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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.

Objective\ud The therapeutic benefits of poly(ADP-ribose) polymerase inhibitors highlight the need to evaluate BRCA1/2 defects in tubal/ovarian cancer (OC). We sought to determine the pattern and disease characteristics associated with tumor BRCA1/2 mutations and BRCA1 methylation in women with OC.\ud Methods\ud We obtained 111 OC specimens from 2 university hospitals and assessed BRCA1/2 mutations and BRCA1 methylation in tumor DNA. The frequency and pattern of BRCA1/2 defects were examined. Associations between patient/disease characteristics and BRCA1/2 defects were ascertained (Fisher’s exact test). Platinum-free interval (PFI), progression-free survival (PFS), and overall survival (OS) based on the underlying BRCA1/2 defect were determined (Kaplan-Meier analysis [log-rank test]).\ud Results\ud We observed a BRCA1/2 dysfunction rate of 40% (28/70) in high-grade serous tubal/ovarian cancer (HGSC), including 14.3% BRCA1 methylation (n=10), 7.1% BRCA1 mutation (n=5), and 18.6% BRCA2 mutation (n=13). Defects in BRCA1/2 genes were associated with stage III/IV HGSC (BRCA1 methylation: P=0.005 [stage III/IV] and P=0.004 [HGSC]; BRCA1/2 mutation: P=0.03 [stage III/IV] and P

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.

False negatives from nasopharyngeal swabs (NPS) using reverse transcriptase PCR (RT-PCR) in SARS-CoV-2 are high. Exhaled breath condensate (EBC) contains lower respiratory droplets that may improve detection. We performed EBC RT-PCR for SARS-CoV-2 genes (E, S, N, ORF1ab) on NPS-positive (n=16) and NPS-negative/clinically positive COVID-19 patients (n=15) using two commercial assays. EBC detected SARS-CoV-2 in 93.5% (29/31) using the four genes. Pre-SARS-CoV-2 era controls (n=14) were negative. EBC was positive in NPS negative/clinically positive patients in 66.6% (10/15) using the identical E and S (E/S) gene assay used for NPS, 73.3% (11/15) using the N/ORF1ab assay and 14/15 (93.3%) combined.

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.

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.

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.

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.

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.

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.

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.

A subacute inflammatory phenotype is implicated in the pathology of insulin resistance (IR) and type 2 diabetes mellitus. Interleukin (IL)-1α and IL-1β are produced by innate immune cells, including macrophages, and mediate their inflammatory response through the IL-1 type I receptor (IL-IRI). This study sought to understand the transcriptomic signature of adipose tissue in obese IL-1RI(-/-) mice. Following dietary intervention, markers of insulin sensitivity and inflammation in adipose tissue were determined, and gene expression was assessed with microarrays. IL-1RI(-/-) mice fed a high-fat diet (HFD) had significantly lower plasma inflammatory cytokine concentrations than wild-type mice. Metabolic network analysis of transcriptomic effects identified up-regulation and co-expression of genes involved in lipolysis, lipogenesis and tricarboxylic acid (TCA) cycle. Further assessment of gene expression in a network of protein interactions related to innate immunity highlighted Stat3 as a potential transcriptional regulator of IL-1 signalling. The complex, downstream effects of IL-1 signalling through the IL-1RI receptor remain poorly defined. Using network-based analyses of transcriptomic signatures in IL-1RI(-/-) mice, we have identified expression changes in genes involved in lipid cycling and TCA cycle, which may be more broadly indicative of a restoration of mitochondrial function in the context of HFD. Our results also highlight a potential role for Stat3 in linking IL-1 signalling to adipogenesis and IR.

