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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.

We review the use of anthracyclines, their associated cardiotoxicity, and the role of taxanes in the treatment of early breast cancer. The efficacy of anthracyclines has been proven in multiple large cohort trials and meta-analyses. The addition of a taxane to an anthracycline-based regimen in the adjuvant setting has improved the disease-free survival and overall survival in patients with early breast cancer. The use of the monoclonal antibody trastuzumab combined with an anthracycline and taxane regimen has had significant benefit both in disease-free survival and overall survival in patients with human epidermal growth factor receptor 2(HER2)–positive disease. However, the development of significant cardiotoxicity related to anthracyclines and the emergence of newer agents that appear to limit the cardiotoxicity but with similar anticancer efficacy have called the role of anthracyclines into doubt. Taxane/trastuzumab-based chemotherapy without anthracyclines is now a widely accepted adjuvant regimen in patients with HER2–positive early breast cancer. Indeed, the use of taxanes in early breast cancer has overtaken the use of anthracyclines, particularly in women with HER2–positive breast cancer. Concerns regarding anthracycline cardiotoxicity and positive data from taxane-based regimens likely have contributed to this change in practice. Ongoing trials will determine whether taxane-based chemotherapy has equivalent efficacy to anthracycline-based regimens in adjuvant therapy of HER2–negative early breast cancer. Moving forward, the use of anthracyclines as initial chemotherapy in early breast cancer may continue to be replaced by taxane-based and novel regimens in the future.

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

The dipyrrinato iron catalyst reacts with organic azides to generate a reactive, high-spin imido radical intermediate, distinct from nitrenoid or imido species commonly observed with low-spin transition metal complexes. The unique electronic structure of the putative group-transfer intermediate dictates the chemoselectivity for intermolecular nitrene transfer. The mechanism of nitrene group transfer was probed via amination and aziridination of para-substituted toluene and styrene substrates, respectively. The Hammett analysis of both catalytic amination and aziridination reactions indicate the rate of nitrene transfer is enhanced with functional groups capable of delocalizing spin. Intermolecular amination reactions with olefinic substrates bearing allylic C–H bonds give rise to exclusive allylic amination with no apparent aziridination products. Amination of substrates containing terminal olefins give rise exclusively to allylic C–H bond abstraction, C–N recombination occurring at the terminal C with transposition of the double bond. A similar reaction is observed with cis-β-methylstyrene where exclusive amination of the allylic position is observed with isomerization of the olefin to the trans-configuration. The high levels of chemoselectivity are attributed to the high-spin electronic configuration of the reactive imido radical intermediate, while the steric demands of the ligand enforce regioselective amination at the terminal position of linear α-olefins.

Background In the phase III RTOG 9802 trial, median overall survival (OS) increased from 7.8 years with radiotherapy alone to 13.3 years (P = .002) with adjuvant procarbazine, lomustine, and vincristine (PCV) following surgery for low grade glioma. Although the PCV regimen improved survival, there were significant toxicities. We hypothesised more frequent toxicities would be observed in clinical practice. We aimed to identify and quantify toxicities in the real world setting in a National Neuro-Oncology centre. Methods Patients with low grade glioma who received PCV following radiotherapy from Nov 2014 to Nov 2018 were identified from Institutional databases. Data on toxicities, dose reductions, dose delays and treatment discontinuation were collected from paper and electronic patient records. Results A total of 41 patients with low grade glioma were identified. Patients were predominantly male (n = 28) and the median age at diagnosis was 41. Of these, 28/41 patients had a diagnosis of oligodendroglioma and 13/41 had astrocytoma. The majority of patients had tumours with favourable molecular features such as IDH mutations (n = 38) and 1p19q co deletions (n = 23). Only 4 patients completed 6 cycles of chemotherapy without any dose modification or delay. 16 patients completed PCV with dose reductions and dose delays at various levels. The dose intensity was as follows: procarbazine: 69%, lomustine: 71% and vincristine: 68%. Neutropaenia and thrombocytopaenia (table) were the major causes of treatment discontinuation (14/21 patients). Three patients had disease progression during chemotherapy and two patient’s treatment was discontinued due to abnormal LFTs. Three patients opted not to continue chemo due to intolerability in context of grade 3 to 4 fatigue and GI symptoms. At median follow up of 17 months, 9/41 patients have had disease progression. Table: 430P . Our Study % RTOG 9802% Patient Completed PCV 48 56 Median Number of Cycles Completed 3 3.5 Thrombocytopenia G2 (50-75) 71 9 G3 (25-50) 17 18 G4 ( 2 0 Neutropenia G2(1-1.5) 87 9 G3(0.5-0.1) 31 35 G4( 7 8 Conclusions Frequent toxicities are observed with PCV in clinical practice. Our data suggest it might be preferable to adjust doses from the start of chemotherapy to improve tolerability. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

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

Background HER2 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.

