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Abstract There is currently an urgent need to identify factors predictive of immunogenicity in colorectal cancer (CRC). Mucinous CRC is a distinct histological subtype of CRC, associated with a poor response to chemotherapy. Recent evidence suggests the commensal facultative anaerobe Fusobacterium may be especially prevalent in mucinous CRC. The objectives of this study were to assess the association of Fusobacterium abundance with immune cell composition and prognosis in mucinous CRC. Our study included two independent colorectal cancer patient cohorts, The Cancer Genome Atlas (TCGA) cohort, and a cohort of rectal cancers from the Beaumont RCSI Cancer Centre (BRCC). Multiplexed immunofluorescence staining of a tumour microarray (TMA) from the BRCC cohort was undertaken using Cell DIVE technology. Our cohorts included 87 cases (13.3%) of mucinous and 565 cases (86.7%) of non-mucinous CRC. Mucinous CRC in the TCGA dataset was associated with an increased proportion of CD8 + lymphocytes (p = 0.018), regulatory T-cells (p = 0.001) and M2 macrophages (p = 0.001). In the BRCC cohort, mucinous RC was associated with enhanced CD8 + lymphocyte (p = 0.022), regulatory T-cell (p = 0.047), and B-cell (p = 0.025) counts. High Fusobacterium abundance was associated with an increased proportion of CD4 + lymphocytes (p = 0.031) and M1 macrophages (p = 0.006), whilst M2 macrophages (p = 0.043) were under-represented in this cohort. Patients with increased Fusobacterium relative abundance in our mucinous CRC TCGA cohort tended to have better clinical outcomes (DSS: likelihood ratio p = 0.04, logrank p = 0.052). Fusobacterium abundance may be associated with improved outcomes in mucinous CRC, possibly due to a modulatory effect on the host immune response. Key messages • Increased Fusobacterium relative abundance was not found to be associated with microsatellite instability in mucinous CRC. • Increased Fusobacterium relative abundance was associated with an M2/M1 macrophage switch, which is especially significant in mucinous CRC, where M2 macrophages are overexpressed. • Increased Fusobacterium relative abundance was associated with a significant improvement in disease specific survival in mucinous CRC. • Our findings were validated at a protein level within our own in house mucinous and non-mucinous rectal cancer cohorts.

Supplementary Table 4: Differentially expressed gene list contrasting tumour region of origin between Lymph Node and Invasive Front.

Purpose: A number of independent gene expression profiling studies have identified transcriptional subtypes in colorectal cancer with potential diagnostic utility, culminating in publication of a colorectal cancer Consensus Molecular Subtype classification. The worst prognostic subtype has been defined by genes associated with stem-like biology. Recently, it has been shown that the majority of genes associated with this poor prognostic group are stromal derived. We investigated the potential for tumor misclassification into multiple diagnostic subgroups based on tumoral region sampled.Experimental Design: We performed multiregion tissue RNA extraction/transcriptomic analysis using colorectal-specific arrays on invasive front, central tumor, and lymph node regions selected from tissue samples from 25 colorectal cancer patients.Results: We identified a consensus 30-gene list, which represents the intratumoral heterogeneity within a cohort of primary colorectal cancer tumors. Using a series of online datasets, we showed that this gene list displays prognostic potential HR = 2.914 (confidence interval 0.9286–9.162) in stage II/III colorectal cancer patients, but in addition, we demonstrated that these genes are stromal derived, challenging the assumption that poor prognosis tumors with stem-like biology have undergone a widespread epithelial–mesenchymal transition. Most importantly, we showed that patients can be simultaneously classified into multiple diagnostically relevant subgroups based purely on the tumoral region analyzed.Conclusions: Gene expression profiles derived from the nonmalignant stromal region can influence assignment of colorectal cancer transcriptional subtypes, questioning the current molecular classification dogma and highlighting the need to consider pathology sampling region and degree of stromal infiltration when employing transcription-based classifiers to underpin clinical decision making in colorectal cancer. Clin Cancer Res; 22(16); 4095–104. ©2016 AACR.See related commentary by Morris and Kopetz, p. 3989

This Supplementary Table indicates the sample source for the study

This Supplementary Figure indicates the project plan

Supplementary Table 3: Differentially expressed gene list contrasting tumour region of origin between Central Tumor and Lymph Node.

