Search

Background: The addition of trastuzumab (H) to pre-operative chemotherapy in HER2+ breast cancer (H+BC) increases the rate of pathological complete response (pCR). TCH is a widely used adjuvant regimen in early stage H+BC. Lapatinib (L) is a small molecule HER2 antagonist that produces responses following H failure, and has been reported to augment H activity in combination in vitro. We compared neo-adjuvant docetaxel, carboplatin (TC) + H v TCL v TCHL in pts with H+BC. Methods: Pts with stages Ic–III H+BC were randomized to receive neo-adjuvant TCH, TCL or TCHL (ICORG/CTRIAL-IE 10-05, NCT01485926 www.clinicaltrials.gov). Pts subsequently underwent surgery and received H post-operatively for 1 year from the first dose of H. The primary endpoint of the trial was pCR. Secondary objectives were overall survival (OS) and relapse-free survival (RFS) and molecular and pharmacological markers of response. This study was supported by GSK, Novartis, The Cancer Clinical Research Trust and The Caroline Foundation. Results: When the NCIC CTG MA.31 trial reported inferior outcomes for L compared to H, we discontinued the TCL arm. This abstract reports data on the 76 pts recruited to the TCH and TCHL arms only, who included stage II (69.7%; n=53) or stage III disease (21.1%; n=16, with stage unknown for 9.2% (n=7). The final analysis set numbered 75 pts. Clinicopathological characteristics were well balanced between arms. The pCR rate of the two arms TCHL and TCH were virtually identical at 51.6% and 52.8% respectively (Fisher’s test p-value: 1.000). TCHL pts had significantly superior 5 year relapse-free survival (relative risk (RR) 0.171, 95% CI 0.041 – 0.713; log-rank test p-value = 0.009). OS was comparable between the TCH and TCHL groups (RR 0.205, 95% CI 0.025 – 1.675); log-rank test p-value = 0.2). Median RFS and OS were not reached. The most frequent serious adverse event was diarrhoea which occurred in 21.3% (n=16/75) pts (Grade 3 diarrhoea 13/16). One pt (TCH arm) who did not have protocol-mandated prophylactic G-CSF had a Grade 5 toxicity. One TCHL pt had a self-limiting diverticular perforation. There was a significantly higher frequency of severe diarrhoea in pts who received the TCHL regimen (Grade 3+, 32.4% vs 10.5%, p=0.038). The use of prophylactic loperamide reduced the frequency of diarrhoea in both the TCHL arm (86.2% vs 44.4%, p=0.004) and in the TCH arm (58.8% vs 24%, p=0.009). A post lock audit with minimum 9-year follow-up showed relapse rates of 15% TCHL vs 33% TCH. Conclusions: The study did not meet its primary pCR endpoint possibly due to a high TCH pCR rate, and small numbers. TCHL produced a statistically significant improvement in RFS compared to TCH. TCHL produced a higher rate of gastro-intestinal toxicity, but the use of loperamide significantly reduced the frequency of diarrhoea. These data suggest that anti-HER2 TKIs merit further investigation in the neo-adjuvant treatment of early stage H+BC. Table 1. pCR rates, 5 year RFS and OS results for ICORG/CTRIAL-IE 10-05 (NCT01485926) study pts. * significant, p Citation Format: John Crown, Denis M. Collins, Alex J. Eustace, Maccon Keane, Linda Coate, John Kennedy, Seamus O’Reilly, Catherine Kelly, Miriam O’Connor, Michael J. Martin, Conleth Murphy, Karen Duffy, Janice Walshe, Thamir Mahgoub, Giuseppe Gullo, Brian Moulton, Alberto Alvarez-Iglesias, Imelda Parker, Bryan Hennessy. Five year follow up of a randomized phase II comparison of neo-adjuvant docetaxel, carboplatin, trastuzumab with or without lapatinib in HER-2 positive breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-11-09.

All ADCC ratios from Figure 3 and trastuzumab/pertuzumab ADCC comparison

Densitometry analysis for Figure 6B

Purpose:Antibody-dependent cell-mediated cytotoxicity (ADCC) is one mechanism of action of the monoclonal antibody (mAb) therapies trastuzumab and pertuzumab. Tyrosine kinase inhibitors (TKIs), like lapatinib, may have added therapeutic value in combination with mAbs through enhanced ADCC activity. Using clinical data, we examined the impact of lapatinib on HER2/EGFR expression levels and natural killer (NK) cell gene signatures. We investigated the ability of three TKIs (lapatinib, afatinib, and neratinib) to alter HER2/immune-related protein levels in preclinical models of HER2-positive (HER2+) and HER2-low breast cancer, and the subsequent effects on trastuzumab/pertuzumab-mediated ADCC.Experimental Design:Preclinical studies (proliferation assays, Western blotting, high content analysis, and flow cytometry) employed HER2+ (SKBR3 and HCC1954) and HER2-low (MCF-7, T47D, CAMA-1, and CAL-51) breast cancer cell lines. NCT00524303 provided reverse phase protein array–determined protein levels of HER2/pHER2/EGFR/pEGFR. RNA-based NK cell gene signatures (CIBERSORT/MCP-counter) post-neoadjuvant anti-HER2 therapy were assessed (NCT00769470/NCT01485926). ADCC assays utilized flow cytometry–based protocols.Results:Lapatinib significantly increased membrane HER2 levels, while afatinib and neratinib significantly decreased levels in all preclinical models. Single-agent lapatinib increased HER2 or EGFR levels in 10 of 11 (91%) tumor samples. NK cell signatures increased posttherapy (P = 0.03) and associated with trastuzumab response (P = 0.01). TKI treatment altered mAb-induced NK cell–mediated ADCC in vitro, but it did not consistently correlate with HER2 expression in HER2+ or HER2-low models. The ADCC response to trastuzumab and pertuzumab combined did not exceed either mAb alone.Conclusions:TKIs differentially alter tumor cell phenotype which can impact NK cell–mediated response to coadministered antibody therapies. mAb-induced ADCC response is relevant when rationalizing combinations for clinical investigation.

Densitometry for Figure 1D

Densitometry analysis for Figure 6D/E

Flow cytometry MFI and % positive cells data for fluorescently labelled trastuzumab and pertuzumab in SKBR3, HCC1954, T47D and MCF-7 treated with TKIs

Rituximab ADCC negative control

Flow cytometry control scatter plots for trastuzumab-488 and pertuzumab-550

MFI comparison at T0 and T6 for T47D/MCF-7

Densitometry for Figure 2B

Innovation in both detection and treatment of cancer is necessary for the constant improvement in therapeutic strategies, especially in patients with novel or resistant variants of cancer. Cancer mortality rates have declined by almost 30% since 1991, however, depending on the cancer type, acquired resistance can occur to varying degrees. To combat this, researchers are looking towards advancing our understanding of cancer biology, in order to inform early detection, and guide novel therapeutic approaches. Through combination of these approaches, it is believed that a more complete and thorough intervention on cancer can be achieved. Here, we will discuss the advances and approaches in both detection and treatment of cancer, presented at the 58th Irish Association for Cancer Research (IACR) annual conference.

Triple negative breast cancer (TNBC) remains a therapeutic challenge due to the lack of targetable genetic alterations and the frequent development of resistance to the standard cisplatin-based chemotherapies. Here, we have taken a systems biology approach to investigate kinase signal transduction networks that are involved in TNBC resistance to cisplatin. Treating a panel of cisplatin-sensitive and cisplatin-resistant TNBC cell lines with a panel of kinase inhibitors allowed us to reconstruct two kinase signalling networks that characterise sensitive and resistant cells. The analysis of these networks suggested that the activation of the PI3K/AKT signalling pathway is critical for cisplatin resistance. Experimental validation of the computational model predictions confirmed that TNBC cell lines with activated PI3K/AKT signalling are sensitive to combinations of cisplatin and PI3K/AKT pathway inhibitors. Thus, our results reveal a new therapeutic approach that is based on identifying targeted therapies that synergise with conventional chemotherapies.

Background Pre-treatment tumour-associated lymphocytes (TILs) and stromal lymphocytes (SLs) are independent predictive markers of future pathological complete response (pCR) in HER2-positive breast cancer. Whilst studies have correlated baseline lymphocyte levels with subsequent pCR, few have studied the impact of neoadjuvant therapy on the immune environment. Methods We performed TIL analysis and T-cell analysis by IHC on the pretreatment and ‘On-treatment’ samples from patients recruited on the Phase-II TCHL (NCT01485926) clinical trial. Data were analysed using the Wilcoxon signed-rank test and the Spearman rank correlation. Results In our sample cohort (n = 66), patients who achieved a pCR at surgery, post-chemotherapy, had significantly higher counts of TILs (p = 0.05) but not SLs (p = 0.08) in their pre-treatment tumour samples. Patients who achieved a subsequent pCR after completing neo-adjuvant chemotherapy had significantly higher SLs (p = 9.09 × 10–3) but not TILs (p = 0.1) in their ‘On-treatment’ tumour biopsies. In a small cohort of samples (n = 16), infiltrating lymphocyte counts increased after 1 cycle of neo-adjuvant chemotherapy only in those tumours of patients who did not achieve a subsequent pCR. Finally, reduced CD3 + (p = 0.04, rho = 0.60) and CD4 + (p = 0.01, rho = 0.72) T-cell counts in 'On-treatment' biopsies were associated with decreased residual tumour content post-1 cycle of treatment; the latter being significantly associated with increased likelihood of subsequent pCR (p Conclusions The immune system may be ‘primed’ prior to neoadjuvant treatment in those patients who subsequently achieve a pCR. In those patients who achieve a pCR, their immune response may return to baseline after only 1 cycle of treatment. However, in those who did not achieve a pCR, neo-adjuvant treatment may stimulate lymphocyte influx into the tumour.

