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Human endogenous retrovirus K (HERV-K) belongs to a family of endogenous retroviruses that are present in our genome with similarities to present day exogenous retroviruses. This virus can express several proteins but our knowledge of HERV-K expression in human cancers is mainly limited to the envelope (Env) protein. Elevated HERV-K env protein expression has been shown in breast cancer both in in vitro and in vivo studies. This project aimed to decipher mechanisms of HERV-K induction in triple negative breast cancer (TNBC), and the impact of HERV-K targeting shRNA mediated knockdown on TNBC characteristics including cell migration, invasion and proliferative capacity in 2D and 3D cell culture models. Our results show that a role for inducible nitric oxide synthase (iNOS) in the induction of HERV-K in TNBC cell lines. RNA seq and bioinformatics analysis was performed to identify key molecular processes regulated by HERV-K and nitric oxide (NO) in MDA-MB-231 TNBC cells. Reduced rates of migration, invasion and proliferation in HERV-K knockdowns points towards the essential role of HERV-K in tumorigenesis and metastasis. HERV-K knockdown also modulated key gene expression signatures traditionally associated with the basal and mesenchymal phenotypes in breast cancer, cellular senescence and MHC class gene regulation. The co-expression of iNOS and HERV-K was assessed in over 150 TNBC cases and the association with patient outcomes assessed. Taken together, our findings indicate that NO and HERV-K may be a useful molecular target for the treatment of TNBC. Citation Format: Dibyangana Bhattacharyya, Eoin Dervan, Sharon Glynn, Grace Callagy. Unravelling the role of human endogenous retrovirus K (HERV-K) in inducible nitric oxide synthase (iNOS) mediated triple negative breast cancer progression [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-25.

Chromosome 17 alterations affect the assessment of HER2 gene amplification in breast cancer (BC), but its clinical significance remains unclear. This study aimed to identify the prevalence of centromere enumeration probe 17 (CEP17) alterations, and its correlation with response to neoadjuvant therapy (NAT) in BC patients with human epidermal growth factor receptor 2 (HER2) immunohistochemistry-equivocal score.A large BC cohort (n = 6049) with HER2 immunohistochemistry score 2+ and florescent in-situ hybridisation (FISH) results was included to assess the prevalence of CEP17 alterations. Another cohort (n = 885) with available clinicopathological data was used to evaluate the effect of CEP17 in the setting of NAT. HER2-amplified tumours with monosomy 17 (CEP17 copy number 1.5 per nucleus), normal 17 (CEP17 1.5- 3.0) and polysomy 17 (CEP17 ≥ 3.0) were observed in 16, 59 and 25%, respectively, compared with 3, 74 and 23%, respectively, in HER2-non-amplified tumours. There was no significant relationship between CEP17 alterations and pathological complete response (pCR) rate in both HER2-amplified and HER2-non-amplified tumours. The independent predictors of pCR were oestrogen (ER) negativity in HER2-amplified tumours [ER negative versus positive; odds ratio (OR) = 11.80; 95% confidence interval (CI) = 1.37-102.00; P = 0.02], and histological grade 3 in HER2 non-amplified tumours (3 versus 1, 2; OR = 5.54; 95% CI = 1.61-19.00; P = 0.007).The impacts of CEP17 alterations are not as strong as those of HER2/CEP17 ratio and HER2 copy number. The hormonal receptors status and tumour histological grade are more useful to identify BC patients with a HER2 immunohistochemistry-equivocal score who would benefit from NAT.

