Abstract S5-01: Whole exome sequencing of pre-treatment biopsies from the neoALTTO trial to identify DNA aberrations associated with response to HER2-targeted therapies
Authors: Pusztai L , Shi W , Jiang T , Nuciforo P , Holmes E , Harbeck N , Sotiriou C , Rimm D , Hatzis C , de la Peña L , Armour A , Piccart-Gebhart M , Baselga J 
Published: May 2016

Abstract
Background: We examined if alterations in nucleic acid variants, genes, pathways, and overall mutational load and clonal entropy are associated with pathologic complete response (pCR) and survival after neoadjuvant anti-HER2 therapies in the NeoALTTO trial.
Methods: Whole exome sequencing was performed of 203 baseline biopsies with outcome information. The mean nucleotide coverage was 150x with &gt;90% of target bases showing &gt; 30x coverage in &gt; 99% of samples. Somatic mutations were called by MuTect and indels by Strelka, using pooled reference normal DNA. Significantly mutated genes (FDR&lt;10%) were identified by MutSigCV. Mutations in 714 canonical biological pathways were assessed and mutational load and genome clonal entropy (MATH) were calculated. Association with pCR and survival were evaluated by logistic regression adjusted for ER status and Cox-proportional hazards regression.
Results: Only 12 genes had mutation rates significantly above background and among these only PI3KCA was associated with lower pCR rate (OR=0.42, p=0.019). Genes with somatic mutations in more than 10 patients were also assessed, but none were associated with pCR or survival. Clonal entropy or adjusted mutation load also did not correlate with response. Mutations in 33 pathways showed significant association with response in the entire cohort. In the trastuzumab arm, 23 of the 33 pathways showed an association with response but none was independent of PIK3CA mutation. We constructed "PIK3CA-gene network" that included all unique genes (n=439) from theese 23 pathways. Of the 66 patients in the trastuzumab arm, 50 carried at least one mutation in one of the 439 genes and among these only 2 achieved pCR (4%) compared to 9 of 16 pCR (56%) among the wild type (OR=0.035; p &lt; 0.001). The same genes/mutations had little impact on pCR in the lapatinib arm (pCR 20%). In the lapatinib arm, mutations in 3 pathways conferred higher probability of pCR. The "Regulation of RhoA activity" pathway, had the most significant association with pCR in the entire cohort (OR=3.77, p=0.0009) and in the lapatininb (pCR 67% vs 17%, OR=14.8, p=0.008) and lapatinib + trastuzumab (OR=3.0, p=0.06) arms, but not in the trastuzumab arm (OR=1.4, p=0.7). Event free and overall survival were also significantly higher in patients who had mutations in this pathway. Twenty seven of the 48 genes in this pathway had mutations affecting 33 patients but different genes were affected in different individuals.
Conclusions: There are no high frequency recurrent single mutations associated with response to HER2-targeted therapies, other than PIK3CA. We identified several biological pathways, including RhoA activity, and a network of PIK3CA associated genes that are significantly associated with response when affected by mutations, however, different genes are mutated in different individuals.
Citation Format: Pusztai L, Shi W, Jiang T, Nuciforo P, Holmes E, Harbeck N, Sotiriou C, Rimm D, Hatzis C, de la Peña L, Armour A, Piccart-Gebhart M, Baselga J. Whole exome sequencing of pre-treatment biopsies from the neoALTTO trial to identify DNA aberrations associated with response to HER2-targeted therapies. [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 S5-01.