Effect of Somatic Mutations in the Four Genes of the HER Family on Occurrence in HER2-positive Breast Cancer, Cell Proliferation Rates, and Resistance to HER2-targeted Therapies in Vitro.

615 Background: Somatic mutations in the 4 HER family genes (EGFR, HER2, 3, 4) occur in HER2-positive breast cancer (HER2+ BC). (Cancer Genome Atlas N. Comprehensive molecular portraits of human breast tumors. Nature 2012;490(7418): 61-70.) We investigated their frequency in a large set of HER2+ BCs and their therapeutic relevance. Methods: Tumors from 246 HER2+ BC patients who received adjuvant trastuzumab-based therapy after surgery were reviewed by a pathologist for tumor content. DNA from 48 matched normal and cancerous samples was sequenced using the Agilent kinome library and a MiSeq. Somatic HER gene mutations identified in this way had their frequency determined by Sequenom MassArray (n=246). HER gene mutations were correlated with patient outcomes. Constructs of 2 hotspot HER3/4 mutations were created and transfected into BT474, HCC1569 and HCC1954 cells. Doubling time, proliferation, Boyden chamber assays and RPPA were used to determine effects of mutations on cell growth, invasion, signalling and response to HER2-targeted therapies. Results: 12 somatic deleterious mutually exclusive mutations in the kinase and furin like domains of the 4 HER genes (3 EGFR, 2 HER2, 2 HER3, 5 HER4 mutations) were identified in 6.5 % of HER2+ BCs including 2 hotspot mutations in HER3/4. HER gene mutations did not impact OS or RFS after adjuvant trastuzumab. HER3/4 hotspot mutations increased doubling rates in vitro and induced resistance to anti-HER2/EGFR TKIs (Table) but not trastuzumab. Transfected cells were sensitive to the PI3K inhibitor copanlisib and copanlisib also restored sensitivity to these TKIs in these cell lines. Conclusions: HER family mutations occur in 6.5% of HER2+BC and affect tumour growth and response to HER2-targeted therapies. Effects of 2 hotspot HER3/4 mutations on response to TKI’s (nM) in vitro in transfected (MUT) vs. wild type (WT) cell lines. ‘*’ indicates a p-value of < 0.05. HER3 MUT IC50 HER3 WT IC50 Lapatinib (BT474) 58.3 +/- 5.2 * 42.7 +/- 4.1 Afatinib (HCC1569) 129.8 +/- 8.8 * 38.4 +/- 3.9 Afatinib (HCC1954) 81.5 +/- 8.0 * 52.8 +/- 3.2 HER4 MUT IC50 HER4 WT IC50 Afatinib (HCC1569) 76.5 +/- 1.3* 44.1 +/- 4.1