Background: Human endogenous retrovirus K (HERV-K) belongs to a family of endogenous retroviruses that are present in our genome with similarities to present day exogenous retroviruses. HERV-K, like other endogenous retroviruses, is transmitted vertically in a Mendelian fashion through the human genome. This virus can express several proteins but our knowledge of HERV-K expression in human cancers is mainly limited to the envelope (Env) protein. Elevated HERV-K env protein expression has been shown in breast cancer both in in vitro and in vivo studies. Previous work from this laboratory has shown increased HERV-K expression in blood is predictive of a prostate cancer diagnosis. Aims & Experimental Approach: This project aimed to decipher whether individual HERV-K proteins (Env and Gag) display differential association with histological type, molecular subtype and patient outcomes in an Irish cohort of triple negative breast cancer patients (n=177). Additionally we aimed to assess the impact of HERV-K targeting shRNA mediated knockdown on TNBC characteristics including cell migration, invasion and proliferative capacity in 2D and 3D cell culture models. RNA seq and bioinformatics analysis was performed to identify key molecular processes regulated by HERV-K in TNBC. Results: HERV-K Env expression showed a trend towards an association with poor survival in TNBC (Log rank test, p=0.105), while HERV-K Gag was not found to be associated with survival. HERV-K Env positive metastatic patients had an increased involvement of the bone and lungs compared to HERV-K Env negative patients, while HERV-K gag positive patients showed reduced liver metastasis involvement. Knockdown of HERV-K in TNBC cell lines MDA-MB-231 and MDA-MB-468 reduced their proliferative, migratory and invasive potential. Preliminary screens using a cancer drug target array shows significantly reduced expression of HDAC4, a regulator of cell growth and the anti-apoptotic protein. GSEA analysis of RNAseq data from MDA-MB-231 HERV-K shRNA knockdown demonstrated enrichment for genes associated with both the Charafe downregulated in Luminal A versus Mesenchymal gene signature (p= 5.14x10-133), and the Charafe downregulated in Luminal A versus Basal gene signature (p= 7.03x10-110), indicating that HERV-K may play an important role in mesenchymal and basal phenotype TNBC. Additionally pathway analysis points toward a role in the regulation of many different pathways and cellular process, including focal adhesion (p= 9.77x10-7), cellular senescence (p= 7.63x10-7) and viral signalling pathways (p= 6.86x10-14). Discussion: Reduced rates of migration, invasion and proliferation in HERV-K knockdowns points towards the essential role of HERV-K in tumorigenesis and metastasis. HERV-K knockdown also modulated key gene expression signatures traditionally associated with the basal and mesenchymal phenotypes in breast cancer. Taken together, our findings indicate that HERV-K may be a useful molecular target for the treatment of TNBC. Citation Format: Sharon A Glynn, Dibyangana Bhattacharyya, Shauna Lambe, Emma Kerr, Simon McDade, Faizan H Khan, Eoin Dervan, Feng Wang-Johanning, Sean Hynes, Grace Callagy. Human endogenous retrovirus-K (HERV-K) is aberrantly expressed in triple negative breast cancer (TNBC) and associated with increased distant metastasis: Impact of HERV-K knockdown on gene expression patterns and invasive potential of mesenchymal TNBC [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-06-05.

Peer reviewed: True, Funder: Egyptian Cultural and Educational Bureau, The assessment of PD-L1 expression in TNBC is a prerequisite for selecting patients for immunotherapy. The accurate assessment of PD-L1 is pivotal, but the data suggest poor reproducibility. A total of 100 core biopsies were stained using the VENTANA Roche SP142 assay, scanned and scored by 12 pathologists. Absolute agreement, consensus scoring, Cohen's Kappa and intraclass correlation coefficient (ICC) were assessed. A second scoring round after a washout period to assess intra-observer agreement was carried out. Absolute agreement occurred in 52% and 60% of cases in the first and second round, respectively. Overall agreement was substantial (Kappa 0.654-0.655) and higher for expert pathologists, particularly on scoring TNBC (6.00 vs. 0.568 in the second round). The intra-observer agreement was substantial to almost perfect (Kappa: 0.667-0.956), regardless of PD-L1 scoring experience. The expert scorers were more concordant in evaluating staining percentage compared with the non-experienced scorers (R2 = 0.920 vs. 0.890). Discordance predominantly occurred in low-expressing cases around the 1% value. Some technical reasons contributed to the discordance. The study shows reassuringly strong inter- and intra-observer concordance among pathologists in PD-L1 scoring. A proportion of low-expressors remain challenging to assess, and these would benefit from addressing the technical issues, testing a different sample and/or referring for expert opinions.

