Abstract P3-06-21: Next generation sequencing to find predictors for chemotherapy response in triple negative breast cancer
Authors: Lips Esther H , Michaut Magali , Hoogstraat Marlous L , Mulder Lennart , Besselink Nicolle , Koudijs Marco J , Voest Emile E , Bernards Rene , Nederlof Petra M , Wesseling Jelle , Rodenhuis Sjoerd , Wessels Lodewyk FA 
Published: May 2015

Abstract
Background: Neoadjuvant chemotherapy is the standard of care for locally advanced triple negative breast cancer (TNBC). A complete remission after chemotherapy is associated with a good prognosis. However, patients with a poor response often relapse and die of metastatic disease. Biomarkers are urgently needed to predict which patients will respond to standard chemotherapy. In the current study we deep-sequenced responding and non-responding tumors to find response prediction markers. In addition, the encountered mutations might provide clues for targeted treatment options.
Methods: Next generation sequencing (NGS) was performed for 2,000 genes involved in oncogenesis with an average coverage of 150 reads. DNA from 56 pre-treatment TNBC-biopsies was sequenced, as was non-tumor DNA from each patient for comparison. Biopsies were obtained from patients scheduled to receive neoadjuvant chemotherapy with doxorubicin/cyclophosphamide. The median follow up was 2.5 years. The presence of a BRCA1 germline mutation or BRCA1 promoter methylation was also known. We assessed three variables. First, we compared samples with a pathological complete remission (pCR) with those that did not achieve a pCR, to find mutations predicting chemotherapy response. Second, we performed the same comparison with relapse free survival as outcome measure. Third, we compared tumors with BRCA mutations or BRCA1 promoter methylation with tumors with a functional copy of the BRCA genes. The goal of this analysis was to detect mutations associated with BRCAness.
Results: The mutations observed were diverse and few recurrent mutations were detected. Most mutations were in TP53, TTN, and PIK3CA (57%, 22%, 9%). The mutation rates were similar between responders and non-responders (average mutation rate of 13 versus 16 mutations per tumor, p=0.27). The analysis of individual genes did not reveal significant differences between responders and non-responders. NOTCH4 mutations showed an association with relapse free survival, with three out of nine relapsing patients bearing a mutation and no mutations occurring in the relapse-free patients (p=0.008). Interestingly, PIK3CA mutations were only observed in patients with a functional BRCA gene (5/26 (19%) versus 0/30, p=0.017). Tumors with BRCA impairment may develop via different routes than tumors with functioning BRCA genes, and the PI3K pathway may play a role in the latter. This finding has also been observed in an independent dataset (Severson et al, 2014, submitted).
Conclusions: Although few recurrent mutations were found, two interesting leads for follow up studies were identified. First, three out of nine tumors with a relapse had a NOTCH4 mutation while NOTCH4 mutations were not occurring in the relapse free group. Second, PIK3CA mutations were associated with BRCA proficient tumors. After validation in larger series, triple negative tumors with PI3K or NOTCH4 mutations can be candidates for agents targeting these oncogenic pathways. In this manner, a more individualized treatment of triple negative breast cancer might become possible in the near future.
Citation Format: Esther H Lips, Magali Michaut, Marlous L Hoogstraat, Lennart Mulder, Nicolle Besselink, Marco J Koudijs, Emile E Voest, Rene Bernards, Petra M Nederlof, Jelle Wesseling, Sjoerd Rodenhuis, Lodewyk FA Wessels. Next generation sequencing to find predictors for chemotherapy response in triple negative breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-06-21.