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Background We tested the hypothesis that inhibitor of apoptosis family (IAP) proteins may be altered in BRCA1-mutated ovarian cancers and that could affect the sensitivity to IAP inhibitors. Methods The levels of IAP proteins were evaluated in human cancers and cell lines. Cell lines were used to determine the effects of IAP inhibitors. The in vivo effects of treatments were evaluated in PDX mouse models. Results Expression of X-linked inhibitor of apoptosis (XIAP) is increased in BRCA1-mutated cancers and high levels are associated with improved patient outcomes after platinum chemotherapy. XIAP overexpression is mediated by NF-kB activation and is associated with an optimisation of PARP. BRCA1-mutated cell lines are particularly sensitive to IAP inhibitors due to an inhibitory effect on PARP. Both a BRCA1-mutated cell line with acquired resistance to PARP inhibitors and one with restored BRCA1 remain sensitive to IAP inhibitors. Treatment with IAP inhibitors restores the efficacy of PARP inhibition in these cell lines. The IAP inhibitor LCL161 alone and in combination with a PARP inhibitor, exhibited antitumour effects in PDX mouse models of resistant BRCA2 and 1-mutated ovarian cancer, respectively. Conclusion A clinical trial may be justified to further investigate the utility of IAP inhibitors.

Background: De novo and acquired resistance to HER2 directed therapy is frequently encountered. Upregulation of the phosphatidylinositol-3-kinase (PI3K) pathway is an important mediator of treatment resistance. This can occur through an activating mutation of the PIK3CA gene or PTEN loss. PIK3CA mutations are present in approximately 20% of HER2 positive breast cancers and as such, the PI3K pathway has emerged as an attractive target for restoring sensitivity to HER2 directed therapy. Methods: We performed a single arm, multicentre, open label Phase Ib/II trial. Patients (pts) with advanced HER2-positive breast cancer whose disease had progressed on at least 1 line of Trastuzumab/T-DM1 based treatment in the metastatic setting were eligible if they met following criteria: ECOG PS ≤ 2 and adequate organ function. Pts with treated, controlled brain metastases were permitted to enrol. Exclusion criteria included uncontrolled hypertension or diabetes mellitus. Pts on Phase IB were treated according to a 6+6 study design with a dose escalation schedule of Copanlisib IV (level 1 = 45mg, level 2 = 60mg) on Day 1, 8 and 15 of a 28-day cycle along with a fixed dose of Trastuzumab 2mg/kg weekly. Phase II treatment was the MTD (maximum tolerated dose) of Copanlisib in combination with Trastuzumab. Archival tumour tissue, voluntary biopsies and serial plasma samples were collected for genomic sequencing. Primary endpoints were MTD (Phase I) and clinical benefit rate (CBR) which was defined as complete response (CR) or partial response (PR) at any time point; or stable disease (SD) lasting at least 24 weeks (Phase II). Secondary endpoints included safety and tolerability, tumor response rate, duration of response, time to treatment failure (TTF) and progression free (PFS) and overall survival (OS). Results: Twelve pts were enrolled in Phase IB. No dose limiting toxicity was observed. The MTD was established as Copanlisib 60mg and Trastuzumab 2mg/kg. Fourteen pts were enrolled in Phase II (6 pts treated at the MTD in Phase IB were included in the final Phase II analysis resulting in a total of 20 pts). The median number of lines of prior treatment in the metastatic setting was 3 (1-8). The most common grade 3-4 toxicities encountered in the Phase Ib/II cohorts included hypertension (n=7, 27%), hyperglycaemia (n=2, 8%) and vomiting (n=2, 8%). Three pts discontinued treatment due to toxicity. The median follow-up for the Phase II cohort was 7.5 months (95% CI 6.0-14.5). PR was observed in 4 pts (20%) and SD (at any time point) was seen in 8 pts (40%). The CBR was 30% (n=6). The duration of response was 15.0 weeks (95% CI 4.9 - 16.1). The median TTF was 11.9 weeks (95% CI 7.5 - 21.1). The median PFS was 3.0 mo (95% CI 0.2 - 5.8) and OS was 14.0 mo (95% CI 5.2-22.8). At the time of analysis, 9 of 20 patients were alive. PIK3CA mutations were detectable in the archival tissue of 11 of 26 pts (42%). PIK3CA hotspot mutations (H1047R, E542K and E545K) were detectable in the plasma of all 26 pts at various points throughout treatment. Pre and post treatment biopsies of 2 pts in the Phase IB trial revealed somatic mutations in DNAH3 and TRRAP, the latter of which encodes a PI3K-like protein kinase. Targeted next generation sequencing was performed on the circulating tumour DNA of 20 pts in the Phase II cohort taken before, during and after treatment to further validate these findings and to assess for other mechanisms of response or resistance. The final translational results will be presented at the meeting. Conclusions: The combination of Copanlisib and Trastuzumab is a safe and tolerable regimen and is associated with clinical efficacy in a heavily pre-treated metastatic HER2-positive breast cancer population. Translational studies may have identified novel resistance biomarkers in this pt cohort. Citation Format: Lisa Prior, Niamh M Keegan, Simon J Furney, Janice M Walshe, Giuseppe Gullo, John Crown, M John Kennedy, Diarmuid Smith, John McCaffrey, Catherine M Kelly, Keith Egan, Jennifer Kerr, Mark Given, Niall Sheehy, Peter O'Donovan, Andres Hernando, Ausra Teiserskiene, Imelda Parker, Elaine Kay, Ray McDermott, Maccon M Keane, Seamus O'Reilly, Liam Grogan, Oscar Breathnach, Patrick G Morris, Sinead Toomey, Bryan T Hennessy. Phase Ib/II trial evaluating safety and efficacy of copanlisib (PI3K inhibitor) and trastuzumab in pre-treated advanced HER2-positive breast cancer: Results from the PantHER study [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-16-08.

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

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

Small diagnostic tissue samples can be inadequate in testing an expanding list of validated oncogenic driver alterations and fail to reflect intratumour heterogeneity (ITGH) in lung cancer. Liquid biopsies are non-invasive and may better reflect ITGH. Most liquid biopsies are performed in the context of circulating tumour DNA (ctDNA) in plasma but Exhaled Breath Condensate (EBC) shows promise as a lung-specific liquid biopsy.In this prospective, proof-of-concept study we carried out targeted Next Generation Sequencing (NGS) on diagnostic tissue samples from 125 patients with lung cancer and compared results to plasma and EBC for 5 oncogenic driver mutations (EGFR, KRAS, PIK3CA, ERBB2, BRAF) using an ultrasensitive PCR technique (UltraSEEK™ Lung Panel on the MassARRAY® System, Agena Bioscience, San Diego, CA, USA).There was a significantly higher failure rate due to unamplifiable DNA in tissue NGS (57/125, 45.6%) compared to plasma (27/125, 21.6%, p 0.001 and EBC (26/125,20.8%, p ≤ 0.001. Consequently, both plasma and EBC identified higher number of mutations compared to tissue NGS. Specifically, there were significantly higher numbers of mutations detected in EGFR, KRAS and PIK3CA in plasma (p = 9.82 × 10The results suggest that EBC is effective in identifying clinically relevant alterations in patients with lung cancer using UltraSEEK™ and has a potential role as an adjunct to plasma testing.