Background: The addition of trastuzumab (H) to pre-operative chemotherapy in H+BC increases the rate of pathological complete remission (pCR). H causes cardiac toxicity, especially when given with anthracyclines (Anth). TCH is a widely used adjuvant regimen with decreased cardiac toxiciy. We reported that TCH produces pCR in 40% of non-randomised pts with H+BC. Lapatinib is an alternative HER2 antagonist, which produces responses following trastuzumab failure, and which has been reported to augment H activity in combination. We studied the non-Anth regimens TCH v TCL v TCHL in pts with H+BC. The primary objective of this study was pCR. Secondary endpoints were toxicity and translational. Methods: Eligibility criteria included: primary BC, HER-2 +, node + disease (histologically or cytologically confirmed) or node-negative with >T1, normal left ventricle ejection fraction, no active/uncontrolled cardiovascular disease, normal organ and marrow function. Treatment consisted of 6 cycles of D (75mg/m2) + C (AUC 6) q3 weekly and H (8 mg/kg on cycle 1 day 1 and 6 mg/kg q3weekly thereafter for one year) ± L (1000mg OD) for up to 1 week before surgery. A sample size of 36 evaluable pts is required to detect an absolute 25% difference in the pCR rate between the hypothesised 65% pCR rate vs the historical-control pCR rate of 40%. Results: Following presentation of NCIC MA31 we decided to suspend accrual on our TCL arm.78 female pts were accrued to TCH/TCHL in 11 ICORG sites between 12/2010- 06/2013. Of 40 patients accrued to TCHL, only 18 pts completed 6 cycles. 17pts came off study early due to toxicity, 3 pts after cycle 3, 2 pts after cycle 2, 12pts after cycle 1. (1 patient was also registered but never started). Of 38 pts accrued to the TCH arm,33 pts completed 6 cycles,2 pts completed 5 cycles and 2 pts w/d after cycle 1. 3 TCHL & 1 TCH pt still remain on Rx. 2 pts have not yet had surgery. 52 SAEs occurred on study, 49 involving hospital admission, & 3 of medical significance. The most frequent SAEs were diarrhoea (10), febrile neutropenia (4), nausea (4), neutropenic sepsis (3), dehydration (2), wound infection (2), vomiting (2) neutropenia (2) decreased haemoglobin (2), GI perforation (1). There was 1 fatality on the TCH arm due to Neutropenic sepsis and typhlitis. One TCHL pt suffered GI perforation at cycle 1. pCR rates were 48% (16/33) for the TCH arm and 44% (7/16) for the TCHL arm. Translational studies are underway. Conclusions: TCH containing treatment produces a high rate of pCR. TCHL will not produce a statistically higher rate of pCR in this sample. The addition of lapatinib to TCH results in substantial GI toxicity. TCHL appears to be less tolerable than other active chemotherapy +H+L regimens such as that used in Neo-ALLTO. ICORG is currently leading an international study of paclitaxel+H +/- L in metastatic BC. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-12-25.

Platelet hyperreactivity is associated with an increased risk of thrombosis. Cancer patients are at an increased risk of thrombosis, a risk that increases with disease progression. While cancer patients show evidence of platelet activation in vivo, few studies have extensively assessed whether these patients display platelet hyperreactivity. We hypothesized that patients with metastatic cancer would display platelet hyperreactivity, reflecting their associated high risk of thrombosis. In a cohort of patients with metastatic cancer (n = 13), we assessed platelet function using well-established assays of platelet reactivity (agonist-induced platelet aggregation, spontaneous platelet aggregation, and agonist-induced P-selectin expression). In comparison with healthy controls (n = 10), patients with metastatic cancer displayed global platelet hyperreactivity. Agonist-induced platelet aggregation responses to ADP (adenosine diphosphate), epinephrine, collagen, arachidonic acid, and PAR-1 (protease-activated receptor-1) activating peptide, as well as spontaneous platelet aggregation, were significantly increased in patients with metastatic cancer. Furthermore, agonist-induced platelet P-selectin expression was also significantly increased within the patient cohort. We demonstrate that patients with metastatic cancer are characterized by global platelet hyperreactivity, a factor that may contribute to their increased risk of thrombosis. We assessed platelet function in a cohort of patients with metastatic cancer (n = 13) using well-established assays of platelet reactivity. Agonist-induced platelet aggregation and activation in response to platelet agonists, as well as spontaneous platelet aggregation, was significantly increased in cancer patients compared with healthy controls. We demonstrate that patients with metastatic cancer are characterized by global platelet hyperreactivity, a factor that may contribute to their increased risk of thrombosis.

