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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.

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.

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.

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 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.

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.

Sentinel lymph node biopsy (SLNB) is now widely used as an alternative to axillary lymph node dissection to stage the axilla in women with early-stage breast cancer. The detailed pathological work-up of sentinel nodes has presented new challenges in the field of pathology. For example, many patterns of low volume disease – isolated tumour cells (ITCs) and micrometastasis (MI) – are difficult to classify and current classification systems are only moderately reproducible. The significance of ITCs and MI in terms of predicting non-SLN involvement and overall outcome is also unclear. The optimal methodology for evaluating the SLN is also unresolved. In this review, we give an overview of these controversial areas in SLN pathology, including the classification, reproducibility and clinical significance of low volume disease; pathological evaluation of the SLN; and methods of intraoperative assessment.

Purpose: Prognostication of breast cancer using clinicopathologic variables, although useful, remains imperfect. Many reports suggest that gene expression profiling can refine the current approach. Alternatively, it has been shown that panels of proteins assessed by immunohistochemistry might also be useful in this regard. We evaluate the prognostic potential of a panel of markers by immunohistochemistry in a large case series to establish if either a single marker or a panel could improve the prognostic power of the Nottingham Prognostic Index (NPI). We validated the results in an independent series.Experimental Design and Results: The expression of 13 biomarkers was evaluated in 930 breast cancers on a tissue microarray. Eight markers [estrogen receptor (ER), progesterone receptor (PR), Bcl-2, cyclin E, p53, MIB-1, cytokeratin 5/6, and HER2] showed a significant association with survival at 10 years on univariate analysis. On multivariate analysis that included these eight markers and the NPI, only the NPI [hazard ratio (HR), 1.35; 95% confidence interval (95% CI), 1.16-1.56; P = 0.0005], ER (HR, 0.59; 95% CI, 0.39-0.88; P = 0.011), and Bcl-2 (HR, 0.68; 95% CI, 0.46-0.99; P = 0.055) were significant. In a subsequent multivariate analysis that included the NPI, ER, and Bcl-2, only Bcl-2 (HR, 0.62; 95% CI, 0.44-0.87; P = 0.006) remained independent of NPI (HR, 1.50; 95% CI, 1.16-1.56; P = 0.004). In addition, Bcl-2, used as a single marker, was more powerful than the use of a panel of markers. Based on these results, an independent series was used to validate the prognostic significance of Bcl-2. ER and PR were also evaluated in this validation series. Bcl-2 (HR, 0.83; 95% CI, 0.71-0.96; P = 0.018) retained prognostic significance independent of the NPI (HR, 2.04; 95% CI, 1.67-2.51; P < 0.001) with an effect that was maximal in the first 5 years.Conclusion: Bcl-2 is an independent predictor of breast cancer outcome and seems to be useful as a prognostic adjunct to the NPI, particularly in the first 5 years after diagnosis.

Gene amplification, an important mechanism of oncogene activation in breast cancer, can have both prognostic and therapeutic implications. In this work, an attempt is made to identify amplified genes that can be used to improve prognostication in breast cancer. A series of 52 node-negative tumours was screened for genomic gains at 57 loci by array-CGH. A subset of these genes was identified that could divide the series into two divergent outcome groups of either long-term survivors or early disease-related deaths (p = 0.01) using a combination of k-means clustering and statistical analysis. The prognostic significance of amplification of four of the genes (EMS1, TOP2A, CCNE1, and ERBB2) was then evaluated, using fluorescent in situ hybridization on a tissue microarray, in a second larger 'validation' series of 232 tumours with a median follow-up of 4.8 years. Adverse disease-related outcome was associated with amplification of TOP2A (p = 0.004); ERBB2 (p = 0.002); and with the combined amplification of TOP2A, ERBB2, and EMS1 (p = 0.01). EMS1 amplification was more common (26% of cases) than previously reported but, in isolation, had no prognostic significance. Amplification of CCNE1, seen in only 6% of cases, had no prognostic role. These results indicate that the complementary use of array-CGH and tissue microarrays has the potential to help in the identification and validation of molecular markers that can be used to classify breast cancers into different prognostic groups.