Purpose: Antibody-dependent cell-mediated cytotoxicity (ADCC) is one mechanism of action of the monoclonal antibody (mAb) therapies trastuzumab and pertuzumab. Tyrosine kinase inhibitors (TKIs), like lapatinib, may have added therapeutic value in combination with mAbs through enhanced ADCC activity. Using clinical data, we examined the impact of lapatinib on HER2/EGFR expression levels and natural killer (NK) cell gene signatures. We investigated the ability of three TKIs (lapatinib, afatinib, and neratinib) to alter HER2/immune-related protein levels in preclinical models of HER2-positive (HER2+) and HER2-low breast cancer, and the subsequent effects on trastuzumab/pertuzumab-mediated ADCC. Experimental Design: Preclinical studies (proliferation assays, Western blotting, high content analysis, and flow cytometry) employed HER2+ (SKBR3 and HCC1954) and HER2-low (MCF-7, T47D, CAMA-1, and CAL-51) breast cancer cell lines. NCT00524303 provided reverse phase protein array–determined protein levels of HER2/pHER2/EGFR/pEGFR. RNA-based NK cell gene signatures (CIBERSORT/MCP-counter) post-neoadjuvant anti-HER2 therapy were assessed (NCT00769470/NCT01485926). ADCC assays utilized flow cytometry–based protocols. Results: Lapatinib significantly increased membrane HER2 levels, while afatinib and neratinib significantly decreased levels in all preclinical models. Single-agent lapatinib increased HER2 or EGFR levels in 10 of 11 (91%) tumor samples. NK cell signatures increased posttherapy (P = 0.03) and associated with trastuzumab response (P = 0.01). TKI treatment altered mAb-induced NK cell–mediated ADCC in vitro, but it did not consistently correlate with HER2 expression in HER2+ or HER2-low models. The ADCC response to trastuzumab and pertuzumab combined did not exceed either mAb alone. Conclusions: TKIs differentially alter tumor cell phenotype which can impact NK cell–mediated response to coadministered antibody therapies. mAb-induced ADCC response is relevant when rationalizing combinations for clinical investigation.

Background The MYC oncogene is one of the most frequently altered driver genes in cancer. MYC is thus a potential target for cancer treatment as well as a biomarker for the disease. However, as a target for treatment, MYC has traditionally been regarded as “undruggable” or difficult to target. We set out to evaluate the efficacy of a novel MYC inhibitor known as MYCMI-6, which acts by preventing MYC from interacting with its cognate partner MAX. Methods MYCMI-6 response was assessed in a panel of breast cancer cell lines using MTT assays and flow cytometry. MYC gene amplification, mRNA and protein expression was analysed using the TCGA and METABRIC databases. Results MYCMI-6 inhibited cell growth in breast cancer cell lines with IC50 values varying form 0.3 μM to >10 μM. Consistent with its ability to decrease cell growth, MYCMI-6 was found to induce apoptosis in two cell lines in which growth was inhibited but not in two cell lines that were resistant to growth inhibition. Across all breast cancers, MYC was found to be amplified in 15.3% of cases in the TCGA database and 26% in the METABRIC database. Following classification of the breast cancers by their molecular subtypes, MYC was most frequently amplified and exhibited highest expression at both mRNA and protein level in the basal subtype. Conclusions Based on these findings, we conclude that for patients with breast cancer, anti-MYC therapy is likely to be most efficacious in patients with the basal subtype.

Introduction Triple negative breast cancer (TNBC) represents a heterogeneous subtype of breast cancer that carries a poorer prognosis. There remains a need to identify novel drivers of TNBC, which may represent targets to treat the disease. c-Met overexpression is linked with decreased survival and is associated with the basal subtype of breast cancer. Cpd A, a kinase inhibitor selective/specific for Met kinase has demonstrated preclinical anti-cancer efficacy in TNBC. We aimed to assess the anti-cancer efficacy of Cpd A when combined with Src kinase, ErbB-family or hepatocyte growth factor (HGF) inhibitors in TNBC cell lines. Methods We determined the anti-proliferative effects of Cpd A, rilotumumab, neratinib and saracatinib tested alone and in combination in a panel of TNBC cells by acid phosphatase assays. We performed reverse phase protein array analysis of c-Met and IGF1Rβ expression and phosphorylation of c-Met (Y1234/1235) in TNBC cells and correlated their expression/phosphorylation with Cpd A sensitivity. We examined the impact of Cpd A, neratinib and saracatinib tested alone and in combination on invasive potential and colony formation.Results TNBC cells are not inherently sensitive to Cpd A, and neither c-Met expression nor phosphorylation are biomarkers of sensitivity to Cpd A. Cpd A enhanced the anti-proliferative effects of neratinib in vitro; however, this effect was limited to cell lines with innate sensitivity to Cpd A. Cpd A had limited anti-invasive effects but it reduced colony formation in the TNBC cell line panel.Conclusions Despite Cpd A having a potential role in reducing cancer cell metastasis, identification of strong predictive biomarkers of c-Met sensitivity would be essential to the development of a c-Met targeted treatment for an appropriately selected cohort of TNBC patients.

Background: Increased tumour infiltrating lymphocytes (TILs) are associated with a better prognosis in HER2+ BC patients treated with neo-adjuvant chemotherapy. The signalling mechanisms associated with increased TIL levels are not fully understood. Chemotactic cytokines (chemokines) and their respective receptors have a major role to play in tumour immune cell infiltrate. Analysis of plasma chemokine levels and TIL levels in HER2+ BC patients treated in the neo-adjuvant setting has identified three chemokines of interest - CCL26, CCL17 and CCL19. Examination of tumor mRNA expression levels of their corresponding receptors CCR3, CCR4, and CCR7 in publicly available datasets reveals a significant association with overall survival in PAM50-defined HER2-enriched BC patients. Methods: Pre-treatment (n=43) and post-treatment (n=29) (2 weeks pre-surgery) blood samples were collected from patients enrolled in ICORG 10-05 (neo-adjuvant chemotherapy (docetaxel/carboplatin) +/- trastuzumab, lapatinib or trastuzumab/lapatinib). Patients were classified as having a pathological complete response (CR) or a non-CR (nCR). Plasma chemokine levels were determined by Luminex xMAP assay and validated by ELISA. TIL levels were determined from H and E-, AE1/AE3- and CD45- stained FFPE tissue. Chemokine receptor mRNA expression was interrogated in publicly available datasets using BreastMark (http://glados.ucd.ie/BreastMark/index.html). The PAM50 HER2-enriched molecular signature and a median cut-off was used for all analyses. Results: Circulating CCL17 levels were significantly lower in patients achieving CR, pre- (p=0.015) and post-treatment (p=0.012). Baseline CCL17 levels correlated with baseline TIL count (r=0.582, p=0.011) for CR but not nCR. There was no association between pre- or post-treatment CCL19 and CCL26 plasma levels and treatment response. However, baseline CCL26 levels were inversely correlated with baseline TILs for patients achieving CR (r= -0.49, p=0.028) but not nCR. Baseline CCL19 displayed a similar trend that did not reach significance (r=0.414, p=0.077). Analysis of publicly available datasets reveals tumor mRNA expression of CCR3 (Hazard ratio (HR)=1.9, p=0.001) and CCR7 (HR=0.53, p=0.002) are associated with overall survival in patients with a HER2-enriched molecular signature. Conclusions: Our results suggest circulating chemokine levels may have value as biomarkers of response and TIL status in HER2+ BC. The strong correlation of chemokine receptors with overall survival in tumors with a HER2-enriched molecular signature suggests further examination of chemokine/chemokine receptor axes is warranted in a larger cohort of HER2+ BC patients. Citation Format: Denis Martin Collins, Alacoque Browne, Stephen F Madden, Nicola Gaynor, Elaine W Kay, Joanna Fay, Katherine Sheehan, Sinead Toomey, Alex J Eustace, William M Gallagher, Bryan T Hennessy, John Crown. Examination of CCL26, CCL17 and CCL19 chemokines as biomarkers in HER2+ breast cancer (BC) in the neo-adjuvant setting [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-10-13.

Triple-negative breast cancer (TNBC) is characterized as an aggressive form of breast cancer (BC) associated with poor patient outcomes. For the majority of patients, there is a lack of approved targeted therapies. Therefore, chemotherapy remains a key treatment option for these patients, but significant issues around acquired resistance limit its efficacy. Thus, TNBC has an unmet need for new targeted personalized medicine approaches. Calcium (Ca2+) is a ubiquitous second messenger that is known to control a range of key cellular processes by mediating signalling transduction and gene transcription. Changes in Ca2+ through altered calcium channel expression or activity are known to promote tumorigenesis and treatment resistance in a range of cancers including BC. Emerging evidence shows that this is mediated by Ca2+, modulation supporting the function of tumour suppressor genes (TSGs) and oncogenes. This review provides insight into the underlying alterations in calcium signalling and how it plays a key role in promoting disease progression and therapy resistance in TNBC which harbours mutations in tumour protein p53 (TP53) and the PI3K/AKT pathway.

