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Stromal tumour infiltrating lymphocytes (sTILs) are a strong prognostic marker in triple negative breast cancer (TNBC). Consistency scoring sTILs is good and was excellent when an internet-based scoring aid developed by the TIL-WG was used to score cases in a reproducibility study. This study aimed to evaluate the reproducibility of sTILs assessment using this scoring aid in cases from routine practice and to explore the potential of the tool to overcome variability in scoring. Twenty-three breast pathologists scored sTILs in digitized slides of 49 TNBC biopsies using the scoring aid. Subsequently, fields of view (FOV) from each case were selected by one pathologist and scored by the group using the tool. Inter-observer agreement was good for absolute sTILs (ICC 0.634, 95% CI 0.539–0.735, p &lt, 0.001) but was poor to fair using binary cutpoints. sTILs heterogeneity was the main contributor to disagreement. When pathologists scored the same FOV from each case, inter-observer agreement was excellent for absolute sTILs (ICC 0.798, 95% CI 0.727–0.864, p &lt, 0.001) and good for the 20% (ICC 0.657, 95% CI 0.561–0.756, p &lt, 0.001) and 40% (ICC 0.644, 95% CI 0.546–0.745, p &lt, 0.001) cutpoints. However, there was a wide range of scores for many cases. Reproducibility scoring sTILs is good when the scoring aid is used. Heterogeneity is the main contributor to variance and will need to be overcome for analytic validity to be achieved.

Breast cancer is stratified into four distinct clinical subtypes, using three key biomarkers (Her2/Neu gene status, Estrogen and Progesterone receptor status). However, each subtype is a heterogeneous group, displaying significant variation in survival rates and treatment response. New biomarkers are required to provide more precise stratification of breast cancer cohorts to inform personalised treatment options/predict outcomes. Tip60 is a member of the MYST sub-family of histone acetyltransferases (HATs), and is directly involved in genome maintenance, gene regulation and DNA damage response/repair pathways (key chemotherapeutic influencing mechanisms). We aimed to determine if quantifying Tip60 staining patterns improved breast cancer stratification. We defined Tip60 protein in vivo, quantifying location (cytoplasmic, nuclear), percent of cells and staining intensity in a breast cancer tissue microarray (n = 337). A significant association of specific Tip60 staining patterns with breast cancer subtype, ER or PR status and Tumour grade was found. Importantly, low Tip60 mRNA expression correlated with poor overall survival and relapse free survival. We found Tip60 is a biomarker able to stratify breast cancer patients, and low Tip60 expression is a significant risk factor indicating a higher chance of disease reoccurrence. This work highlights Tip60 regulation as a key factor influencing the development of breast cancer.