Geminin inhibits DNA replication by preventing Cdt1 from loading minichromosome maintenance (MCM) proteins onto DNA. The present study has investigated whether the frequency of geminin expression predicts clinical outcome in breast cancer. Immunohistochemistry was used first to examine geminin expression in normal and malignant breast tissue (n = 67). Correlations with cell-cycle parameters, pathological features, and clinical outcome were then determined using an invasive breast carcinoma tissue microarray (n = 165). Breast carcinomas were scanned for mutations (n = 61) and copy number imbalances (n = 241) of the geminin gene. Finally, the cell cycle distribution of geminin in breast cancer cells was investigated in vivo and in vitro. Despite a putative tumour suppressor function, it was found that increased geminin expression is a powerful independent indicator of adverse prognosis in invasive breast cancer. Both poor overall survival (p = 0.0002) and the development of distant metastases (p = 0.005) are predicted by high geminin expression, which performs better in this patient cohort than traditional factors currently used to determine prognosis and appropriate therapy. No mutations or deletions of the geminin gene and no evidence that a high frequency of protein expression is related to gene amplification were found. It is shown that geminin is expressed from S to M phase in breast carcinoma tissue and cell lines, disappearing at the metaphase--anaphase transition. While MCM proteins identify all non-quiescent cells, geminin identifies the sub-fraction that have entered S phase, but not exited mitosis, thereby indicating the rate of cell-cycle progression. It is suggested that this explains its unexpected value as a prognostic marker in breast cancer.

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

The steroid hormone oestrogen is central to normal female physiology, reproduction and behaviour, through its effects on cellular processes including cell proliferation and cell survival. The effects of oestrogen are mediated by nuclear ERs (oestrogen receptors). ER status is important for the development, progression and treatment of breast cancer. miRNAs (microRNAs) are small non-coding RNAs that bind the 3′-UTR (untranslated region) of target mRNAs to reduce their stability and/or translation. miRNAs participate in oestrogen signalling by regulating oestrogen-responsive genes and pathways. Interestingly expression and maturation of miRNAs can also be regulated by ER signalling at multiple levels. In addition to regulating the expression of miRNAs at the transcriptional level, ER appears to be able to regulate the biogenesis of miRNAs. In the present review, we summarize recent findings on miRNA biogenesis and describe various mechanisms by which oestrogen signalling can modulate the production of miRNAs.

Primary objective. To investigate the relationship between human epidermal growth factor receptor (HER)-2/neu and the gene encoding topoisomerase IIα (TOP2A) in breast cancer, while elucidating their association with clinicopathological variables. Methods. Real-time quantitative polymerase chain reaction (RQ-PCR) was performed on a 96-patient study group to assess gene amplification, and levels were determined using the comparative cycle threshold approach and Taqman assays. An immunohistochemistry (IHC) microarray (n = 76) was then employed to check for correlation between gene amplification and protein expression levels. Results. Amplification levels of TOP2A did not differ significantly according to HER-2/neu status by either RQ-PCR or IHC microarray. Of the HER-2/neu− patients, 29.1% demonstrated levels of TOP2A above the third quartile, whereas 22.9% of the HER-2/neu+ patients had values in the first quartile (log TOP2A Conclusions. Our results indicate that amplification of TOP2A in breast cancer is not confined to those who are concomitantly HER-2/neu+, and suggest that a significant proportion of HER-2/neu− patients exhibit high levels of TOP2A.