Background: Anti-HER2 therapies such as trastuzumab used for the treatment of patients with HER2+ metastatic breast cancer (MBC) have led to significant improvements to disease progression. We previously identified cases from the “Thousand Patient HER2 database” project at Saint Vincent’s University Hospital (SVUH) Dublin, of HER2+ MBC long-term durable complete responders to trastuzumab, and reported that Copy Number Aberration (CNA) burden may represent a novel prognostic predictor to trastuzumab response from the exome analysis of in HER2+ MBC “exceptional responders’’ (ExRs). However, whole-genome sequencing (WGS) allows a better understanding of how CNA affects the MBC genome and to-date, the complete genome of this “exceptional” cohort has never been described. Methods: We performed WGS analysis to characterise the CNA profiles of 9 ExRs from our HER2+ MBC cohort treated with trastuzumab. Samples were obtained from patients who never progressed/relapsed for more than 5 years (OS > 60 months). DNA was sequenced from tumours (primary or metastases) and matching control (blood or normal tissue) at a mean depth of 60X and 30X, respectively (18 samples). Somatic single nucleotide variants (SNV) were detected using GATK4 Mutect2 and CNA were identified using Control-FREEC. Results: Eighty-five HER2+ MBC were identified with OS > 60 months, of which 28 were ExRs with bone, lung, liver and lymph metastasis who responded exceptionally to trastuzumab, with a mean OS of 108 months (range 61-236 months). This cohort includes patients who were diagnosed between 31 and 80 years old (median=51). WGS analysis revealed CNA in chr6p21 with amplification of CCND3 and in chr17q12 with amplification of RAD51D. SNV were identified in genes involved in the DNA damage repair (DDR) pathway such as ATM, BRCA2, RAD50 and FANCA. On-going analysis will allow the CNA profiles of all ExRs to be presented and their CNA burden calculated in order to investigate the relationship between whole genome CNA burden and HER2+ MBC patient survival. Conclusion: To our knowledge, this is the first study to sequence the whole genome of HER2+ MBC, never relapse exceptional responders. The identification of the genomic aberrations of these metastatic patients increases our understanding of the mechanisms involved in MBC progression. CNA burden may represent a novel prognostic predictor to trastuzumab response and new outcomes for patients, particularly as MBC is generally termed incurable. Citation Format: Charlotte Andrieu, Laura P. Ivers, Jose Javier Berenguer Pina, Darko Skrobo, Jo Ballot, Alex J. Eustace, Cecily Quinn, Giuseppe Gullo, Naomi Walsh, John P. Crown. Whole genome sequencing of long-term, never relapse exceptional responders HER2+ advanced metastatic breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-24.

Introduction: Computational approaches have aided in estimating cellular composition of the tumour microenvironment. The evaluation of immune composition in tumours before treatment may predict pathologic complete response (pCR). The aim of the study was to perform a meta-analysis of HER2-positive breast cancer subjects who received neoadjuvant trastuzumab to detect associations between immune cells measured by CIBERSORT and ESTIMATE and pCR. Methods: PubMed was used to identify transcriptomic data of HER2-positive breast cancer patients who received neoadjuvant trastuzumab. Baseline data from eight neoadjuvant studies (N=338) was downloaded from GEO. Data from each study was background corrected and quantile normalised using ‘limma’ or ‘oligo’ packages in R. Immune profiles per sample was generated using computational softwares CIBERSORT and ESTIMATE, and were then linked to pCR status. Correlations between immune contexture and pCR for each study were interpreted using statistical testing. Meta-analysis by a logistic regression model was conducted on studies which passed assumptions to identify CIBERSORT immune subsets robust to pCR. Results: CIBERSORT results showed that three studies had reduced T follicular helper cells (Tfh) (Brodsky p=0.38, CHER-LOB p=0.17, TransNOAH p=0.25) and two studies had reduced plasma cells (CHER-LOB p=0.15, Brodsky p=0.38) in the pCR group, but was not significant after multiple correction. ESTIMATE analysis showed that data from two studies had elevated immune infiltration in pCR (Brodsky p=0.19, CHER-LOB p=0.10) but was not significant. A meta-analysis of pooled data from four studies (TRIO-US B07, 03-311, TransNOAH, CHER-LOB) showed that low Tfh (p=0.053, OR=0.04, CI [0.0012-0.99]) and high memory B-cells (p=0.008, OR=2126.9, CI [8.12-7.65 × 10+5]) prior to trastuzumab treatment may be associated with a better chance of achieving pCR. Conclusion: Results from our meta-analysis proposed that memory B- and T follicular helper subsets may predict a role in achieving pCR. Incorporating studies with larger sample cohorts such as the CALGB-40601 (N=265) study can achieve statistical power of this analysis. Citation Format: Dalal AlSultan, Alex J. Eustace, Stephen F. Madden, John Crown. In-silico approaches that detect immune contexture to trastuzumab response in neo-adjuvant studies [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-10.

Background: The “Thousand Patient HER-2 database” project at Saint Vincent’s University Hospital (SVUH) Dublin has been used to identify HER2+ BC patients with durable complete response (never relapsed) to trastuzumab-based therapy. ~10% of met HER2+ BC patients achieve a durable complete response to trastuzumab, meaning the majority of patients progress on treatment. Higher stromal tumour immune infiltrate has been associated with longer OS in met HER2+ BC. There is limited tumor immune profile and PD-1 expression data available for patients with met HER2+ BC with short OS. Using the SVUH database, we have identified a preliminary cohort of 21 met HER2+ BC patients that received trastuzumab and had an OS < 30 months. This study examines the levels of pan T cell marker CD3, cytotoxic T cell marker CD8, Natural Killer (NK) cell marker CD56 and immune checkpoint PD-1 by immunohistochemistry (IHC) in this preliminary cohort. Methods: Formalin-fixed, paraffin-embedded (FFPE) biopsy specimens (n=21 primary, n=7 matched metastatic biopsies) and associated clinico-pathological data were curated. Tumor biopsies were processed for IHC staining of CD8 (Agilent IR62361-2), CD3 (Agilent IR50361-2), CD56 (Agilent IR62861-2) and PD-1 (Roche 07099029001). PD-1 staining was available for 20/21 samples. Staining was performed using the DAKO Link 48 Autostainer as per the manufacturer’s instructions using positive (tonsil tissue) and negative controls (isotype controls). Slides were processed using the Aperio AT2 Digital Slide Scanner (Leica Biosystems), reviewed using Aperio ImageScope 12.4 software (Leica Biosystems) and analyzed in QuPath (University of Edinburgh). Images were annotated to outline tumor areas and an algorithm was trained to identify cells and classify them as either tumor or stromal. Data was expressed as number of positively stained cells/mm2 breast tumor or stromal tissue. Survival studies utilized the Kaplan Meier method. The paired Student’s T test was utilized for primary vs metastatic site comparisons. Results: Designating samples with > 1 stained cell/mm2 breast tumor as positive (pos) and zero stained cells as negative (neg), 19/21 (90.5%) primary samples were pos for CD3, 15/21 (71.4%) for CD56, 14/21 (66.6%) for CD8, and 10/20 (50%) for PD-1. Within the stromal compartment, 20/21 (95.2%) primary samples were pos for CD8, 18/21 (85.7%) for CD56, 16/21 (76.2%) for CD3 and 8/20 (40%) for PD-1. PD-1 expression in the primary tumor (median OS PD-1pos 7.85 mo vs PD-1neg 5.39 mo, hazard ratio (HR) 0.642 (95% CI 0.256-1.613), p=0.346) or the stroma (median OS PD-1pos 8.84 mo vs PD-1neg 5.39 mo, HR 0.495 (95% CI 0.197-1.244), p=0.135) was not significantly associated with OS. When comparing matched primary and metastatic samples (n=7), increased stromal levels of CD3 (4/7), CD8 (4/7), CD56 (5/7) and PD-1 (4/7) were observed. Increased levels of CD3 and CD8 were observed for 2/7 samples, and increased levels of CD56 and PD-1 for 4/7 samples. With the exception of tumor CD8 levels which decreased, mean values for tumor and stromal CD3, CD56, PD-1 and stromal CD8 levels were higher in metastatic sites but all differences were not found to be significant (p>0.05). Conclusions: Our results suggest that met HER2+ BC patients with < 30 months OS have significant T cell and NK cell presence in the tumor and stromal compartments in both primary and metastatic sites. Further expansion of this limited dataset is planned to gain greater insight in to the immune cell profiles and PD-1 status of met HER2+ BC patients with short OS. Citation Format: Denis M. Collins, Janet McCormack, Laura P. Ivers, Jose Javier Berenguer Pina, Jo Ballot, Cecily Quinn, Darko Skrobo, Alex J. Eustace, Naomi Walsh, Aurelie Fabre, John Crown. Immune cell profile of tumors from patients with metastatic (met) HER2+ breast cancer (BC) with < 30 months overall survival (OS). [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-11.