167 Background: Multiple activating genetic mutations in the phosphatidylinositol-3 kinase (PI3K) and the mitogen activated protein kinase (MAPK) pathways have been implicated in the development of resistance to anti-cancer therapies. Ribociclib has limited activity as a single agent in CRC. However, combining Ribociclib with targeted therapies of the MAPK and PI3K pathways may be a promising treatment strategy for CRC. Methods: We explored the in vitro efficacy of drug combinations Ribociclib (R) and Alpelisib (A) in four CRC cell lines with different mutational status; CACO2 ( PIK3CA/KRAS wild-type), LS1034 ( KRAS mutated), SNUC4 ( PIK3CA mutated) and DLD1 ( PIK3CA/KRAS mutated). Drug combination index (CI) was calculated using Calcusyn Biosoft software. We used a western immunoblotting method for protein analysis. The Chick Chorio-Allantoic Membrane (CAM) assay was used for in-vivo analysis of the effect of the drug combination. Results: IC50s for R and A were calculated for all four cell lines. CACO2 and DLD1 cells were resistant to Ribociclib (IC50 > 15µM). The cell lines had varying sensitivity to both drugs. Drug combination analysis showed that the combination of R and A has a synergistic anti-proliferative effect in all CRC cell lines tested. The combination of R and A is highly synergistic in LS1034 cells which harbour a KRAS mutation (CI = 0.16). Relative expression of the proteins Cyclin D1, E2F-1, p-BCL-2, p-AKT, p-Rb and p-S6 was determined by measuring the density of each band from the western blot experiments and normalizing to β-actin. Combined inhibition of CDK4/6 and PI3Kα caused a simultaneous reduction of p-RB, p-AKT and p-S6 and a more complete inhibition of the PI3K/AKT/mTOR pathway in all four cell lines tested. There was also an increase in the expression of the apoptotic marker pBCL2 in cells treated with the combination of R and A compared to vehicle control(VC). We observed a macroscopic reduction of visible tumour in all cell lines treated with the R and A combination in vivo as compared to VC. Microscopic examination also showed less Ki67 staining and negative cytokeratin staining in combination treated groups. Conclusions: We see a synergistic response to treatment with the combination of R and A in all cell lines and this combination may be a rational treatment strategy in colorectal cancer.

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.

Background Activation of the phosphoinositide -3 kinase (PI3K) pathway is a resistance mechanism to anti-HER2 targeted therapy. This trial was conducted to determine the maximum tolerated dose (MTD) of copanlisib, an oral pan-class I PI3K inhibitor, combined with trastuzumab for patients with advanced HER2 positive breast cancer resistant to anti-HER2 therapy. Patients and Methods In this phase Ib open label dose escalation study, using a 6 + 6 design, patients with advanced HER2-positive breast cancer who had disease progression following at least one prior line of HER2 therapy in the metastatic setting were treated with a dose escalation schedule of copanlisib (dose level 1 =45mg or dose level 2 = 60mg) IV on days 1, 8 and 15 of a 28 day cycle along with a fixed dose of trastuzumab 2mg/kg weekly after a loading dose of 4mg/kg in cycle 1. Archival tumour tissue, voluntary serial tumour biopsies and serial plasma samples were collected for genomic sequencing. Results Twelve patients were enrolled. MTD was determined as copanlisib 60mg plus trastuzumab 2mg/kg weekly. There was no dose limiting toxicity. The most common treatment-related adverse events (AE) of any grade experienced in more than 2 patients were hyperglycemia (58%), fatigue (58%), nausea (58%) and hypertension (50%). Confirmed stable disease at 16 weeks was observed in 6 (50%) participants. PIK3CA mutations were detected in archival tumour tissue of 6 (50%) patients and did not appear to influence likelihood of clinical benefit. PIK3CA mutations were detected in serial plasma ctDNA of all 12 patients and fluctuated over the course of treatment. Next-Generation Sequencing (NGS) analysis identified novel somatic mutations in the TTRAP gene, which encodes a PI3K-like protein kinase, detected only in tumour samples obtained at metastatic time points. Additionally, NGS analysis demonstrated clear temporal genomic heterogeneity with decreasing PIK3CA mutation variant allele frequency (VAF) post therapy Conclusions The combination of copanlisib and trastuzumab was safely administered with good overall tolerability in this trial. Preliminary anti-tumour stability was observed in patients with heavily pre-treated metastatic HER2 positive breast cancer. Translational studies identified a number of potential biomarkers for further study in the now initiated phase 2 clinical trial. Citation Format: Niamh M Keegan, Simon Furney, Janice Walshe, Giuseppe Gullo, John Kennedy, Kyran Bulger, John McCaffrey, Catherine M Kelly, Keith Egan, P O'Donovan, Andres Hernando, Ausra Teiserskiene, Imelda Parker, Angela M Farrelly, Aoife Carr, Giulio Calzaferri, Ray McDermott, Maccon M Keane, Liam Grogan, Oscar S Breathnach, Patrick G Morris, Sinead Toomey, Bryan T Hennessy. A phase Ib trial of copanlisib in combination with trastuzumab in pretreated recurrent or metastatic HER2-positive breast cancer “PantHER” [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 P1-19-24.

Simple Summary Patients with HER-2 positive breast cancer who progress through available HER2-targeted therapy, at present, have few effective treatment options. PIK3CA is mutated in approximately 20% of HER2 positive breast cancers, contributes to HER-2 therapy resistance and may be predictive of response to PI3K inhibitors, including copanlisib. PIK3CA gene mutations were assessed in archival tumour tissue and serially in plasma circulating tumour DNA over the course of treatment with copanlisib. Disease stabilisation (stable disease ≥16 weeks) was seen with copanlisib and trastuzumab in a proportion of participants (n = 6, 50%). PIK3CA mutation detected in archival tumour tissue did not appear to predict tumour response to copanlisib and trastuzumab in this small, heavily pre-treated cohort. Notably, PIK3CA circulating tumour DNA mutations were detected in the plasma of all trial participants, including those who tested negative for the mutation in tissue. This study established a dosing strategy for the novel combination of the PI3K inhibitor copanlisib with trastuzumab and suggested clinical activity for the combination in heavily pre-treated HER-2 positive advanced breast cancer. Further evaluation in a phase 2 study in patients with HER2 therapy resistant tumours is ongoing (NCT02705859). Abstract Background: Activation of the phosphoinositide-3 kinase (PI3K) pathway is a resistance mechanism to anti-human epidermal growth factor receptor 2 (HER2) therapy. This phase Ib trial was conducted to determine the maximum tolerated dose (MTD) of copanlisib, an intravenous (IV) pan-class I PI3K inhibitor, combined with trastuzumab. Methods: Patients with advanced HER2-positive breast cancer and disease progression following at least one prior line of HER2 therapy in the metastatic setting were treated with copanlisib (45 or 60 mg) IV on days 1, 8 and 15 of a 28-day cycle with a fixed dose of trastuzumab 2 mg/kg weekly. Results: Twelve patients were enrolled. The MTD was determined as copanlisib 60 mg plus trastuzumab 2 mg/kg weekly. The most common adverse events of any grade occurring in more than two patients were hyperglycaemia (58%), fatigue (58%), nausea (58%) and hypertension (50%). Stable disease was confirmed at 16 weeks in six participants (50%). PIK3CA mutations were detected in archival tumour of six participants (50%). PIK3CA hotspot mutations, were detectable in pre- and on-treatment plasma of all participants. Pre- and post-treatment tumour biopsies for two patients identified temporal genomic heterogeneity, somatic mutations in the TRRAP gene, which encodes a PI3K-like protein kinase, and emergent somatic mutations related to protein kinase signalling. Conclusion: Copanlisib and trastuzumab can be safely administered with fair overall tolerability. Preliminary evidence of tumour stability was observed in patients with heavily pre-treated, metastatic HER2 positive breast cancer. Several potential biomarkers were identified for further study in the current phase 2 clinical trial. NCT: 02705859.