The last UK breast cancer (BC) human epidermal growth factor receptor 2 (HER2) testing guideline recommendations were published in 2015. Since then, new data and therapeutic strategies have emerged. The American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) published a focused update in 2018 that reclassified in situ hybridisation (ISH) Group 2 (immunohistochemistry (IHC) score 2+andHER2/chromosome enumeration probe 17 (CEP17) ratio ≥2.0 andHER2 copy number

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.

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.

Pathology is the study of disease and is an important component in medical education. However, with medical curriculum reform, its role and contribution to medical courses is under potential threat. We surveyed the status of pathology in all six Irish medical schools. Information was received from five direct undergraduate and four graduate entry programmes. Pathology was recognisable as a core subject in all but one of the medical schools, was generally taught in years two or three, and the greatest contact hours were for histopathology (44–102 hours). Lectures were the most common teaching modality, and all used single best or extended matching answer multiple-choice questions as part of assessments. Currently, pathology is very visible in Irish medical education but needs to remain relevant with the move to theme and case-based teaching. There is heavy reliance on lectures and on non-academic/full-time hospital staff to deliver teaching, which may not be sustainable.

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.

Simple Summary Breast cancers that lack expression of the predictive markers oestrogen receptor, progesterone receptor and human epidermal growth factor receptor-2 are known as triple-negative breast cancers (TNBCs) and are generally considered to have a poor prognosis. As available targeted treatments are not effective, aggressive chemotherapy is frequently advocated for patients with TNBC. It is now becoming apparent that TNBC is not one entity but constitutes a range of malignancies with different clinical behaviour. This paper reviews 7 distinct histological subtypes of TNBC where the overall prognosis is favourable, and aggressive systemic treatment is generally not indicated. Their recognition and separation from the larger group of no special type TNBC are important. The members of the European Working Group for Breast Screening Pathology review the morphology, known molecular features and reported outcomes, and formulate a consensus statement regarding the approach to the subtypes that are associated with a favourable prognosis. Abstract Triple-negative breast cancers (TNBC), as a group of tumours, have a worse prognosis than stage-matched non-TNBC and lack the benefits of routinely available targeted therapy. However, TNBC is a heterogeneous group of neoplasms, which includes some special type carcinomas with a relatively indolent course. This review on behalf of the European Working Group for Breast Screening Pathology reviews the literature on the special histological types of BC that are reported to have a triple negative phenotype and indolent behaviour. These include adenoid cystic carcinoma of classical type, low-grade adenosquamous carcinoma, fibromatosis-like metaplastic carcinoma, low-grade mucoepidermoid carcinoma, secretory carcinoma, acinic cell carcinoma, and tall cell carcinoma with reversed polarity. The pathological and known molecular features as well as clinical data including treatment and prognosis of these special TNBC subtypes are summarised and it is concluded that many patients with these rare TNBC pure subtypes are unlikely to benefit from systemic chemotherapy. A consensus statement of the working group relating to the multidisciplinary approach and treatment of these rare tumour types concludes the review.