Background: Neratinib is an irreversible pan-HER tyrosine kinase inhibitor with demonstrated clinical activity in HER2+ and HER2-mutated breast cancers. The main toxicity of neratinib is diarrhea, which is common in the absence of prophylaxis. Preclinical models suggest that neratinib-associated diarrhea may involve inflammatory, bile acid malabsorption and secretory factors. The phase II CONTROL study is currently investigating the prophylactic efficacy of the opioid receptor antagonist loperamide in combination with budesonide (a corticosteroid used for inflammatory gastrointestinal conditions) or colestipol (bile acid sequestrant) on neratinib-associated diarrhea in early-stage HER2+ breast cancer (NCT02400476). This in vitro study examines the impact of loperamide and budesonide on the anti-proliferative activity of neratinib or trastuzumab and pertuzumab in HER2+ or HER2-low breast cancer cell lines. Methods: HER2+ breast cancer cell lines SKBR3 (estrogen receptor [ER]–), BT474 (ER+), HCC1569 (ER–) and HER2-low, pertuzumab-sensitive MDA-MB-175-VII (ER+) breast cancer cells were investigated using a 5-day acid phosphatase-based proliferation assay to determine the concentrations required to inhibit growth by 50% (IC50). Fixed ratios of drugs were utilised in combination assays to generate Combination Index (CI) values (Calcusyn®) where available. Clinically relevant levels of neratinib, trastuzumab and pertuzumab were utilised in all experiments. Physiologically relevant levels of budesonide (˜4.2 nM) and loperamide (˜2.5 nM) were exceeded to provide IC50 values for these compounds. Results: All cell lines tested had neratinib IC50 values in the nM range (Table). Trastuzumab and the trastuzumab/pertuzumab combination did not exceed 50% inhibition in the HER2+ cell lines. In the HER2+ breast cancer cell lines tested, loperamide had no impact on neratinib activity in BT474, enhanced neratinib activity in SKBR3, and the combination of loperamide and neratinib proved synergistic in HCC1569 (CI = 0.77 +/– 0.2). Budesonide produced strong synergism in combination with neratinib in SKBR3 (CI = 0.27 +/– 0.03), had no impact on neratinib activity in BT474 and improved response to neratinib in HCC1569. Loperamide and budesonide improved the activity of trastuzumab and pertuzumab in all three HER2+ models tested, and had no impact on pertuzumab activity in MDA-MB-175-VII. Interestingly, neratinib proved synergistic in combination with pertuzumab in MDA-MB-175-VII (CI = 0.75 +/– 0.5 nM). Table.Anti-proliferative effects of agents testedBreast cancer cell lineNeratinib IC50, nMLoperamide IC50, nMBudesonide IC50, nMPertuzumab IC50, nMTrastuzumab (% inhibition, 2.5μg/ml)Trastuzumab/Pertuzumab (% inhibition, 2.5μg/ml)SKBR32.8 +/– 0.47.7 +/– 0.52.7 +/– 0.2NA26.3 +/– 1.3NABT4741.4 +/– 0.12.6 +/– 0.27 +/– 0.6NA40.1 +/– 4.3NAHCC156917.3 +/– 0.79.3 +/– 1.728.7 +/– 0.5No effectNo effect26.1 +/– 2.8MDA-MB-175-VII3 +/– 0.3> 20> 201.2 +/– 0.2NANANA = not acquired. Conclusions: Our preclinical results suggest that budesonide and loperamide do not antagonise the anti-proliferative activity of neratinib or HER2-directed monoclonal antibodies in HER2+ breast cancer cell lines. Citation Format: Collins DM, Gaynor N, Conlon N, Gullo G, Eustace AJ, Crown J. Budesonide and loperamide do not impact the cytotoxicity of neratinib or HER2-directed monoclonal antibodies in HER2+ breast cancer cell lines [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-07-08.

Introduction: Tumor infiltrating lymphocytes (TILs) have a significant effect on tumor progression and survival outcome, and serve as a potential biomarker for pathologic complete response (pCR) in neoadjuvant HER2-positive breast cancer therapy. The assessment of TIL abundance and organization prior to treatment initiation has been associated with improved survival and response in breast cancer. This study aims to assess immune contexture using in silico approaches in a meta-analysis of neoadjuvant trastuzumab therapy. Methods: A literature search was conducted on baseline and on-treatment mRNA data from neoadjuvant studies that enrolled patients who received HER2-targeted therapies. A total of 322 baseline samples from eight neoadjuvant datasets were retrieved from Gene Expression Omnibus (GEO) repository (0. Of these studies, two had available on-treatment samples (03-311; n=50, TRIO-US B07 (n=64). The raw data were pre-processed by normalization and background correction using ‘limma’ and ‘oligo’ packages in R. mRNA profiles of baseline and paired samples were analyzed using in silico approaches CIBERSORT and ESTIMATE. Response information was assigned to each sample to observe the proportion of immune cell contexture in pCR and non-responder (NR) groups in baseline analysis. Generalized linear mixed model (GLMM) analyses on baseline data from CIBERSORT was conducted using the ‘lme4’ package in R to investigate whether a systemic relationship of appropriate immune cell signatures occur across the pooled data. Statistical hypothesis on baseline data was applied to observe differences between pCR and NR groups. Testing on paired data was conducted to observe the differences in immune contexture between baseline and on-treatment. Results: Preliminary results of baseline analysis using CIBERSORT revealed a non-significant trend in reduced follicular helper T-cells and plasma cells in the pCR group amongst three neoadjuvant studies (Brodsky, CHER-LOB and TransNOAH). ESTIMATE analysis observed a trend in elevated immune infiltrate in pCR groups from two studies (Brodsky and CHER-LOB), but was not significant after multiple correction. Paired data analysis using CIBERSORT revealed that single-agent trastuzumab reduced activated natural killer cells (p Citation Format: Dalal AlSultan, Alex J. Eustace, Stephen F. Madden, John Crown. Exploring immune contexture using computational approaches in HER2-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2717.

Background The MYC oncogene is one of the most frequently altered driver genes in cancer. MYC is thus a potential target for cancer treatment as well as a biomarker for the disease. However, as a target for treatment, MYC has traditionally been regarded as "undruggable" or difficult to target. We set out to evaluate the efficacy of a novel MYC inhibitor known as MYCMI-6, which acts by preventing MYC from interacting with its cognate partner MAX. Methods MYCMI-6 response was assessed in a panel of breast cancer cell lines using MTT assays and flow cytometry. MYC gene amplification, mRNA and protein expression was analysed using the TCGA and METABRIC databases. Results MYCMI-6 inhibited cell growth in breast cancer cell lines with IC

Introduction Triple negative breast cancer (TNBC) represents a heterogeneous subtype of breast cancer that carries a poorer prognosis. There remains a need to identify novel drivers of TNBC, which may represent targets to treat the disease. c-Met overexpression is linked with decreased survival and is associated with the basal subtype of breast cancer. Cpd A, a kinase inhibitor selective/specific for Met kinase has demonstrated preclinical anti-cancer efficacy in TNBC. We aimed to assess the anti-cancer efficacy of Cpd A when combined with Src kinase, ErbB-family or hepatocyte growth factor (HGF) inhibitors in TNBC cell lines. Methods We determined the anti-proliferative effects of Cpd A, rilotumumab, neratinib and saracatinib tested alone and in combination in a panel of TNBC cells by acid phosphatase assays. We performed reverse phase protein array analysis of c-Met and IGF1Rβ expression and phosphorylation of c-Met (Y1234/1235) in TNBC cells and correlated their expression/phosphorylation with Cpd A sensitivity. We examined the impact of Cpd A, neratinib and saracatinib tested alone and in combination on invasive potential and colony formation.Results TNBC cells are not inherently sensitive to Cpd A, and neither c-Met expression nor phosphorylation are biomarkers of sensitivity to Cpd A. Cpd A enhanced the anti-proliferative effects of neratinib in vitro; however, this effect was limited to cell lines with innate sensitivity to Cpd A. Cpd A had limited anti-invasive effects but it reduced colony formation in the TNBC cell line panel.Conclusions Despite Cpd A having a potential role in reducing cancer cell metastasis, identification of strong predictive biomarkers of c-Met sensitivity would be essential to the development of a c-Met targeted treatment for an appropriately selected cohort of TNBC patients.

Background An increasing number of anti-cancer therapeutic agents target specific mutant proteins that are expressed by many different tumor types. Successful use of these therapies is dependent on the presence or absence of somatic mutations within the patient’s tumor that can confer clinical efficacy or drug resistance. Methods The aim of our study was to determine the type, frequency, overlap and functional proteomic effects of potentially targetable recurrent somatic hotspot mutations in 47 cancer-related genes in multiple disease sites that could be potential therapeutic targets using currently available agents or agents in clinical development. Results Using MassArray technology, of the 1300 patient tumors analysed 571 (43.9%) had at least one somatic mutation. Mutations were identified in 30 different genes. KRAS (16.5%), PIK3CA (13.6%) and BRAF (3.8%) were the most frequently mutated genes. Prostate (10.8%) had the lowest number of somatic mutations identified, while no mutations were identified in sarcoma. Ocular melanoma (90.6%), endometrial (72.4%) and colorectal (66.4%) tumors had the highest number of mutations. We noted high concordance between mutations in different parts of the tumor (94%) and matched primary and metastatic samples (90%). KRAS and BRAF mutations were mutually exclusive. Mutation co-occurrence involved mainly PIK3CA and PTPN11, and PTPN11 and APC. Reverse Phase Protein Array (RPPA) analysis demonstrated that PI3K and MAPK signalling pathways were more altered in tumors with mutations compared to wild type tumors. Conclusions Hotspot mutational profiling is a sensitive, high-throughput approach for identifying mutations of clinical relevance to molecular based therapeutics for treatment of cancer, and could potentially be of use in identifying novel opportunities for genotype-driven clinical trials.