Background:The phosphoinositide 3 kinase (PI3K) pathway is important in the oncogenic function of HER2. Aberrant activation of PI3K is implicated in resistance to trastuzumab and other HER2-targeted therapies and is frequent, with up to 22% of HER2 positive breast cancer having a PIK3CA mutation. Copanlisib is a pan-class 1 PI3K inhibitor administered i.v. with low nanomolar activity against both PI3Kα and PI3Kβ. Copanlisib has been shown to re-sensitise trastuzumab resistant cell lines to trastuzumab with synergism seen in some cell lines between copanlisib and HER2 targeted therapy. Trial design: This is a phase Ib open label, single arm adaptive, multi-centre trial of copanlisib in combination with T-DM1. Eligible patients will receive T-DM1 at 3.6mg/kg i.v. on day 1 of a 21-day cycle plus copanlisib. Copanlisib will be administered i.v. according to the dose escalation scheme (dose level 1 is 45mg on days 1 and 8, dose level 2 is 60mg on days 1 and 8, dose level 3 is 60mg on days 1, 8, and 15). Dose level -1 will be 45 mg on day 1 in case dose de-escalation is needed. We will enrol 3 to 6 patients per dose level. All patients in each level must have completed at least the first cycle of therapy before enrolment in the next dose level. Patients not completing the first cycle for a reason other than toxicity will be replaced. Dose escalation and determination of the Maximum Tolerated Dose (MTD) will be based on the occurrence of Dose Limiting Toxicities (DLT). Eligibility criteria:Eligible patients are those with unresectable locally advanced or metastatic HER2-positive BC who previously received trastuzumab and a taxane, separately or in combination. Participants must have adequate organ function and ECOG PS ≤ 2 Objectives:The primary objective is to determine the MTD for copanlisib in combination with T-DM1 in patients with pre-treated unresectable locally advanced or metastatic HER2-positive BC. Secondary objectives include evaluating the safety, efficacy and cardiotoxicity in patients treated with this regimen. Exploratory objectives include examining for predictive biomarkers in tumour tissue and blood or plasma and to examine molecular tumour adaptation to clinical trial therapy. Statistical methods: Patients will be accrued in cohorts of 3 patients according to a standard 3+3 algorithm, with dose escalation and determination of MTD based on the occurrence of DLT, using the usual threshold probability of 33%. The final dose level will be expanded to include a total of 6 additional patients (expansion cohort). Present accrual and target accrual:The trial will start accrual in October 2018. Maximum of 24 patients will be enrolled. Citation Format: Hassan A, Gullo G, O'Reilly S, Ruiz-Borrego M, Toomey S, Grogan L, Breathnach O, Morris PG, Walshe JM, Crown J, O'Mahony D, Falcon A, Egan K, Hernando A, Teiserskiene A, Kelly CM, Coate L, Hennessy BT. Phase Ib clinical trial of coPANlisib in combination with Trastuzumab emtansine (T-DM1) in pre-treated unresectable locally advanced or metastatic HER2-positive breAst cancer (BC) “PANTHERA”-CTRIAL-IE 17-13 [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 OT3-06-01.

Standard treatment for locally advanced rectal cancer (LARC) is neoadjuvant chemoradiotherapy (NACRT), followed by surgical resection. However, &gt, 70% of patients do not achieve a complete pathological response and have higher rates of relapse and death. There are no validated pre- or on-treatment factors that predict response to NACRT besides tumour stage and size. We characterised the response of 33 LARC patients to NACRT, collected tumour samples from patients prior to, during and after NACRT, and performed whole exome, transcriptome and high-depth targeted sequencing. The pre-treatment LARC genome was not predictive of response to NACRT. However, in line with the increasing recognition of microbial influence in cancer, RNA analysis of pre-treatment tumours suggested a greater abundance of Fusobacteria in intermediate and poor responders. In addition, we investigated tumour heterogeneity and evolution in response to NACRT. While matched pre-treatment, on-treatment and post-treatment tumours revealed minimal genome evolution overall, we identified cases in which microsatellite instability developed or was selected for during NACRT. Recent research has suggested a role for adaptive mutability to targeted therapy in colorectal cancer cells. We provide preliminary evidence of selection for mismatch repair deficiency in response to NACRT. Furthermore, pre-NACRT genomic landscapes do not predict treatment response but pre-NACRT microbiome characteristics may be informative.

e15041 Background: The treatment of HER2+ breast cancer (BC) has improved substantially with the use of targeted therapies, and patients achieving pathological complete response (pCR) following neoadjuvant therapy have significantly improved disease free survival. However, there is currently no way to determine which patients are most likely to respond to neoadjuvant HER2-targeting treatment. Growing evidence indicates tumour infiltrating lymphocytes influence HER2+ BC outcome. We hypothesize that a deeper investigation of the immune landscape of HER2+ patient tumours may improve patient stratification and identify an immune profile associated with pCR. Methods: 3plex immunohistochemistry panels identifying immune cell subpopulations and tertiary lymphoid structures were applied to HER2+ BC tumors (pre-treatment N=19, post-treatment N=9) of patients of the neoadjuvant TCHL clinical trial (NCT01485926), who received HER2-targeting therapy. Quantitative analysis of CD3+, CD20+, DC-Lamp+, CD4+, and CD8+ immune cells on multi-labelled whole-slide images of TCHL tumor sections was performed using artificial intelligence image analysis models. Patient-specific densities of each cell type in tumor regions were determined to facilitate assessment of pre-treatment differences in target immune subpopulations in complete, partial, and non-responders. Results: Statistical analysis demonstrated a trend for higher densities of each immune subpopulation in pre-treatment biopsies of pCR vs. non-pCR patients. Patient categorisation based on median threshold demonstrated patients achieving pCR are more frequently high for each immune cell type assessed, while non-pCR patients are more frequently in the low category. Investigation of association of pre-treatment immune infiltrate with clinicopathological features demonstrated trends for higher levels of T-cell infiltration in later stage and hormone receptor negative patients. Conclusions: Multiplex IHC is an optimal technique for the concurrent assessment of multiple target markers in patient tissue, which can be quantitatively analyzed in a high throughput manner using AI models. The findings of this preliminary study support recent evidence regarding the role of tumor infiltrating immune cells in HER2+ BC outcome and warrant further investigation in a larger cohort.

The EGFR T790M somatic mutation is the most common mechanism of resistance to tyrosine kinase inhibitors in NSCLC. Patients with advanced disease are not always amenable to repeat biopsy for further molecular analysis. Developing noninvasive methods to detect T790M in cell-free DNA in the absence of tissue is being actively investigated. Unfortunately, the low sensitivity of plasma for detection of T790M has limited its clinical use. Exhaled breath condensate (EBC) is an easily collected sample that is known to harbor cell-free DNA, including lung cancer mutations. This report details the potential utility of exhaled breath condensate in the detection of the EGFR T790M mutation.

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.