Closing the Cycle Cyclic hydrocarbons that incorporate nitrogen in the ring are among the most heavily investigated compounds in medicinal chemistry. Hennessy and Betley (p. 591 ) demonstrate an iron catalyst that forms a range of such cyclic compounds by inducing linear alkyl azides to curl back on themselves, inserting the nitrogen at one end into a carbon-hydrogen bond further down the chain. The reaction furthers a trend of C-H bond activation chemistry that forms elaborate products from relatively simple precursors, without the need to install activating groups at unreactive sites.

Mutations in BRCA1/2 increase the risk of developing breast and ovarian cancer. Germline BRCA1/2 mutations occur in 8.6-13.7% of unselected epithelial ovarian cancers, somatic mutations are also frequent. BRCA1/2 mutated or dysfunctional cells may be sensitive to PARP inhibition by synthetic lethality. The aim of this study is to comprehensively characterise the BRCA1/2 status of a large panel of ovarian cancer cell lines available to the research community to assist in biomarker studies of novel drugs and in particular of PARP inhibitors. The BRCA1/2 genes were sequenced in 41 ovarian cell lines, mRNA expression of BRCA1/2 and gene methylation status of BRCA1 was also examined. The cytotoxicity of PARP inhibitors olaparib and veliparib was examined in 20 cell lines. The cell line SNU-251 has a deleterious BRCA1 mutation at 5564G > A, and is the only deleterious BRCA1/2 mutant in the panel. Two cell lines (UPN-251 and PEO1) had deleterious mutations as well as additional reversion mutations that restored the protein functionality. Heterozygous mutations in BRCA1/2 were relatively common, found in 14.6% of cell lines. BRCA1 was methylated in two cell lines (OVCAR8, A1847) and there was a corresponding decrease in gene expression. The BRCA1 methylated cell lines were more sensitive to PARP inhibition than wild-type cells. The SNU-251 deleterious mutant was more sensitive to PARP inhibition, but only in a long-term exposure to correct for its slow growth rate. Cell lines derived from metastatic disease are significantly more resistant to veliparib (2.0 fold p = 0.03) compared to those derived from primary tumours. Resistance to olaparib and veliparib was correlated Pearsons-R 0.5393, p = 0.0311. The incidence of BRCA1/2 deleterious mutations 1/41 cell lines derived from 33 different patients (3.0%) is much lower than the population incidence. The reversion mutations and high frequency of heterozygous mutations suggest that there is a selective pressure against BRCA1/2 in cell culture similar to the selective pressure seen in the clinic after treatment with chemotherapy. PARP inhibitors may be useful in patients with BRCA1 deleterious mutations or gene methylation.

Advanced or recurrent low-grade serous ovarian cancers (LGSC) are resistant to conventional systemic treatments. LGSC carry mutations in RAS or RAF, leading to several clinical trials evaluating MEK inhibitors (MEKi). As LGSC cell lines and xenografts have been difficult to establish, little is known about the efficacy and on-target activity of MEKi treatment in this disease. We compared four different MEKi (trametinib, selumetinib, binimetinib and refametinib) in novel LGSC patient-derived cell lines. Molecular characterization of these cells included copy-number variation and hotspot mutational analysis. Proliferation, apoptosis and cell viability assays were used to study drug efficacy. MEKi on-target efficacy was measured using western blotting and isoelectric point focusing for ERK1/2 phosphorylation. Ten LGSC cell lines were derived from 7 patients with advanced/recurrent disease. Copy number variation showed significant heterogeneity among cell lines, however all samples showed deletions in chromosome 9p21.3, and frequent copy number gains in chromosomes 12 and 20. Mutations in KRAS/NRAS were identified in 4 patients (57%) and RAS mutation status was not associated with higher baseline levels of ERK phosphorylation. Different degrees of MEKi sensitivity were observed in the LGSC cell lines. Two cell lines, both with KRAS mutations, were highly sensitive to MEKi. Drug anti-proliferative efficacy correlated with the degree of inhibition of ERK phosphorylation, with trametinib being the most potent agent. Differences in MEKi efficacy were observed in LGSC cell lines. Trametinib showed the greatest anti-proliferative effects. This study serves as a basis for much needed future research on MEKi drug efficacy in LGSC.

Carbonyls and alkenes, two of the most common functional groups in organic chemistry, generally do not react with one another. Now, a simple Lewis acid has been shown to catalyse metathesis between alkenes and ketones in a new carbonyl olefination reaction.