// Sinead Toomey 1, * , Stephen F. Madden 2, * , Simon J. Furney 3 , Yue Fan 4 , Mark McCormack 5 , Carragh Stapleton 5 , Mattia Cremona 1 , Gianpiero L. Cavalleri 5 , Malgorzata Milewska 1 , Naomi Elster 1 , Aoife Carr 1 , Joanna Fay 6 , Elaine W. Kay 6 , Susan Kennedy 7 , John Crown 7 , William M. Gallagher 4 , Bryan T. Hennessy 1, 8, ** , Alex J. Eustace 1, ** 1 Medical Oncology Group, Department of Molecular Medicine, Royal College of Surgeons in Ireland, Ireland 2 Population Health Sciences Division, Royal College of Surgeons in Ireland, Ireland 3 School of Medicine, University College Dublin, Ireland 4 University College Dublin, UCD School of Biomolecular and Biomedical Science, Conway Institute of Biomolecular and Biomedical Research, Ireland 5 Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, Ireland 6 Department of Histopathology, Beaumont Hospital, Dublin, Ireland 7 St Vincent’s University Hospital, Dublin, Ireland 8 Department of Oncology, Beaumont Hospital, Dublin, Ireland * These authors contributed equally to this work ** Joint senior authors Correspondence to: Alex J. Eustace, email: alexeustace@rcsi.ie Keywords: ERBB-family germline mutations, single nucleotide polymorphisms, high depth sequencing, survival impact Received: May 03, 2016 Accepted: October 12, 2016 Published: October 20, 2016 ABSTRACT Background: Trastuzumab treatment for women with HER2-positive breast cancer (BC) resulted in the significant improvement of both relapse free survival (RFS) and overall survival (OS). However, many women who are classified as HER2-positive do not respond. Many studies have focused on the role of somatic mutations rather than germline polymorphisms in trastuzumab resistance. Results: We completed an Agena MassArray screen of 10 ERBB-family single nucleotide polymorphisms (SNPs) in 194 adjuvant trastuzumab treated HER2-positive BC patients. SNPs in EGFR genes have a significant association with RFS and OS. Patients with the minor allele of EGFR N158N had significantly worse OS (hazard ratio (HR) = 4.01, (confidence interval (CI) = 1.53– 10.69), p = 0.05) relative to those with either the heterozygous or wild-type (WT) allele. Patients with the minor allele of EGFR T903T (HR = 3.52, (CI = 1.38– 8.97), p = 0.05) had worse RFS relative to those with either the heterozygous or WT allele. Patients and methods: Using next generation sequencing (NGS) we identified ERBB-family (EGFR, HER2, HER3 and HER4) single nucleotide polymorphisms (SNPs) that occurred in 2 or more patients of a 32 HER2-positive BC patient cohort. Agena MassArray analysis confirmed the frequency of these SNPs in 194 women with HER2-positive BC who received trastuzumab in the adjuvant setting. Using Kaplan-Meier estimates and Cox regression analysis we correlated the presence of ERBB-family SNPs with both RFS and OS. Conclusions: The presence of germline ERBB-family SNPs may play an important role in how a patient responds to adjuvant trastuzumab, and clinical assessment of these SNPs by targeted genetic screening of patients’ blood may be important to stratify patients for treatment.

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

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.

The WHO estimate that >1×106deaths in Europe annually can be attributed to diseases related to excess body weight, and with the rising global obesity levels this death rate is set to drastically increase. Obesity plays a central role in the metabolic syndrome, a state of insulin resistance that predisposes patients to the development of CVD and type 2 diabetes mellitus. Obesity is associated with low-grade chronic inflammation characterised by inflamed adipose tissue with increased macrophage infiltration. This inflammation is now widely believed to be the key link between obesity and development of insulin resistance. In recent years it has been established that activation of pro-inflammatory pathways can cross talk with insulin signalling pathways via a number of mechanisms including (a) down-regulation of insulin signalling pathway proteins (e.g. GLUT4 and insulin receptor substrate (IRS)-1), (b) serine phosphorylation of IRS-1 blocking its tyrosine phosphorylation in response to insulin and (c) induction of cytokine signalling molecules that sterically hinder insulin signalling by blocking coupling of the insulin receptor to IRS-1. Long-chain (LC)n-3 PUFA regulate gene expression (a) through transcription factors such as PPAR and NF-κB and (b) via eicosanoid production, reducing pro-inflammatory cytokine production from many different cells including the macrophage. LCn-3 PUFA may therefore offer a useful anti-inflammatory strategy to decrease obesity-induced insulin resistance, which will be examined in the present review.