Background The phosphoinositide 3 kinase (PI3K) pathway is important in the oncogenic function of HER2. Aberrent activation of PI3K is implicated in resistance to trastuzumab and other HER2-targeted therapies and is frequent, with up to 22% of HER2 positive breast cancer having a PIK3CA mutation. Copanlisib is a pan-class 1 PI3K inhibitor that shows particular activity against PI3Kα, the isoform encoded by the PIK3CA gene. Copanlisib has been shown to re-sensitise trastuzumab resistant cell lines to trastuzumab with synergism seen in some cell lines between copanlisib and HER2 targeted therapy. Trial design The study is a phase Ib/II open label, single arm adaptive, multi-centre trial of copanlisib in combination with trastuzumab. Eligible patients are treated with a dose escalation schedule of copanlisib IV on Days 1, 8 and 15 of a 28 day cycle with trastuzumab 2 mg/kg weekly (loading dose of 4 mg/kg in cycle 1). The phase II dose will be based on the maximum tolerated dose (MTD) established in Phase Ib. Patients are treated until radiologic or symptomatic progression, unacceptable toxicity, consent withdrawal or physician's decision. Eligibility criteria Eligible patients must have recurrent incurable or metastatic HER2-positive breast cancer that has progressed on at least one prior line of trastuzumab or T-DM1-based treatment regimen in this setting. Patients with treated and controlled brain metastases are eligible. Participants must have adequate organ function and ECOG PS ≤ 2. Patients recruited for the Phase II part of the study must have a PIK3CA mutation. Patients with uncontrolled arterial hypertension, uncontrolled diabetes or recent clinically serious infections are excluded. Specific aims The primary end point for the phase Ib part of this study is to determine the MTD for the combination. For the phase II study is anti-tumour efficacy, measured by Clinical Benefit Rate (CBR). Secondary end points are evaluation of safety and tolerability, progression-free survival, time to treatment failure, duration of response and overall survival. Incorporated translational endpoints include examination of molecular tumor adaptation in tissue and blood. Given the role of PI3K in cellular glucose metabolism, an additional exploratory objective is to determine if quantitive reduction in metabolic signal on Positron Emission Tomography-Computed Tomography (PET-CT) is predictive of benefit from therapy. Statistical methods To establish the MTD, we use a modified 3+3 design where 3 additional patients will be accrued even if the first 3 patients accrued experience no dose limiting toxicities (DLT) in sequential cohorts for a planned 12 patients. To determine the CBR, a one sample exact binomial test with a one sided significance level of 5%, 19 evaluable patients will provide >80% power to detect a difference between the null hypothesis proportion of 30% for CBR versus the alternative hypothesis proportion of 65%. Present accrual and target accrual There are 9 patients recruited so far to the phase Ib part of this study. Target accrual is 12 and for phase II is 19 patients. Contact information for people with a specific interest in the trial Prof Bryan Hennessy, Beaumont Hospital, Dublin Ireland Funded by Bayer Citation Format: Keegan NM, Walshe J, Gullo G, Kennedy J, Bulger K, Kelly CM, Crown J, Toomey S, Egan K, Kerr J, Given M, Hernando A, Teiserskiene A, Grogan L, Breathnach O, Morris PG, Keane M, Hennessy BT. A phase Ib/II trial of coPANlisib in combination with tratuzumab in pretreated recurrent or metastatic HER2-positive breast cancer “PantHER” [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 OT3-06-05.

// 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.

The treatment landscape for metastatic melanoma has been revolutionised by the introduction of immunotherapy and targeted therapies. Despite these advances, some patients exhibit primary or acquired resistance to treatment. We present the case of a resected mucosal melanoma that on relapse underwent transformation to a dedifferentiated state. The relapsed tumour was phenotypically disparate and demonstrated loss of all typical melanoma-associated immunohistochemical markers. Furthermore, it demonstrated aggressive biological behaviour and immunotherapy resistance. We performed genomic profiling of the original and relapsed tumour to further elucidate the mechanisms underlying this rare phenomenon. Mass spectrometry-based single-nucleotide polymorphism genotyping technology was used to screen for mutations in the original and recurrent tumour. Whole-exome sequencing was performed on the original tumour, recurrent tumour and blood. Both the original and recurrent tumour shared a NRAS mutation, a similar aneuploidy profile and proportion of somatic single-nucleotide variants. However, in contrast to the original tumour, the recurrent tumour demonstrated a lower mutational burden and deletions in the CDKN2A/CDKN2B and CHEK2 genes. The genomic similarity between the original and recurrent tumour attests to a common ancestry and the possible existence of nongenomic drivers inciting phenotype plasticity. In contrast, the low mutational load and potential inactivation of tumour suppressor genes in the recurrent tumour may underlie its rapid proliferative rate and immunoresistance. Dynamic treatment models are desired in the future to track the genomic and epigenetic evolution of a tumour to guide optimal therapy choice and sequencing.

Purpose: Apoptosis is essential for chemotherapy responses. In this discovery and validation study, we evaluated the suitability of a mathematical model of apoptosis execution (APOPTO-CELL) as a stand-alone signature and as a constituent of further refined prognostic stratification tools. Experimental Design: Apoptosis competency of primary tumor samples from patients with stage III colorectal cancer (n = 120) was calculated by APOPTO-CELL from measured protein concentrations of Procaspase-3, Procaspase-9, SMAC, and XIAP. An enriched APOPTO-CELL signature (APOPTO-CELL-PC3) was synthesized to capture apoptosome-independent effects of Caspase-3. Furthermore, a machine learning Random Forest approach was applied to APOPTO-CELL-PC3 and available molecular and clinicopathologic data to identify a further enhanced signature. Association of the signature with prognosis was evaluated in an independent colon adenocarcinoma cohort (TCGA COAD, n = 136). Results: We identified 3 prognostic biomarkers (P = 0.04, P = 0.006, and P = 0.0004 for APOPTO-CELL, APOPTO-CELL-PC3, and Random Forest signatures, respectively) with increasing stratification accuracy for patients with stage III colorectal cancer. The APOPTO-CELL-PC3 signature ranked highest among all features. The prognostic value of the signatures was independently validated in stage III TCGA COAD patients (P = 0.01, P = 0.04, and P = 0.02 for APOPTO-CELL, APOPTO-CELL-PC3, and Random Forest signatures, respectively). The signatures provided further stratification for patients with CMS1-3 molecular subtype. Conclusions: The integration of a systems-biology–based biomarker for apoptosis competency with machine learning approaches is an appealing and innovative strategy toward refined patient stratification. The prognostic value of apoptosis competency is independent of other available clinicopathologic and molecular factors, with tangible potential of being introduced in the clinical management of patients with stage III colorectal cancer. Clin Cancer Res; 23(5); 1200–12. ©2016 AACR.

ObjectiveThe mitochondrial apoptosis pathway is controlled by an interaction of multiple BCL-2 family proteins, and plays a key role in tumour progression and therapy responses. We assessed the prognostic potential of an experimentally validated, mathematical model of BCL-2 protein interactions (DR_MOMP) in patients with stage III colorectal cancer (CRC).DesignAbsolute protein levels of BCL-2 family proteins were determined in primary CRC tumours collected from n=128 resected and chemotherapy-treated patients with stage III CRC. We applied DR_MOMP to categorise patients as high or low risk based on model outputs, and compared model outputs with known prognostic factors (T-stage, N-stage, lymphovascular invasion). DR_MOMP signatures were validated on protein of n=156 patients with CRC from the Cancer Genome Atlas (TCGA) project.ResultsHigh-risk stage III patients identified by DR_MOMP had an approximately fivefold increased risk of death compared with patients identified as low risk (HR 5.2, 95% CI 1.4 to 17.9, p=0.02). The DR_MOMP signature ranked highest among all molecular and pathological features analysed. The prognostic signature was validated in the TCGA colon adenocarcinoma (COAD) cohort (HR 4.2, 95% CI 1.1 to 15.6, p=0.04). DR_MOMP also further stratified patients identified by supervised gene expression risk scores into low-risk and high-risk categories. BCL-2-dependent signalling critically contributed to treatment responses in consensus molecular subtypes 1 and 3, linking for the first time specific molecular subtypes to apoptosis signalling.ConclusionsDR_MOMP delivers a system-based biomarker with significant potential as a prognostic tool for stage III CRC that significantly improves established histopathological risk factors.