Background Lapatinib has clinical efficacy in the treatment of trastuzumab-refractory HER2-positive breast cancer. However, a significant proportion of patients develop progressive disease due to acquired resistance to the drug. Induction of apoptotic cell death is a key mechanism of action of lapatinib in HER2-positive breast cancer cells. Methods We examined alterations in regulation of the intrinsic and extrinsic apoptosis pathways in cell line models of acquired lapatinib resistance both in vitro and in patient samples from the NCT01485926 clinical trial, and investigated potential strategies to exploit alterations in apoptosis signalling to overcome lapatinib resistance in HER2-positive breast cancer. Results In this study, we examined two cell lines models of acquired lapatinib resistance (SKBR3-L and HCC1954-L) and showed that lapatinib does not induce apoptosis in these cells. We identified alterations in members of the BCL-2 family of proteins, in particular MCL-1 and BAX, which may play a role in resistance to lapatinib. We tested the therapeutic inhibitor obatoclax, which targets MCL-1. Both SKBR3-L and HCC1954-L cells showed greater sensitivity to obatoclax-induced apoptosis than parental cells. Interestingly, we also found that the development of acquired resistance to lapatinib resulted in acquired sensitivity to TRAIL in SKBR3-L cells. Sensitivity to TRAIL in the SKBR3-L cells was associated with reduced phosphorylation of AKT, increased expression of FOXO3a and decreased expression of c-FLIP. In SKBR3-L cells, TRAIL treatment caused activation of caspase 8, caspase 9 and caspase 3/7. In a second resistant model, HCC1954-L cells, p-AKT levels were not decreased and these cells did not show enhanced sensitivity to TRAIL. Furthermore, combining obatoclax with TRAIL improved response in SKBR3-L cells but not in HCC1954-L cells. Conclusions Our findings highlight the possibility of targeting altered apoptotic signalling to overcome acquired lapatinib resistance, and identify potential novel treatment strategies, with potential biomarkers, for HER2-positive breast cancer that is resistant to HER2 targeted therapies. Electronic supplementary material The online version of this article (10.1186/s12885-018-4852-1) contains supplementary material, which is available to authorized users.

Background: CXCL16 is a pro-inflammatory chemokine associated with chemotaxis of CXCR6-expressing lymphocytes (T cells, NKT cells, NK cells) and the promotion of breast cancer cell proliferation, migration, and invasion in vitro. CXCL16 exists in a transmembrane or a cleaved, soluble form. There is limited clinically relevant data available on the CXCL16/CXCR6 signaling axis in HER2+ BC. This preliminary study examines tumor CXCL16 and CXCR6 mRNA expression and patient outcome in publicly available datasets and examines soluble CXCL16 in the plasma of 32 HER2+ BC patients enrolled in ICORG 10-05 (neo-adjuvant chemotherapy (docetaxel/carboplatin) +/- trastuzumab, lapatinib or trastuzumab/lapatinib). Methods: CXCL16 and CXCR6 mRNA expression was interrogated in publicly available datasets using BreastMark, a web-based tool for preliminary assessment of putative biomarkers in breast cancer.A median cut-off was used for all analyses. Pre-treatment and post-treatment (2 weeks pre-surgery) blood samples were collected from patients enrolled in ICORG 10-05. Plasma CXCL16 levels were determined by Luminex xMAP assay. Pre- and post-treatment levels of CXCL16 were compared directly or based on response (pathological complete response, CR, n=14 or non-pathological complete response, nCR, n=18). Stromal lymphocyte (SL) and tumor-infiltrating lymphocyte (TIL) levels were determined from Haematoxylin and Eosin-, AE1/AE3- and CD45FFPE- stained formalin-fixed paraffin embedded tissue. Pre-treatment lymphocyte levels were correlated with pre- and post-treatment levels of CXCL16 (Pearson's product-moment correlation). Results: In BC as a whole, analysis of publicly available datasets reveals tumor CXCL16 expression is not associated with outcome (n=1238, HR=0.9953, p=0.9516) but high tumor CXCR6 expression is (n=2652, HR=1.127, p=0.0476). Within a HER2+ cohort, inverse correlative analysis suggests high CXCR6/low CXCL16 tumor expression is significantly associated with a worse outcome (n=61, HR=2.731, p=0.01). For ICORG 10-05 patients, circulating CXCL16 plasma levels were significantly higher post-treatment (p Conclusions: Our preliminary results suggest tumor levels of CXCL16/CXCR6 are associated with patient outcome and circulating levels of soluble CXCL16 are altered by treatment and correlate with tumor immune infiltrate in HER2+ BC patients. Further examination of tumor CXCL16/CXCR6 expression and circulating CXCL16 as potential biomarkers of response is warranted in a larger cohort of HER2+ BC patients. Citation Format: Collins DM, Madden SF, Eustace AJ, Toomey S, Kay EW, Fay J, O'Donovan N, Gallagher WM, Hennessy B, Crown J. Tumor CXCL16/CXCR6 expression and soluble CXCL16 in HER2+ breast cancer (BC) [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-11-03.

As HER2 is a client protein of the molecular chaperone Hsp90, targeting Hsp90 may be beneficial in HER2-positive breast cancer. In this study, the activity of the Hsp90 inhibitor NVP-AUY922 was assessed in HER2 overexpressing breast cancer cell lines, including two cell line models of acquired trastuzumab-resistance. The seven HER2-positive breast cancer cell lines tested showed significant sensitivity to NVP-AUY922 in vitro, with IC50 values between 6 and 17 nM. Combining NVP-AUY922 with chemotherapy did not improve response. NVP-AUY922 in combination with trastuzumab, significantly enhanced growth inhibition in three of the seven cell lines tested. In conclusion, our data shows that NVP-AUY922 displays potent anti-cancer activity in HER2-positive and trastuzumab-resistant breast cancer cells, and supports further testing of NVP-AUY922 in patients with HER2-positive breast cancer.

// John O’Shea 1 , Mattia Cremona 1 , Clare Morgan 1 , Malgorzata Milewska 1 , Frankie Holmes 2 , Virginia Espina 3 , Lance Liotta 3 , Joyce O’Shaughnessy 4 , Sinead Toomey 1 , Stephen F. Madden 5 , Aoife Carr 1 , Naomi Elster 1 , Bryan T. Hennessy 1, * and Alex J. Eustace 1, * 1 Department of Medical Oncology, Royal College of Surgeons in Ireland, Beaumont Hospital, Beaumont, Ireland 2 Texas Oncology-Memorial Hermann Memorial City, US Oncology Research, Houston, TX, USA 3 George Mason University, Manassas, VA, USA 4 Baylor-Sammons Cancer Center, Texas Oncology, US Oncology, Dallas, TX, USA 5 Data Science Centre, Royal College of Surgeons in Ireland, Dublin, Ireland * These authors have contributed equally to this work Correspondence to: Bryan T. Hennessy, email: bryanhennessy74@gmail.com Keywords: MEK inhibitor, HER2-positive breast cancer, acquired resistance to HER2-targeted therapies, reverse phase protein array Received: August 25, 2016 Accepted: June 02, 2017 Published: July 22, 2017 ABSTRACT Purpose: The MEK/MAPK pathway is commonly activated in HER2-positive breast cancer, but little investigation of targeting this pathway has been undertaken. Here we present the results of an in vitro preclinical evaluation of refametinib, an allosteric MEK1/2 inhibitor, in HER2-positive breast cancer cell lines including models of acquired resistance to trastuzumab or lapatinib. Methods: A panel of HER2-positive breast cancer cells were profiled for mutational status and also for anti-proliferative response to refametinib alone and in combination with the PI3K inhibitor (PI3Ki) copanlisib and the HER2-targeted therapies trastuzumab and lapatinib. Reverse phase protein array (RPPA) was used to determine the effect of refametinib alone and in combination with PI3Ki and HER2-inhibitors on expression and phosphorylation of proteins in the PI3K/AKT and MEK/MAPK pathways. We validated our proteomic in vitro findings by utilising RPPA analysis of patients who received either trastuzumab, lapatinib or the combination of both drugs in the NCT00524303/LPT109096 clinical trial. Results: Refametinib has anti-proliferative effects when used alone in 2/3 parental HER2-positive breast cancer cell lines (HCC1954, BT474), along with 3 models of these 2 cell lines with acquired trastuzumab or lapatinib resistance (6 cell lines tested). Refametinib treatment led to complete inhibition of MAPK signalling. In HCC1954, the most refametinib-sensitive cell line (IC 50 = 397 nM), lapatinib treatment inhibits phosphorylation of MEK and MAPK but activates AKT phosphorylation, in contrast to the other 2 parental cell lines tested (BT474-P, SKBR3-P), suggesting that HER2 may directly activate MEK/MAPK and not PI3K/AKT in HCC1954 cells but not in the other 2 cell lines, perhaps explaining the refametinib-sensitivity of this cell line. Using RPPA data from patients who received either trastuzumab, lapatinib or the combination of both drugs together with chemotherapy in the NCT00524303 clinical trial, we found that 18% (n=38) of tumours had decreased MAPK and increased AKT phosphorylation 14 days after treatment with HER2-targeted therapies. The combination of MEK inhibition (MEKi) with refametinib and copanlisib led to synergistic inhibition of growth in 4/6 cell lines tested (CI @ED 75 = 0.39-0.75), whilst the combinations of lapatinib and refametinib led to synergistic inhibition of growth in 3/6 cell lines (CI @ED 75 = 0.39-0.80). Conclusion: Refametinib alone or in combination with copanlisib or lapatinib could represent an improved treatment strategy for some patients with HER2-positive breast cancer, and should be considered for clinical trial evaluation. The direct down-regulation of MEK/MAPK but not AKT signalling by HER2 inhibition (e.g. by lapatinib or trastuzumab), which we demonstrate occurs in 18% of HER2-positive breast cancers may serve as a potential biomarker of responsiveness to the MEK inhibitor refametinib.