// Pablo Garrido 1, 7 , Aliaa Shalaby 1 , Elaine M. Walsh 1 , Nessa Keane 1 , Mark Webber 1 , Maccon M. Keane 2 , Francis J. Sullivan 3 , Michael J. Kerin 4 , Grace Callagy 1 , Aideen E. Ryan 5, 6 and Sharon A. Glynn 1, 3, 7 1 Discipline of Pathology, Lambe Institute for Translational Research, School of Medicine, National University of Ireland Galway, Galway, Republic of Ireland 2 Medical Oncology, Galway University Hospital, Galway, Republic of Ireland 3 Prostate Cancer Institute, National University of Ireland Galway, Galway, Republic of Ireland 4 Discipline of Surgery, Lambe Institute for Translational Research, School of Medicine, National University of Ireland Galway, Galway, Republic of Ireland 5 Discipline of Pharmacology and Therapeutics, Lambe Institute for Translational Research, School of Medicine, National University of Ireland Galway, Galway, Republic of Ireland 6 Regenerative Medicine Institute (REMEDI), Biomedical Sciences, National University of Ireland Galway, Galway, Republic of Ireland 7 Apoptosis Research Centre, National University of Ireland Galway, Galway, Republic of Ireland Correspondence to: Sharon A. Glynn, email: sharon.glynn@nuigalway.ie Keywords: triple negative breast cancer, EGFR, inflammation, metastasis, nitric oxide Received: January 17, 2017 Accepted: July 03, 2017 Published: July 26, 2017 ABSTRACT Inflammation is implicated in triple negative breast cancer (TNBC) progression. TNBC carries a worse prognosis than other breast cancer subtypes, and with the clinical and molecular heterogeneity of TNBC, there is a lack of effective therapeutic targets available. Identification of molecular targets for TNBC subtypes is crucial towards personalized patient stratification. Inducible nitric oxide synthase (iNOS) has been shown to induce p53 mutation accumulation, basal-like gene signature enrichment and transactivation of the epidermal growth factor receptor (EGFR) via s-nitrosylation. Herein we report that iNOS is associated with disease recurrence, distant metastasis and decreased breast cancer specific survival in 209 cases of TNBC. Employing TNBC cell lines representing normal basal breast, and basal-like 1 and basal-like 2 tumors, we demonstrate that nitric oxide (NO) induces EGFR-dependent ERK phosphorylation in basal-like TNBC cell lines. Moreover NO mediated cell migration and cell invasion was found to be dependent on EGFR and ERK activation particularly in basal-like 2 TBNC cells. This occurred in conjunction with NF-κB activation and increased secretion of pro-inflammatory cytokines IL-8, IL-1β and TNF-α. This provides substantial evidence for EGFR as a therapeutic target to be taken into consideration in the treatment of a specific subset of basal-like TNBC overexpressing iNOS.

Background Argonaute-2 (Ago2) is an essential component of microRNA biogenesis implicated in tumourigenesis. However Ago2 expression and localisation in breast cancer remains undetermined. The aim was to define Ago2 expression (mRNA and protein) and localisation in breast cancer, and investigate associations with clinicopathological details. Methods Ago2 protein was stained in breast cancer cell lines and tissue microarrays (TMAs), with intensity and localization assessed. Staining intensity was correlated with clinicopathological details. Using independent databases, Ago2 mRNA expression and gene alterations in breast cancer were investigated. Results In the breast cancer TMAs, 4 distinct staining intensities were observed (Negative, Weak, Moderate, Strong), with 64.2% of samples stained weak or negatively for Ago2 protein. An association was found between strong Ago2 staining and, the Her2 positive or basal subtypes, and between Ago2 intensity and receptor status (Estrogen or Progesterone). In tumours Ago2 mRNA expression correlated with reduced relapse free survival. Conversely, Ago2 mRNA was expressed significantly lower in SK-BR-3 (HER2 positive) and BT-20 (Basal/Triple negative) cell lines. Interestingly, high levels of Ago2 gene amplification (10–27%) were observed in breast cancer across multiple patient datasets. Importantly, knowledge of Ago2 expression improves predictions of breast cancer subtype by 20%, ER status by 15.7% and PR status by 17.5%. Conclusions Quantification of Ago2 improves the stratification of breast cancer and suggests a differential role for Ago2 in breast cancer subtypes, based on levels and cellular localisation. Further investigation of the mechanisms affecting Ago2 dysregulation will reveal insights into the molecular differences underpinning breast cancer subtypes. Electronic supplementary material The online version of this article (10.1186/s12885-019-5884-x) contains supplementary material, which is available to authorized users.

Purpose The rate of pathological complete response (pCR) for patients with triple negative breast cancer (TNBC) is increased when carboplatin is added to neo-adjuvant chemotherapy (NACT). However, while phase III trial data showing a survival benefit are awaited, carboplatin is not yet standard-of-care for TNBC. The aim of this study was to examine the rate of pCR and the outcome for those treated with carboplatin and to examine the predictors of response to therapy. Methods The retrospective series comprised 333 consecutive patients with TNBC (median follow-up time, 43 months). Adjuvant chemotherapy was given to 51% (n = 168) of patients and 29% (n = 97) received anthracycline–taxane NACT with carboplatin given to 9% (n = 31) of patients. Results Overall, 25% (n = 78) of patients experienced a breast cancer recurrence and 22% (n = 68) died from disease. A pCR breast and pCR breast/axilla was more common in those who received carboplatin (n = 18, 58% and n = 17, 55%, respectively) compared those who did not (n = 23, 36% and n = 18, 28%, respectively) (p = 0.041 and p = 0.011, respectively). By multivariable analysis, carboplatin and high tumor grade were independent predictors of pCR breast/axilla (ORnon-pCR = 0.17; 95% CI 0.06–0.54; p = 0.002; and ORnon-pCR = 0.05, 95% CI 0.01–0.27; p