Aim To investigate the prevalence of alcohol in unnatural deaths in the West of Ireland between 2003 and 2007. Methods The reports of 1669 postmortem examinations carried out at Galway University Hospitals were reviewed; 379 non-homicidal unnatural deaths were eligible for the study. Alcohol levels were measured in blood and/or urine in 311 cases. For each case, gender, age, cause of death and toxicology results were recorded. Results Alcohol was detected in 162 out of 311 cases (52%); 133 (82%) cases were men and 29 (18%) were women. Alcohol levels >150 mg/100 ml were found in 99 cases (61%), most commonly in 18–49-year-olds (n=74; 75%). Road traffic crashes (RTCs) (n=38; 23%), drownings (n=38; 23%) and hangings (n=25; 15%) were common unnatural deaths associated with alcohol. The majority of RTC deaths involved the driver (n=27; 71%). The alcohol level was higher than the legal driving limit of 80 mg/100 ml in 82% (n=22) and >150 mg/100 ml in 59% (n=16) of these. Mortality of passengers (n=6; 16%) and pedestrians (n=5; 13%) was less common. Conclusions Alcohol remains a major contributor to unnatural deaths in the West of Ireland, particularly with respect to mortality in young people. Young men are especially vulnerable. Deaths in RTCs and by drowning and hanging are commonly associated with alcohol. Many driver fatalities involve alcohol levels far above legal limits. Alcohol measurement in all unnatural deaths would facilitate more accurate determination of its role.

Carcinoma of the male breast is an infrequent and poorly understood disease (Sinha et al. in Ann R Coll Surg Engl 88(5):W3-W5, 2006). It differs from female breast cancer in both demographic and histopathological characteristics.Herein we describe a case of intracystic papillary carcinoma in situ in a male breast with a review of the literature.Although rare, in situ carcinoma constitutes a larger proportion of male than female breast cancer in a non-screened population. It is characteristically of the papillary and micropapillary subtype. Intracystic papillary carcinoma is a noncomedo intraductal carcinoma constituting about 0.5% of female breast cancers and is associated with a slightly inferior prognosis than other noncomedo intraductal carcinomas (Lefkowitz et al. in Hum Pathol 25:802-809, 1994).

Breast cancers with nodal isolated tumour cells (ITC) and micrometastases are categorised as node-negative and node-positive, respectively, in the tumour node metastasis (TNM) classification. Two recently published interpretations of the TNM definitions were applied to cases of low-volume sentinel lymph node (SLN) involvement and their corresponding non-SLNs for reclassification as micrometastasis or ITC. Of the 517 cases reviewed, 82 had ITC and 435 had micrometastasis on the basis of one classification, and the number of ITC increased to 207 with 310 micrometastases on the basis of the other. Approximately 24% of the cases were discordantly categorised. The rates of non-SLN metastases associated with SLN ITCs were 8.5% and 13.5%, respectively. Although the second interpretation of low-volume nodal stage categories has better reproducibility, it may underestimate the rate of non-SLN involvement. The TNM definitions of low-volume nodal metastases need to be better formulated and supplemented with visual information in the form of multiple sample images.

The classic triad of symptoms seen in chronic mesenteric ischaemia is post-prandial pain, sitophobia (fear of food) and progressive weight loss. Patients with mesenteric ischaemia secondary to a prothrombotic state such as that rendered by the Factor V Leiden mutation, are substantially younger than the typical elderly patient in whom embolic disease triggered by atrial fibrillation is the main underlying cause. This is one such case report documenting arterial thrombosis in a 37-year-old male with a subsequently identified heterozygous Factor V Leiden mutation. Factor V Leiden mutation is a contributing risk factor in cases of small bowel infarction.

Aims There is a paucity of published data on prospectively identified rates of out-of-hospital sudden cardiac death (SCD). We sought to determine the incidence, survival and aetiology of out-of-hospital SCD in the West of Ireland for the year 2005. Methods and results Data from emergency room resuscitation records were collected throughout the year from all hospitals in the West of Ireland and recorded according to pre-specified criteria. Hospital records of survivors were analysed. Simultaneously, autopsy reports from all pathology laboratories in the region were systematically reviewed and cases of SCD identified. Cardiac arrest associated with non-cardiac pathology was excluded. The population base was 414 277. There were 212 recorded cases of out-of-hospital SCD; 160 (75.5%) were male and the mean age was 63.3 years. The incidence rate was 51.2/100 000/year. The most common aetiology was coronary artery disease (161 cases; 75.9%). The majority of cases occurred in the home (152, 71.7%). Thirteen (6.1%) patients survived to admission of whom eight (3.8%) were alive at discharge. All survivors had ventricular fibrillation as the presenting rhythm. Conclusion The burden of SCD in the West of Ireland is considerable. The vast majority of cases occur in the home. Survival rates in this rural population cohort remain low.

