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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

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.

Triple Negative Breast Cancer (TNBC), a subtype of breast cancer, has fewer successful therapeutic therapies than other types of breast cancer. Insulin-like growth factor receptor 1 (IGF1R) and the Insulin receptor (IR) are associated with poor outcomes in TNBC. Targeting IGF1R has failed clinically. We aimed to test if inhibiting both IR/IGF1R was a rationale therapeutic approach to treat TNBC. We showed that despite IGF1R and IR being expressed in TNBC, their expression is not associated with a negative survival outcome. Furthermore, targeting both IR/IGF1R with inhibitors in multiple TNBC cell lines did not inhibit cell growth. Linsitinib, a small molecule inhibitor of both IGF1R and IR, did not block tumour formation and had no effect on tumour growth in vivo. Cumulatively these data suggest that while IGF1R and IR are expressed in TNBC, they are not good therapeutic targets. A potential reason for the limited anti-cancer impact when IR/IGF1R was targeted may be because multiple signalling pathways are altered in TNBC. Therefore, targeting individual signalling pathways may not be sufficient to inhibit cancer growth.

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: 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: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 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.

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.

Background: HER2-positive breast cancer (BC) accounts for approximately 15% of all BC. HER2-targeted therapies, such as trastuzumab and lapatinib, have significantly improved the outcome for these patients. However, HER2-targeted therapy resistance is a common clinical issue. We have previously shown that protein phosphatase 2A (PP2A) plays a role in mediating acquired lapatinib resistance in HER2-positive BC and that response to lapatinib is enhancedin vitroby the lab-grade PP2A inhibitor, okadaic acid. The aim of this study was to examine the in vitro and in vivo efficacy of LB-100, a PP2A inhibitor that has completed phase I clinical testing (NCT01837667), in models of HER2-positive BC with acquired resistance to lapatinib. Methods: HER2-positiveSKBR3 and HCC1954 BC cell lines were treated with 250 nM or 1 μM lapatinib, respectively, for 6 months to generate lapatinib-resistant SKBR3-L and HCC1954-L cell lines. In vitro sensitivity to lapatinib and LB-100 was assessed by 2D acid phosphatase assay. Combination index (CI) values were generated to identify synergistic combinations. Propidium iodide staining was used to determine cell cycle arrest and apoptosis. In order to examine the in vivo efficacy of LB-100, HCC1954-L cells were implanted into the mammary fat pad of BALB/c nude mice and treated with vehicle, lapatinib, LB-100, or lapatinib plus LB-100. To examine the prevention of the development of lapatinib resistance, SKBR3 and HCC1954 cells were treated twice weekly with lapatinib, LB-100 or the combination and stained with crystal violet when confluent. Results: SKBR3-L and HCC1954-L cells were resistant to lapatinib at clinically relevant concentrations (IC50values = 2.37 ± 0.58 μM and 1.67 ± 0.34 μM). This represents a 46- and 5.2-fold decrease in lapatinib sensitivity. LB-100 had a greater anti-proliferative effect in the lapatinib-resistant SKBR3-L and HCC1954-L cell lines compared to their respective parental cell lines (IC50values = 2.12 ± 0.2 μM v 5.38 ± 0.6 μM, and 2.31 ± 0.19 μM v 5.32 ± 0.82 μM, respectively). LB-100 overcame lapatinib resistance in both models, as lapatinib plus LB-100 was synergistic in both cell lines (CI values = 0.56 ± 0.13 and 0.68 ±0.26). LB-100 caused cell death through the induction of apoptosis in SKBR3- L (p = 0.019) and HCC1954-L (p = 0.046) and the addition of lapatinib to LB-100 increased apoptotic induction in HCC1954-L cells (p=0.046).Lapatinib plus LB-100 was well tolerated in vivo. The HCC1954-L cell line maintained resistance to lapatinib in vivo and the combination of lapatinib and LB-100 significantly reduced HCC1954-L tumour volume compared to all other treatment arms (p = 0.0006). Interestingly, in vitro short-term resistance assays showed that the addition of LB-100 to lapatinib could also block the emergence of lapatinib resistance in both parental SKBR3 and HCC1954 cell lines. Conclusions: This study indicates that LB-100 has in vitro and in vivo efficacy against lapatinib-resistant HER2-positive BC cell line models and justifies further investigation into its potential to circumvent or prevent lapatinib resistance in HER2-positive BC. Citation Format: Conlon N, McDermott M, Browne B, Roche S, O'Neill F, Meiller J, Browne A, Eustace A, Collins DM, O'Donovan N, Crown J. Pre-clinical investigation of PP2A inhibitor LB-100 in overcoming and preventing lapatinib resistance in HER2-positive breast cancer [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 P5-03-02.