Background: The Insulin-like growth factor (IGF) pathway plays a role in tumour development and progression. In vivo, IGF1 activity is regulated by the IGF binding proteins (IGFBPs). IGFBP4 inhibits the activity of IGF1 but proteolytic cleavage by pregnancy-associated plasma protein-A (PAPP-A) releases active IGF1. A modified IGFBP4, dBP4, which was resistant to PAPP-A cleavage but retained IGF1 binding capacity, was engineered, expressed in Human Embryonic Kidney (HEK) 293 cells and purified. This study examined the effects of dBP4 on IGF1-induced cell migration, invasion and angiogenesis in vitro. The effect of intra-tumour injections of dBP4 on tumour angiogenesis and metastasis was examined using the 4T1.2luc orthotopic model of breast cancer. Methods: PAPP-A resistance and IGF binding capacity of dBP4 were characterized by Western blot and surface plasmon resonance, respectively. 4T1.2luc are mouse mammary adenocarcinoma cells transfected with luciferase to allow in vivo imaging. The effect of dBP4 on IGF1-induced Akt activation in 4T1.2luc cells was assessed by Western blot. Cell migration and invasion assays were performed using 4T1.2luc cells. Angiokit™ assays and Matrigel® implants were used to assess the effects of dBP4 on angiogenesis in vitro and in vivo, respectively. An orthotopic breast cancer model - 4T1.2luc cells implanted in the mammary fat pad of BALB/c mice - was used to assess the effect of intra tumour injection of purified dBP4 on tumour angiogenesis and metastasis. Tumour growth and lung metastasis were examined by in vivo imaging and tumour angiogenesis was evaluated by CD31 immunohistochemistry. Results: Our engineered, PAPP-A resistant IGFBP4 (dBP4) retained IGF1 binding capacity and inhibited IGF1 activation of Akt as well as IGF1-induced migration and invasion by 4T1.2 mammary adenocarcinoma cells. dBP4 inhibited IGF1-induced angiogenesis in vitro and in Matrigel implants in vivo. Direct intra-tumour injection of soluble dBP4 reduced angiogenesis in 4T1.2 luc mammary tumours tumour and reduced lung metastasis. Conclusion: A PAPP-A resistant IGFBP4, dBP4, inhibits angiogenesis and metastasis in 4T1.2 mammary fat pad tumours. This study highlights the therapeutic potential of dBP4 as an approach to block the tumour-promoting actions of IGF1.

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

Because of advances in targeted therapies, the clinical evaluation of cutaneous melanoma is increasingly based on a combination of traditional histopathology and molecular pathology. Therefore, it is necessary to expand our knowledge of the molecular events that accompany the development and progression of melanoma to optimize clinical management. The central objective of this study was to increase our knowledge of the mutational events that complement melanoma progression. High-throughput genotyping was adapted to query 159 known single nucleotide mutations in 33 cancer-related genes across two melanoma cohorts from Ireland (n = 94) and Belgium (n = 60). Results were correlated with various clinicopathological characteristics. A total of 23 mutations in 12 genes were identified, that is - BRAF, NRAS, MET, PHLPP2, PIK3R1, IDH1, KIT, STK11, CTNNB1, JAK2, ALK, and GNAS. Unexpectedly, we discovered significant differences in BRAF, MET, and PIK3R1 mutations between the cohorts. That is, cases from Ireland showed significantly lower (P

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

179 Background: Combinative inhibitors with multiple actions to target downstream effectors are good strategy to reduce resistance occurrence. This is a preclinical study to evaluate efficacy of Palbociclib (P; CDK4/6 inhibitor) in combination with either Gedatolisib (G; P13K/mTOR dual inhibitors) or PD0325901 (PD; MEK 1/2 inhibitor) in CRC cell models. Methods: Five CRC cell lines with hot-spots mutations in PI3K and MAPK pathways genes are used. Cell growth assay is used to evaluate antiproliferative response to P, G and PD alone and in combination. RPPA is used to clarify the role actions of combinative drugs on resistance development and new druggable proteins. Paired t-test is used. Results: All cell lines except LS1034 (IC50=7.2µM) were sensitive to G. LS411N and SNUC4 were sensitive to P whereas Caco2, DLD1 and LS1034 were resistant (IC50>15µM). All cells lines were sensitive to PD. When tested in combination, P+G has synergistic response in all cells lines, except for LS411N (CI=0.9; p=0.02). P+G is highly synergistic in LS1034 with KRAS mutation (CI=0.1; p=0.11). P+G is also synergistic in DLD1 with co-occurring PIK3CA and KRAS mutations (CI=0.6; p=0.04). P+PD is synergistic in all cells lines except for LS411N. In SNUC4 with PIK3CA mutation, both drug combinations have equal synergism (CI=0.5; p [Table: see text]

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.

In locally advanced rectal cancer, neoadjuvant chemoradiotherapy is performed prior to surgery to downstage the tumour. Thirty to 40 % of patients do not respond. Defects in apoptotic machinery lead to therapy resistance; however, to date, no study quantitatively assessed whether B cell lymphoma 2 (BCL2)-dependent regulation of mitochondrial apoptosis, effector caspase activation downstream of mitochondria or a combination of both predicts patient responses. In a cohort of 20 rectal cancer patients, we performed protein profiling of tumour tissue and employed validated ordinary differential equation-based systems models of apoptosis signalling to calculate the ability of cancer cells to undergo apoptosis. Model outputs were compared to clinical responses. Systems modelling of BCL2-signalling predicted patients in the poor response group (p = 0.0049). Systems modelling also demonstrated that rectal cancers depended on BCL2 rather than B cell lymphoma-extra large (BCL(X)L) or myeloid cell leukemia 1 (MCL1) for survival, suggesting that poor responders may benefit from therapy with selective BCL2 antagonists. Dynamic modelling of effector caspase activation could not stratify patients with poor response and did not further improve predictive power. We deliver a powerful patient stratification tool identifying patients who will likely not benefit from neoadjuvant chemoradiotherapy and should be prioritised for surgical resection or treatment with BCL2 antagonists.Modelling BCL2-family proteins identifies patients unresponsive to therapy. Caspase activation downstream of mitochondria cannot identify these patients. Rectal tumours of poor responders are BCL2- but not BCL-XL-dependent. DR_MOMP allows clinicians to identify patients who would not benefit from therapy. DR_MOMP is also a useful patient stratification tool for BCL2 antagonists.