The response of human epidermal growth factor receptor2 (HER2)- positive breast cancer (BC) patients to anti-HER2 targeted therapy is significant. However, the response is not uniform and a proportion of HER2-positive patients do not respond. This study aims to identify predictors of response in the neoadjuvant treatment and to assess the discordance rate of HER2 status between pre- and post-treatment specimens in HER2-positive BC patients. The study group comprised 500 BC patients treated with neoadjuvant chemotherapy (NACT) and/or neoadjuvant anti-HER2 therapy and surgery who had tumours that were 3+ or 2+ with HER2 immunohistochemistry (IHC). HER2 IHC 2+ tumours were classified into five groups by fluorescence in situ hybridisation (FISH) according to the 2018 ASCO/CAP guidelines of which Groups 1, 2 and 3 were considered HER2 amplified. Pathological complete response (pCR) was more frequent in HER2 IHC 3+ tumours than in HER2 IHC 2+/HER2 amplified tumours, when either in receipt of NACT alone (38% versus 13%; p = 0.22) or neoadjuvant anti-HER2 therapy (52% versus 20%; p < 0.001). Multivariate logistic regression analysis showed that HER2 IHC 3+ and histological grade 3 were independent predictors of pCR following neoadjuvant anti-HER2 therapy. In the HER2 IHC 2+/HER2 amplified tumours or ASCO/CAP FISH Group 1 alone, ER-negativity was an independent predictor of pCR following NACT and/or neoadjuvant anti-HER2 therapy. In the current study, 22% of HER2-positive tumours became HER2-negative by IHC and FISH following neoadjuvant treatment, the majority (74%) HER2 IHC 2+/HER2 amplified tumours. Repeat HER2 testing after neoadjuvant treatment should therefore be considered.

A 51-year-old male presented 25 years ago with excessive sweating and haematuria. Blood pressure was labile. CT abdomen showed a large right-sided adrenal mass. Two 24-h urine collections showed elevated urinary catecholamines. Right adrenal resection was performed; a phaeochromocytoma (PC) was confirmed histologically. Two decades later, the patient represented with excessive sweating and measured variable blood pressure readings. Measurement of plasma metanephrines (PMets) showed elevated normetanephrine (NMN) [50,250 (R.I. 0-1180) pmol/L] and metanephrine (MN) [1030 (R.I. 0-510) pmol/L] values. CT abdomen showed a 100 × 90 × 63 mm enhancing mass in the right retroperitoneum. Curative resection was undertaken confirming recurrent PC. Follow-up post-resection, plasma NMN was discordant, 1314 pmol/L (above decision threshold) at 30 min and 911 pmol/L (below decision threshold) at 40 min. Acute clinical awareness of persistent disease mandated the performance of a metaiodobenzylguanidine (MIBG) scan and CT abdomen. These confirmed residual disease in the upper right side of the retroperitoneum. Persistent disease following redo surgery could have been missed if only seated-sampling upper reference limits were applied to PMets collected at 40 min. Our experience with this patient triggered a review of our PMets sampling strategy. There was no statistically significant difference in PMets sampled at 30 and at 40 min seated-rest. Optimum diagnostic test accuracy was achieved using a supine-sampling strategy at a single time point (30 min). Our case highlights the importance of maintaining a high index of clinical suspicion for residual/recurrent disease in the face of inconclusive biochemistry, followed by appropriate targeted radiology using MIBG or PET-CT in patients with PPGL.

// 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.

Background: The androgen receptor (AR) has emerged as a potential therapeutic target for AR-positive triple-negative breast cancer (TNBC). However, conflicting reports regarding AR’s prognostic role in TNBC are putting its usefulness in question. Some studies conclude that AR positivity indicates a good prognosis in TNBC, whereas others suggest the opposite, and some show that AR status has no significant bearing on the patients’ prognosis. Methods: We evaluated the prognostic value of AR in resected primary tumors from TNBC patients from six international cohorts {US (n = 420), UK (n = 239), Norway (n = 104), Ireland (n = 222), Nigeria (n = 180), and India (n = 242); total n = 1407}. All TNBC samples were stained with the same anti-AR antibody using the same immunohistochemistry protocol, and samples with ≥1% of AR-positive nuclei were deemed AR-positive TNBCs. Results: AR status shows population-specific patterns of association with patients’ overall survival after controlling for age, grade, population, and chemotherapy. We found AR-positive status to be a marker of good prognosis in US and Nigerian cohorts, a marker of poor prognosis in Norway, Ireland and Indian cohorts, and neutral in UK cohort. Conclusion: AR status, on its own, is not a reliable prognostic marker. More research to investigate molecular subtype composition among the different cohorts is warranted.