// Sinead Toomey 1, * , Stephen F. Madden 2, * , Simon J. Furney 3 , Yue Fan 4 , Mark McCormack 5 , Carragh Stapleton 5 , Mattia Cremona 1 , Gianpiero L. Cavalleri 5 , Malgorzata Milewska 1 , Naomi Elster 1 , Aoife Carr 1 , Joanna Fay 6 , Elaine W. Kay 6 , Susan Kennedy 7 , John Crown 7 , William M. Gallagher 4 , Bryan T. Hennessy 1, 8, ** , Alex J. Eustace 1, ** 1 Medical Oncology Group, Department of Molecular Medicine, Royal College of Surgeons in Ireland, Ireland 2 Population Health Sciences Division, Royal College of Surgeons in Ireland, Ireland 3 School of Medicine, University College Dublin, Ireland 4 University College Dublin, UCD School of Biomolecular and Biomedical Science, Conway Institute of Biomolecular and Biomedical Research, Ireland 5 Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, Ireland 6 Department of Histopathology, Beaumont Hospital, Dublin, Ireland 7 St Vincent’s University Hospital, Dublin, Ireland 8 Department of Oncology, Beaumont Hospital, Dublin, Ireland * These authors contributed equally to this work ** Joint senior authors Correspondence to: Alex J. Eustace, email: alexeustace@rcsi.ie Keywords: ERBB-family germline mutations, single nucleotide polymorphisms, high depth sequencing, survival impact Received: May 03, 2016 Accepted: October 12, 2016 Published: October 20, 2016 ABSTRACT Background: Trastuzumab 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. Results: We 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 methods: Using 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. Conclusions: The 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.

As there is now evidence that switching clinical nonresponders early in primary systemic therapy to alternate treatment regimens can enhance survival in some breast cancer patients, the need for a robust intermediate endpoint that can guide treatment response across all tumor subtypes is urgent. Recently, chemotherapy drugs have been shown to induce a decrease in RNA quality in tumor cells from breast cancer biopsies in some patients at midtherapy, and that this has been associated with subsequent achievement of pathological complete response (pCR). The decrease in RNA quality has been shown to be associated with RNA disruption; aberrant RNA bands visualized by RNA electrophoresis have been associated with subsequent tumor cell death. The objectives of these studies are to show that a new assay based on induction of RNA disruption in tumor cells by chemotherapy can stratify at midtherapy, pCR responders from non-pCR responders irrespective of clinical response and to present early evidence that clinically useful RNA disruption can be detected as early as 14 days after initiation of treatment.RNA disruption in tumor cells was quantified by analysis of the RNA electrophoresis banding pattern and expressed as an RNA disruption index (RDI). To develop the RNA disruption assay (RDA), RDI was correlated with clinical outcome (pCR) from the NCIC-CTG MA.22 breast cancer clinical trial (ClinicalTrials.gov NCT00066443). RDA Zones were established by stratifying patients using RDI values into Zone 1, Zone 2, and Zone 3. Zone 3 included seven out of eight pCR responders, whereas Zone 1 contained no pCR responders. An intermediate zone (Zone 2) was established which contained one pCR. Subsequently, to determine early drug response, RNA disruption was examined by RDI after 14 days exposure to trastuzumab, zoledronic acid, or letrozole + cyclophosphamide ± sorafenib therapy.In MA.22, RDA stratified 23 of 85 patients in Zone 1 as pCR nonresponders, 24 patients in Zone 2, an intermediate zone, and 38 patients in Zone 3, pCR responders and non-pCR patients who share RDI comparable to those achieving pCR. In the early response studies, after 14 days exposure to chemotherapy, some RNA disruption as measured by RDI elevation could be detected in 3/12 trastuzumab, 7/15 zoledronic acid, 5/29 letrozole + cyclophosphamide, and 5/23 letrozole + cyclophosphamide + sorafenib patients.RDA is a novel intermediate endpoint that has promise for clinical utility for breast cancers early in response-guided primary systemic therapy.