Background HER2 is overexpressed in 10-25% of gastric cancers (GCs). The association of PI3K pathway activating mutations with HER2 overexpression, or the impact of aberrant PI3K pathway signalling on response to trastuzumab (Tr) in GC has not been described. In HER2-positive breast cancer, targeting the PI3K pathway can overcome resistance to HER2-targeted therapies; however the role of PI3K inhibitors in sensitizing HER2-positive GCs to Tr or in overcoming Tr resistance is unknown. Methods Tumour samples from 69 GC patients were stratified into HER2-positive (n = 29) and HER2-negative (n = 40) groups. Mass spectrometry-based genotyping (Agena Bioscience) was used to analyse 105 hotspot somatic mutations in PIK3CA, EGFR, ERBB2, ERBB3 and ERBB4 in the tumours and in a panel of HER2-positive GC cell lines (N87, OE19, ESO26 and SNU16). The anti-proliferative response to the PI3K inhibitor copanlisib alone and in combination with the HER2-targeted therapies Tr and lapatinib was assessed in vitro. Results Patients with HER2-positive GC had significantly poorer overall survival compared to HER2-negative patients (15.9 months vs. 35.7 months; p = 0.0032). PIK3CA, EGFR and ERBB mutations occurred more frequently in HER2-negative tumours than HER2-positive tumours, but had no impact on progression free or overall survival. In vitro OE19 cells were resistant to copanlisib, while all other cell lines were sensitive, independent of PIK3CA mutation status, with IC50s ranging from 23.4nm (N87) to 93.8nm (SNU16). All cell lines except SNU16 were sensitive to lapatinib with IC50s ranging from 0.04µM to 1.5µM. OE19 and SNU16 were resistant to Tr. The combination of lapatinib and copanlisib is synergistic in ESO-26 and OE-19 cells (ED50: 0.83±0.19 and 0.88±0.13, respectively) and additive in N87 cells (ED50:1.01±0.55). The combination of copanlisib and Tr significantly improved growth inhibition compared to either therapy alone in N87, ESO26 and OE19 cells (p Conclusions Copanlisib is an effective monotherapy in some HER2-positive GC cell lines. Combinations of copanlisib and Tr offer greater benefit than either drug alone, and may restore sensitivity to Tr in cells with intrinsic resistance to Tr. The addition of copanlisib to HER2 targeted therapy warrants further investigation in HER2-positive GC. Legal entity responsible for the study The authors. Funding North East Cancer Research and Education Trust (NECRET). Disclosure All authors have declared no conflicts of interest.

// 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.

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 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.

1036Background: PI3K pathway activation is implicated in resistance to trastuzumab (T) therapy in breast cancer (BC). Copanlisib (C) is a pan-class I PI3K inhibitor with particular activity against...

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.

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.

BACKGROUND: We have shown that ERBB (EGFR, ERBB2, ERBB and ERBB4) germline single nucleotide polymorphisms (SNPs) have a negative impact on the outcome of trastuzumab treated HER2-positive breast cancer (BC) patients. Currently TCH (taxotere, platinum and trastuzumab) based therapy is used to treat early stage HER2-positive BC. We investigate the importance of germline SNPs in ERBB genes and those genes involved in homologous recombination deficiency (HRD), on how patients respond to TCH therapy. PATIENTS AND METHODS: ERBB/HRD SNPs were identified in a panel of 32 HER2-positive BC patients by next generation sequencing (NGS). Agena MassArray analysis confirmed the genotype of these SNPs in a further 157 women. Kaplan-Meier estimates and Cox regression analysis identified that both ERBB/HRD SNPs were associated with relapse free survival (RFS) in patients who received a TCH based treatment versus those who received alternate therapies. Protein extracted from formalin fixed paraffin embedded tumours (n=60), was run on an RPPA platform to measure expression and phosphorylation of proteins (69 antibodies). Logistical regression identified protein levels associated with the presence/absence of ERBB/HRD SNPs that were significantly associated with RFS. RESULTS: Ten ERBB/HRD SNPs were profiled in 157 trastuzumab treated HER2-positive BC patients. The minor alleles of the ERBB2 (rs1136201), ERBB3 (rs2229046) and BARD1 (rs2070096) SNPs significantly associated with a worse RFS in patients who received TCH based therapy relative to those who had the reference allele (ERBB2: HR=2.67 (CI=1.05-6.78), p=0.04; ERBB3 rs2229046: HR=4.95 (CI=1.91-12.79), p=9.75x10-4; BARD1: HR=3.27 (CI =1.16-9.17), p=0.02). The impact of ERBB/HRD SNPs on RFS was not observed in patients who did not receive TCH treatment. The minor allele of the RNF8 rs2284922 SNP is associated with a worse RFS (RNF8: HR=12.42 (CI =2.00-77.19), p=6.89x10-3) relative to those who had the reference allele only in patients who did not receive TCH treatment. RPPA analysis identified that patients who received TCH therapy and had the minor allele of the ERBB3 SNPs were significantly associated with the expression of HER2, p27 and MEK1/2 (rs2229046; minor allele associated with low expression of p27 (p=7.22x10-3) and with high expression of HER2 (p=6.53x10-3); rs773123, minor allele associated with low expression of p27 (p=5.38x10-4) and with high expression of MEK1/2 (p=6.24x10-3). CONCLUSIONS: The presence of germline ERBB/HRD SNPs may play an important role in how a patient responds to TCH based therapy, and clinical assessment of these SNPs by targeted genetic screening of patients' blood may allow for stratification of patients prior to treatment. Citation Format: Stephen F. Madden, Sinead Toomey, Simon Furney, Malgorzata Milewska, Joanna Fay, Elaine W. Kay, John Crown, Susan Kennedy, Bryan T. Hennessy, Alex J. Eustace. The impact of germline single nucleotide polymorphisms (SNPs) in ERBB-family genes and genes associated with homologous recombination deficiency (HRD) on response to taxotere, platinum and trastuzumab (TCH) based therapy in the treatment of HER2-positive breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3402. doi:10.1158/1538-7445.AM2017-3402

3616 Background: Locally advanced rectal cancer, LARC (T3/4 and/or N+) is currently treated with neoadjuvant chemoradiotherapy (NACRT), however clinicopathological response is variable. Monitoring clonal evolution in response to NACRT may identify mutations driving therapeutic resistance or tumor growth after treatment. Methods: Fresh-frozen pre- and post-NACRT tumor and matched normal tissue from LARC patients were stratified into good (RCPath A), intermediate (RCPath B) and poor (RCPath C) responders. Following histological review, targeted exome capture was performed using an Agilent SureSelect Human all Exome V3 kit. Samples were sequenced to a minimum of 100X coverage on an Illumina HiSeq2000, and clonal evolution was assessed in matched pre- and post-NACRT tumor samples. Results: The median somatic mutation burden in pre-treatment samples was 114 (IQR 19-207). Two tumors were microsatellite (MSI) unstable and had elevated mutational burdens. The least evolution occurred in the poor responders, where there was little change in clonal composition after treatment, and driver mutations in genes including TP53 and APC were retained. On average 79% of pre-treatment mutations were retained post-treatment in poor responders and 33% of mutations were retained in intermediate responders. Many of the intermediate responders had loss of driver mutations including TP53 from the pre-treatment sample, but also shared a number of mutations in genes including PIK3CA and BRAF between pre- and post-treatment samples. There was also increased frequency in the post-treatment samples of clones that were not present in the pre-treatment samples. In one intermediate responder, all 47 mutations that were present in the pre-treatment sample including the driver mutations TP53 and APC were absent in the post-treatment sample, while 10 completely new mutations were identified. Conclusions: Dynamic mutational processes occur in LARC following selective pressures of exposure to NACRT, including changes in somatic mutation presence or frequency after treatment, owing to persistence or loss of sub-clones. As NACRT can profoundly affect the LARC genome, monitoring molecular changes during treatment may be clinically useful.