Background: Combining bevacizumab and chemotherapy produced superior response rates compared with chemotherapy alone in metastatic breast cancer. As bevacizumab may cause hypertension (HTN) and increase the risk of cardiac failure, we performed a pilot study to evaluate the feasibility and toxicity of a non-anthracycline-containing combination of docetaxel with cyclophosphamide and bevacizumab in early stage breast cancer patients. Methods: Treatment consisted of four 3-weekly cycles of docetaxel and cyclophosphamide (75/600 mg/m2). Bevacizumab was administered 15 mg/kg intravenously on day 1, and then every 3 weeks to a total of 18 cycles of treatment. Serum biomarker concentrations of vascular endothelial growth factor (VEGF), cardiac troponin-I (cTnI), myeloperoxidase (MPO), and placental growth factor (PlGF) were quantified using enzyme-linked immunosorbent assay (ELISA) in 62 patients at baseline and whilst on treatment to determine their utility as biomarkers of cardiotoxicity, indicated by left ventricular ejection fraction (LVEF). Results: A total of 106 patients were accrued in nine sites. Median follow up was 65 months (1–72 months). Seventeen protocol-defined relapse events were observed, accounting for an overall disease-free survival (DFS) rate of 84%. The DFS rates for hormone receptor positive (HR+) and triple-negative (TN) patients were 95% versus 43%, respectively. The median time to relapse was 25 (12–54) months in TN patients versus 38 (22–71) months in HR+ patients. There have been 13 deaths related to breast cancer . The overall survival (OS) rate was 88%. The 5-year OS rate in HR+ versus TN was 95% versus 57%. None of the measured biomarkers predicted the development of cardiotoxicity. Conclusions: We observed a low relapse rate in node-positive, HR+ patients; however, results in TN breast cancer were less encouraging. Given the negative results of three large phase III trials, it is unlikely that this approach will be investigated further. Trial Registration ClinicalTrials.gov Identifier: NCT00911716.

In pre-clinical studies, triple-negative breast cancer (TNBC) cells have demonstrated sensitivity to the multi-targeted kinase inhibitor dasatinib, however, clinical trials with single-agent dasatinib showed limited efficacy in unselected populations of breast cancer, including TNBC. To study potential mechanisms of resistance to dasatinib in TNBC, we established a cell line model of acquired dasatinib resistance (231-DasB). Following an approximately three-month exposure to incrementally increasing concentrations of dasatinib (200 nM to 500 nM) dasatinib, 231-DasB cells were resistant to the agent with a dasatinib IC50 value greater than 5 &mu, M compared to 0.04 &plusmn, 0.001 &micro, M in the parental MDA-MB-231 cells. 231-DasB cells also showed resistance (2.2-fold) to the Src kinase inhibitor PD180970. Treatment of 231-DasB cells with dasatinib did not inhibit phosphorylation of Src kinase. The 231-DasB cells also had significantly increased levels of p-Met compared to the parental MDA-MB-231 cells, as measured by luminex, and resistant cells demonstrated a significant increase in sensitivity to the c-Met inhibitor, CpdA, with an IC50 value of 1.4 &plusmn, 0.5 &micro, M compared to an IC50 of 6.8 &plusmn, 0.2 &micro, M in the parental MDA-MB-231 cells. Treatment with CpdA decreased p-Met and p-Src in both 231-DasB and MDA-MB-231 cells. Combined treatment with dasatinib and CpdA significantly inhibited the growth of MDA-MB-231 parental cells and prevented the emergence of dasatinib resistance. If these in vitro findings can be extrapolated to human cancer treatment, combined treatment with dasatinib and a c-Met inhibitor may block the development of acquired resistance and improve response rates to dasatinib treatment in TNBC.

Background:Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited therapeutic options. Epidermal growth factor receptor (EGFR) has been shown to be over-expressed in TNBC and represents a rational treatment target.Methods:We examined single agent and combination effects for afatinib and dasatinib in TNBC. We then determined IC50and combination index values using Calcusyn. Functional analysis of single and combination treatments was performed using reverse phase protein array and cell cycle analysis. Finally, we determined the anticancer effects of the combination in vivo.Results:A total of 14 TNBC cell lines responded to afatinib with IC50values ranging from 0.008 to 5.0 µM. Three cell lines, belonging to the basal-like subtype of TNBC, were sensitive to afatinib. The addition of afatinib enhanced response to the five other targeted therapies in HCC1937 and HDQP1 cells. The combination of afatinib with dasatinib caused the greatest growth inhibition in both cell lines. The afatinib/dasatinib combination was synergistic and/or additive in 13/14 TNBC cell lines. Combined afatinib/dasatinib treatment induced G1 cell cycle arrest. Reverse phase protein array results showed the afatinib/dasatinib combination resulted in efficient inhibition of both pERK(T202/T204) and pAkt(S473) signalling in BT20 cells, which was associated with the greatest antiproliferative effects. High baseline levels of pSrc(Y416) and pMAPK(p38) correlated with sensitivity to afatinib, whereas low levels of B-cell lymphoma 2 (Bcl2) and mammalian target of rapamycin (mTOR) correlated with synergistic growth inhibition by combined afatinib and dasatinib treatment. In vivo, the combination treatment inhibited tumour growth in a HCC1806 xenograft model.Conclusions:We demonstrate that afatinib combined with dasatinib has potential clinical activity in TNBC but warrants further preclinical investigation.

Introduction: The phase II neoadjuvant clinical trial ICORG10-05 (NCT01485926) compared chemotherapy (docetaxel, carboplatin) in combination with trastuzumab, trastuzumab and lapatinib or lapatinib alone in HER2+ breast cancer patients. Lapatinib is a dual HER2/EGFR tyrosine kinase inhibitor. Trastuzumab is a monoclonal antibody which targets HER2 and is capable of eliciting antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by immune effector cells. Studies have shown that tumour infiltrating lymphocyte (TIL) levels and the cytotoxic capacity of circulating immune cells can correlate with response to trastuzumab. Less is known regarding the effect of chemotherapy on the immune response to trastuzumab. This study examines the effects of neo-adjuvant chemotherapy on the phenotype and cytotoxic capacity of peripheral blood mononuclear cells (PBMCs) of HER2+ breast cancer patients. Methods: Matched blood samples were taken pre- and post-neoadjuvant treatment. PBMCs were isolated by density centrifugation and frozen. Direct PBMC-mediated cytotoxicity and trastuzumab-ADCC levels were assessed against the HER2+ breast cancer cell line (SKBR3) and non-MHC class I-restricted leukemic cell line (K562) using a FACS based assay (n=19 matched pre-and post-treatment samples). The immunophenotype of 17 pre-treatment and 13 post-treatment samples was also determined using the DURAClone IM antibody panel (CD16, CD56, CD19, CD14, CD4, CD8, CD3, CD45) on the CytoFLEX platform. Analysis of both data sets was performed using FCS Express and MedCalc. Results: The cytotoxic capacity of PBMCs was reduced following neo-adjuvant chemotherapy. Direct cytotoxicity elicited against the K562 cell line was reduced post-treatment (p=0.04). ADCC elicited against the SKBR3 cell line was also decreased in post-treatment samples (p=0.009). A decrease in cytotoxic capacity was associated with alterations in the immunophenotype of the post-treatment PBMC samples. The proportion of CD56+ NK cells (p=0.001), CD19+ B cells (p=0.001) and CD14+ monocytes (p=0.03) decreased significantly post-treatment. CD56+ NK cells are hypothesised to be the main ADCC effector cell population in peripheral blood. Interestingly, the proportion of CD3+ T cells (p=0.002), including CD4+ (p=0.007) and CD8+ (p=0.035) T cell populations, were increased post-treatment. Conclusion: These results indicate that neo-adjuvant chemotherapy reduces the cytotoxic capacity of circulating immune cells and alters the proportion of adaptive and innate immune cell subsets. These effects warrant further investigation particularly with the emergence of immunotherapies in HER2+ breast cancer. Citation Format: Nicola Gaynor, Alfonso Blanco, Alexandra Canonici, Alexander J. Eustace, Martina McDermott, Barry Moran, Jean M. Fletcher, Norma O'Donovan, John Crown, Denis M. Collins. The effect of neo-adjuvant chemotherapy on immune cell phenotype and cytotoxic capacity in HER2+ breast cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 951.