Background/Aims: This study is aimed at analyzing the survival rates and prognostic factors of stage IV colorectal cancer patients from 3 European cohorts undergoing combination chemotherapy with bevacizumab. Methods: Progression free-survival (PFS) and overall survival (OS) were analyzed in 172 patients using the Kaplan-Meier method and uni- and multivariable Cox proportional hazards regression models. Results: The median PFS was 9.7 and the median OS 27.4 months. Patients treated at centers in Germany (n = 97), Ireland (n = 32), and The Netherlands (n = 43) showed a median PFS of 9.9, 9.2, and 9.7 months, OS of 34.0, 20.5, and 25.1 months, respectively. Patients >65 years had a significantly shorter PFS (9.5 vs. 9.8 months) but not OS (27.4 vs. 27.5 months) than younger patients. High tumor grade (G3/4) was associated with a shorter PFS, T4 classification with both shorter PFS and OS. Fluoropyrimidine (FP) chemotherapy backbones (doublets and single) had comparable outcomes, while patients not receiving FP backbones had a shorter PFS. In multivariable analysis, age and non-FP backbone were associated with inferior PFS, T4 classification and therapy line >2nd were significantly associated with poor PFS and OS. Conclusion: The observed survival rates confirm previous studies and demonstrate reproducible benefits of combination bevacizumab regimens. Classification T4, non-FP chemotherapy backbone, and age >65 were associated with inferior outcome.

// 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.

Purpose: BRCA1/2-mutated and some sporadic triple-negative breast cancers (TNBC) have DNA repair defects and are sensitive to DNA-damaging therapeutics. Recently, three independent DNA-based measures of genomic instability were developed on the basis of loss of heterozygosity (LOH), telomeric allelic imbalance (TAI), and large-scale state transitions (LST). Experimental Design: We assessed a combined homologous recombination deficiency (HRD) score, an unweighted sum of LOH, TAI, and LST scores, in three neoadjuvant TNBC trials of platinum-containing therapy. We then tested the association of HR deficiency, defined as HRD score ≥42 or BRCA1/2 mutation, with response to platinum-based therapy. Results: In a trial of neoadjuvant platinum, gemcitabine, and iniparib, HR deficiency predicted residual cancer burden score of 0 or I (RCB 0/I) and pathologic complete response (pCR; OR = 4.96, P = 0.0036; OR = 6.52, P = 0.0058). HR deficiency remained a significant predictor of RCB 0/I when adjusted for clinical variables (OR = 5.86, P = 0.012). In two other trials of neoadjuvant cisplatin therapy, HR deficiency predicted RCB 0/I and pCR (OR = 10.18, P = 0.0011; OR = 17.00, P = 0.0066). In a multivariable model of RCB 0/I, HR deficiency retained significance when clinical variables were included (OR = 12.08, P = 0.0017). When restricted to BRCA1/2 nonmutated tumors, response was higher in patients with high HRD scores: RCB 0/I P = 0.062, pCR P = 0.063 in the neoadjuvant platinum, gemcitabine, and iniparib trial; RCB 0/I P = 0.0039, pCR P = 0.018 in the neoadjuvant cisplatin trials. Conclusions: HR deficiency identifies TNBC tumors, including BRCA1/2 nonmutated tumors more likely to respond to platinum-containing therapy. Clin Cancer Res; 22(15); 3764–73. ©2016 AACR.

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 Approximately 15%–23% of breast cancers overexpress human epidermal growth factor receptor 2 (HER2), which leads to the activation of signaling pathways that stimulate cell proliferation and survival. HER2-targeted therapy has substantially improved outcomes in patients with HER2-positive breast cancer. However, both de novo and acquired resistance are observed. Design A literature search was performed to identify proposed mechanisms of resistance to HER2-targeted therapy and identified novel targets in clinical development for treating HER2-resistant disease. Results Proposed HER2-resistance mechanisms include impediments to HER2-inhibitor binding, signaling through alternative pathways, upregulation of signaling pathways downstream of HER2, and failure to elicit an appropriate immune response. Although continuing HER2 inhibition beyond progression may provide an additional clinical benefit, the availability of novel therapies targeting different mechanisms of action could improve outcomes. The developmental strategy with the most available data is targeting the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (mTOR) pathway. The oral mTOR inhibitor everolimus has shown promising activity in combination with chemotherapy and trastuzumab in trastuzumab-refractory, advanced breast cancer. Conclusions Non-HER2-targeted therapy is a promising means of overcoming resistance to HER2-targeted treatment. Ongoing clinical studies will provide additional information on the efficacy and safety of novel targeted therapies in HER2-resistant advanced breast cancer.