583 Background: In the TCHL trial (NCT01485926) 78 women with HER2-positive breast cancer (BC) underwent neo-adjuvant treatment with either TCH (Docetaxel, Carboplatin, Trastuzumab) or TCHL (TCH + Lapatinib) therapy. Of the 78 patients, 24 consented to an optional on-treatment biopsy 20 days after 1 cycle of therapy. We analysed the impact of tumour infiltrating lymphocytes (TILs) on pathological complete response (pCR) and also determined the impact of TCH/TCHL therapy on immune cell modulation after 20 days of treatment. Methods: We assessed TIL and stromal lymphocytes (SL) counts using immunohistochemical staining with Haemotoxalyin+Eosin, AE1/AE3 and CD45 in formalin fixed paraffin embedded (FFPE) baseline biopsy samples and in fresh frozen (FF) biopsies taken 20-days post cycle 1 (Day-20) of TCH/TCHL. RNA libraries were generated, using the Truseq mRNA library prep kit on the Neoprep platform and sequenced on the NextSeq 500. We measured the transcriptomic profile of 8 pre and on-treatment sample pairs and then used the Microenvironment Cell Populations (MCP)-counter method to measure the abundance of 10 immune cell populations (T cells, CD8 T cells, cytotoxic lymphocytes, NK cells, B lineage, myeloid dendritic cells, neutrophils, endothelial cells and fibroblasts). Results: We found that higher baseline levels of TILs (p = 0.045) but not SL were associated with an increased likelihood of a patient achieving a pCR to TCH/L based therapy. We found in day 20 on-treatment biopsies of women that subsequently went onto have a pCR that levels of SLs but not TILs were significantly higher (p = 0.049) than in those women who did not have a pCR. Finally we found significant increases in the level of monocytes (p = 0.05) and fibroblasts (p = 0.01), but not other immune cell populations, in the day 20 on-treatment biopsies in comparison with the mutated pre-treatment biopsies. Conclusions: In our study baseline TILs but not SLs have a predictive role in the likelihood of a patient achieving a pCR. We also found that TCHL based therapy significantly altered both monocytes and fibroblasts, indicating a possible role for these immune subtypes in response to TCHL therapy.

9032 Background: The EGFR-T790M somatic mutation is the most common mechanism of resistance to Tyrosine Kinase Inhibitors (TKI) in NSCLC. However patients with advanced disease are not always amenable to repeat biopsy for further molecular analysis. Developing non-invasive methods to detect T790M in cell-free DNA (cfDNA), in the absence of tissue is being actively investigated. Furthermore these 'liquid biopsies' may also overcome the problem of tumour genomic heterogeneity. Unfortunately the sensitivity of plasma for T790M detection has been disappointing with a significant chance of a false negative result. Exhaled breath condensate (EBC) is an easily collected sample and is known to harbour cfDNA, including lung cancer mutations. We explored the potential of EBC as a novel method of T790M detection. Methods: We recruited 26 patients who were either 1) known T790M positive pre/during Osimertinib therapy or 2) other mEGFR positive patients on 1st/2nd generation TKI. We collected matched plasma and EBC samples in the majority of cases. EBC samples were collected using the RTube device. Plasma was collected using standard EDTA tubes and extracted within 90 minutes. Using UltraSEEK chemistry, a targeted PCR for ultra-sensitive somatic mutation profiling on the MassARRAY system (Agena Bioscience), we compared the performance of EBC to plasma for the detection of T790M. Results: See Table. Conclusions: In this pilot study we describe the first ever report of the successful and consistent detection of T790M in the EBC of patients with EGFR mutated NSCLC, using a commercially available targeted assay. Our results suggest EBC is responsive to recognised dynamic molecular changes that occur on TKI treatment. We believe this makes EBC analysis an attractive avenue for future research, to optimise the detection of T790M mutations in liquid biopsies. [Table: see text]

11587 Background: The advent of immunotherapy represents a paradigm shift in the treatment of NSCLC compared to conventional chemotherapy. Recent studies have shown higher mts burden assessed by exome sequencing are associated with improved objective response and clinical benefit. We performed this study to evaluate the impact of ML assessment by LTP, correlating with PD-L1 exp and clinicopathological variables in resected NSCLC. Methods: NSCLC patients(pts) who underwent curative resection between 1998 and 2006 at our institution were included. PD-L1 status was assessed using Ventana SP124 antibody on archival FFPE surgical tumour specimens cores. PD-L1 was scored positive if membranous staining was present in >1% of tumour cells aggregated across the replicate cores to address heterogeneity. In collaboration with the Lung Cancer Genomics Ireland Study a targeted panel of 49 genes were assessed by Sequenom MassArray including genes in MAPK and PI3K pathways. Clinical data was obtained from hospital electronic database. Results: Ninety-one pts were included, of which 51 (56.0%) were males, with a median age of 65 years (range: 42 – 82). 51.6%, n=47 with squamous histological subtypes, 46.2%, n=42 were ex-smoker and 49.5%, n=45 had Stage I disease. 23.1%, n=21 had PD-L1 positivity. 149 mts were identified of which, 32(21.5%) with PHLPP2, 31(20.9%) with PIK3R1 and 21(14.1%) with TP53. The presence of PI3K and TP53 mts are associated with positive PD-L1 status (see table). An inverse correlation of PD-L1 positivity with ML of (1 vs 2 vs 3: 53.8% vs 30.8% vs 15.4%) was noted. Conclusions: We did not identify higher PD-L1 exp with higher ML assessed by a LTP widely used in clincial practice. However, positive PD-L1 exp was correlated with PIK3R1 and TP53 mts , warranting further investigation as potential modulators or surrogates of positve PD-L1 expression. [Table: see text]

The PI3K pathway is a key mechanism of trastuzumab resistance, but early attempts to indirectly target this pathway with mTOR inhibitors have had limited success. We present the results of a preclinical study of the selective alpha/delta isoform dominant PI3K inhibitor BAY 80-6946 tested alone and in combination with HER2-targeted therapies in HER2-positive cell lines, including models with acquired resistance to trastuzumab and/or lapatinib. A panel of HER2-positive breast cancer cells were profiled for their mutational status using Sequenom MassARRAY, PTEN status by Western blot, and anti-proliferative response to BAY 80-6946 alone and in combination with the HER2-targeted therapies trastuzumab, lapatinib and afatinib. Reverse phase protein array was used to determine the effect of BAY 80-6946 on expression and phosphorylation of 68 proteins including members of the PI3K and MAPK pathways. The Boyden chamber method was used to determine if BAY 80-6946 affected cellular invasion and migration. BAY 80-6946 has anti-proliferative and anti-invasive effects when used alone in our panel of cell lines (IC50s 3.9–29.4 nM). BAY 80-6946 inhibited PI3K signalling and was effective in cells regardless of their PI3K, P53 or PTEN status. The combination of HER2-targeted therapies and BAY 80-6946 inhibited growth more effectively than either therapy used alone (with clear synergism in many cases), and can restore sensitivity to trastuzumab and lapatinib in cells with acquired resistance to either trastuzumab and/or lapatinib. The addition of BAY 80-6946 to HER2-targeted therapy could represent an improved treatment strategy for patients with refractory metastatic HER2-positive breast cancer, and should be considered for clinical trial evaluation.

Angiogenesis or new vessel formation is essential for tumour growth and progression. Therefore, targeting angiogenesis has been an attractive strategy in the treatment ofcancer. Bevacizumab is a recombinant humanized monoclonal IgG1 antibody thattargets vascular endothelial growth factor-A (VEGF-A) - a key molecular player inangiogenesis. Bevacizumumab has shown clinical efficacy in phase III clinical trials inseveral advanced solid malignancies. The clinical efficacy of bevacizumumab isprimarily due to its antiangiogenic effects; however, there are direct antitumor effectsand immunomodulatory effects. Enhancing the immune system to restore itsantitumour activity has been utilized successfully in clinical setting. In this article we willdiscuss the possible immunomodulatory effects of the most clinically usedantiangiogenic agent; bevacizumumab.