Comparative genomic hybridization (CGH) is a powerful screening technique that can identify regions of gain and loss within the whole genome in a single experiment. The combination of laser capture microdissection, whole-genome amplification, and CGH permits genomic screening with high specificity and sensitivity. This complement of techniques has enabled analysis of focal regions and subpopulations of cells within a tissue, which has previously been difficult, providing insight into disease progression and heterogeneity. This chapter outlines the techniques involved in producing labeled probes from DNA extracted from laser capture microdissected material and the methods for hybridization of these probes to metaphase chromosomes. This protocol can also be applied to the preparation of probes for CGH arrays.

Comparative genomic hybridization (CGH) is a powerful screening technique that can identify regions of gain and loss within the whole genome in a single experiment. The combination of laser capture microdissection, whole-genome amplification, and CGH permits genomic screening with high specificity and sensitivity. This complement of techniques has enabled analysis of focal regions and subpopulations of cells within a tissue, which has previously been difficult, providing insight into disease progression and heterogeneity. This chapter outlines the techniques involved in producing labeled probes from DNA extracted from laser capture microdissected material and the methods for hybridization of these probes to metaphase chromosomes. This protocol can also be applied to the preparation of probes for CGH arrays.

Most studies of genomic rearrangements in common cancers have focused on regional gains and losses, but some rearrangements may break within specific genes. We previously reported that five breast cancer cell lines have chromosome translocations that break in the NRG1 gene and that could cause abnormal NRG1 expression. NRG1 encodes the Neuregulins 1 (formerly the Heregulins), ligands for members of the ErbB/epidermal growth factor-receptor family, which includes ErbB2/HER2. We have now screened for breaks at NRG1 in paraffin sections of breast tumors. Tissue microarrays were screened by fluorescence in situ hybridization, with hybridization probes proximal and distal to the expected breakpoints. This screen detects breaks but does not distinguish between translocation or deletion breakpoints. The screen was validated with array-comparative genomic hybridization on a custom 8p12 high-density genomic array to detect a lower copy number of the sequences that were lost distal to the breaks. We also precisely mapped the breaks in five tumors with different hybridization probes. Breaks in NRG1 were detected in 6% (19 of 323) of breast cancers and in some lung and ovarian cancers. In an unselected series of 213 cases with follow-up, breast cancers where the break was detected tended to be high-grade (65% grade III compared with 28% of negative cases). They were, like breast tumors in general, mainly ErbB2 low (11 of 13 were low) and estrogen receptor positive (11 of 13 positive).

Purpose: To test the hypothesis that prognostic information in breast cancer may be derived from an accurate assessment of epithelial cell cycle entry, as indicated by expression of minichromosome maintenance (MCM) proteins. Materials and Methods: We used immunohistochemistry to examine the distribution of Mcm-2 in breast tissue. Power calculations based on a pilot study of 67 whole tissue sections led to selection of an independent 347-core breast carcinoma tissue microarray validation set. We tested for associations between Mcm-2 (and Ki-67) labeling index (LI) and various clinicopathologic parameters. Results: Mcm-2 was expressed more frequently than the standard proliferation marker Ki-67 in whole tissue sections of normal breast (P = .0003) and breast carcinoma (P < .0001). In 221 assessable cores of invasive carcinoma, the Mcm-2 LI showed a positive association with tumor size (P = .002), mitotic index (P < .0001), histologic grade (P < .0001), and the Nottingham Prognostic Index (NPI) score (P < .0001). Using a cutoff value of 50%, Mcm-2 LI was associated with overall survival (P = .0007), disease-free interval (P = .0002), and with the development of regional recurrence (P = .011) and distant metastases (P = .0016). Cox regression analysis suggested that the Mcm-2 LI is a strong prognostic factor in breast cancer that is independent and superior to histologic grade, lymph node stage, and Ki-67 LI, but not the NPI score. Conclusion: Mcm-2 may be of utility as a prognostic marker to refine the prediction of outcome in breast cancer, for example when combined with parameters currently used in the NPI.