Background: Defects in BRCA1, BRCA2, and other members of the homologous recombination pathway have potential therapeutic relevance when used to support agents that introduce or exploit double-stranded DNA breaks. This study examines the association between homologous recombination defects and genomic patterns of loss of heterozygosity (LOH). Methods: Ovarian tumours from two independent data sets were characterised for defects in BRCA1, BRCA2, and RAD51C, and LOH profiles were generated. Publically available data were downloaded for a third independent data set. The same analyses were performed on 57 cancer cell lines. Results: Loss of heterozygosity regions of intermediate size were observed more frequently in tumours with defective BRCA1 or BRCA2 (P=10−11). The homologous recombination deficiency (HRD) score was defined as the number of these regions observed in a tumour sample. The association between HRD score and BRCA deficiency was validated in two independent ovarian cancer data sets (P=10−5 and 10−29), and identified breast and pancreatic cell lines with BRCA defects. Conclusion: The HRD score appears capable of detecting homologous recombination defects regardless of aetiology or mechanism. This score could facilitate the use of PARP inhibitors and platinum in breast, ovarian, and other cancers.

Sarcoidosis is a multisystem granulomatous disease of unknown origin with pulmonary and extrapulmonary manifestations. Worldwide it is most often diagnosed in the third and fourth decades and most often affects Swedish, Danish and black patients. The association between malignancy and sarcoidosis has not been conclusively proven. Cancer can eventually occur in patients who have an established diagnosis of sarcoidosis for example, in sarcoidosis-lymphoma syndrome. Sarcoidosis can also subsequently develop in an oncology patient. There are multiple obstacles to confirming epidemiologically the linkage between sarcoidosis and malignancy. Histological verification and clinical acumen are needed to avoid misdiagnosis. The 18 fluorodeoxyglucose (18-FDG) PET has failed to provide a non invasive diagnostic method to differentiate neoplasia from benign sarcoid lesions and tissue diagnosis is essential before commencing a new therapeutic intervention in patients with lymphoma. We report 3 cases of co-diagnosis of sarcoidosis and lymphoma that were seen in an oncology unit in Drogheda, Co. Louth. Our patients varied in the temporal association between the diagnosis of sarcoidosis and lymphoma as well as their demographic characteristics. These cases help to demonstrate the need for careful clinical, histological and radiological assessment.

There is increased understanding of the heterogeneity of breast tumors, with greater emphasis now being placed on histological and molecular profiles and, in particular, their implications for prognosis and therapy. This review addresses breast cancers of unusual histological subtype with an approximate incidence ≤1%. Given the rarity of these tumors, the literature contains primarily case reports, small series, and population-based studies. Data are heterogeneous and almost entirely retrospective, frequently gathered over long time periods, in the context of changing pathological techniques and reporting. In addition, our understanding of the disease biology and therapeutic context has also evolved significantly over this time. There is often limited information about the specific therapies used and the rationale for choosing such an approach. Meaningful comparisons of treatment modalities are not feasible and it is not possible to define management guidelines. Instead, this review correlates the available information to give an impression of how each subgroup behaves—of the favored surgical technique, responses to therapy, and prognosis—as well as the emerging molecular data, highlighting new research areas for potential target in clinical trials. Each tumor subtype described represents a small but real cohort of patients with breast cancer, and although inferences may be made from this review, we are mindful of the paucity of data. The management of each patient must be considered in the context of their unique clinical presentation and correlated with the evidence-based principles that apply to more common breast cancer histologies.

PTEN (phosphatase and tensin homolog) is a tumor suppressor that negatively regulates the PI3K-AKT signaling pathway, which is implicated in the pathogenesis of endometrial carcinoma. Sanger sequencing has been considered to be the gold standard for detection of PTEN sequence abnormalities. However, this approach fails to address the epigenetic mechanisms that contribute to functional PTEN loss. Using a study cohort of 154 endometrioid and non-endometrioid endometrial carcinomas, we performed full-length PTEN sequencing and PTEN immunohistochemistry on each tumor. PTEN sequence abnormalities were detected in a significantly lower proportion of cases (43%) than PTEN protein loss (64%, P=0.0004). Endometrioid tumors had a significantly higher proportion of PTEN sequence abnormalities and PTEN protein loss than non-endometrioid tumors. Within the latter group, PTEN sequence abnormalities and PTEN protein loss were most frequent in undifferentiated carcinomas, followed by mixed carcinomas; they were least frequent in carcinosarcomas. Overall, at least one PTEN sequence abnormality was detected in each exon, and the greatest number of sequence abnormalities was detected in exon 8. Pure-endometrioid tumors had a significantly higher frequency of sequence abnormalities in exon 7 than did the non-endometrioid tumors (P=0.0199). Importantly, no mutational hotspots were identified. While PTEN protein loss by immunohistochemistry was identified in 89% of cases with a PTEN sequence abnormality, PTEN protein loss was detected by immunohistochemistry in 44% of cases classified as PTEN wild type by sequencing. For the first time, we demonstrate that PTEN immunohistochemistry is able to identify the majority of cases with functional PTEN loss. However, PTEN immunohistochemistry also detects additional cases with PTEN protein loss that would otherwise be undetected by gene sequencing. Therefore, for clinical purposes, immunohistochemistry appears to be a preferable technique for identifying endometrial tumors with loss of PTEN function.