Introduction: Neratinib is an irreversible, small molecule pan-HER tyrosine kinase inhibitor that has shown clinical activity against HER2-amplified (HER2+) and HER2-mutated breast cancer. However, there are limited data on potential mechanisms of resistance to neratinib. This study aimed to develop HER2+ breast cancer cell line models of acquired neratinib resistance, and to elucidate the effects of acquired resistance on HER family signalling and drug response. As neratinib has activity in both estrogen receptor positive (ER+) and ER- HER2+ breast cancer, neratinib-resistant cell lines were generated and characterised from breast cancer cell lines of both subtypes. Methods: HER2+, ER- HCC1569 cells were continuously exposed to 150 nM neratinib for six months to generate the neratinib-resistant HCC1569 (HCC1569-N) cell line. HER2+ ER+ BT474 cells were continuously exposed to escalating doses of 5-80 nM neratinib for six months to generate the BT474-N cell line. Sensitivity to HER2-targeted therapies (neratinib, lapatinib and trastuzumab) and chemotherapeutics (5’dFUR, carboplatin, and vinorelbine) were assessed by 5-day acid phosphatase based proliferation assay. IC50 and combination index (CI) values were calculated using CalcuSyn software. Total and phosphorylated levels of HER2, EGFR, ERK, and Src were assessed by Western blotting following neratinib treatment (150 nM), to determine changes in HER2 signalling. Results: The HCC1569-N and BT474-N cells had a 50.3-fold and 105-fold increase in neratinib resistance. In addition, both cell lines developed cross-resistance to lapatinib and were resistant at clinically relevant concentrations (> 1 µM). HCC1569-Par cells are innately resistant to trastuzumab, which was not altered in HCC1569-N cells. However, neratinib resistance also caused resistance to trastuzumab in the BT474-N cell line, compared to its trastuzumab-sensitive parental cell line (Table 1). The HCC1569-N cell line did not show any significant difference in sensitivity to 5’dFUR, carboplatin, or vinorelbine compared to parental HCC1569, but the addition of vinorelbine to neratinib was synergistic (CI value = 0.28 ± 0.19). HCC1569-N cells displayed no changes in total protein or phosphorylated levels of HER2 or EGFR compared to parental cells. Additionally, neratinib treatment still resulted in inhibition of HER2, EGFR, and downstream ERK signalling, indicating a lack of reliance on HER2 signalling in HCC1569-N cells. Phosphorylated Src levels were elevated (p = 6.3 × 10−4) in HCC1569-N compared to parental cells. Conclusions: Neratinib resistance in HCC1569-N and BT474-N cells confers cross-resistance to other HER2-targeted therapies. Increased Src activity may be implicated in the neratinib-resistant HER2+ ER- phenotype. Cell line10 µg/mL Trastuzumab (%Growth)NeratinibIC50 valueLapatinibIC50 valueHCC1569-Par97.6 ± 2.57.8 ± 5.8653.0 ± 144.0HCC1569-N92.7 ± 2.9393.0 ± 77.1> 1000BT474-Par44.7 ± 1.81.2 ± 0.323.9 ± 19.6BT474-N97.8 ± 3.3126.8 ± 53.5> 1000 Citation Format: Neil T Conlon, Alacoque Browne, Laura Breen, Alex J Eustace, John Crown, Denis M Collins. The development and characterization of novel HER2-positive breast cancer models of acquired neratinib resistance [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-03-20.

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.

Background Insulin Receptor (INSR) signalling may play a role in resistance to insulin-like growth factor receptor (IGF1R) therapy. Although IGF1R is frequently expressed in triple negative breast cancer (TNBC), we found that an IGF1R monoclonal antibody did not inhibit the growth of TNBC cells. Thus, we hypothesised that dual targeting of IGF1R and INSR may be more effective in TNBC. Methods Relative mRNA expression of INSR and IGFIR were analysed across different subtypes of breast cancer using the BreastMark database. INSR-A, INSR-B, IGF1R and insulin like growth factor 2 (IGFII) was quantified by qRT-PCR in a panel of 11 TNBC cell lines. Proliferation assays were performed on TNBC cell lines treated with the dual IR/INSR inhibitor Linsitinib (OSI-906). Combinations were performed by testing Linsitinib with Xentuzumab (BI-836845), cisplatin, docetaxel, or the PI3K inhibitor Dactolisib (NVP-BEZ235). We also tested the anti-proliferative effect of Linsitinib in combination with Xentuzumab when stimulated with IGF-I and IGF-II following serum-starvation for 24 hours. Finally, Linsitinib was tested in HCC-1143 xenografts to assess the effect of low-dose treatment on tumour formation. Results No significant differences were observed between the basal-like and non-basal-like cell lines for IR or IGF1R mRNA expression. However, IGF-II mRNA was more frequently detected in non-basal-like (80%, 4/5) compared to basal-like cell lines (20%, 1/5). Only three of the 12 cell lines tested showed sensitivity to Linsitinib with IC50 values less than 10 µM. The two most sensitive cell lines, HDQ-P1 and HCC1143, expressed detectable levels of IGF1R, INSR-A and INSR-B mRNAs. Although Linsitinib blocked IGF-I and IGF-II stimulated proliferation in both HCC1143 and HDQ-P1 cells, addition of Linsitinib to either chemotherapy or Dactolisib did not enhance therapeutic response. Linisitinib did not reduce tumour formation or tumour growth in an in vivo TNBC cell-line xenograft model. Conclusions Although IGF1R has been shown to be frequently expressed in TNBC, our in vitro and in vivo data suggest that it may not be a good therapeutic target in the TNBC subtype. Legal entity responsible for the study The authors. Funding Boehringer Ingleheim. Disclosure N. O’Donovan: Research grant / Funding (institution): Boehringer Ingleheim. J. Crown: Honoraria (self), Speaker Bureau / Expert testimony, Research grant / Funding (institution): Boehringer Ingleheim; Shareholder / Stockholder / Stock options, Full / Part-time employment: OncoMark Limited; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Eisai; Honoraria (self): Amgen; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Puma Biotechnology; Honoraria (self), Advisory / Consultancy: Seattle Genetics; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: Pfizer; Honoraria (self), Advisory / Consultancy: Vertex; Honoraria (self), Speaker Bureau / Expert testimony: Genomic Health; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Roche; Honoraria (self), Travel / Accommodation / Expenses: MSD Oncology; Travel / Accommodation / Expenses: AstraZeneca; Travel / Accommodation / Expenses: Abbvie. All other authors have declared no conflicts of interest.

Despite recent advances in targeted therapies and immunotherapies metastatic melanoma remains only rarely curable. The objective of the present study was to identify novel therapeutic targets for metastatic melanoma. A library of 160 well-characterised and potent protein kinase inhibitors was screened in the BRAF mutant cell line Sk-Mel-28, and the NRAS mutant Sk-Mel-2, using proliferation assays. Of the 160 inhibitors tested, 20 achieved >50% growth inhibition in both cell lines. Six of the 20 were cyclin dependent kinase (CDK) inhibitors, including two CDK4 inhibitors. Fascaplysin, a synthetic CDK4 inhibitor, was further tested in 8 melanoma cell lines. The concentration of fascaplysin required to inhibit growth by 50% (IC50 value) ranged from 0.03 to 0.22 μM. Fascaplysin also inhibited clonogenic growth and induced apoptosis. Sensitivity to PD0332991, a therapeutic CDK4/6 inhibitor was also evaluated in the melanoma cell lines. PD0332991 IC50 values ranged from 0.13 to 2.29 μM. Similar to fascaplysin, PD0332991 inhibited clonogenic growth of melanoma cells and induced apoptosis. Higher levels of CDK4 protein correlated with lower sensitivity to PD0332991 in the cell lines. Combined treatment with PD0332991 and the BRAF inhibitor PLX4032, showed additive anti-proliferative effects in the BRAF mutant cell line Malme-3M. In summary, targeting CDK4 inhibits growth and induces apoptosis in melanoma cells in vitro, suggesting that CDK4 may be a rational therapeutic target for metastatic melanoma.

Background The Cancer Genome Atlas analysis revealed that somatic EGFR, receptor tyrosine-protein kinase erbB-2 (ERBB2), Erb-B2 receptor tyrosine kinase 3 (ERBB3) and Erb-B2 receptor tyrosine kinase 4 (ERBB4) gene mutations (ERBB family mutations) occur alone or co-occur with somatic mutations in the gene encoding the phosphatidylinositol 3-kinase (PI3K) catalytic subunit (PIK3CA) in 19% of human epidermal growth factor receptor 2 (HER2)-positive breast cancers. Because ERBB family mutations can activate the PI3K/AKT pathway and likely have similar canonical signalling effects to PI3K pathway mutations, we investigated their combined impact on response to neoadjuvant HER2-targeted therapies. Methods Baseline tumour biopsies were available from 74 patients with HER2-positive breast cancer who were enrolled in the phase II TCHL neoadjuvant study (ICORG 10-05) assessing TCH (docetaxel, carboplatin, trastuzumab) (n = 38) versus TCL (docetaxel, carboplatin, lapatinib) (n = 10) versus TCHL (docetaxel, carboplatin, trastuzumab, lapatinib) (n = 40), each for six cycles. Activating mutations in PIK3CA and ERBB family genes were identified using mass spectrometry-based genotyping. Phosphatase and tensin homolog (PTEN) expression was assessed by immunohistochemistry. Results PIK3CA and/or ERBB family mutations were detected in 23 (31.1%) tumour samples tested, whereas PTEN expression was low in 31.1% of cases tested. Mutation frequency was similar in each treatment arm (31.3% in TCH arm, 30% in TCL arm and 31.3% in TCHL arm) and was not influenced by oestrogen receptor (ER) status (27.6% in ER-negative patients, 33.3% in ER-positive patients) or progesterone receptor (PR) status (32.6% in PR-negative patients, 29% in PR-positive patients). There was no significant difference in pathological complete response (pCR) rates between 47 patients with wild-type (WT) tumours and 22 patients whose tumours carried mutations (in either PIK3CA or ERBB family genes) (42.5% vs. 54.5%; p = 0.439). Similarly, there was no significant difference in pCR rates between patients with PIK3CA/ERBB family mutated/PTEN-low (i.e., PI3K-activated) tumours and patients without PI3K activation (50% vs. 44%; p = 0.769). However, in the TCHL (but not the TCH) group, the pCR rate was higher for 9 patients with PIK3CA/ERBB family mutated tumours than for 20 patients with PIK3CA/ERBB family WT tumours (77.8% vs. 35%; p = 0.05). Conclusions Our results indicate that patients who receive neoadjuvant TCHL and have PIK3CA/ERBB family mutated tumours may be more likely to have a pCR than patients with WT tumours. Trial registration ClinicalTrials.gov, NCT01485926. Registered on 2 December 2011. Electronic supplementary material The online version of this article (doi:10.1186/s13058-017-0883-9) contains supplementary material, which is available to authorized users.