Aims: To describe a robust pretreatment protocol for preparing paraffin wax embedded tissues on tissue microarrays for fluorescence in situ hybridisation (FISH). The newly developed pretreatment protocol described here was compared with the commonly used sodium thiocyanate based protocol and two different heating methods used in standard antigen unmasking protocols for immunohistochemistry (pressure cooking and microwaving in citrate acid buffer). Methods: Dewaxed tissue sections were incubated in 10mM citric acid buffer at 80°C for 30 minutes to two hours, followed by a short pepsin digestion (1–5 mg/ml). Pretreated tissues were co-denatured with DNA probes at 80°C for 10 minutes, followed by hybridisation at 37°C for 48–72 hours. Results: The three protocols using citrate acid buffer produced FISH signals with superior signal to noise ratios compared with sodium thiocyanate pretreatment. Most importantly, the best tissue attachment was achieved using the newly developed pretreatment protocol: on tissue microarrays less than 1% of cores were lost. To date, a total of 30 probes have been successfully hybridised on to breast tissue and multi-tissue microarrays. Conclusion: This pretreatment protocol is easy, reproducible, and facilitates FISH on tissue microarrays, with potential for widespread application in cancer research.

The histopathologic classification of breast cancer stratifies tumors based on tumor grade, stage, and type. Despite an overall correlation with survival, this classification is poorly predictive and tumors with identical grade and stage can have markedly contrasting outcomes. Recently, breast carcinomas have been classified by their gene expression profiles on frozen material. The validation of such a classification on formalin-fixed paraffin-embedded tumor archives linked to clinical information in a high-throughput fashion would have a major impact on clinical practice. The authors tested the ability of tumor tissue microarrays (TMAs) to sub-classify breast cancers using a TMA containing 107 breast cancers. The pattern of expression of 13 different protein biomarkers was assessed by immunohistochemistry and the multidimensional data was analyzed using an unsupervised two-dimensional clustering algorithm. This revealed distinct tumor clusters which divided into two main groups correlating with tumor grade (P0.001) and nodal status (P = 0.04). None of the protein biomarkers tested could individually identify these groups. The biological significance of this classification is supported by its similarity with one derived from gene expression microarray analysis. Thus, molecular profiling of breast cancer using a limited number of protein biomarkers in TMAs can sub-classify tumors into clinically and biologically relevant subgroups.

Telepathology is a means of practicing pathology at a distance, viewing images on a computer display rather than directly through a microscope. Without compression, images take too long to transmit to a remote location and are very expensive to store for future examination. However, to date the use of compressed images in pathology remains controversial. This is because commercial image compression algorithms such as JPEG achieve data compression without knowledge of the diagnostic content. Often images are lossily compressed at the expense of corrupting informative content. None of the currently available lossy compression techniques are concerned with what information has been preserved and what data has been discarded. Their sole objective is to compress and transmit the images as fast as possible. By contrast, this paper presents a novel image compression technique, which exploits knowledge of the slide diagnostic content. This 'content based' approach combines visually lossless and lossy compression techniques, judiciously applying each in the appropriate context across an image so as to maintain 'diagnostic' information while still maximising the possible compression. Standard compression algorithms, e.g. wavelets, can still be used, but their use in a context sensitive manner can offer high compression ratios and preservation of diagnostically important information. When compared with lossless compression the novel content-based approach can potentially provide the same degree of information with a smaller amount of data. When compared with lossy compression it can provide more information for a given amount of compression. The precise gain in the compression performance depends on the application (e.g. database archive or second opinion consultation) and the diagnostic content of the images.