Background Pleomorphic and Florid Lobular carcinoma in situ (P/F LCIS) are rare variants of LCIS, the exact nature of which is still debated. Aim To collect a large series of P/F LCIS diagnosed on preoperative biopsies and evaluate their association with invasive carcinoma and high grade duct carcinoma in situ (DCIS). Data obtained were compared with those reported in the literature. Methods A multi-institutional series of P/F LCIS was retrieved. All cases were diagnosed on pre-operative biopsies, which was followed by an open surgical excision. Data on post-operative histopathology were available. A literature review was performed. Results A total of 117 cases were collected; invasive carcinoma and/or DCIS was present in 78/117 cases (66.7%). Seventy cases of P/F LCIS were pure on biopsy and 31 of these showed pathological upgrade in post-surgical specimens. Pre-operative biopsy accuracy was 47/78 (60.3%); pre-operative biopsy underestimation of cancer was 31/78 (39,7.%). In the literature review papers, invasive carcinoma or DCIS was associated with 274 of 418 (65.5%) cases of P/F LCIS. Pre-operative biopsy accuracy was 66% (181/274) whereas pre-operative biopsy underestimation of cancer was 33.9% (93/274). Conclusions The data presented here indicate that P/F LCIS is frequently associated with invasive carcinoma or high grade DCIS and that pre-operative biopsy is associated with an underestimation of malignancy. Open surgery is indicated when P/F LCIS is diagnosed pre-operatively.

XBP1 is a multitasking transcription factor and a key component of the unfolded protein response (UPR). Despite the wealth of knowledge about the role of XBP1 in luminal/ER-positive breast cancer, not much is known about the effectors of XBP1 in this context. Here we show that NCOA3 is a transcriptional target of XBP1. We observed increased expression of NCOA3 during conditions of UPR and oestrogen (E2) stimulation. Further investigations revealed a role for the IRE1-XBP1 axis in the induction of NCOA3 during UPR and oestrogen signalling. We identify a novel role for NCOA3 in activation of PERK-ATF4 axis during UPR where knockdown of NCOA3 compromised the optimal activation of the PERK-ATF4 pathway. We found that NCOA3 is required for induction of XBP1 during E2 stimulation and uncover a positive feedback regulatory loop that maintains high levels of NCOA3 and XBP1 in breast cancer. Furthermore, upregulated NCOA3 was required for XBP1-mediated resistance to antihormonal agents. Increased expression of NCOA3 was associated with poor prognosis and higher levels of XBP1-S in breast cancer tissues. Our results uncover a novel steroid hormone-independent role for NCOA3 in UPR signalling. Further we identify a positive feedback regulatory loop consisting of XBP1 and NCOA3 that maintains high levels of NCOA3 and XBP1 expression in breast cancer tissues. Taken together our data identify XBP1-NCOA3 axis that regulates cell fate decisions in ER-positive breast cancer cells.

