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Background: Lymphocyte predominant breast cancers (LPBC) account for 5-30% of TNBCs; 65-80% of TNBCs have low/moderate levels of sTILs and 15-20% have no lymphocyte infiltration. TNBCs with high stromal tumor infiltrating lymphocytes (sTILs) have higher rates of pathological complete response (pCR) with anthracycline-taxane (AT) neoadjuvant chemotherapy (NACT), but the effect of carboplatin (Cb) is unclear. In GeparSixto, Cb increased pCR rates in LPBC but there was no interaction between LPBC and Cb in TNBCs. This study examines the predictive role of sTILs in TNBCs, focusing on the association between sTILs and pCR with and without Cb based NACT. Methods: Patients diagnosed with TNBC and treated with NACT in University Hospital Galway, Ireland from 2004-2016 were identified. Patients were treated with a carboplatin-anthracycline-taxane regimen (Cb-AT) (n=30) or an AT regimen (n=54). As per the International TILs Working Group, sTILs were scored on digitized slides of full-face tumor sections from diagnostic needle core biopsies. sTILs were categorized as 0-10%; 11-25%; 26-49%; ≥50% (LPBC). Odds ratio (OR) was calculated using non-pCR as the baseline. pCR was defined as ypT0/isN0. Results: Eighty-eight (n=88) cases with sufficient sTILs for analysis were identified. The median sTIL count was 12.5% (range 0-75%): 50% of cases (n=44) had sTILs 0-10%; 25% (n=22) had sTILs 11-25%; 14% (n=12) had sTILs 26-49% and 11% (n=10) had sTILs ≥50%. Cases with sTILs >10% had higher rates of pCR compared to those with sTILs ≤10% (52% vs 29%; p=0.021). In cases treated with an AT regimen, tumors with sTILs >10% had higher pCR rates than those with sTILs ≤10% (46% vs 12%; p=0.005). In cases treated with a Cb-AT regimen, there was no difference in pCR rates between sTILs >10% tumors and sTILs ≤10% tumors (60% vs 53%; p=0.500). In this cohort, pCR rates were higher in LPBC compared to non-LPBCs (80% vs 36%; p=0.010). In cases treated with AT, pCR rates were higher in LPBC than non-LPBC (83% vs 23%; p=0.007). However, in cases treated with Cb-AT, there were no differences in pCR rates between LPBC and non-LPBC (67% vs 56%; p=0.603). In LPBC, the choice of chemotherapy did not affect pCR rates: 67% vs 83% for Cb-AT and AT regimens respectively (p=0.583). In non-LPBC, pCR rates increased with Cb: 56% vs 23% for Cb-AT and AT regimens respectively (p=0.005). By univariate analysis, there was a significant association between sTILs and pCR. The association was greatest in LPBC (OR 0.14; p=0.016) and remained significant in those treated with AT (OR 0.06; p=0.014) but not in cases treated with Cb-AT (OR 0.63; p=0.714). In a multivariable model, with tumor grade and Cb use as co-variables, LPBC was independently associated with pCR (OR0.14; p=0.030). In cases treated with AT, sTILs were associated with pCR (OR 0.18, p=0.019 for sTILs >10%; OR 0.08, p=0.029 for LPBC). In cases treated with Cb-AT, sTILs were not associated with pCR (p=0.372 and p=0.622 for sTILs >10% and LPBC respectively). Conclusions: In TNBC, sTILs are predictive of response to NACT: as sTILs increase, pCR rates increase. The likelihood of a pCR increased by 86% for LPBC vs non-LPBC. The effect of sTILs on pCR was most notable in LPBC treated with AT based NACT where the odds of a pCR increased by 94%. In cases treated with Cb-AT, there was no association between sTILs and pCR. This study suggests that tumors with high sTILs are more chemosensitive than tumors with low sTILs. High sTIL tumors had high pCR rates with AT based NACT but the addition of Cb did not increase pCR rates. Cb-AT NACT could be reserved for patients with low sTIL tumors, in order to increase the rates of pCR in that subgroup, who had low rates of pCR in this study. In the future, sTILs could be used as a predictive biomarker to guide chemotherapy selection. Citation Format: Elaine M Walsh, Mark O'Loughlin, Aliaa Shalaby, Mark Webber, Michael J Kerin, Sharon A Glynn, Grace Callagy, Maccon M Keane. Pathological complete response after carboplatin-based neoadjuvant chemotherapy in triple negative breast cancer: The importance of stromal tumor infiltrating lymphocytes as a predictive biomarker [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 P1-10-11.

A method was developed to expedite the imaging of diatoms by optimizing their orientation. This quick technique puts grooves into a SEM stub so that as the diatoms are laid down they either land on the flat surface or in a variety of positions in the grooves, thereby reducing time required to capture taxonomically critical features.

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.