Not all neuropathic pain patients gain relief from current therapies that include the anticonvulsant, gabapentin, thought to modulate calcium channel function. We report a neural circuit that is permissive for the effectiveness of gabapentin. Substance P-saporin (SP-SAP) was used to selectively ablate superficial dorsal horn neurons expressing the neurokinin-1 receptor for substance P. These neurons project to the brain as shown by retrograde labelling and engage descending brainstem serotonergic influences that enhance spinal excitability via a facilitatory action on 5HT(3) receptors. We show the integrity of this pathway following nerve injury contributes to the behavioural allodynia, neuronal plasticity of deep dorsal horn neurons and the injury-specific actions of gabapentin. Thus SP-SAP attenuated the tactile and cold hypersensitivity and abnormal neuronal coding (including spontaneous activity, expansion of receptive field size) seen after spinal nerve ligation. Furthermore the powerful actions of gabapentin after neuropathy were blocked by either ablation of NK-1 expressing neurones or 5HT(3) receptor antagonism using ondansetron. Remarkably, 5HT(3) receptor activation provided a state-dependency (independent of that produced by neuropathy) allowing GBP to powerfully inhibit in normal uninjured animals. This circuit is therefore a crucial determinant of the abnormal neuronal and behavioural manifestations of neuropathy and importantly, the efficacy of gabapentin. As this spino-bulbo-spinal circuit contacts areas of the brain implicated in the affective components of pain, this loop may represent a route by which emotions can influence the degree of pain in a patient, as well as the effectiveness of the drug treatment. These hypotheses are testable in patients.

Oncotype DX is an RT-PCR assay used to predict which patients with ER-positive node-negative (NN) disease will benefit from chemotherapy. Each patient is stratified into a risk category based on a recurrence score (RS) and the TAILORx trial is determining the benefit of chemotherapy for patients with mid-range RSs. We tested if Oncotype DX and TAILORx risk categories could be predicted by standard pathological features and protein markers corresponding to 10 genes in the assay (ER, PR, Ki67, HER2, BCL2, CD68, Aurora A kinase, survivin, cyclin B1 and BAG1) on 52 patients who enrolled on TAILORx. Immunohistochemistry for the protein markers was performed on whole tissue sections. Classification and regression tree (CART) analysis correctly classified 69% of cases into Oncotype DX risk categories based on the expression of PR, survivin and nuclear pleomorphism. All tumours with PR staining (Allred score ≥ 2) and marked nuclear pleomorphism were in the high-risk category. No case with PR