Background We seek to further elucidate mechanisms by which inflammatory mediators promote estrogen receptor (ER)-negative breast cancer progression and poor survival. We previously reported association between inducible nitric oxide synthase (NOS2) and poor outcome in ER-negative tumours. In tumours aberrant NOS2 induction facilitates tissue remodelling and stimulates neovascularisation. Also involved in inflammation and wound healing is cyclooxygenase-2 (COX2). We demonstrated that COX2 is associated with Akt activation and poor outcome in ER-negative tumours. We hypothesise that co-expression COX2 with NOS2 in ER-negative tumours amplifies effects of NOS2 on poor outcome, via EGFR and TLR4 signalling loop activation, and polarization of the tumour immune-compartment to pro-tumorigenic M2 phenotype. Methodology We determined the association of NOS2 and COX2 co-expression on breast cancer specific survival in ER-negative and triple negative breast tumours (N=102), via immunohistochemistry and cox regression statistical analysis. To explore the mechanism of NOS2 induction of COX2 through transactivation of EGFR, NO donors in combination with EGFR inhibitors were used to determine if NO exposure results in amplified EGFR and PGE2 pro-survival and pro-metastatic signalling in triple negative breast cancer cell lines. Finally, we explored the ability of NO to modify the ability of triple negative breast cancer secretome to induce polarisation of macrophages to a pro-tumorigenic M2 phenotype. Results Co-expression of NOS2 and COX2 in triple negative breast cancer results in poor outcome, via activation of pro-survival signalling and modification of the immune compartment to a pro-tumorigenic M2 associated phenotype. NO induces activation of growth factor signalling pathways and secretion of M2 promoting cytokines that induce THP1 macrophage polarization to an M2 phenotype. Citation Format: Glynn S, Garrrido-Cuesta P, Wink D, Ridnour L, Ambs S, Keane M, Walsh E, Callagy G. NOS2&COX2 activation of TLR4 & EGFR signalling causes poor outcome in oestrogen receptor-negative breast cancer via pro-survival signals and immune polarisation. [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 P2-05-15.

Microinvasion is the smallest morphologically identifiable stage of invasion. Its presence and distinction from in situ carcinoma may have therapeutic implications, and clinical staging also requires the recognition of this phenomenon. Microinvasion is established on the basis of several morphological criteria, which may be difficult and not perfectly reproducible among pathologists. The aim of this study was to assess the consistency of diagnosing microinvasion in the breast on traditional haematoxylin and eosin (HE) stained slides and to evaluate whether immunohistochemistry (IHC) for myoepithelial markers could improve this. Digital images were generated from representative areas of 50 cases stained with HE and IHC for myoepithelial markers. Cases were specifically selected from the spectrum of in situ to microinvasive cancers. Twenty-eight dedicated breast pathologists assessed these cases at different magnifications through a web-based platform in two rounds: first HE only and after a washout period by both HE and IHC. Consistency in the recognition of microinvasion significantly improved with the use of IHC. Concordance rates increased from 0.85 to 0.96, kappa from 0.5 to 0.85, the number of cases with 100 % agreement rose from 9/50 to 25/50 with IHC and the certainty of diagnosis also increased. The use of IHC markedly improves the consistency of identifying microinvasion. This corroborates previous recommendations to use IHC for myoepithelial markers to clarify cases where uncertainty exists about the presence of microinvasion. Microinvasive carcinoma is a rare entity, and seeking a second opinion may avoid overdiagnosis.

