/28 OS events vs 28 DFS/14 OS events]. Anthracycline treatment improved significantly overall survival [HR=0.42; p=0.008]. Risk recurrence increased continuously over 5 year and 10 years DFS/OS with increasing PITX2 DNA methylation in the anthracycline-treated population but not in the untreated patients over 5 years DFS/OS and only marginally over 10 years DFS/OS. PITX2 methylation identifies at the cut-off of PMR 2 a patient population with poor vs good overall survival with statistical significance [HR=3.96, p=0.011] in the treated patient cohort but did not identify patients with poor vs good outcome in the untreated patient cohort [HR=1.55, p=0.259].
Summary: In this hypothesis generating study DNA-methylation of PITX2 identifies with high statistical significance anthracycline-treated patients with poor vs good survival. Recurrence risk increases with increasing PITX2 DNA-methylation. PITX2 DNA-methylation shows no prognostic, but significant predictive value for anthracycline treatment in TNBC patients in this study. To confirm conclusively the predictive value of PITX2 DNA-methylation, the results warrant a confirmatory study with tumor specimens from a prospective trial of anthracycline-treated patients vs. anthracycline-free treated patients.
Citation Format: Marion Kiechle, Gabriele Schricker, Rudolf Napieralski, Michaela Aubele, Gert Auer, Kurt Ulm, Jonathan Perkins, Stefan Paepke, Moritz Hamann, Olaf G. Wilhelm. PITX2 DNA methylation: A prognostic/predictive biomarker for anthracycline-based chemotherapy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-08-65.