8 Abstract Approximately 20 % of human breast cancers 9 (BC) overexpress HER2 protein, and HER2-positivity is 10 associated with a worse prognosis. Although HER2-tar-11 geted therapies have significantly improved outcomes for 12 HER2-positive BC patients, resistance to trastuzumab-13 based therapy remains a clinical problem. In order to better 14 understand resistance to HER2-targeted therapies in HER2-15 positive BC, it is necessary to examine HER family sig-16 nalling as a whole. An extensive literature search was 17 carried out to critically assess the current knowledge of 18 HER family signalling in HER2-positive BC and response 19 to HER2-targeted therapy. Known mechanisms of trast-20 uzumab resistance include reduced receptor-antibody 21 binding (MUC4, p95HER2), increased signalling through 22 alternative HER family receptor tyrosine kinases 23 (RTK), altered intracellular signalling involving loss of 24 PTEN, reduced p27kip1, or increased PI3 K/AKT activity 25 and altered signalling via non-HER family RTKs such as 26 IGF1R. Emerging strategies to circumvent resistance to 27 HER2-targeted therapies in HER2-positive BC include co-28 targeting HER2/PI3 K, pan-HER family inhibition, and 29 novel therapies such as T-DM1. There is evidence that 30 immunity plays a key role in the efficacy of HER-targeted 31 therapy, and efforts are being made to exploit the immune 32 system in order to improve the efficacy of current anti-HER 33 therapies. With our rapidly expanding understanding of 34 HER2 signalling mechanisms along with the repertoire of 35 HER family and other targeted therapies, it is likely that the 36 near future holds further dramatic improvements to the 37 prognosis of women with HER2-positive BC. 38 39 Keywords Trastuzumab · HER2 · Breast cancer · 40 PI3 K 41 Introduction 42 BC is the second most common cancer in the world, and the 43 fifth highest cause of cancer mortality worldwide [1]. 20 % of 44 human BC's overexpress HER2, and HER2-positivity is 45 associated with a significantly worse prognosis. HER2 first 46 became targetable in patients with trastuzumab (Herceptin, 47 ™ Genentech/Roche), a monoclonal antibody that has sig-48 nificantly improved outcomes for patients with HER2-49 positive BC, but the efficacy of trastuzumab is limited in 50 some patients by acquired and de novo resistance [2]. 51 HER family signalling 52There are 20 known RTK families: since members of over 53 half of these have been found to be mutated or overex-54 pressed in diseases marked by abnormal proliferation, 55 RTK's have been considered potential targets for cancer 56 therapy. HER2, a type 1 transmembrane protein RTK, and 57 an oncogenic driver of the growth of HER2-positive BC, is 58 associated with a shorter time to relapse and decreased 59 overall survival (OS 44Author Proof 26] evidence shows that lapatinib is effective against 151 trastuzumab-resistant HER2-positive BC, and it is cur-152 rently used as subsequent therapy for patients with disease 153 that has progressed on trastuzumab. Lapatinib inhibits 15...

11 Abstract The PI3K pathway is a key mechanism of 12 trastuzumab resistance, but early attempts to indirectly target 13 this pathway with mTOR inhibitors have had limited suc-14 cess. We present the results of a preclinical study of the 15 selective alpha/delta isoform dominant PI3K inhibitor BAY 16 80-6946 tested alone and in combination with HER2-tar-17 geted therapies in HER2-positive cell lines, including mod-18 els with acquired resistance to trastuzumab and/or lapatinib. 19 A panel of HER2-positive breast cancer cells were profiled 20 for their mutational status using Sequenom MassARRAY, 21 PTEN status by Western blot, and anti-proliferative response 22 to BAY 80-6946 alone and in combination with the HER2-23 targeted therapies trastuzumab, lapatinib and afatinib. 24 Reverse phase protein array was used to determine the effect 25 of BAY 80-6946 on expression and phosphorylation of 68 26 proteins including members of the PI3K and MAPK path-27 ways. The Boyden chamber method was used to determine if 28 BAY 80-6946 affected cellular invasion and migration. BAY 29 80-6946 has anti-proliferative and anti-invasive effects when 30 used alone in our panel of cell lines (IC 50 's 3.9-29.4 nM Author Proof 5, 6] and has recently been associated with lapatinib resis-64 tance [7, 8] 114 Proliferation assays 115For all resistant cell lines, drug was removed from the cells 116 at least 7-days prior to starting assays, and no P/S was 117 added to media during proliferation assays. 3 9 10 4 cells/ 118 well were seeded in 96-well plates, apart from BT474 and 119 BT474-Res which were seeded at 5 9 10 4 cells/well. 120 Plates are incubated overnight at 37°C to allow cells to 121 adhere. Drugs were added to the plates at specific con-122 centrations and incubated at 37°C. Following 5-day 123 incubation, during which control cells attained 80-90 % 124 confluence, all media were removed from the plates, and 125 washed once with PBS. Proliferation was measured using 126 the acid phosphatase assay as previously described [16]. 127 Invasion and migration assays 128Invasion and migration assays were performed using the 129 Boyden chamber method as previously described [16].130 After 24 h, the plates were removed from the incubator. 44Author Proof 192 BT474 = 3.9 ± 0.8 nM; BT474-Res = 6.25 ± 0.8 nM).193 Treatment with BAY 80-6946 (at a non-lethal dose) for 194 24 h significantly decreased invasion in both cell lines 195 (BT474 p = 0.008; MDAMB453 p = 0.008) but had no 196 effect on migration ( Supplementary Fig. 1).197 We studied the effect of lapatinib and afatinib (dual 198 EGFR/HER2 inhibitors) in our panel of cell lines. Lapati-199 nib IC 50 s range from 33.3 ± 9.5 nM in BT474 cells up to 200 greater than 500 nM in cells with acquired lapatinib 201 resistance. The cell lines with acquired lapatinib resistance 202 SKBR3-L, -TL and HCC1954-L did not achieve an IC 50 at Supplementary Fig. 2). In SKBR3 and BT474 cells which 224 had diminished AKT activation (S473) in response to BAY 225 80-6946 treatment, a corresponding decr...