Supplementary Table 2: Differentially expressed gene list contrasting tumour region of origin between Central Tumor and Invasive Front. Supplementary Table 3: Differentially expressed gene list contrasting tumour region of origin between CT-LN. Supplementary Table 4: Differentially expressed gene list contrasting tumour region of origin between LN-IF.

Mucinous rectal cancer is associated with a higher incidence of microsatellite instability, and a poorer response to neoadjuvant chemoradiotherapy compared to other subtypes of rectal adenocarcinoma. Immune checkpoint inhibitors are an emerging family of anti-cancer therapeutics associated with highly variable outcomes in colorectal cancer. Though the immune landscape of mucinous rectal cancer has not been fully explored, the presence of mucin is thought to act as a barrier preventing immune cell infiltration.The aim of this study was to determine the immune properties of mucinous rectal cancer and investigate the degree of lymphocyte infiltration in this cohort.This is a retrospective cohort study which involved, multiplexed immunofluorescence staining of tumor microarrays.Samples originated from a single university teaching hospital.Our cohort included 15 cases of mucinous and 43 cases of non-mucinous rectal cancer.Immune cells were classified and quantified. Immune cell counts were compared between mucinous and non-mucinous cohorts. Immune marker expression within tumor epithelial tissue was evaluated to determine degree of lymphocyte infiltration.Cytotoxic (p = 0.022), and regulatory T-cells (p = 0.010) were found to be overrepresented in the mucinous cohort compared to the non-mucinous group. PD-1 expression was also found to be significantly greater in the mucinous group (p = 0.001). CD3 (p = 0.001) and CD8 (p = 0.054) expression within tumor epithelium was also higher in the mucinous group, suggesting adequate immune infiltration despite the presence of mucin. Microsatellite instability status was not found to be a predictor of immune marker expression in our analysis.The relatively small size of the cohort.Mucinous rectal cancer is associated with an immune rich tumor microenvironment, which was not associated with microsatellite instability status. See Video Abstract at http://links.lww.com/DCR/C65.

There is currently an urgent need to identify factors predictive of immunogenicity in colorectal cancer (CRC). Mucinous CRC is a distinct histological subtype of CRC, associated with a poor response to chemotherapy. Recent evidence suggests the commensal facultative anaerobe Fusobacterium may be especially prevalent in mucinous CRC. The objectives of this study were to assess the impact of Fusobacterium prevalence on immune cell expression and prognosis in mucinous CRC. Our study included two independent colorectal cancer patient cohorts, The Cancer Genome Atlas (TCGA) cohort, and a cohort of rectal cancers from the Beaumont RCSI Cancer Centre (BRCC). Multiplexed immunofluorescence staining of a tumor microarray (TMA) from the BRCC cohort was undertaken using Cell DIVE technology. Our cohorts included 87 cases (13.3%) of mucinous and 565 cases (86.7%) of non-mucinous CRC. Mucinous CRC in the TCGA dataset was associated with increased CD8 + lymphocyte (p = 0.018), regulatory T-cell (p = 0.001) and M2 macrophage (p = 0.001) expression. Similarly in the BRCC cohort, mucinous RC was associated with enhanced CD8 + lymphocyte (p = 0.022), regulatory T-cell (p = 0.047), and B-cell (p = 0.025) counts. Elevated Fusobacterium expression was associated with increased CD4+ (p = 0.031) and M1 macrophage (p = 0.006) expression, whilst M2 macrophages (p = 0.043) were under-expressed in the TCGA cohort. Increased Fusobacterium relative abundance in mucinous CRC was associated with improved clinical outcomes in our TCGA cohort despite having no association with MSI status (DSS: likelihood ratio p = 0.04, logrank p = 0.052). Fusobacterium abundance is associated with improved outcomes in mucinous CRC, possibly due its modulatory effect on the host immune response.