Locally advanced rectal cancer (LARC: T3/4 and/or node-positive) is treated with preoperative/neoadjuvant chemoradiotherapy (CRT), but responses are not uniform. The phosphatidylinositol 3-kinase (PI3K), MAP kinase (MAPK), and related pathways are implicated in rectal cancer tumorigenesis. Here, we investigated the association between genetic mutations in these pathways and LARC clinical outcomes. We genotyped 234 potentially clinically relevant nonsynonymous mutations in 33 PI3K and MAPK pathway–related genes, including PIK3CA, PIK3R1, AKT, STK11, KRAS, BRAF, MEK, CTNNB1, EGFR, MET, and NRAS, using the Sequenom platform. DNA samples were extracted from pretreatment LARC biopsy samples taken from 201 patients who were then treated with long-course neoadjuvant CRT followed by surgical resection. Sixty-two mutations were detected in 15 genes, with the highest frequencies occurring in KRAS (47 %), PIK3CA (14 %), STK11 (6.5 %), and CTNNB1 (6 %). Mutations were detected in BRAF, NRAS, AKT1, PIK3R1, EGFR, GNAS, MEK1, PDGFRA, ALK, and TNK2, but at frequencies of

587Background: ‘TCHL’ an ICORG phase-II neo-adjuvant study in HER2+ve Breast cancer (BC) assessed the impact of TCH (Docetaxel, Carboplatin, Trastuzumab) versus TCHL (Docetaxel, Carboplatin, Trastu...

591Background: TCGA analysis revealed that somatic EGFR, ERBB2, ERBB3 and ERBB4 mutations (ERBB family mutations) occur alone or co-occur with somatic PIK3CA mutations in 19% of HER2-positive breas...

Introduction: Non-small cell lung cancer (NSCLC) is the leading cause of cancer morbidity and mortality globally in males, with rates increasing in females in recent years. New therapeutic intervention strategies will be crucial in improving the current survival rates for patients with this disease, which currently stand at less than 15% at 5 years. The PI3K-Akt- mTOR pathway regulates cell growth and proliferation and is often dysregulated in cancers including NSCLC, making it an attractive new therapeutic target in this setting. GDC-0980 is a selective dual inhibitor of PI3K and mTOR, which is currently in Phase II clinical trials for solid tumours. As with all targeted therapies, acquired resistance to GDC-0980 is anticipated to be a major hurdle in the success of this drug. The aim of this project is to develop and characterise four cell line models of resistance to GDC-0980, each representing a different molecular subtype of NSCLC, in order to identify biomarkers of response/resistance to the drug that may dictate beneficial treatment strategies. Methods: H460, A549, H1975 and SKMES-1 cells were cultured in GDC-0980 at IC50 concentrations over a period of several months, along with matched ‘parent’ cell lines. Development of resistance was assessed by monthly BrdU proliferation assays. Cell growth patterns were compared across the sensitive and resistant cell lines in real time using the xCELLigence platform. Cell lines were then interrogated for alterations in DNA (Sequenom sequencing of 547 mutations in 46 genes), mRNA (SABiosciences arrays profiling expression of >150 genes), miRNA (Exiqon expression profiling of 2100 miRNAs) and protein (R&D Phospho Kinase array expression profiling of 43 kinases and 2 associated total proteins, bottoms-up label-free mass spectrometry and Western blot analysis). Results: H1975 cells (PIK3CA mutant and activated pAkt (Ser473/Thr308), pmTOR, pS6R) were most sensitive to GDC-0980, however they were the first to develop resistance to the drug. Results obtained from xCELLigence studies identified H1975 resistant cells as having the highest cell index out of all parent and resistant cell lines after 100 hours of cell growth, suggesting that these are the most aggressive cells. A 33 miRNA signature was identified contrasting H1975 parent and resistant cells. 1,200 proteins were found to be differentially expressed between H1975 parent and resistant cells (p < 0.05, fold change >2) by bottoms-up label-free mass spectrometry. Preliminary results based on these screens have identified dysregulated metabolism and IGF-1 pathway signalling as potential mechanisms of resistance to GDC-0980 in H1975 cells. Comparison of Nanog, Sox2, Klf4, Oct4 and c-Myc expression highlighted a putative stem-like signature in all resistant cell lines. Several dysregulated genes and proteins will be presented including a kinase which plays a key role in cell proliferation and cancer progression and is targetable by dual and triple kinase inhibitors. Discussion and Conclusion: While the panel of four NSCLC cell lines all responded well to GDC-0980 treatment initially, resistance to the drug developed rapidly in 3 of the cell lines, within 4-6 months. Initial data highlights IGF-1 signalling pathway proteins and the emergence of a stem-like signature as potential mediators of resistance to the drug. Of particular interest is the identification of a specific kinase which is targetable by third generation small molecule inhibitors. Further elucidation of these bypass strategies are under investigation and are crucial to the design of optimal treatment protocols for patients. Citation Format: Susan Heavey, Paul Dowling, Sinéad Toomey, Aoife Carr, Bryan Hennessy, Michael O'Neill, Martin P Barr, Stephen Finn, Sinéad Cuffe, Kenneth J. O'Byrne, Kathy Gately. In depth molecular characterization of novel PI3K-mTOR inhibitor resistant NSCLC cell lines. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A45.

Background: The success of PI3K targeted inhibition has been hampered by a high rate of drug resistance, which may be facilitated by MEK pathway signaling. As such, investigation of PI3K-MEK co-targeted inhibition is warranted. With high morbidity and mortality rates and a clear need for new therapeutic intervention strategies, non-small cell lung cancer (NSCLC) is an ideal setting to investigate the effectiveness of this co-targeted inhibition strategy. GDC-0941 is a pan-PI3K inhibitor which has been investigated in combination with GDC-0973, a MEK inhibitor, in early clinical studies. No data has been published to date on the combination of GDC-0980 (a dual PI3K/mTOR inhibitor) and GDC-0973, which we believe may induce improved responses. We aim to elucidate the role of mutation status in response to this co-targeted inhibition approach in vitro, as well as correlating the frequency/type of PI3K/ MEK pathway mutations to PI3K pathway signaling in a well characterized NSCLC patient cohort. Methods: The effects of GDC-0941, GDC-0980 and GDC-0973 on proliferation, apoptosis and protein expression were examined in a panel of 4 NSCLC cell lines by BrdU Assay, HCA, PathScan protein array and Western blot. DNA was extracted from 120 NSCLC patient tissue samples, and screened for 547 clinically relevant mutations in 46 genes using the Sequenom platform. Total protein extracts, from freshly resected matched tumor/normal tissues from PIK3CA mutation positive versus wild-type patients, were subjected to phosphoproteomic analysis using Pathscan arrays (CST). Results: GDC-0941 and GDC-0980 treatment induced dose-dependent anti-proliferative and pro-apoptotic responses across all 4 NSCLC cell lines, while GDC-0973 treatment induced only anti-proliferative responses. GDC-0980 & GDC-0973 combination treatment induced significantly greater phosphoprotein inhibition than treatment with either inhibitor alone, most notably in cell lines harboring PIK3CA mutations. This combined treatment approach also led to significant increases in apoptosis and synergistic reductions in proliferation across the panel of cell lines as calculated using the Chou Talalay method. NSCLC patient mutational profiling identified a PIK3CA mutation frequency of >11%. Differences in PI3K pathway signaling were quantified in PIK3CA mutant versus wild-type patient matched tumor/normal tissues and results will be presented. Conclusion: This research underpins the importance of mutation status and pathway activation in sensitivity to targeted therapies. Combined inhibition of the PI3K and MEK pathways is a promising strategy for treatment of NSCLC, although efficacy is dependent on molecular subtype. In the era of personalized medicine, patient genotyping is crucial to improve patient survival and reduce toxicities. Citation Format: Susan Heavey, Sinead Toomey, Aoife Carr, Sinead Cuffe, Stephen Finn, Bryan Hennessy, John Crown, Martin Barr, Kenneth O'Byrne, Kathy Gately. PI3K targeted inhibition in NSCLC: An in vitro evaluation and correlation of mutation status to activated PI3K pathway signaling in NSCLC patients. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B25.