BackgroundTrastuzumab treatment for women with HER2-positive breast cancer (BC) resulted in the significant improvement of both relapse free survival (RFS) and overall survival (OS). However, many women who are classified as HER2-positive do not respond. Many studies have focused on the role of somatic mutations rather than germline polymorphisms in trastuzumab resistance.ResultsWe completed an Agena MassArray screen of 10 ERBB-family single nucleotide polymorphisms (SNPs) in 194 adjuvant trastuzumab treated HER2-positive BC patients. SNPs in EGFR genes have a significant association with RFS and OS. Patients with the minor allele of EGFR N158N had significantly worse OS (hazard ratio (HR) = 4.01, (confidence interval (CI) = 1.53– 10.69), p = 0.05) relative to those with either the heterozygous or wild-type (WT) allele. Patients with the minor allele of EGFR T903T (HR = 3.52, (CI = 1.38– 8.97), p = 0.05) had worse RFS relative to those with either the heterozygous or WT allele.Patients and methodsUsing next generation sequencing (NGS) we identified ERBB-family (EGFR, HER2, HER3 and HER4) single nucleotide polymorphisms (SNPs) that occurred in 2 or more patients of a 32 HER2-positive BC patient cohort. Agena MassArray analysis confirmed the frequency of these SNPs in 194 women with HER2-positive BC who received trastuzumab in the adjuvant setting. Using Kaplan-Meier estimates and Cox regression analysis we correlated the presence of ERBB-family SNPs with both RFS and OS.ConclusionsThe presence of germline ERBB-family SNPs may play an important role in how a patient responds to adjuvant trastuzumab, and clinical assessment of these SNPs by targeted genetic screening of patients' blood may be important to stratify patients for treatment.

Background:Somatic mutations in the ERBB genes (epidermal growth factor receptor: EGFR, ERBB2, ERBB3, ERBB4) promote oncogenesis and lapatinib resistance in metastatic HER2+ (human epidermal growth factor-like receptor 2) breast cancer in vitro. Our study aimed to determine the frequency of mutations in four genes: EGFR, ERBB2, ERBB3 and ERBB4 and to investigate whether these mutations affect cellular behaviour and therapy response in vitro and outcomes after adjuvant trastuzumab-based therapy in clinical samples.Methods:We performed Agena MassArray analysis of 227 HER2+ breast cancer samples to identify the type and frequency of ERBB family mutations. Of these, two mutations, the somatic mutations ERBB4-V721I and ERBB4-S303F, were stably transfected into HCC1954 (PIK3CA mutant), HCC1569 (PIK3CA wildtype) and BT474 (PIK3CA mutant, ER positive) HER2+ breast cancer cell lines for functional in vitro experiments.Results:A total of 12 somatic, likely deleterious mutations in the kinase and furin-like domains of the ERBB genes (3 EGFR, 1 ERBB2, 3 ERBB3, 5 ERBB4) were identified in 7% of HER2+ breast cancers, with ERBB4 the most frequently mutated gene. The ERBB4-V721I kinase domain mutation significantly increased 3D-colony formation in 3/3 cell lines, whereas ERBB4-S303F did not increase growth rate or 3D colony formation in vitro. ERBB4-V721I sensitized HCC1569 cells (PIK3CA wildtype) to the pan class I PI3K inhibitor copanlisib but increased resistance to the pan-HER family inhibitor afatinib. The combinations of copanlisib with trastuzumab, lapatinib, or afatinib remained synergistic regardless of ERBB4-V721I or ERBB4-S303F mutation status.Conclusions:ERBB gene family mutations, which are present in 7% of our HER2+ breast cancer cohort, may have the potential to alter cellular behaviour and the efficacy of HER- and PI3K-inhibition.