The development of insulin resistance in the obese is associated with chronic, low-grade inflammation. We aimed to identify novel links between obesity, insulin resistance and the inflammatory response by comparing C57BL/6 with type I interleukin-1 receptor knockout (IL-1RI(-/-)) mice, which are protected against diet-induced insulin resistance. Mice were fed a high-fat diet for 16 wk. Insulin sensitivity was measured and proteomic analysis was performed on adipose, hepatic and skeletal muscle tissues. Despite an equal weight gain, IL-1RI(-/-) mice had lower plasma glucose, insulin and triacylglycerol concentrations, compared with controls, following dietary treatment. The higher insulin sensitivity in IL-1RI(-/-) mice was associated with down-regulation of antioxidant proteins and proteasomes in adipose tissue and hepatic soluble epoxide hydrolase, consistent with a compromised inflammatory response as well as increased glycolysis and decreased fatty acid beta-oxidation in their muscle. Their lower hepatic triacylglycerol concentrations may reflect decreased flux of free fatty acids to the liver, decreased hepatic fatty acid-binding protein expression and decreased lipogenesis. Correlation analysis revealed down-regulation of classical biomarkers of ER stress in their adipose tissue, suggesting that disruption of the IL-1RI-mediated inflammatory response may attenuate cellular stress, which was associated with significant protection from diet-induced insulin resistance, independent of obesity.

Adipose tissue may be the source of insulin desensitizing proinflammatory molecules that predispose to insulin resistance. This study investigated whether dietary fatty acids could attenuate the proinflammatory insulin-resistant state in obese adipose tissue. The potential antidiabetic effect of cis-9, trans-11–conjugated linoleic acid (c9,t11-CLA) was determined, focusing on the molecular markers of insulin sensitivity and inflammation in adipose tissue of ob/ob C57BL-6 mice. Feeding a c9,t11-CLA–enriched diet reduced fasting glucose (P < 0.05), insulin (P < 0.05), and triacylglycerol concentrations (P < 0.01) and increased adipose tissue plasma membrane GLUT4 (P < 0.05) and insulin receptor (P < 0.05) expression compared with the control linoleic acid–enriched diet. Interestingly, after the c9,t11-CLA diet, adipose tissue macrophage infiltration was less, with marked downregulation of several inflammatory markers in adipose tissue, including reduced tumor necrosis factor-α and CD68 mRNA (P < 0.05), nuclear factor-κB (NF-κB) p65 expression (P < 0.01), NF-κB DNA binding (P < 0.01), and NF-κB p65, p50, c-Rel, p52, and RelB transcriptional activity (P < 0.01). To define whether these observations were direct effects of the nutrient intervention, complimentary cell culture studies showed that c9,t11-CLA inhibited tumor necrosis factor-α–induced downregulation of insulin receptor substrate 1 and GLUT4 mRNA expression and promoted insulin-stimulated glucose transport in 3T3-L1 adipocytes compared with linoleic acid. This study suggests that altering fatty acid composition may attenuate the proinflammatory state in adipose tissue that predisposes to obesity-induced insulin resistance.

Background— Cyclooxygenase (COX) activity is induced in human atherosclerosis, and the products formed may modify the disease directly or through an effect on platelets. We examined the role of COX-1 and -2 on platelet vessel wall interactions and development of atherosclerosis in a murine model. Methods and Results— Apolipoprotein E–deficient (apoE −/− ) mice fed a 1% cholesterol diet were treated with a selective COX-1 inhibitor (SC-560), a selective COX-2 inhibitor (SC-236), or vehicle. Urinary prostacyclin and thromboxane metabolites (2,3-dinor-6-keto-PGF 1α and 2,3-dinor-TXB 2 ) were increased in the apoE −/− knockout mouse. There was also induction of both COX isoforms in the vascular lesions formed, which stained for CD41, a platelet-specific marker, and for CD40L. Selective inhibition of COX-2 had no effect on lesion formation and, despite selective reduction in prostacyclin generation, had no effect on platelet activity, as measured by thromboxane formation or platelet deposition. Selective inhibition of COX-1 reduced 2,3-dinor-TXB 2 generation and lesion formation. However, platelet deposition on the vessel wall persisted, with well-defined monolayers seen. There was also persistent expression of the macrophage marker CD68 and increased expression of the cell death protein Bax. In contrast to lesion development, the selective COX-1 inhibitor had no effect on the regression of evolving lesions. Conclusions— COX-1 plays an important role in the early stages of lesion development in the apoE −/− knockout model of atherosclerosis, preventing gross lesion formation in the face of continued vascular injury and inflammation. Despite the inhibition of prostacyclin, COX-2 inhibition had no effect on lesion development or platelet–vessel wall interactions.