The frequency of lymphocytes infiltrating tumors is a known prognostic in estrogen receptor (ER) negative cancers. ER+ disease is putatively believed to be immune cold, however, there exists a subset of ER+ tumors with high immune infiltrate and with a significant spatial heterogeneity. The clinic impact of such infiltrate - especially between the Oncotype Dx Recurrence Score risk categories - remains unclear. Moreover, the distribution of tumor and stromal tissues, while noted as significantly heterogenous, is still ill-defined and not yet clinically used prognostically, despite evidence to support its utility. Using a cohort (n=450) of serial sections taken from early-stage, ER+/HER2- breast tumors of the Irish arm of the TAILORx clinical trial, we aimed to investigate the tumor architecture and spatial distribution of tumor immune infiltrate and proliferating tumor cells using digital image analysis. Antibodies against Ki67 (proliferation marker) and CD45 (leukocyte common antigen), and a routine Haematoxylin and Eosin stain were applied to serial sections of 450 full-face tumors via chromogenic immunohistochemistry, as outlined previously [1]. Digital image analysis was performed using open-source software, QuPath [2]. Pixel classifiers were trained and validated against an expert pathologist in order to define observed lymphocytes as tumor or stromal-infiltrating, and to establish a classifier to quantify the tumor-stroma ratio (TSR) and infiltrating tumor area. Distances of CD45-positive cells from tumor were computed, along with autocorrelation statistics [3,4] of CD45 and Ki67 hotspots; firstly in order to quantify spatial heterogeneity, and secondly to examine whether Ki67 as a component gene in the Oncotype Dx assay has a foundation in tumor biology or is being confounded by potentially Ki67-positive lymphocytes. Subdividing by Oncotype Dx risk categories, no significant difference in TSR was observed (p=0.09799), neither for intermediary risk patients receiving hormone therapy alone or in combination with chemotherapy (p=0.3873). While there was an observed trend overall (p=0.092), no significance was found for recurrence between intermediary risk subcategories (HT alone: p=0.393, HT+CT: p=0.288). However, in the cohort as a whole, median TSR was 0.3215 (range 0 - 5.023), with statistically significant differences in recurrence risk observed (cohort high v low by median TSR. HR: 6.356, 95CI: 2.263-17.84, p Citation Format: Zak Kinsella, Anna Blümel, Mairi Lucas, Andreas Lindner, Claudia A. Gonzalez, Arman Rahman, Joanna Fay, Tony O'Grady, Verena Murphy, John Crown, Cathy Kelly, William Gallagher, Darran O'Connor. Modelling the spatial heterogeneity of CD45-positive tumor infiltrating lymphocytes in early-stage, estrogen receptor-positive breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5787.