Insulin-like growth factor-1 receptor (IGF1R) signalling is implicated in resistance to trastuzumab. However, the benefit of co-targeting HER2 and IGF1R has not been extensively studied, and the relationship between activated IGF1R and clinical response to trastuzumab has not been reported. This study aimed to evaluate the combination of trastuzumab with IGF1R tyrosine kinase inhibitors (TKIs) in a panel of HER2-positive breast cancer cell lines, and to examine the relationship between IGF1R expression and activation and response to trastuzumab in HER2-positive breast cancer patients. The anti-proliferative effects of trastuzumab combined with IGF1R TKIs BMS-536924 or NVP-AEW541 were measured in nine HER2-positive cell lines. IGF1R and phosphorylated IGF1R/insulin receptor (pIGF1R/IR) were measured by immunohistochemistry in 160 tumour samples from trastuzumab-treated patients (ICORG 06-22). The HER2-positive cell lines displayed varying sensitivity to IGF1R TKIs alone (IC(50)s: 0.7 to10 μM). However, when combined with trastuzumab, a significantly enhanced effect was observed in five cell lines treated with BMS-536924, and three with NVP-AEW541. While IGF1R levels correlated with reduced response to NVP-AEW541 alone, neither IGF1R nor pIGF1R were predictive of response to BMS-536924 or NVP-AEW541 in combination with trastuzumab. Low HER2 levels correlated with response to BMS-536924 in combination with trastuzumab. Akt levels correlated with improved response to trastuzumab and NVP-AEW541 (P = 0.039). Cytoplasmic IGF1R staining was observed in all tumours, membrane IGF1R was detected in 13.8 %, and pIGF1R/IR was detected in 48.8 %. Although membrane IGF1R staining was associated with larger tumour size (P = 0.041), and lower tumour grade (P = 0.024), no association between IGF1R or pIGF1R/IR and patient survival was observed. In conclusion, while neither IGF1R expression nor activation was predictive of response to trastuzumab, these pre-clinical data provide evidence that co-targeting HER2 and IGF1R may be beneficial in some HER2-amplified breast cancers.

Breast cancer is the leading cause of cancer related deaths in women worldwide and is classified into subtypes based on the cancer’s receptor status. Of these subtypes, those expressing the human epidermal growth factor receptor 2 (HER2) receptor were traditionally associated with poor prognosis. Several advances have been made in the treatment of HER2-positive breast cancer, yet issues of resistance and poor response to therapy remains prevalent. In this study we explored the impact of HER-family and homologous recombination deficiency SNPs on response to patients who received TCH-based (docetaxel (T), carboplatin (C), and trastuzumab (H)) treatment versus those who received other treatment regimens. Using Cox regression analysis, we identified 6 SNPs that correlate with recurrence free survival in our patients and supported our findings using support vector machines. We also used reverse phase protein array analysis to examine the impact ERBB3 SNPs may have on both the PI3K/AKT and MAPK/ERK signaling pathways. Finally, using cell line models, we correlated SNP status with sensitivity to platinum based drugs and docetaxel. We found that patients on a TCH based regimen with the minor allele of the ERBB3 (rs2229046 and rs773123) and BARD1 (rs2070096) SNPs, were significantly more likely to relapse than those women who were not. Additionally, we observed that patients with these ERBB3 SNPs had shown elevated protein expression/phosphorylation of Src kinase, c-MET (Y1234/1235), GSK-3β (S9) and p27, indicating that these SNPs are associated with non-PI3K/AKT signaling. Finally, using cell line models, we demonstrate that the BARD1 SNP (rs2229571) is associated with greater sensitivity to both carboplatin and cisplatin. The BARD1 and ERBB3 SNPs can potentially be used to determine those patients that will have a worse response to TCH based treatment, an effect that may arise from the SNPs impact on altered cellular signaling.