Background TNBC comprises 10-20% of breast cancers. Lehmann et al. identified 6 TNBC subtypes by gene expression profiling: BL1, BL2, IM, M, MSL & LAR. Despite the diversity of TNBC, standard of care is combination chemotherapy as in non TNBCs. Identification of chemosensitive TNBC subtypes is necessary. Chemoresistant TNBCs need alternative targets to improve treatment strategies. Aims 1. To validate an IHC biomarker panel to define molecular subtypes of TNBC 2. To correlate molecular subtypes with prognosis to identify appropriate therapy 3. To improve diagnostic tools to individualize therapy based on TNBC subtypes Methods A TMA was constructed of 197 TNBCs diagnosed from 1999 - 2014. An 8-protein IHC panel was developed to identify TNBC subtypes on FFPE tissue. The panel includes markers for key pathways to discriminate between 6 subtypes: AR, Bcl2, c-myc, TIE1, PDGFC, MMP2, Il2R, MSH2. To date, AR & Bcl2 have been stained and assessed, together with p53 & Ki67. 10% was used as the cut off for positivity. Clinical data were obtained from hospital records and incorporated in the database. Results On initial observation, AR+ tumors had an older age at diagnosis than AR- (60 v 56), lower rates of family history (36 v 87%) and longer DFS (31 v 21 months). Bcl2+ tumors had a younger age at diagnosis than Bcl2- (55 v 59), lower recurrence rates (25 v 31%) and longer DFS (33 v 15 months). High Ki67 tumors had a younger age at diagnosis than low Ki67 (56 v 65), higher rates of family history (30 v 14%), lower recurrence rates (16 v 25%) and longer DFS (30 v 2 months). By the time of presentation, the entire panel of 8 proteins will be analyzed. The clinicopathological association of specific TNBC subtypes and the impact of TNBC subtype on chemotherapy response will be statistically assessed. Analysis will include response, duration, DFS and OS. This study will ultimately correlate TNBC molecular subtypes with prognosis to aid clinical decision making, individualize therapies and improve patient outcomes. n = 197AR +AR -Bcl2 +Bcl2 -Ki67 >10%Ki67 Citation Format: Elaine M. Walsh, Aliaa Shalaby, Laura Murillo, Mark Webber, Michael Kerin, Sharon Glynn, Grace Callaghy, Helen Ingoldsby, Maccon Keane. Identification of triple negative breast cancer (TNBC) subtypes by an immunohistochemistry (IHC) panel with impact on clinical outcomes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1708. doi:10.1158/1538-7445.AM2015-1708

Background: Dicer, an RNase III-type endonuclease, is the key enzyme involved in RNA interference and microRNA pathways. Aberrant expression of Dicer is reported in several human cancers. Our aim was to assess the prognostic role of Dicer in breast cancer. Methods: The entire series comprised 666 invasive breast cancers (IBCs), 480 DCIS cases (397 associated with IBC and 83 pure DCIS) and 305 lymph node metastases. Cytoplasmic Dicer expression by immunohistochemistry was scored as negative (no staining) and positive (weak, moderate or strong staining). Results: Dicer staining was assessable in 446 IBC, 128 DCIS and 101 lymph node metastases. Expression of Dicer was observed in 33% (145/446) of IBCs, 34% (44/128) of DCIS and 57% (58/101) of lymph node metastases. Dicer expression was increased in nodal metastases compared to primary tumours (p< 0.001); and was associated with ER negativity (p