// Sarah Shields 1 , Emer Conroy 1 , Tony O’Grady 5 , Alo McGoldrick 1 , Kate Connor 4 , Mark P. Ward 4 , Zivile Useckaite 4 , Eugene Dempsey 6 , Rebecca Reilly 1 , Yue Fan 1 , Anthony Chubb 4 , David Gomez Matallanas 2, 3 , Elaine W. Kay 5 , Darran O’Connor 4 , Amanda McCann 1, 2 , William M. Gallagher 1 and Judith A. Coppinger 1, 4 1 UCD School of Biomolecular and Biomedical Science, University College Dublin, Dublin 4, Ireland 2 UCD School of Medicine, University College Dublin, Dublin 4, Ireland 3 Systems Biology Ireland, University College, Dublin 4, Ireland 4 Royal College of Surgeons in Ireland, Dublin 2, Ireland 5 Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin 9, Ireland 6 School of Biology and Environmental Science, University College Dublin, Dublin 4, Ireland Correspondence to: Judith A. Coppinger, email: judithcoppinger@rcsi.ie Keywords: TNBC; BAG3; EGFR; signalling; proliferation Received: May 25, 2017 Accepted: February 21, 2018 Epub: February 28, 2018 Published: March 20, 2018 ABSTRACT Triple-negative breast cancer (TNBC), is a heterogeneous disease characterised by absence of expression of the estrogen receptor (ER), progesterone receptor (PR) and lack of amplification of human epidermal growth factor receptor 2 (HER2). TNBC patients can exhibit poor prognosis and high recurrence stages despite early response to chemotherapy treatment. In this study, we identified a pro-survival signalling protein BCL2- associated athanogene 3 (BAG3) to be highly expressed in a subset of TNBC cell lines and tumour tissues. High mRNA expression of BAG3 in TNBC patient cohorts significantly associated with a lower recurrence free survival. The epidermal growth factor receptor (EGFR) is amplified in TNBC and EGFR signalling dynamics impinge on cancer cell survival and disease recurrence. We found a correlation between BAG3 and EGFR expression in TNBC cell lines and determined that BAG3 can regulate tumour cell proliferation, migration and invasion in EGFR expressing TNBC cells lines. We identified an interaction between BAG3 and components of the EGFR signalling networks using mass spectrometry. Furthermore, BAG3 contributed to regulation of proliferation in TNBC cell lines by reducing the activation of components of the PI3K/AKT and FAK/Src signalling subnetworks. Finally, we found that combined targeting of BAG3 and EGFR was more effective than inhibition of EGFR with Cetuximab alone in TNBC cell lines. This study demonstrates a role for BAG3 in regulation of distinct EGFR modules and highlights the potential of BAG3 as a therapeutic target in TNBC.

Introduction Junctional Adhesion Molecule-A (JAM-A) is a cell-cell adhesion protein that regulates physiological adhesion in epithelial and endothelial cells. Our previous work has shown that increased JAM-A expression on breast tumour epithelial tissue associates with poor prognosis in patients with invasive breast cancer, and identified a correlation between high JAM-A expression and levels of the important oncogene human epidermal growth factor receptor-2 (HER2). An important role for HER2 in gastro-oesophageal cancer is emerging, however the specific role of JAM-A in this setting is unclear. Material and methods In a pilot study, gastric cancer patient tissue sections (n=11) were stained for JAM-A expression. A novel, weighted ranking system was developed to semi-quantitatively score the extremely heterogeneous JAM-A membranous staining in gastric tissues; ultimately ranking tissues as low, moderate or high JAM-A expression. JAM-A scores were also correlated with HER2 status in each section. In parallel, a panel of gastro-oesophageal cell lines was used to investigate potential molecular relationships between JAM-A and HER2. Finally, HER2 protein expression was examined following transient JAM-A gene silencing in the gastro-oesophageal junction cancer cell line ESO26. Results and discussions The pilot study revealed that high JAM-A expression significantly correlated with HER2 positivity, and noted a novel staining pattern for JAM-A in diffuse gastric cancers. Molecular studies showed that JAM-A gene silencing reduced HER2 protein expression in gastric cancer cells. These data suggests that JAM-A may act as a novel upstream regulator of HER2 in gastro-oesophageal cancer cells, and warrant further investigation. Current studies are focussed on elucidating the mechanisms underpinning regulation of HER2 expression by JAM-A. Conclusion Promising preliminary indications that JAM-A plays a role in regulating HER2 expression in non-breast cancers may suggest it as both an unfavourable biomarker, as well as a novel putative pharmacological target for HER2-positive gastro-oesophageal cancers. This abstract has emanated from research conducted with the financial support of Science Foundation Ireland (SFI) under grant number 13/IA/1994 (to AMH).