The 2012 IMPAKT task force investigated the medical usefulness of current methods for the classification of breast cancer into the 'intrinsic' molecular subtypes (luminal A, luminal B, basal-like and HER2). A panel of breast cancer and/or gene expression profiling experts evaluated the analytical validity, clinical validity and clinical utility of two approaches for molecular subtyping of breast cancer: the prediction analysis of microarray (PAM)50 assay and an immuno-histochemical (IHC) surrogate panel including oestrogen receptor (ER), HER2 and Ki67. The panel found the currently available evidence on the analytical validity and clinical utility of Ki67 based on a 14% cut-off and PAM50 to be inadequate. The majority of the working group members found the available evidence on the analytical validity, clinical validity and clinical utility of ER/HER2 to be convincing. The panel concluded that breast cancer classification into molecular subtypes based on the IHC assessment of ER, HER2 and Ki67 with a 14% cut-off and on the PAM50 test does not provide sufficiently robust information to modify systemic treatment decisions, and recommended the use IHC for ER and HER2 for the identification of clinically relevant subtypes of breast cancers. Methods for breast cancer classification into molecular subtypes should, however, be incorporated into clinical trial design.

BACKGROUND For Estrogen Receptor (ER) positive, early stage breast cancer, the 21 gene Recurrence Score (RS) has clinical use both as a prognostic tool and to predict chemotherapy benefit. The availability of this tool in Ireland has led to a reduction in the use of adjuvant chemotherapy for women with lymph node (LN) negative disease. However, the RS is not routinely funded for patients with LN positive (LN+) breast cancer in Ireland. In addition, there are limited international data on the use of this tool in the preoperative setting. In this prospective observational study, we are investigating whether access to the 21 gene RS leads to a reduction in the receipt of chemotherapy for patients with ER+, LN+ breast cancer, and to correlate the 21 gene RS with response to preoperative systemic therapy. TRIAL DESIGN This is a national, multi-site, prospective, observational study that will examine the impact of the 21 gene RS on chemotherapy recommendations in both the neoadjuvant and adjuvant setting. Prior to and following tumor testing with the 21 gene RS, Physicians will complete a questionnaire which details type and strength of systemic therapy recommendations. ELIGIBILITY Cohort 1 (postoperative) will include patients with ER+ tumors of any size with involvement of 1-3 lymph nodes (N1 including micrometastases). Cohort 2 (preoperative) will include patients with ER+, T2-T4 tumors with biopsy proven nodal metastases. Both cohorts will have ECOG PS 0 or 1 and be fit for consideration of chemotherapy as determined by the Investigator. SPECIFIC AIMS The primary endpoint is the percentage reduction in the number of patients for whom treating physicians recommend chemotherapy after testing with 21 gene RS. Secondary endpoints include the correlation between the 21 gene RS and residual cancer burden score, as well as pathological, clinical and radiological response rates. The economic impact of the 21 gene RS in ER+, LN+ will also be assessed. STATISTICAL METHODS The sample size is based on similar decision impact studies conducted in other countries. Physician recommendations for chemotherapy pre 21-gene RS and recommendations post 21-gene RS testing will be compared and percentage change estimated with 95% confidence intervals. For secondary endpoints, the Pearson correlation coefficient (rho) will be used to examine the strength of the correlation between the 21 gene RS category and response. A budget impact model will be used to estimate the cost reduction in adjuvant chemotherapy as a result of 21-gene RS testing. PRESENT ACCRUAL AND TARGET ACCRUAL Target accrual is 75 in each of the neoadjuvant and adjuvant cohorts to total 150 patients. Supported by Genomic Health. Citation Format: Keegan NM, Milewski M, Kelly CM, Murphy V, Chao C, Walsh J, Kennedy MJ, O'Connor M, Murphy C, O'Reilly S, Keane M, Duffy K, Hennessy B, Morris PG. The impact of the 21 gene recurrence score (RS) on chemotherapy prescribing in estrogen receptor (ER) positive, lymph node positive early stage breast cancer in Ireland [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr OT3-04-03.