Background: African American (AA) triple-negative breast cancer (TNBC) patients experience worse clinical outcomes and exhibit 40% higher mortality rate than their European Americans (EAs) counterparts. There are currently no distinctions in inherent tumor biology between the ethnically distinct TNBC patients that serve as risk-predictive markers allowing new tailored treatments. Recently, androgen receptor (AR) has emerged as a new target for treating TNBC. However, the prognostic value of AR in TNBC remains controversial. To reconcile conflicting reports about the impact of AR loss on prognosis of TNBC and uncover the molecular pathways that may underlie the racial disparity in outcomes among AR-negative TNBCs, we determined the prognostic value of AR in diverse TNBC cohorts. Since loss of AR is associated with worse clinical outcome and African and AA women are more prone to aggressive disease course, we hypothesized that AR loss may underlie the global disparate burden in TNBC. Methods: We evaluated AR expression in well-annotated formalin-fixed, paraffin-embedded TNBC resection samples (n=1351) obtained from multiple hospitals from US, UK, Norway, Ireland, Nigeria and India. Samples with ≥1% nuclei staining positive for AR were deemed to be AR-positive. Associations between AR status, clinicopathologic variables and overall survival (OS) were evaluated. We performed gene set enrichment analysis for AR low and high group in AA and EA TNBCs. In vitro experiments were performed to examine whether AR loss increased the metastatic potential of TNBC cells. Results: We observed a significant difference in AR expression among EA and AA TNBCs (p=0.02) with AR loss associated with women of African ancestry (>90%, p14%) (HR=1.72; p=0.095) AA TNBC (n=98) when compared to EA TNBCs (n=80). Furthermore, AR status retained its significant prognostic value (HR=1.549, p=0.036) after controlling for age, grade, Ki67, race and chemotherapy status. Gene set enrichment analysis revealed that Wnt/β-catenin signaling was the top-enriched gene ontology in the AR-low TNBC subgroup. Moreover, β-catenin protein levels are higher in AA AR-low TNBCs compared with AA AR-high TNBCs (p Conclusion: Our study suggests a striking association of AR loss in TNBC with women of African ancestry. Our data offer compelling evidence that oncogenic Wnt/β-catenin signaling may link AR loss to more aggressive disease course and represent actionable biology in AA AR-negative TNBCs for whom no targeted treatments are currently on the horizon. This abstract is also being presented as Poster C102. Citation Format: Shristi Bhattarai, Sergey Klimov, Karuna Mittal, Uma Krishnamurthi, Xiaoxian Bill Li, Deepika Wali, Ceyda Sonmez Wetherilt, Ansa Riaz, Mohammad A. Aleskandarany, Andrew R. Green, Ian O. Ellis, Meenakshi Gupta, Lauren E. McCullough, Upender Manne, Johnson Agboola, Brett Baskovich, Emiel A. Janssen, Grace Callagy, Anurag Mehta, Tanuja Shet, Rakha A. Emad, Padmashree C.G. Rida, Ritu Aneja. Prognostic role of androgen receptor in triple-negative breast cancer: A global multi-institutional experience [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr PR18.

BACKGROUND: Several studies have demonstrated a prognostic role for stromal tumour infiltrating lymphocytes (sTILs) in triple-negative breast cancer (TNBC). The reproducibility of scoring sTILs is variable with potentially excellent concordance being achievable using a software tool. We examined agreement between breast pathologists across Europe scoring sTILs on H&E-stained sections without software, an approach that is easily applied in clinical practice. The association between sTILs and response to anthracycline-taxane NACT was also examined. METHODOLOGY: Pathologists from the European Working Group for Breast Screening Pathology scored sTILs in 84 slides from 75 TNBCs using the immune-oncology biomarker working group guidance in two circulations. There were 16 participants in the first and 19 in the second circulation. RESULTS: Moderate agreement was achieved for absolute sTILs scores (intraclass correlation coefficient (ICC) = 0.683, 95% CI 0.601-0.767, p-value

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.

Triple negative breast cancers (TNBCs), which are defined as estrogen-receptor, progesterone-receptor, and HER2-receptor negative, account for 10-20% of breast cancers, and they are associated with early metastasis, chemotherapeutic resistance, and poor survival rates. One aspect of TNBC that complicates its prognosis and the development of new molecular therapeutic targets is its clinical and molecular heterogeneity. Herein we compare TNBC and basal cytokeratin-positive breast cancers. We examine the different TNBC molecular subtypes, based on gene expression profiling, which include basal-like, mesenchymal, and luminal androgen receptors, in the context of their biology and impact on TNBC prognosis. We explore the potential role of inducible nitric oxide synthase (iNOS) in TNBC tumor biology and treatment responses. 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, all of which are key components of TNBC biology. Moreover, iNOS predicts poor outcome in TNBC, and iNOS inhibitors show efficacy against TNBC when used in combination with chemotherapy. We discuss molecular targeted approaches, including EGFR, PARP, and VEGF inhibitors and immunotherapeutics, that are under consideration for the treatment of TNBC and what role, if any, iNOS may play in their success.

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.