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

e12085 Background: High numbers of sTILs are predictive of pCR in TNBCs. This study examines the role of sTILs as a predictive biomarker for carboplatin (Cb) based NACT. Methods: sTILs were scored on 88 pre NACT TNBC biopsies as per the International TILs Working Group and were scored as continuous and categorical variables: 0-10%; 11-25%; 26-49%; ≥50%. OR was calculated using non-pCR as baseline parameter. Results: Patients with sTILs > 10% had increased pCR rates compared to those with sTILs ≤10%: pCR breast (B) (61% vs 31% p = 0.004); pCR breast/axilla (BA) (52% vs 29% p = 0.021). In those treated with Cb, there were no differences in pCR B between sTILs > 10% or ≤10% (67% vs 53%; p = 0.355) or pCR BA (60% vs 53%; p = 0.500). In those who did not receive Cb, sTILs > 10% had increased pCR B (57% vs 15%; p = 0.002) and pCR BA (46% vs 12%; p = 0.005) compared to sTILs ≤10%. Similar trends were seen with sTILs > 25% and ≥50%: differences were seen in patients who did not receive Cb, but not in those treated with Cb. In LPBCs, the addition of Cb did not significantly increase pCR rates: 67% vs 83% for Cb and non-Cb regimens (p = 0.583). In non-LPBC, pCR rates were increased with Cb: pCR B 59% vs 31% (p = 0.017), and pCR BA 56% vs 23% (p = 0.005) for Cb and non-Cb regimens. The association between sTILs and pCR was significant in multivariable models adjusting for grade and Cb. In patients who did not receive Cb (n = 54), increasing sTILs were associated with increased pCR B (OR 0.15 p = 0.004; OR 0.27 p = 0.039; OR 0.10 p = 0.05 for > 10%, > 25%, ≥50%) and pCR BA (OR 0.18 p = 0.019; OR 0.08 p = 0.029 for > 10%, ≥50%). In patients treated with Cb (n = 30), increasing sTILs were not associated with pCR B (OR 1.97 p = 0.581; OR 0.27 p = 0.334; OR 0.50 p = 0.719) or pCR BA (OR 2.93 p = 0.372; OR 0.79 p = 0.831; OR 0.40 p = 0.622 for > 10%, > 25%, ≥50%). Conclusions: sTILs are predictive of pCR: as sTILs increase, pCR rates increase. Tumors with high sTILs had high pCR rates to anthracycline-taxane (AT) NACT. The effect of increasing sTILs on pCR was most notable in patients who did not receive Cb based NACT, suggesting that tumors with high sTILs are inherently sensitive to AT based chemotherapy. Intensifying NACT with Cb could be used to increase pCR rates in patients with low sTILs. sTILs should be explored as a biomarker to intensify chemotherapy in those with low sTILs, and to de-escalate chemotherapy in tumors with high sTILs.

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.

Background: Triple negative breast cancer (TNBC) is characterised by a lack of expression of oestrogen receptor and progesterone receptor and lack of HER2 overexpression. Using gene expression profiling, TNBC can be subtyped into six subtypes: BL1,BL2,IM,M,MSL&LAR. However, gene profiling is not yet routine in clinical practice. Also, despite the heterogeneity of TNBC, standard of care remains combination chemotherapy without targeted therapy. It is necessary to identify TNBC subtypes with differing responses to chemotherapy. In chemoresistant TNBCs, identifying alternative therapeutic targets will facilitate improved treatment strategies and outcomes. Aims: 1. To develop and validate an IHC panel to facilitate subtyping of TNBC 2. To determine the prognostic impact of TNBC subtypes by assessing clinical features, RFS and OS rates 3. To determine the predictive impact of TNBC subtypes by assessing the sensitivity of TNBC subtypes to differing chemotherapy regimens 4. Ultimately to identify new molecular targets to individualise treatment strategies for TNBC Methods: In order to identify TNBC subtypes on FFPE tissue, an 8-protein IHC protein panel has been developed based on genes enriched in the 6 TNBC subtypes. IHC Protein PanelProteinBL1BL2IMMMSLLARAR-----++MYC++-++/---TIE1------Bcl2--+-+-PDGFC-++-+++-MMP2-++-+++-RAD21++-+/-+/---IL2R--++-+/-- A tissue microarray has been constructed of 301 TNBCs diagnosed from 1999–2014. To date, 196 cases have been stained and scored for androgen receptor (AR) and 199 cases for Bcl2. A database has been constructed incorporating clinical data with pathological and outcome data. Results: n=301 AR+Bcl2Ki67 >10%p53+n30120114121116%10010577361 Median 35-40% Median Age5565545456Range24-9237-9229-8429-9129-92Family History n836353232%27.530312628BRCA Mutation160453BRCA111-232BRCA24-221NACT611141311CR22-11-PR33111118SD2-1-1POD41112Mets at Dx n92222%3101.751.651.75Recurrence n674272728%2220242224Median TTP20.530.5342625Range2-20012-349-2003-2002-95Median OS3944.5342625Range1-3261-1362-3261-3261-163 On initial observation, AR+ TNBCs were diagnosed at an older age than other TNBCs (65 v 55), were less likely to have BRCA mutations, more likely to be metastatic at diagnosis and were associated with longer median OS (44.5 mos). 21% of AR+ cases were Bcl2+. 45% of AR positive cases had Ki67 Bcl2+ TNBCs had low rates of metastatic disease at diagnosis and had the longest median time to progression (34 mos). TNBCs with high Ki67 had the shortest median overall survival (31.5 mos). We propose that at the time of presentation, the entire IHC panel will be stained and scored. Statistical analysis will assess the association of TNBC subtypes on clinicopathological features as well as the impact of subtype on chemotherapy response. Final analysis will also include duration of treatment response, DFS and OS. Our aim is that this study will prognostically and predictively subtype TNBCs so that clinical decision making, therapeutic strategies and patient outcomes can be improved. Citation Format: Walsh EM, Shalaby A, Murillo LS, Webber MJ, Kerin MJ, Glynn SA, Callagy GM, Ingoldsby H, Keane MM. Subtyping of triple negative breast cancer by a novel immunohistochemistry panel: Assessing the correlation between subtypes and clinical outcomes. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-05-09.

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 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.