Introduction: Despite the recent successes of HER2-targeted therapies, drug resistance still represents a significant clinical problem. Neratinib, an irreversible pan-HER tyrosine kinase inhibitor, has been recently FDA approved for the extended adjuvant treatment of early-stage HER2-positive breast cancer and little is known of its mechanism of resistance in breast cancer. Using a neratinib-resistant cell line model of HER2-positive breast cancer, HCC1954-N, the aim of this study was to characterise cross-resistance to other HER-targeting tyrosine kinase inhibitors (TKIs) and investigate the role of Src in acquired neratinib resistance. Methods: The effect of HER-family targeting TKIs neratinib, lapatinib, afatinib and, the Src inhibitor, dasatinib on proliferation of HCC1954-N and HCC1954-Par cells was assessed by acid phosphatase assays and three-dimensional Matrigel assays. Cell cycle progression was examined by flow cytometry using a propidium iodide assay. Caspase 3/7-Glo assay was used to assess apoptosis induction. To assess the prevention of the development of neratinib resistance, HCC1954 cells were treated twice weekly with neratinib, dasatinib, or the combination and stained with crystal violet when confluent. Results: The HCC1954-N cell line was resistant to neratinib and to afatinib and lapatinib compared to parental HCC1954, but neratinib was still the most potent of the three anti-proliferative agents. The combination of neratinib and dasatinib was synergistic in the neratinib-resistant HCC1954-N cell line in both adherent (CI value = 0.1 ± 0.03) and three-dimensional (CI value = 0.36 ± 0.02) conditions. Neratinib plus dasatinib did not result in cell cycle arrest; however, the combination caused a significant increase in the sub-G1 cell cycle population (p value = 0.018), indicating induction of apoptosis. This was further validated by increased activation of caspase 3 and 7 (p value = 0.015) in HCC1954-N cells treated with neratinib plus dasatinib. The addition of dasatinib to neratinib treatment prevented the development of neratinib resistance in parental HCC1954 cells. Conclusions: Further pre-clinical investigation of the combination of neratinib plus dasatinib is warranted as a potential therapeutic intervention for patients with neratinib-resistant HER2-positive breast cancer. Citation Format: Neil Conlon, Michelle Lowry, Susan Breslin, Lorraine O'Driscoll, Alexander J. Eustace, John Crown, Norma O'Donovan, Denis M. Collins. Src inhibition overcomes neratinib resistance in HER2-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1834.

Activating PI3K mutations have been identified in more than 30% of breast cancers (BC). These mutations have been associated with resistance (res) to trastuzumab (Tras) a HER2-binding monoclonal antibody. PIM kinase expression has been shown to be markedly elevated in PI3K treated BC samples suggesting that it could be a major res pathway for PI3K inhibitors in BC, potentially limiting their clinical utility. IBL-302 is a novel molecule that inhibits both PIM and PI3K signalling. This mechanism of action could afford significant benefit in the treatment of BC. We evaluated the preclinical activity of a IBL-302, in a range of BC models. IBL-302 was tested in the Sanger Institute GDSC screening panel of more than 700 different cancer cell lines in a CellTiter Glo anti-proliferation assay (72hr incubation). We subdivided a panel of 36 BC cell lines into their clinical subtype (HER2+ n=6, HR+ n=6 & TNBC n=24). IBL-302 had significantly lower IC50 in TNBC cell lines when compared to HR+ and HER2+ BC cell lines (r = 0.352, p = 0.038). Sensitivity to IBL-302 correlated significantly with PIM 1 expression (r= -0.45, p = 0.0045) and PIM 2 expression (r =- 0.3238, p = 0.0506) but no such relationship was observed for PIM 3.The HER2 gene or protein is amplified or overexpressed in ~25% of BC and promotes an aggressive phenotype, through the activation of the PI3K/AKT pathway. We utilised our HER2+ BC, Tras res cell lines which had previously been utilised in another PI3K / Tras refractory trial (NCT02705859). IBL-302 had anti-proliferative effects in HER2+ lines (SKBR3, HCC1954 and BT474 cells) including matched models of acquired Tras (SKBR-T, BT474-T) or lapatinib (SKBR-L, HCC1954-L) res. IBL-302 achieved IC50 ranging from (33.4 ± 2.9nM) in SKBR3-T cells to (136 ± 35.2nM) in SKBR3-L, but interestingly both acquired Tras res cell lines SKBR3-T and BT474-T cells were significantly more sensitive to IBL-302 than that observed in the matched SKBR3-P (p>0.01) and BT474-P (p>0.01) cells respectively. It was also observed that the combination of Tras and IBL-302 significantly increased the anti-proliferative effect in SKBR3-P (P Citation Format: Sean P. Kennedy, Michael O Neill, Darren Cunningham, Carmen Blanco-Aparicio, Sonia Martinez, Alex Eustace, Bryan Hennessy. Evaluation of dual-acting PIM/PI3K inhibitor IBL-302 in preclinical breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2932.

