Abstract LB-A22: Assessment of mutations with a custom NSABP anti-HER2 resistance AmpliSeq (NAR) panel in NSABP clinical trial FB-7
Authors: Feng Huichen , Wang Ying , Lucas Peter , Kim Seong-Rim , Lipchik Corey , Gavin Patrick , Jacobs Samuel , Srinivasan Ashok , Pogue-Geile Katherine L. 
Published: August 2018

Background: The NSABP FB-7 trial, reported at SABCS 2015 (Jacobs et al., abstract # PD5-04), enrolled HER2-positive breast cancer patients (pts) with locally advanced disease who were randomly assigned to trastuzumab (T) or neratinib (N) or the combination (T+N) with weekly paclitaxel (P) followed by standard AC. Dual anti-HER2 therapy yielded greater pathological complete response (pCR) rates than single targeted therapies in the overall cohort. The purpose of this study was to evaluate the technical qualities of a new custom, targeted AmpliSeq panel and to use it to assess mutations in FB-7 pretreatment biopsies and surgical samples. Methods: To identify mutations that could act as resistance markers to anti-HER2 therapy, we designed a custom panel, termed the NSABP Anti-HER2 Resistance AmpliSeq (NAR) panel, interrogating 117 genes involved in HER2 signaling pathways and/or previously implicated as resistance markers to trastuzumab. The NAR panel, including 3,847 amplicons in 2 pools, was used to sequence HDx™ reference standards with the Ion Torrent sequencing platform. First, the panel and our mutation-calling pipeline were tested for reproducibility and accuracy using the reference samples. Subsequently, DNA from pretreatment biopsies and matched surgical samples (when available) from FB-7 pts were assayed for mutations using the NAR panel. Results: Across the NAR panel, the average target coverage was 94.25%. For the ERBB2 gene, the coverage was 96.0%. HDx™ reference standards, with known allele frequencies as evaluated by ddPCR, were used to assess the accuracy of the NAR panel, using a custom pipeline to filter SNVs. HDx™ reference standards were sequenced with the NAR panel, and the resulting allele frequencies compared with the known allele frequencies. Based on examination of the following mutations, BRAF-V600E, KRAS-G13D, KRAS-G12D, NRAS-Q61K, PIK3CA-H1047R, MET-Del-FS, PDGFRA-G426D, EGF-G719S, we observed a Pearson correlation coefficient of 0.9822. Next, we used our NAR Ampliseq panel to sequence a subset of colon samples collected as part of the NSABP Genome Assessment Guided Medicine (N-GAMe) cohort. Comparison of our NAR AmpliSeq results and those from a CLIA certified Illumina sequencing platform over all ERBB2 polymorphism and mutation sites demonstrated a correlation coefficient of 0.978. We also assessed the association of copy number determined with the Ion Torrent sequencing software with pCR. We confirmed that the pCR rates were higher in patient samples with greater amplification of ERBB2 (P &lt; 0.05). Mutant allele frequencies were compared in the before-treatment biopsies to surgical samples. The mutant allele frequencies of PIK3CA-E545K, MAP2K4-S184L, MTOR-R491Q, and MAP2K4-D150N were increased after treatment, suggesting that these mutations may have provided a selective advantage during treatment, implicating a potential resistance marker. We predicted that N or T+N-treated patients with an ERBB2-V777L mutation would achieve pCR, but such a patient did not achieve pCR. Other mutations in this patient may have been responsible for the failure to achieve pCR. One patient with an ERBB2-S310F mutation and treated with N, did achieve pCR, as predicted. Conclusion: Preliminary analysis of FB-7 suggests that sequence analysis with Ion torrent sequencing using the NAR panel may be clinically useful. Support: Puma Biotechnology and PA state grant, PA DOH disclaims responsibility for analysis, interpretations, or conclusions

Citation Format: Huichen Feng, Ying Wang, Peter Lucas, Seong-Rim Kim, Corey Lipchik, Patrick Gavin, Samuel Jacobs, Ashok Srinivasan, Katherine L. Pogue-Geile. Assessment of mutations with a custom NSABP anti-HER2 resistance AmpliSeq (NAR) panel in NSABP clinical trial FB-7 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-A22.