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.

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...

Conjugated linoleic acid (CLA) is found naturally in meat and dairy products, and represents a potential therapeutic functional nutrient. However, given the discrepancies in isomer composition and concentration, controversy surrounds its proposed antidiabetic, antiobesity effects. This study focused on the effects of CLA-enriched beef (composed predominantly of c9, t11-CLA) in two separate models of metabolic disease: proatherosclerotic ApoE(-/-) mice and diabetic, leptin-deficient ob/ob mice. Animals were fed CLA-enriched beef for 28 days, and markers of the metabolic syndrome and atherosclerosis were assessed. Comprehensive hepatic transcriptomic analysis was completed to understand divergent metabolic effects of CLA. CLA-enriched beef significantly reduced plasma glucose, insulin, nonesterified fatty acid and triacylglycerol and increased adiponectin levels in ob/ob mice. In contrast, plasma lipid profiles and glucose homeostasis deteriorated and promoted atherosclerosis following the CLA-enriched beef diet in ApoE(-/-) mice. Hepatic transcriptomic profiling revealed divergent effects of CLA-enriched beef on insulin signaling and lipogenic pathways, which were adversely affected in ApoE(-/-) mice. This study demonstrated clear divergence in the effects of CLA. CLA-enriched beef improved metabolic flexibility in ob/ob mice, resulting in enhanced insulin sensitivity. However, CLA-enriched diet increased expression of lipogenic genes, resulting in inefficient fatty acid storage which increases lipotoxicity in peripheral organs, and led to profound metabolic dysfunction in ApoE(-/-) mice. While CLA may have potential health effects, in some circumstances, caution must be exercised in presenting this bioactive lipid as a potential functional food for the treatment of metabolic disease.

Understanding the functional relationships between diet, gene expression and phenotype is a key challenge in nutritional science. The LIPGENE project was an international research effort assessing the impact of genotype and dietary fat intake on metabolic health (1) . In the final stage of this project, we generated adipose tissue microarray data in order to examine the effect of habitual dietary components on transcriptomic activity and, subsequently, markers of metabolic health. A sub-cohort of participants in the LIPGENE human intervention study were included in this analysis of adipose tissue transcriptomic signatures (2) . Habitual dietary data included SFA, MUFA, w‐3 and w‐6 PUFA, carbohydrates, protein and alcohol. Blood samples were collected and assessed for 46 markers of metabolic and cardiovascular health including cholesterol, TAG, insulin sensitivity and glucose metabolism (assessed by IVGTT) and markers of inflammation and oxidative stress. Plasma fatty acid composition was also determined as a marker of dietary fat intake, and urinary 8-iso-PGF2a as a marker of oxidative stress. As an alternative to pathway analysis, we have taken a network-based approach to the analysis of dietary, transcriptomic and comprehensive plasma marker profile. Sparse partial least squares and regularised canonical correlation analyses were used to identify strongly correlated diet-gene and plasma marker-gene pairs, which were then mapped to a high-quality reconstruction of the human metabolic network. Diet-sensitive and transcriptionally co-expressed sub-paths were subsequently extracted from the network using an algorithm designed to trace paths of enzymatic metabolite conversion. With this approach we identified a prominent sub-path in the network that was transcriptionally correlated with dietary w-3 PUFA intake and strongly co-expressed within the sub-path. This sub-path interlinked the related processes of glycerophospholipid, arachidonic acid and linoleic acid metabolism ‐ and was shown to correlate with plasma markers of metabolic health including plasma DHA and urinary 8‐9-iso-PGF2a. Promoter analysis further revealed a number of significantly over-represented adipogenic transcription factors (such as pparg and klf4) in the promoter regions of genes that were positively correlated with w-3 PUFA intake. This integrated bioinformatics approach highlighted novel regulatory and metabolic mechanisms relating dietary w-3 PUFA intake to metabolic health via adipogenic transcription factors and interconnected lipid metabolism pathways.