Triple-negative breast cancer (TNBC), is a heterogeneous disease characterised by absence of expression of the estrogen receptor (ER), progesterone receptor (PR) and lack of amplification of human epidermal growth factor receptor 2 (HER2). TNBC patients can exhibit poor prognosis and high recurrence stages despite early response to chemotherapy treatment. In this study, we identified a pro-survival signalling protein BCL2- associated athanogene 3 (BAG3) to be highly expressed in a subset of TNBC cell lines and tumour tissues. High mRNA expression of BAG3 in TNBC patient cohorts significantly associated with a lower recurrence free survival. The epidermal growth factor receptor (EGFR) is amplified in TNBC and EGFR signalling dynamics impinge on cancer cell survival and disease recurrence. We found a correlation between BAG3 and EGFR expression in TNBC cell lines and determined that BAG3 can regulate tumour cell proliferation, migration and invasion in EGFR expressing TNBC cells lines. We identified an interaction between BAG3 and components of the EGFR signalling networks using mass spectrometry. Furthermore, BAG3 contributed to regulation of proliferation in TNBC cell lines by reducing the activation of components of the PI3K/AKT and FAK/Src signalling subnetworks. Finally, we found that combined targeting of BAG3 and EGFR was more effective than inhibition of EGFR with Cetuximab alone in TNBC cell lines. This study demonstrates a role for BAG3 in regulation of distinct EGFR modules and highlights the potential of BAG3 as a therapeutic target in TNBC.

Biobank Ireland Trust (BIT) was established in 2004 to promote and develop an Irish biobank network to benefit patients, researchers, industry, and the economy. The network commenced in 2008 with two hospital biobanks and currently consists of biobanks in the four main cancer hospitals in Ireland. The St. James's Hospital (SJH) Biobank coordinates the network. Procedures, based on ISBER and NCI guidelines, are standardized across the network. Policies and documents—Patient Consent Policy, Patient Information Sheet, Biobank Consent Form, Sample and Data Access Policy (SAP), and Sample Application Form have been agreed upon (after robust discussion) for use in each hospital. An optimum sequence for document preparation and submission for review is outlined. Once consensus is reached among the participating biobanks, the SJH biobank liaises with the Research and Ethics Committees, the Office of the Data Protection Commissioner, The National Cancer Registry (NCR), patient advocate groups, researchers, and other stakeholders. The NCR provides de-identified data from its database for researchers via unique biobank codes. ELSI issues discussed include the introduction of prospective consent across the network and the return of significant research results to patients. Only 4 of 363 patients opted to be re-contacted and re-consented on each occasion that their samples are included in a new project. It was decided, after multidisciplinary discussion, that results will not be returned to patients. The SAP is modeled on those of several international networks. Biobank Ireland is affiliated with international biobanking groups—Marble Arch International Working Group, ISBER, and ESBB. The Irish government continues to deliberate on how to fund and implement biobanking nationally. Meanwhile BIT uses every opportunity to promote awareness of the benefits of biobanking in events and in the media.

Purpose: A number of independent gene expression profiling studies have identified transcriptional subtypes in colorectal cancer with potential diagnostic utility, culminating in publication of a colorectal cancer Consensus Molecular Subtype classification. The worst prognostic subtype has been defined by genes associated with stem-like biology. Recently, it has been shown that the majority of genes associated with this poor prognostic group are stromal derived. We investigated the potential for tumor misclassification into multiple diagnostic subgroups based on tumoral region sampled. Experimental Design: We performed multiregion tissue RNA extraction/transcriptomic analysis using colorectal-specific arrays on invasive front, central tumor, and lymph node regions selected from tissue samples from 25 colorectal cancer patients. Results: We identified a consensus 30-gene list, which represents the intratumoral heterogeneity within a cohort of primary colorectal cancer tumors. Using a series of online datasets, we showed that this gene list displays prognostic potential HR = 2.914 (confidence interval 0.9286–9.162) in stage II/III colorectal cancer patients, but in addition, we demonstrated that these genes are stromal derived, challenging the assumption that poor prognosis tumors with stem-like biology have undergone a widespread epithelial–mesenchymal transition. Most importantly, we showed that patients can be simultaneously classified into multiple diagnostically relevant subgroups based purely on the tumoral region analyzed. Conclusions: Gene expression profiles derived from the nonmalignant stromal region can influence assignment of colorectal cancer transcriptional subtypes, questioning the current molecular classification dogma and highlighting the need to consider pathology sampling region and degree of stromal infiltration when employing transcription-based classifiers to underpin clinical decision making in colorectal cancer. Clin Cancer Res; 22(16); 4095–104. ©2016 AACR. See related commentary by Morris and Kopetz, p. 3989

