Background: Biomarkers predicting response to bevacizumab containing therapy in metastatic breast cancer (MBC) are of urgent need. In a retrospective single-institution analysis we have previously shown that a 3-gene methylation signature (MLH1,POLKand TMBIM6) could discriminate between responders and non-responders to a bevacizumab-based therapy in two independent cohorts of patients with MBC with an AUC of 0.94 and 0.86, respectively (Gampenrieder SP et al. Theranostics. 2018. 8(8):2278-2288). Here, we present the validation of these findings within the prospective phase III trial TANIA (Vrdoljak E et al. Ann Oncol. 2016. 27(11):2046-52) randomizing 494 patients with HER2-negative MBC to chemotherapy plus bevacizumab or chemotherapy alone for two consecutive treatment lines (second- and third-line). All patients had already received bevacizumab-containing therapy in the first-line setting.

Patients and methods: DNA isolated from archival FFPE tumor samples was available from 200 patients consenting to optional translational research within the TANIA trial. Out of these, 176 samples were collected prior to first-line bevacizumab therapy and were analyzed retrospectively. Sufficient DNA for methylation analysis was available from 124 patients: 64 treated with chemotherapy plus bevacizumab and 60 treated with chemotherapy alone. All samples were isolated from the primary tumor. Quantitative methylation analysis was performed by pyrosequencing on the PyroMark Q24 Advanced System (Qiagen). PFS and OS analyses were performed in both study arms comparing “predicted responders” (PRED_R) versus “predicted non-responders” (PRED_NR) based either on median dichotomization or according to the cutoffs for individual CpG and the combined 3-CpG methylation logistic regression model.

Results:Out of the 124 evaluable patients, 32 (25.8%) were classified as PRED_R and 92 as RED_NR by the 3-gene methylation signature. PRED_R did not have a significantly different second-line PFS (HR 0.95, 95%CI 0.57-1.57; P = 0.84) or OS (HR 0.91, 95%CI 0.51-1.60; P = 0.73) when treated in the bevacizumab-containing study arm compared to PRED_NR. In addition, PRED_R did not show a longer PFS when treated with bevacizumab compared to PRED_R treated with chemotherapy alone (HR 0.95, 95%CI 0.59-1.54; P = 0.83). Furthermore, there was no difference in third-line PFS and the combination of second- and third-line PFS between PRED_R and PRED-NR in the bevacizumab arm. In the control arm, PRED_NR showed a statistically significant shorter PFS compared to PRED_R (HR 0.50, 95%CI 0.22-0.77; P = 0.006), but not OS (HR 0.95, 95%CI 0.51-1.77; P = 0.86).

Conclusion: Our 3-gene methylation signature was not confirmed as predictive biomarker for bevacizumab efficacy in metastatic breast cancer.

(This research project was partially supported by ROCHE Austria GmbH)

Citation Format: Gampenrieder SP, Angela R, Rinnerthaler G, Hackl H, Steiner M, Pulverer W, Weinhaeusel A, Klinglmayr E, Karl T, Ilic S, Hufnagl C, Hauser-Kronberger C, Egle A, Greil R. A 3-gene DNA methylation signature fails to predict response to bevacizumab in metastatic breast cancer patients treated within the TANIA phase III trial [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-10-07.