Conjugated linoleic acid (CLA) refers to a group of positional and geometric isomers of linoleic acid and has been shown to suppress the development of atherosclerosis in experimental models. However, the mechanism involved is unclear although it is believed it may act as a cyclooxygenase inhibitor or as an agonist of the nuclear receptors, peroxisome proliferator activated receptors (PPARs). In this study, we examined the effect of cis-9,trans-11:trans-10,cis-12-CLA (80:20 blend) on the regression of pre-established atherosclerosis. ApoE(-/-) mice fed a 1% cholesterol diet were randomized at 8 weeks to continue receiving the diet supplemented with 1% control saturated fat or 1% CLA blend for a further 8 weeks. CLA supplementation did not simply prevent progression but induced almost complete resolution of atherosclerosis. Although CLA inhibited platelet deposition, as detected by staining of platelet glycoprotein alpha11b beta111a, it did not inhibit COX-mediated generation of prostaglandins in this model. However, PPARalpha and PPARgamma expression was increased in the aorta of the CLA-treated animals. This was coincident with decreased macrophage accumulation and decreased expression of the macrophage scavenger receptor CD36 and increased apoptosis in the aorta in vivo. CLA induces the resolution of atherosclerosis by negatively regulating the expression of pro-inflammatory genes and inducing apoptosis in the atherosclerotic lesion.

Conjugated linoleic acid (CLA) refers to a group of positional and geometric isomers of linoleic acid that has been shown to suppress the development of atherosclerosis in a rabbit model. We investigated whether CLA acts as a cyclo-oxygenase (COX) inhibitor or as an agonist of the peroxisome-proliferator-activator receptor (PPAR) γ in the ApoE−/− mouse model. In vitro, a 9-cis, 11-trans isomer of CLA inhibited prostaglandin formation and oxygen consumption by both isoforms of COX, with no evidence by MS of alternative products being generated. In vivo, supplementation with CLA was found to induce resolution of atherosclerosis. The effect of CLA in vivo could not be explained by COX inhibition alone, as urinary prostaglandin levels were unchanged in animals receiving CLA supplementation, and administration of selective COX inhibitors did not induce lesion regression. There was however induction of PPARγ, a known response to agonists of this nuclear orphan receptor.

Background: Aberrant activity of the mitogen-activated protein kinase (MAPK) signalling pathway represents a critical factor in the initiation and development of melanoma. Activation can be the result of largely mutually exclusive somatic mutations of KIT, NRAS, or more frequently, BRAF(V600E). The FDA and EMA approval of a BRAF inhibitor (vemurafenib) that blocks the function of the BRAFV600E protein established a new paradigm for targeted drug development. Currently, BRAF inhibitors are one of the most promising approaches to treat metastatic melanoma. Methods: In this study, we applied mass spectrometry-based SNP genotyping (Sequenom) to detect single nucleotide mutations in 33 cancer-related genes including BRAF, NRAS, KIT, CTNNB1, GNAS, and MET. PCR and extension primers for the multiplexed assay were designed with the Sequenom Assay Design software. Samples were processed as recommended by the manufacturer. Matrix chips were assayed on a Sequenom MassArray MALDI-TOF Mass Array system. Visual inspection and Sequenom Typer software were used to perform genotyping based on mass spectra. We compared two independent melanoma cohorts, one from Ireland (SVUH cohort; n=97) and the other from Belgium (Leuven cohort; n=60). Results: Intriguingly, patients from Ireland showed significant lower BRAFV600E mutation rates (19.0%) when compared to the mutation frequency seen with the Belgian cohort (43.3%) or with the mutation frequency reported by other studies on non-Irish populations (50-70%). In addition, BRAFV600K mutation is rare in the SVUH cohort (1.0%) compared with the Leuven cohort (6.7%). Mutations in BRAFV600M and V600R were Conclusion: Our observation of reduced BRAFV600E mutation rates in Irish patients will directly impact future treatment strategies for these patients. The identification of substantial mutation rates in MET opens up opportunities for targeting a significant subset of melanoma patients with MET inhibitors. Citation Format: Balazs Balint, Karin van den Hurk, Sinead Toomey, Louise Unwin, Kieran Sheahan, Enda McDermott, Ian Murphy, Joost van den Oord, Mairin Rafferty, Bryan Hennessy, William M. Gallagher. Low incidence of BRAFV600E mutation among melanoma patients in Ireland. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 23. doi:10.1158/1538-7445.AM2013-23