International and national guidelines highlight the importance of accuracy, reproducibility, and quality control of in situ hybridization (ISH) methods for testing breast carcinomas. However, few guidelines cover the reporting of ISH cases with "unusual" signal patterns, including, eg, heterogeneity and loss of chromosome enumeration probe or gene signals. These cases are, in fact, relatively frequent, and there is a need for developing evidence- or consensus-based reporting guidelines to ensure consistency of treatment. Following an audit of cases from a single center (including >1,700 cases) we show that approximately 10% of ISH results reflect unusual signal patterns. We illustrate the most common of these patterns and provide reporting guidelines for diagnosticians and recommendations for future research. Our goal is to ensure that in the future such "rogues" are reported in a consistent manner that, ultimately, will be supported by molecular and biochemical evidence.

The effect of selective and non-selective cyclo-oxygenase inhibition on tumour growth and metastasis in an orthotopic model of breast cancer was investigated. 4T1 mammary adenocarcinoma cells were injected into the mammary fat pad of female BALB/c mice. When tumours reached a mean tumour diameter of 8.4±0.4 mm, mice were randomised into three groups (n=6 per group) and received daily intraperitoneal injections of the selective cyclo-oxygenase-2 inhibitor, SC-236, the non selective cyclo-oxygenase inhibitor, Indomethacin, or drug vehicle. Tumour diameter was recorded on alternate days. From 8 days after initiation of treatment, tumour diameter in animals treated with either SC-236 or indomethacin was significantly reduced relative to controls. Both primary tumour weight and the number of lung metastases were significantly reduced in the SC-236 and indomethacin treated mice. Microvessel density was reduced and tumor cell apoptosis increased in the primary tumour of mice treated with either the selective or non-selective cyclo-oxygenase inhibitor. In vitro, cyclo-oxygenase inhibition decreased vascular endothelial growth factor production and increased apoptosis of tumour cells. Our results suggest that cyclo-oxygenase inhibitors will be of value in the treatment of both primary and metastatic breast cancer. British Journal of Cancer (2002) 87, 231–237. doi:10.1038/sj.bjc.6600462 www.bjcancer.com © 2002 Cancer Research UK

