Abstract P2-10-04: Targeted sequencing in early breast cancer: Identification of novel candidate mutations predictive of anthracycline benefit
Authors: Spears M , Kalatskaya I , Trinh QM , Liao L , Chong TM , Crozier C , Dion D , Heisler L , Timms L , Stein LD , Pritchard KI , Levine MN , Shepherd L , Twelves CJ , Bartlett JMS 
Published: February 2018

Background The use of chemotherapies such as anthracyclines and taxanes have improved overall and disease free survival in breast cancer. For all patients, anthracyclines can have significant toxicities including cardiotoxicity and leukemia. It is therefore essential to select the subset of patients who will receive the optimal overall benefit from anthracycline therapy and to identify molecular pathways driving resistance. To fully understand the impact of mutations in the context of current breast cancer therapy, requires a comprehensive mapping of key molecular events in the context of treatment. We sequenced 101 genes, that were prioritized based on not only gene frequency, but also taking into account the importance of amino acid substitution, type of mutation and network connectivity, in 692 primary tumours to both identify driver genes and pathway cassettes and to understand their clinical significance in response to anthracycline treatment.

Methods We performed targeted sequencing in patients from the BR9601 (n=374) and CCTG MA.5 (n=703) clinical trials. The BR9601 and MA.5 clinical trials examined the effectiveness of combination chemotherapy consisting of CMF (cyclophosphamide, methotrexate and 5-fluorouracil) with or without epirubicin. DNA was extracted, samples were sequenced using AmpliSeq Technology adapted to Illumina and somatic mutations were called using a novel mutation calling pipeline (ISOWN). A priori analyses were performed using distant recurrence free survival (DRFS) as the primary endpoint.

Results: In 692 successfully analysed samples 509 (73.6%) samples exhibited at least one single nucleotide mutation (range 0-54). 94/101 genes were mutated in at least one patient. Only variants in PIK3CA, TP53, CDH1, TLE6, MLL3 and USH2A were detected in 5% or more of samples. TSC22D1, RB1 and ZNF565 were associated with increased risk of distant relapse in multivariate analyses corrected for clinic-pathological variables. No single genes were predictive of anthracycline treatment compared to CMF in multivariate analyses corrected for clinic-pathological variables. Signaling cassettes/modules were designed based on the pathway database, Reactome. Within the signaling cassettes one module was predictive of anthracycline failure. Patients with one or more mutations in this module had an increased risk of distant relapse (HR 0.52, 95% CI 0.29-0.95, p=0.034) when treated with an anthracycline containing chemotherapy regimen compared to CMF (HR 1.34 95% CI 1.05-1.72, p=0.019).

Conclusions: We successfully performed a signaling pathway-based targeted sequencing analysis within predefined signaling modules. We identified a single signaling cassette linked to anthracycline resistance in early breast cancer. However, further work to validate this study in a separate clinical trial is warranted.

Citation Format: Spears M, Kalatskaya I, Trinh QM, Liao L, Chong TM, Crozier C, Dion D, Heisler L, Timms L, Stein LD, Pritchard KI, Levine MN, Shepherd L, Twelves CJ, Bartlett JMS. Targeted sequencing in early breast cancer: Identification of novel candidate mutations predictive of anthracycline benefit [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-10-04.