The conventional approach to performance evaluation for image compression in telemedicine is simply to measure compression ratio, signal-to-noise ratio and computational load. Evaluation of performance is however a much more complex and many sided issue. It is necessary to consider more deeply the requirements of the applications. In telemedicine, the preservation of clinical information must be taken into account when assessing the suitability of any particular compression algorithm. In telemedicine the metrication of this characteristic is subjective because human judgement must be brought in to identify what is of clinical importance. The assessment must therefore take into account subjective user evaluation criteria as well as objective criteria. This paper develops the concept of user based assessment techniques for image compression used in telepathology. A novel visualization approach has been developed to show and explore the highly complex performance space taking into account both types of measure. The application considered is within a general histopathology image management system; the particular component is a store-and-forward facility for second opinion elicitation. Images of histopathology slides are transmitted to the workstations of consultants working remotely to enable them to provide second opinions.

Objective. CD44, an integral membrane glycoprotein, may have an important role in early tumorigenesis, specifically, facilitating early tumor progression. Reports of the expression of CD44 in early uterine cervical squamous carcinogenesis are conflicting. We examined the expression of CD44 in microinvasive carcinoma of the cervix (MIC), as yet unreported, and compared it to that in cervical intraepithelial neoplasia (CIN) 1 and CIN 3 to further elucidate its role in early squamous carcinogenesis. Methods. Seventeen cases of CIN 1, 24 cases of CIN 3, and 20 cases of MIC were stained with antibodies to CD44s, CD44v5, and CD44v6. Only membranous staining was considered positive. Results. Positive membranous staining (>50% cells) was observed in 97% of cases of CIN 1 using all three antibodies. In CIN 3, positive staining was seen more often with CD44v6 (18/24) and CD44v5 (19/24) than with CD44s (6/24). Expression of CD44v6 was retained more often in MIC (16/20) compared with CD44s (3/20) and CD44v5 (9/20). Those cases of CIN 3 and MIC that failed to meet our criteria for positive staining showed either heterogeneous or absent staining. Conclusion. There is a qualitative and quantitative reduction in expression of CD44 in MIC and CIN 3 compared with CIN 1. Down-regulation of CD44 variants may occur later in neoplastic progression than CD44s. This pattern may reflect their important biological function in early progression by cervical cancer cells. Patchy and heterogeneous staining in more advanced lesions limits the usefulness of CD44 and its variants in the assessment of microinvasion.

Genetic haemochromatosis constitutes a high risk factor for the development of hepatocellular carcinoma. It is widely accepted that venesection prevents the evolution of cirrhosis in haemochromatosis and indirectly protects against the development of hepatocellular carcinoma. Clinical, pathological and radiological data are presented on three patients who did not conform to the 'siderosis-cirrhosis-carcinoma' sequence and in whom prompt and adequate iron depletion did not prevent the development of cancer. This is the first report of hepatocellular carcinoma intervening in non-cirrhotic liver in two siblings with genetic haemochromatosis. The current literature on the subject is reviewed. The direct oncogenic role of iron remains to be elucidated. Hepatocellular carcinoma should be considered as a differential diagnosis in patients with non-cirrhotic genetic haemochromatosis who present with clinical deterioration during the course of an otherwise uneventful venesection programme.