Blanaid Mee,1 Eoin Gaffney,2,3 Sarah McGarrigle,4 Sharon A Glynn,5,6 Elisabeth M Connolly,7 Paul H McCormick,7 Delia Flannery,7 Katrina O'Connor,7 Margaret Lawson,1 Lisa Ryan,1 Timothy Nugent,1Ronan Ward,1Francis J Sullivan,6Joanna Fay,8Tony O'Grady,8Elaine Kay,8Joe Eustace,9Louise Burke,9Stephen Finn,1Richard Flavin1 1Department of Pathology, St James's Hospital, 2Trinity College Dublin, Dublin, 3National University of Ireland Galway, Galway, 4Department of Surgery, School of Medicine, Faculty of Health Sciences, Trinity College Dublin, Dublin, 5Department of Pathology, National University of Ireland Galway, 6Prostate Cancer Institute, National University of Ireland Galway, Galway, 7Department of Surgery, St James's Hospital, 8Department of Pathology, Beaumont Hospital, Dublin, 9Department of Pathology, Cork University Hospital, Cork, IrelandAbstract: Ireland has an established reputation in specialized global pharmaceutical manufacturing. However, simple high-volume manufacturing will not sustain the Irish pharmaceutical industry, and government agencies recommend a greater focus on innovation and research and development (R&D). Biobank Ireland Trust sought the views of the Irish pharmaceutical industry on the potential benefits of a national biobank network (NBN), national biobank web portal (NBWP), and center for translational molecular oncologic pathology (CTOP). Questionnaires were sent to 19 companies and eleven responded. Questionnaire A was completed by six companies presently engaged in R&D in Ireland – three pharmaceutical companies, two spin outs, and one contract research organization. Six of six respondents reported that: a NBN would benefit their company; the development of a NBWP was important; and finally, they forecast that the requirement for biobanked material would continue to increase. While three of six predicted that a NBN would facilitate an expansion of current R&D activities. The relative importance of accessing biobanked material and data varied. An associated NBWP was considered essential to enable researchers to rapidly determine the content of the NBN for research, including preclinical studies. Individual companies had requirements for biobanked material from a wide variety of cancer sites, sample types, and sample derivatives. Questionnaire B was completed by five pharmaceutical companies currently not engaged in R&D in Ireland. Four of five reported that a CTOP would benefit their company. All five stated that a CTOP could cultivate industry–academic collaborations. All five also determined that NBN–NBWP–CTOP infrastructure would assist in promoting Ireland as an R&D center. Finally, four of five indicated that an NBN would make Ireland more competitive for new clinical trials. This pilot survey suggests that an NBN with associated infrastructure would greatly facilitate research conducted by the pharmaceutical sector in Ireland.Keywords: pharmaceutical industry, Irish biobanks, NBN, CTOP, NBWP, biobanked material

The heat shock protein, HSP70, is over-expressed in many tumours and acts at the crossroads of key intracellular processes in its role as a molecular chaperone. HSP70 associates with a vast array of peptides, some of which are antigenic and can mount adaptive immune responses against the tumour from which they are derived. The pool of peptides associated with HSP70 represents a unique barcode of protein metabolism in tumour cells. With a view to identifying unique protein targets that may be developed as tumour biomarkers, we used purified HSP70 and its associated peptide pool (HSP70–peptide complexes, HSP70-PCs) from different human breast tumour cell lines as targets for phage display biopanning. Our results show that HSP70-PCs from each cell line interact with unique sets of peptides within the phage display library. One of the peptides, termed IST, enriched in the biopanning process, was used in a ‘pull-down’ assay to identify the original protein from which the HSP70-associated peptides may have been derived. The eukaryotic translation initiation factor 3 (eIF-3), a member of the elongation factor EF1α family, and the HSP GRP78, were pulled down by the IST peptide. All of these proteins are known to be up-regulated in cancer cells. Immunohistochemical staining of tumour tissue microarrays showed that the peptide co-localised with HSP70 in breast tumour tissue. The data indicate that the reservoir of peptides associated with HSP70 can act as a unique indicator of cellular protein activity and a novel source of potential tumour biomarkers.

Objective Type 2 diabetes has a long pre clinical asymptomatic phase. Early detection may delay or arrest disease progression. The Diabetes Mellitus and Vascular health initiative (DMVhi) was initiated as a prospective longitudinal cohort study on the prevalence of undiagnosed Type 2 diabetes and prediabetes, diabetes risk and cardiovascular risk in a cohort of Irish adults aged 45-75 years. Research Design and Methods Members of the largest Irish private health insurance provider aged 45 to 75 years were invited to participate in the study. Exclusion criteria: already diagnosed with diabetes or taking oral hypoglycaemic agents. Participants completed a detailed medical questionnaire, had weight, height, waist and hip circumference and blood pressure measured. Fasting blood samples were taken for fasting plasma glucose (FPG). Those with FPG in the impaired fasting glucose (IFG) range had a 75gm oral glucose tolerance test performed. Results 122,531 subjects were invited to participate. 29,144 (24%) completed the study. The prevalence of undiagnosed diabetes was 1.8%, of impaired fasting glucose (IFG) was 7.1% and of impaired glucose tolerance (IGT) was 2.9%. Dysglycaemia increased among those aged 45-54, 55-64 and 65-75 years in both males (10.6%, 18.5%, 21.7% respectively) and females (4.3%, 8.6%, 10.9% respectively). Undiagnosed T2D, IFG and IGT were all associated with gender, age, blood pressure, BMI, abdominal obesity, family history of diabetes and triglyceride levels. Using FPG as initial screening may underestimate the prevalence of T2D in the study population. Conclusions This study is the largest screening study for diabetes and prediabetes in the Irish population. Follow up of this cohort will provide data on progression to diabetes and on cardiovascular outcomes.