Introduction BRCA1-methylated OC (BMOC) are specifically sensitive to platinums and PARPi, though acquired resistance to these agents eventually develops. Elucidating underlying druggable resistance mechanisms is needed to enable novel therapeutic options in BMOC. Material and methods We developed two PARPi resistant (olaparib and talazoparib) and one carboplatin resistant OC cell line models (named OVC8RO, OVC8RT and OVC8RC, respectively) derived from the BRCA1-methylated cell line OVCAR8, following continuous (PARPi) or pulsed (carboplatin) drug exposure. Fold resistance (FR) to the parent drug (as determined by the ratio of the resistant cell line IC50 to the parent cell line IC50) suggested clinically relevant resistance models in OVC8RC (FR=4.80±0.43) and OVC8RO (FR=5.71±0.21). OVC8RT displayed higher level resistance (FR=45.61±11.10). We obtained tissue from 5 matched primary and recurrent (post platinum) BMOC patient tumours. Reverse phase protein array (RPPA) was used to examine differential expression and phosphorylation levels of 63 proteins between parent/resistant cell lines, and primary/matched recurrent tumours. 5 day acid phosphatase cytotoxicity assays were used to determine the IC50 of drugs. Synergy in drug combination assays was determined as per the Chou-Talalay method. Results and discussions Significant increases in PI3K p110a (except in OVC8RT) and AKT S473, along with a significant decrease in PTEN were seen in all resistant cell lines, relative to the parent cell line’s baseline levels (p Conclusion The addition of copanlisib to carboplatin or PARPi could represent a novel therapeutic strategy in BMOC that has acquired resistance to either carboplatin or PARPi.

e13021Background: We have previously shown that protein phosphatase 2A (PP2A) may mediate acquired lapatinib resistance and inhibition of PP2A with okadaic acid enhances lapatinib response in vitro...