Approximately 20 % of human breast cancers (BC) overexpress HER2 protein, and HER2-positivity is associated with a worse prognosis. Although HER2-targeted therapies have significantly improved outcomes for HER2-positive BC patients, resistance to trastuzumab-based therapy remains a clinical problem. In order to better understand resistance to HER2-targeted therapies in HER2-positive BC, it is necessary to examine HER family signalling as a whole. An extensive literature search was carried out to critically assess the current knowledge of HER family signalling in HER2-positive BC and response to HER2-targeted therapy. Known mechanisms of trastuzumab resistance include reduced receptor-antibody binding (MUC4, p95HER2), increased signalling through alternative HER family receptor tyrosine kinases (RTK), altered intracellular signalling involving loss of PTEN, reduced p27kip1, or increased PI3K/AKT activity and altered signalling via non-HER family RTKs such as IGF1R. Emerging strategies to circumvent resistance to HER2-targeted therapies in HER2-positive BC include co-targeting HER2/PI3K, pan-HER family inhibition, and novel therapies such as T-DM1. There is evidence that immunity plays a key role in the efficacy of HER-targeted therapy, and efforts are being made to exploit the immune system in order to improve the efficacy of current anti-HER therapies. With our rapidly expanding understanding of HER2 signalling mechanisms along with the repertoire of HER family and other targeted therapies, it is likely that the near future holds further dramatic improvements to the prognosis of women with HER2-positive BC.

Dasatinib has anti-proliferative and anti-invasive effects in melanoma cell lines. However clinical trials have shown modest activity for dasatinib in metastatic melanoma. Although dasatinib targets SRC kinase, neither expression nor phosphorylation of SRC appears to predict response to dasatinib. Identification of predictive biomarkers for dasatinib may facilitate selection of melanoma patients who are more likely to respond to dasatinib. We correlated the anti-proliferative effects of dasatinib in 8 melanoma cell lines with expression of a previously identified 6-gene biomarker panel. We examined the relationship between response to dasatinib and expression of each gene at both the mRNA and protein level. Dasatinib inhibited growth in 3 of the 8 cell lines tested. mRNA expression of the panel of 6 biomarkers did not correlate with response, whilst elevated protein expression of ANXA1, CAV-1 and EphA2 correlated significantly with response to dasatinib in the panel of cell lines. Expression of ANXA1, CAV-1 and EphA2 were analysed in 124 melanoma samples by immunohistochemistry. ANXA1 protein was detected in 81 % (97/120) of tumours, CAV-1 in 44 % (54/122) of tumours and EphA2 in 74 % (90/121) of tumours. Thirty one % (35/113) of tumours tested expressed all three markers and 19 % (21/112) had moderate or strong expression of ANXA1, CAV-1 and EphA2. Seventeen percent (19/112) of melanoma samples were positive for SRC kinase expression, combined with high expression of ANXA1, CAV-1 and EphA2. This subgroup may represent a population of melanoma patients who would be more likely to derive clinical benefit from dasatinib treatment.

Treatment with tyrosine kinase inhibitors (TKIs) including trastuzumab has revolutionized the management of HER2-positive breast cancer. Recent evaluation of clinical trial data suggests that a subset of HER2/ER double-positive cancers may not receive significant benefit from the TKI therapy. Here we investigate the cross talk between HER2 and ER in breast cancer and monitor the effect of trastuzumab on the tyrosine kinase effector transcription factor Myc. In HER2-positive breast cancer patients treated with neoadjuvant trastuzumab, steroid receptor-negative status (ER and PR negative) of pre-treatment biopsies predicted pathological complete response (pCR) (n=31 patients, P=0.0486), whereas elevated Myc protein inversely associated with pCR (P=0.0446). Liquid chromatography mass spectrometry identified the corepressor SMRT as a novel Myc-interacting protein. Trastuzumab treatment enhanced Myc-SMRT interactions in HER2-overexpressing breast cancer cells (LCC1) and inhibited expression of the Myc target gene survivin. In HER2-low, ER-positive steroid-dominant cells (MCF7), trastuzumab therapy repressed Myc-SMRT interactions and upregulated survivin expression. Trastuzumab treatment induced ER-CBP interactions, enhanced ER transcriptional activity and upregulated expression of the ER target gene pS2. The absence of pS2 expression in pre-treatment biopsies predicted pCR to neoadjuvant trastuzumab in breast cancer patients (n=25, P=0.0089) and pS2 expression associated with residual cancer burden (P=0.0196). Furthermore, metastatic tissues from patients who had failed trastuzumab therapy were pS2 positive. In HER2-overexpressing cells, trastuzumab treatment can repress Myc transcriptional activity and clinical response is favorable. However, with co-expression of the steroid pathway, this inhibition is lost and response to treatment is often poor.

Background Neratinib (NER) is an irreversible pan-HER kinase inhibitor with demonstrated clinical activity for HER2-positive and HER2-mutated breast cancers (BC). Mechanisms of resistance to NER are poorly understood. The Src/Abl inhibitor dasatinib (DAS) has shown ability to overcome resistance to HER2-targeted agents such as lapatinib (LAP) and TRAS in vitro. This pre-clinical study investigated the efficacy of DAS in combination with NER to overcome or prevent NER resistance in BC models. Methods Anti-proliferative effects of NER, LAP, TRAS, DAS, and NER plus DAS were assessed in five NER-resistant HER2+ BC cell lines (HCC1954-N, HCC1569-N, EFM192A-N, BT474-N, and SKBR3-N) by acid phosphatase assay. IC50values and Combination index (CI) values were calculated to determine synergy (CI 150 nM NER. Apoptosis induction was assessed by Caspase 3/7-Glo assay. Reverse phase protein array was used to determine changes in key signalling pathways in HCC1954-N cells. BC cells were treated twice weekly with NER and/or DAS and crystal violet stained when confluent to examine resistance development. Results All NER resistant cell lines examined had significantly reduced response to NER (11-83 fold increase in IC50values versus parental cells), as well as LAP and TRAS, compared to their parental cells.The combination of NER and DAS displayed synergy in all five NER resistant cell lines (CI = 0.1-0.6). NER plus DAS caused a strong induction of apoptosis in the HCC1954-N cells (p = 0.015). NER/DAS treatment caused changes in 23 phospho- or total protein levels, including suppression of Akt, MAPK, Src, p38 and AMPK signalling. NER alone (9 phospho- and 1 total proteins) and DAS alone (3 phospho- and 3 total proteins) altered fewer targets. The addition of DAS to NER prevented the emergence of NER resistance in parental HCC1954 and HCC1569 cells. Conclusions This study provides pre-clinical rationale for the combination of NER and DAS in NER-resistant HER2+ BC and shows that this combination warrants further investigation. Legal entity responsible for the study The authors. Funding Puma Biotechnology. Disclosure N. Conlon: Research grant/Funding (institution): Puma Biotechnology. J. Crown: Full/Part-time employment: OncoMark; Honoraria (self), Advisory/Consultancy, Speaker Bureau/Expert testimony, Research grant/Funding (institution): Eisai; Honoraria (self): Amgen; Honoraria (self), Advisory/Consultancy, Research grant/Funding (institution): Puma Biotechnology; Honoraria (self): Seattle Genetics; Honoraria (self), Advisory/Consultancy, Speaker Bureau/Expert testimony, Travel/Accommodation/Expenses: Pfizer; Honoraria (self), Advisory/Consultancy: Vertex; Honoraria (self), Advisory/Consultancy, Research grant/Funding (institution), Travel/Accommodation/Expenses: Roche; Honoraria (self), Travel/Accommodation/Expenses: MSD Oncology; Travel/Accommodation/Expenses: AstraZeneca; Travel/Accommodation/Expenses: Abbvie; Honoraria (self), Advisory/Consultancy, Research grant/Funding (institution): Boehringer Ingelheim; Honoraria (self), Speaker Bureau/Expert testimony: Genomic Health. D.M. Collins: Research grant/Funding (institution): Puma Biotechnology; Research grant/Funding (institution): Roche. All other authors have declared no conflicts of interest.