Capecitabine monotherapy is considered standard treatment in anthracycline- and taxane-pretreated metastatic breast cancer and has proven efficacy in this setting. Randomized studies and retrospective analyses have shown that, in patients who received capecitabine monotherapy, or in combination with docetaxel, dose modification of capecitabine is effective in the management of adverse events without compromising efficacy. Dose adjustment of capecitabine is easy to implement due to its twice-daily oral administration. This article reports the findings of a retrospective review of a large data set to consolidate the information about the impact of capecitabine dose modification on efficacy and safety outcomes in patients with metastatic breast cancer. Data on dose modification and outcomes were available from 4 phase II capecitabine monotherapy trials, 1 phase III capecitabine/docetaxel combination trial, and an analysis of consecutive patients who received capecitabine outside of a clinical trial (n = 971). Dose reductions were required in 41% of patients who received monotherapy (n = 131) and 65% of patients who received capecitabine/docetaxel (80% of these required dose reductions of both agents) (n = 163). Time to disease progression and overall survival were similar, or even slightly longer, among patients who received lower vs. full-dose capecitabine in all of the studies reviewed. Reduced capecitabine doses were associated with a lower incidence of treatment-related adverse events, specifically hand-foot syndrome, diarrhea, and stomatitis. Together, these data support the practice of dose-reducing capecitabine, including the possibility of starting at a lower dose (

Anti-mitotic chemotherapeutic agents such as taxanes activate the spindle assembly checkpoint (SAC) to arrest anaphase onset, but taxane-exposed cells eventually undergo slippage to exit mitosis. The therapeutic efficacy of taxanes depends on whether slippage after SAC arrest culminates in continued cell survival, or in death by apoptosis. However, the mechanisms that determine these outcomes remain unclear. Here, we identify a novel role for cyclin G1 (CCNG1), an atypical cyclin. Increased CCNG1 expression accompanies paclitaxel-induced, SAC-mediated mitotic arrest, independent of p53 integrity or signaling through the SAC component, BUBR1. CCNG1 overexpression promotes cell survival after paclitaxel exposure. Conversely, CCNG1 depletion by RNA interference delays slippage and enhances paclitaxel-induced apoptosis. Consistent with these observations, CCNG1 amplification is associated with significantly shorter post-surgical survival in patients with ovarian cancer who have received adjuvant chemotherapy with taxanes and platinum compounds. Collectively, our findings implicate CCNG1 in regulating slippage and the outcome of taxane-induced mitotic arrest, with potential implications for cancer therapy. Oncogene (2012) 31, 2450-2460; doi: 10.1038/onc.2011.431; published online 7 November 2011

Dipyrromethene ligand scaffolds were synthesized bearing large aryl (2,4,6-Ph(3)C(6)H(2), abbreviated Ar) or alkyl ((t)Bu, adamantyl) flanking groups to afford three new disubstituted ligands ((R)L, 1,9-R(2)-5-mesityldipyrromethene, R=aryl, alkyl). While high-spin (S=2), four-coordinate iron complexes of the type ((R)L)FeCl(solv) were obtained with the alkyl-substituted ligand varieties (for R=(t)Bu, Ad and solv=THF, OEt(2)), use of the sterically encumbered aryl-substituted ligand precluded binding of solvent and cleanly afforded a high-spin (S=2), three-coordinate complex of the type ((Ar)L)FeCl. Reaction of ((Ad)L)FeCl(OEt(2)) with alkyl azides resulted in the catalytic amination of C-H bonds or olefin aziridination at room temperature. Using a 5% catalyst loading, 12 turnovers were obtained for the amination of toluene as a substrate, while greater than 85% of alkyl azide was converted to the corresponding aziridine employing styrene as a substrate. A primary kinetic isotope effect of 12.8(5) was obtained for the reaction of ((Ad)L)FeCl(OEt(2)) with adamantyl azide in an equimolar toluene/toluene-d(8) mixture, consistent with the amination proceeding through a hydrogen atom abstraction, radical rebound type mechanism. Reaction of p-(t)BuC(6)H(4)N(3) with ((Ar)L)FeCl permitted isolation of a high-spin (S=2) iron complex featuring a terminal imido ligand, ((Ar)L)FeCl(N(p-(t)BuC(6)H(4))), as determined by (1)H NMR, X-ray crystallography, and (57)Fe Mössbauer spectroscopy. The measured Fe-N(imide) bond distance (1.768(2) Å) is the longest reported for Fe(imido) complexes in any geometry or spin state, and the disruption of the bond metrics within the imido aryl substituent suggests delocalization of a radical throughout the aryl ring. Zero-field (57)Fe Mössbauer parameters obtained for ((Ar)L)FeCl(N(p-(t)BuC(6)H(4))) suggest a Fe(III) formulation and are nearly identical with those observed for a structurally similar, high-spin Fe(III) complex bearing the same dipyrromethene framework. Theoretical analyses of ((Ar)L)FeCl(N(p-(t)BuC(6)H(4))) suggest a formulation for this reactive species to be a high-spin Fe(III) center antiferromagnetically coupled to an imido-based radical (J = -673 cm(-1)). The terminal imido complex was effective for delivering the nitrene moiety to both C-H bond substrates (42% yield) as well as styrene (76% yield). Furthermore, a primary kinetic isotope effect of 24(3) was obtained for the reaction of ((Ar)L)FeCl(N(p-(t)BuC(6)H(4))) with an equimolar toluene/toluene-d(8) mixture, consistent with the values obtained in the catalytic reaction. This commonality suggests the isolated high-spin Fe(III) imido radical is a viable intermediate in the catalytic reaction pathway. Given the breadth of iron imido complexes spanning several oxidation states (Fe(II)-Fe(V)) and several spin states (S=0→(3)/(2)), we propose the unusual electronic structure of the described high-spin iron imido complexes contributes to the observed catalytic reactivity.