Abstract 1914 Poster Board I-937 Introduction: Abnormal proliferation is associated with poor outcome in patients with diffuse large B cell lymphoma (DLBCL), with Ki-67 a well established prognostic marker. More recently gene expression profiling has shown high MYC expression, a marker of proliferation, to be associated with poor outcome in DLBCL. Thus, the cell cycle is an attractive therapeutic target in aggressive lymphoma. Cdc7, a serine/threonine kinase is essential for initiation of DNA replication and cell cycle regulation. Cdc7 phosphorylation of the replicative helicase Mcm2 is a key event in this process. Overexpression of Mcm2 has been linked to increased proliferation and poor outcome in a variety of cancers, including DLBCL. No data exists on the prognostic relevance of the phosphorylated form of Mcm2. Recently, overexpression of Cdc7 and its regulatory subunit Dbf4 has been demonstrated in multiple cancers but again, the prognostic relevance of this is unknown. Emerging data shows that inhibition of Cdc7 activity with a new class of pyrrolopyridinone kinase inhibitors has activity against a broad range of cancers, including lymphoma cell lines, even in cells lacking p53. We hypothesized that increased Cdc7 expression and/or Mcm2 phosphorylation may be associated with poor prognosis in diffuse large B cell lymphoma (DLBCL) and that if confirmed, Cdc7 inhibition could be a potential treatment strategy in DLBCL. Aim: The primary aim of this project was to explore the prognostic significance of Mcm2 phosphorylation and Cdc7 expression in archived diagnostic biopsy material from patients with DLBCL. Methods: All patients with a confirmed diagnosis of treated for DLBCL treated in Galway University Hospital between 2001 and 2009 and having suitable material were selected. Chart review was undertaken to document baseline demographics, International Prognostic Index (IPI), treatment, remission rate, progression free survival (PFS) and overall survival (OS). Two tissue cores (0.6mm diameter) from each specimen were arrayed onto a tissue microarray (TMA). Expression of phospho-Mcm2 and Cdc7 as well as Ki-67, p53, Mcm2, c-myc, standard germinal centre and non-germinal centre markers were evaluated by immunohistochemisty (IHC) on the TMAs. Expression of these markers was evaluated against clinical endpoints. PFS and OS curves were constructed using the method of Kaplan and Meier and evaluated using the log-rank test. The relationship between phospho-Mcm2, Cdc7 and the known prognostic factors: germinal center and non-germinal centre type, Ki-67, and c-myc were explored through Mann-Whitney rank test. Results: Seventy-eight patients including 43 males (median age 62) and 35 females (median age 67) were included. 10% of patients were in the low, 37% in the low-intermediate, 24% in the high —intermediate and 28% in the high IPI group. IPI score was significantly associated with survival (p10% was set as the cut off for high Cdc7. The cumulative OS at 50 months was 57% in the low Cdc7 group and 37% in the high Cdc7 group (p=0.26) for the whole cohort. For the patients with a low and low-intermediate IPI the cumulative OS at 50 months was 68% in the low Cdc7 group and 40% in the high Cdc7 group (p=0.1). For patients with a high and intermediate-high IPI, the cumulative OS at 50 months was 51% in low Cdc7 group and 42 % in high Cdc7 group (p=0.9). The PFS was not significantly different in the whole cohort as well as in IPI subgroups between patient with high and low Cdc7. Interestingly there was a significant correlation between Cdc7 and Ki67 expression (p Conclusion: Despite a relatively small sample size, our results suggest that increased activity of the Cdc7 kinase may be associated with a trend towards worse outcome. This is particularly apparent in low to low-intermediate IPI. Cdc7 could be also a potential therapeutic target in DLBCL. Further studies are needed to characterise prognostic and therapeutic significance of high expression of Cdc7 and pMcm2. Disclosures: No relevant conflicts of interest to declare.

Background A number of protein markers have been investigated as prognostic adjuncts in breast cancer but their translation into clinical practice has been impeded by a lack of appropriate validation. Recently, we showed that BCL2 protein expression had prognostic power independent of current used standards. Here, we present the results of a meta-analysis of the association between BCL2 expression and both disease free survival (DFS) and overall survival (OS) in female breast cancer. Methods Reports published in 1994–2006 were selected for the meta-analysis using a search of PubMed. Studies that investigated the role of BCL2 expression by immunohistochemistry with a sample size greater than 100 were included. Seventeen papers reported the results of 18 different series including 5,892 cases with an average median follow-up of 92.1 months. Results Eight studies investigated DFS unadjusted for other variables in 2,285 cases. The relative hazard estimates ranged from 0.85 – 3.03 with a combined random effects estimate of 1.66 (95%CI 1.25 – 2.22). The effect of BCL2 on DFS adjusted for other prognostic factors was reported in 11 studies and the pooled random effects hazard ratio estimate was 1.58 (95%CI 1.29–1.94). OS was investigated unadjusted for other variables in eight studies incorporating 3,910 cases. The hazard estimates ranged from 0.99–4.31 with a pooled estimate of risk of 1.64 (95%CI 1.36–2.0). OS adjusted for other parameters was evaluated in nine series comprising 3,624 cases and the estimates for these studies ranged from 1.10 to 2.49 with a pooled estimate of 1.37 (95%CI 1.19–1.58). Conclusion The meta-analysis strongly supports the prognostic role of BCL2 as assessed by immunohistochemistry in breast cancer and shows that this effect is independent of lymph node status, tumour size and tumour grade as well as a range of other biological variables on multi-variate analysis. Large prospective studies are now needed to establish the clinical utility of BCL2 as an independent prognostic marker.