Abstract P6-07-03: Genetic heterogeneity revealed by targeted exome sequencing in advanced triple-negative breast cancer
Authors: Pannuti A , Filipovic A , Hicks C , Lefkowitz E , Ptacek T , Miele L , Stebbing J 
Published: May 2016

Abstract
Introduction: Clinical decisions in oncology are increasingly guided by molecular genetics. Next-generation sequencing (NGS) provides a platform for identifying potential therapeutic targets and clinically relevant genetic markers for personalized therapy.
Methodology: We performed sequencing with the SmartGen 421 NGS gene panel, on relapsed triple negative breast cancer (TNBC) specimens (n = 11) by obtaining a biopsy from the most recent site of progressive disease. Custom HaloplexTM reagents were used to capture the regions of interest by hybridization to probes corresponding to target regions. Targets were amplified to enrich DNA libraries. Libraries were sequenced on an Ion Torrent Personal Genome Machine, and sequences compared to the reference genome GRCh37/hg19. The coding regions and +/-5 base pairs of the introns of 421 genes were sequenced. Analytic sensitivity for SNP calls was 94.1% (69.2%-99.7%, at 95% CI), specificity was 100%, for introns and deletions sensitivity was 78.9% (62.2%-89.9%, 95% CI) and specificity was 99.994%. Results: A total of 96 genes contained genetic variants, with each case harbouring 4 to 15 variants. No two cases had identical mutation profiles, indicating significant genetic heterogeneity. We tabulated a) all the genetic variants and b) potentially actionable variants based on predicted effects on protein structure and conservation of mutated amino acids, using COSMIC. Among the identified variants, n = 10/96 have been previously described in COSMIC. The others are previously unreported, de novo mutations of unknown functional significance. Not surprisingly, n = 8/11 tumors contained TP53 mutations, 7 of which previously described. Genes involved in DNA repair and cell stress responses were found frequently mutated (e.g., ATM, ATR, CHEK1, CHEK2, MLH1, MUTYH, PMS1, PMS2, XPC). Interestingly, 5 mutations in the PI3K-AKT-TOR pathway were identified, including two previously described PIK3CA mutations historically linked to estrogen receptor positive disease. We did not observe PTEN mutations often linked to basal-like breast cancer. Among identified genetic variants, n = 23/96 affected genes encoding kinases (receptor and non-receptor tyrosine kinases, serine/threonine kinases, lipid kinases), including 4 involved in DNA damage response. Developmental genes in the Notch, Wnt and Hedgehog pathways were also mutated. Our results emphasize molecular heterogeneity of TNBC and suggest a variety of possible mechanisms for treatment resistance. We must also acknowledge limitation of this study, which include: lack of information on transcript expression levels of the mutated genes, the limited set of genes examined (n = 421/ case), incomplete knowledge of functional and structural effects of genetic variants on each target gene and the contextual effect of coexisting variants, and the possibility that rare cellular clones may be below the limit of detection. Conclusions: We demonstrate that targeted sequencing using NGS is cost-effective, reliable and feasible for clinical use. Multiparamenter molecular profiling (DNA, RNA, protein, metabolism) will eventually provide more complete pictures of individual tumors for truly personalized treatment.
Citation Format: Pannuti A, Filipovic A, Hicks C, Lefkowitz E, Ptacek T, Miele L, Stebbing J. Genetic heterogeneity revealed by targeted exome sequencing in advanced triple-negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-07-03.