To evaluate histological findings in untreated chronic hepatitis C patients at diagnosis 17 years after infection and to assess histological progression on repeat liver biopsy 2 years later.Thirty patients infected with hepatitis C virus (HCV), genotype 1b, by contaminated anti-D immunoglobulin in Ireland in 1977 were studied. These patients were diagnosed in 1994 for the first time. All patients were positive for HCV-RNA by polymerase chain reaction (PCR).Each patient underwent two liver biopsies approximately 2 years apart 17 and 19 years after initial infection. The liver biopsies were scored by two pathologists by the modified histological activity index using a numerical score. At first liver biopsy at time of presentation, eight patients had normal alanine aminotransferase (ALT), four had an ALT of more than 100 IU/I and 18 had an ALT level between 40 and 100 IU/I.In the initial (1994) biopsies, the median grade (inflammation) was 5/18, range 1-9 and the median stage (fibrosis) was 2/6, range 0-6. One patient showed cirrhosis (stage 6/6) and six patients (20%) had developed moderate fibrosis (stage 3-4/6). On the repeat biopsy, 2 years later, median grade (inflammation) was 5/18, range 2-9 and stage (fibrosis) was 1/6, range 0-6.This group of patients, infected with HCV genotype 1b and untreated for 19 years, allows evaluation of the natural history of this virus. The majority of patients showed mild chronic hepatitis. Only one patient had developed cirrhosis. There was no significant histological disease progression between the two biopsy specimens over a 2 year period. The results suggest that the prognosis in such cases could at least be guardedly optimistic and that sequential liver biopsy may be performed less frequently.

We report the case of a middle-aged man who presented de novo with abdominal pain and hepatomegaly and was found to have positive serology for hepatitis C and subsequently a primary hepatic lymphoma. An increased incidence of primary hepatocellular cancer is well characterized in both cirrhotic and non-cirrhotic cases of chronic hepatitis C. The relationship between chronic hepatitis C and primary hepatic lymphoma remains obscure. It has been established that hepatitis C can sustain the clonal B-cell expansion that occurs in associated cryoglobulinaemia, and hepatitis C RNA has been detected within extrahepatic lymphoma tissue. Viral aetiologies for lymphoma are well characterized, such as Epstein-Barr virus (EBV) and human T-cell leukaemia virus (HTLV) I and II. Existing models of chronic infection causing lymphoma within the gastrointestinal tract include that of Helicobacter pylori and mucosa-associated lymphoid tumour of the stomach. Given the relatively low frequency of occurrence it may be prudent to perform a retrospective analysis on past cases of primary hepatic lymphoma in order to determine whether or not hepatitis C was present.

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

of 2,4-dinitrobenzenesulfonic acid (DNBS, 15 mg/rat), to assess systemic [body and spleen weight] and tissue inflammatory parameters [macroscopic and microscopic damage, tumour necrosis factor-α (TNF-α), interleukin-10 (IL-10), superoxide anion production (dihydroethidium fluorescence) and malondialdehyde (MDA) levels]. Animals received ACA (1, 3, 10 or 30 mg/kg/day), DEX (0.1 mg/kg/day) or vehicle intraperitoneally for 6 days, starting 1 day before DNBS administration. Results. Colitis was associated with a decreased body weight (-10±5 g vs vehicle; 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.

Abstract #1057 Background: Microvessel density (MVD) is a commonly used parameter to describe tumour vascularity and has been widely studied as a possible prognosticator in breast cancer. Other descriptors of vascular beds have been developed and have been applied to a variety of systems including the reproductive and nervous systems. These stereological approaches are providing objective unbiased assessment of various vascular beds. Aim: The aim of this pilot study was to use stereological methods to describe the vasculature in invasive ductal breast carcinoma, to test the hypothesis that microvasculature anatomy effects Tumor Grade, Size and Nodal Status. Methods: A cohort of 51 breast cancer patients with pre-menopausal invasive ductal carcinoma and moderate NPI was identified.Sections were stained with CD34 using the automated Ventana Discovery staining platform. A number of stereological parameters were estimated in three tissue compartments (gland,stroma and adipose):Number per Unit area (Na), Length Density (Lv) and Radial Diffusion Distance. Data was analysed using Analyses of Variance methods using Minitab© Statistical software. Results: There was no significant difference in the radial diffusion difference when groups were analysed by Grade Size and Nodal Status. Similar results were found for Na and Lv Conclusion: This study suggests that there is no simple link between tumor vasculature and Grade, size and nodal status in Invasive Ductal Breast Carcinoma. This study illustrates the potential usefulness of the stereological approach in the study of vascular beds in breast cancer studies by providing access to potentially physiologically relevant anatomical parameters of microvasculature beds. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1057.