Introduction: The microenvironment plays a major role in the survival of Chronic Lymphocytic Leukaemia (CLL) cells by promoting CLL cell growth and drug resistance. The traffic of CLL cells that involve migration from the blood and adhesion to the high endothelial venules (HEVs) facilitates the homing of lymphocytes in the lymph nodes to protect CLL cells and offer them the necessary survival signals. L- Selectin mediates adhesion of CLL cells to LN-microenvironment through a multistep process that entails rolling, sticking, and crawling. Acitretin is a retinoid that used in the treatment of severe psoriasis and less frequently in the control of squamous skin cancer. Acitretin activates nuclear retinoic acid receptors (RAR), resulting in induction of cell differentiation, inhibition of cell proliferation, and inhibition of tissue infiltration by inflammatory cells. It is reported that retinoids induce apoptosis in CLL and improve the sensitivity of CLL cells to fludarabine. Method: We used MEC-1 "CLL like" cell line and CLL cells from known CLL patients to investigate the effect of acitretin on the expression of the major markers that control CLL cells traffic including L-selectin (CD62L), CD49d and CXCR4 (CD184) . We treated CLL cells with increasing doses of acitretin (10 nM up to 10 mM). After treatment for 48 to 72 hours, L-selectin, CD49d and CXCR4 expression was measured using flowcytometry. Dimethyl sulfoxide (DMSO) treated cells were used as a negative control. A Leukocyte adhesion assay was done using CytoSelect Leukocyte-endothelium adhesion assay according to the protocol provided. To assess the mechanism through which L-Selectin reduces the adhesion of CLL cells to the endothelium, we used western blot to measure the effect of acitretin on PI3k-p85. Results: Acitretin in a dose as low as 10 nM significantly reduced the expression of L-selectin in primary CLL cells after treatment for 48 hours (P= 0.0475) and 72 hours (P= 0.0048). On the contrary, acitretin did not change the expression of CD49d or CXCR4. Similar significant reduction in L-selectin expression was recognized when MEC-1 cells were treated with 10 nM of acitretin for 48 hours (P= 0.0179) and 72 hours (P= 0.0159). To test the effect of acitretin on the adhesion of MEC-1 cells to the endothelium; we used CytoSelect adhesion assay that showed significant reduction in the adhesion of MEC-1 cells to human umbilical vein endothelial cells (HUVEC) after treatment with acitretin 10 mM for 48 hours (P= 0.0048). Ibrutinib treatment has also significantly reduced the adhesion of MEC-1 cells to HUVEC cells (P= 0.0045) and when combined with acitretin the reduction in adhesion was more pronounced (P= 0.0003). Finally, to understand the mechanism through which acitretin down regulates the expression of L-selectin and reduces the adhesion of MEC-1 cells, we tested the effect of acitretin on the PI3K pathway using western blot. PI3K is required for the cytoskeleton changes during neutrophil rolling on E‐selectin and we investigated if similar interaction takes place between PI3K and L-selectin in CLL cells. Treatment of MEC-1 cells with acitretin 10 mM for 48 hours resulted in significant down regulation of the PI3K-p85 (P= 0.0007). Treatment with Ibrutinib alone or combined with acitretin did not result in significant change in PI3K- p85. This may explain the mechanism through which acitretin reduces the expression of L-selectin and compromises CLL adhesion to the endothelium. Conclusion: We showed that the retinoid acitretin significantly reduced the expression of L-selectin in CLL cells and MEC-1 "CLL like" cells in vitro. The reduction in L-selectin resulted in reduced adhesion of CLL cells to the endothelium hampering homing of CLL cells to lymph nodes. The mechanism through which acitretin exerts this effect can be explained by down regulation of PI3K-p85. The use of acitretin as an adjunct treatment in CLL can be beneficial, however further research including in vivo studies is required. Disclosures Quinn: Janssen: Honoraria.

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.

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