Background: Afatinib is an irreversible pan-HER inhibitor approved for non-small cell lung cancer. We have previously shown that afatinib inhibits growth of HER2-positive breast cancer cells and enhances response to trastuzumab. However, we have also shown that long-term exposure to tyrosine kinase inhibitors leads to the development of acquired resistance. To determine if acquired afatinib resistance develops in HER2-positive breast cancer cells, we exposed a HER2-positive breast cancer cell line to afatinib for 6 months and investigated alterations in the cells following long-term exposure. Methods: SKBR3 cells were treated with 150 nM afatinib twice-weekly for 6 months. Growth response to drug inhibitors was assessed by acid phosphatase assay. Drug sensitivity was examined in four HER2-positive cell lines (SKBR3, EFM192A, BT474 and HCC1954) and three acquired trastuzumab resistant cell lines (SKBR3-T, BT474-T and EFM192A-T). Reverse phase protein array (RPPA) was used to determine alterations in key signaling pathways. Src, p-Src, EGFR, p-EGFR, ERK1/2, p-ERK 1/2 levels were examined by Western blotting. To examine the prevention of the development of afatinib resistance, cells were treated twice weekly with afatinib, dasatinib, or the combination and stained with crystal violet when confluent. Results: Following 6 months of afatinib treatment, the SKBR3-A cells were more resistant to afatinib compared to parental cells (IC50 SKBR3-A 284 ± 28.2 nM vs SKBR3-Par 10.9 ± 3.4 nM). Furthermore, the resistant cells were cross-resistant to lapatinib, neratinib and trastuzumab. RPPA interrogation of the SKBR3-A cells showed alterations in several pathways, including significantly increased levels of p-Src (Y416). SKBR3-A cells were more sensitive to Src inhibition with dasatinib compared to SKBR3-Par cells and the combination of afatinib and dasatinib was highly synergistic in SKBR3-A cells (CI value = 0.09 ± 0.06). The combination of afatinib and dasatinib was also synergistic in the trastuzumab resistant SKBR3-T cells (Table). Afatinib and dasatinib inhibited EGFR and Src activation and ERK 1/2 signalling in SKBR3-A cells. Short-term resistance assays showed that the addition of dasatinib to afatinib blocks the emergence of resistant cells in three of four HER2 positive cell lines tested and two of the three acquired trastuzumab resistant cell lines tetsed. Conclusion: HER2-positive breast cancer cells that are highly sensitive to afatinib can develop acquired resistance to afatinib within six months. Src is a potential target to prevent the development of afatinib resistance and thus combined treatment with afatinib and dasatinib may be beneficial in patients with HER2-positive breast cancer. Percentage growth in HER2-positive cells treated with afatinib and/or dasatinib for 5 days. 20 nM Afatinib40 nM Dasatinib20 nM Afatinib + 40 nM DasatinibSKBR325.2 ± 6.1109.9 ± 6.222.6 ± 6.2SKBR3-A62.1 ± 0.787.3 ± 11.036.3 ± 0.3*SKBR3-T42.6 ± 4.9107.4 ± 9.729.3 ± 3.5*BT47418.2 ± 3.079.5 ± 5.213.3 ± 1.1BT474-T6.9 ± 2.795.3 ± 2.06.2 ± 2.3EFM192A39.1 ± 4.7105.7 ± 3.135.3 ± 2.7EFM192A-T30.4 ± 5.299.8 ± 2.528.5 ± 4.4HCC195461.8 ± 8.180.5 ± 8.219.4 ± 3.6** indicates enhanced anti-proliferative response for the combination compared to the single agents. Citation Format: Conlon N, Canonici A, Morgan C, Cremona M, Hennessey BT, Eustace A, O'Brien N, Slamon D, Crown J, O'Donovan N. Targeting Src kinase blocks development of afatinib resistance in HER2-positive breast cancer [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 P4-03-15.

Background: The application of genomic technologies to patient tumor samples identified groups of signaling pathways which acquire activating mutations. Some cancers are dependent on these mutations and the aberrant proteins resulting from these mutations can be targeted by novel drugs which can eradicate the cancer. Methods: We used www.cbioportal.org to determine the frequency of ERBB mutations in solid tumors. We then determined the sensitivity of a panel of cell lines to clinically available PI3K inhibitors. Using proliferation and apoptosis assays as well as functional interrogation with reverse phase protein arrays we demonstrated the impact of targeting ERBB-mutant cancers with the combination of a PI3K inhibitor and the pan-HER family inhibitor afatinib. Results: In over 14,000 patients we found that 12% of their tumors have an ERBB family gene mutation (EGFR, ERBB2, ERBB3 and ERBB4). In cancers not commonly associated with HER family protein overexpression, such as ovarian, endometrial, melanoma and head and neck cancers ( n = 2116), we found that ERBB family mutations are enriched, occurring at rates from 14% to 34% and commonly co-occur with PIK3CA mutations. Importantly, we demonstrate that ERBB family mutant cancers are sensitive to treatment with PI3K inhibitors. Finally we show that the combination of afatinib and copanlisib represents a novel therapeutic strategy for patients whose cancers harbor both ERBB family and PIK3CA mutation. Conclusions: We demonstrate that ERBB family mutations are common in cancers not associated with overexpression or amplification of HER family proteins. These ERBB family mutant cancers are sensitive to treatment with PI3K inhibitors, and when combined with pan-HER inhibitors have synergistic antiproliferative effects.

We report principal component analysis (PCA) of (1)H NMR spectra recorded for a group of human lung carcinoma cell lines in culture and (1)H NMR analysis of extracts from the same samples. The samples studied were cells of lung tumour origin with different chemotherapy drug resistance patterns. For whole cells, it was found that the statistically significant causes of spectral variation were an increase in the choline and a decrease in the methylene mobile lipid (1)H resonance intensities, which correlate with our knowledge of the level of resistance displayed by the different cells. Similarly, in the (1)H NMR spectra of the aqueous and lipophilic extracts, significant quantitative differences in the metabolite distributions were apparent, which are consistent with the PCA results.