Objectives: intimal hyperplasia (IH) is a major cause of re-stenosis post-vascular intervention. Induction of heat shock proteins (HSPs), by thermal pre-conditioning, reduces IH. Our aim was to investigate the effect of the pharmacological HSP inducer herbimycin A on IH in the rat carotid balloon injury model. Materials and Methods: thirty male Sprague–Dawley rats were randomized into three groups. All groups underwent balloon injury to the left carotid artery. Stress proteins were induced 18 h pre-operatively by heat shock or herbimycin A. Two weeks post-operatively, animals were sacrificed and carotid intima/media area ratio (I/M ratio) calculated using computerized planimetry. Neo-intimal proliferation was assessed immunohistochemically with PCNA (proliferating cell nuclear antigen). Western blot and immunohistochemistry for arterial HSP70 and HSP27 were performed. Results: heat stress and herbimycin significantly reduced the I/M ratio (p < 0.05 vs balloon injury alone). Neo-intimal proliferation was significantly reduced in the heat stress and herbimycin groups (p < 0.05 vs balloon injury alone). Heat stress induced arterial HSP70 and HSP27. Herbimycin A increased arterial HSP27. Conclusion: herbimycin A significantly attenuates IH after balloon injury. HSP27 may be the HSP involved in mediating this response. Pharmacological inducers of HSPs may have a therapeutic role to play in preventing re-stenosis post-vascular intervention.Eur J Vasc Endovasc Surg 25, 40–47 (2003)

Cervical cancer is the second most common cancer amongst women worldwide. It is now accepted that almost all cervical cancers arise through the disruption of the pathways of p53 and the product of the retinoblastoma gene (pRb) by high-risk Human Papilloma Virus (HPV) oncoproteins E6 and E7. In cervical cancer p16INK4a is strongly over expressed when pRb is inactivated by HPV E7. This over expression highlights the potential of p16INK4a as a marker for cervical intraepithelial neoplasia and cervical cancer. Immunocytochemical (ICC) analysis of P16INK4a expression was performed on 30 negative, 30 Borderline, 30 CIN1 and 30 High Grade ThinPrep® cervical samples selected from 1094 consented samples received for routine cervical screening assessment. The results were correlated with HR-HPV status as determined by a PCR assay and the cytology findings. In addition a real time RT-PCR technique was employed to evaluate P16INK4a gene expression in 50 of the 120 study samples. Eighty-five of the 120 cervical samples were found positive by the ICC assay, 13/30 Negative, 23/30 Borderline, 20/30 CIN 1 and 29/30 High Grade. The ICC staining results were supported by the results of the P16INK4a real time RT-PCR analysis and the HR-HPV PCR assay. Interestingly not all HR-HPV positive samples were P16INK4a ICC positive, suggesting the marker may be capable of discriminating between productive viral infections and active cell transformation. In conclusion P16INK4a is a useful marker of HR-HPV mediated cervical dyskaryosis in ThinPrep® samples and may be utilized as an adjunct to the Pap smear to aid in the identification of patients with lesions which are more likely to progress to cervical cancer. Additional studies correlating p16INK4a ICC with viral load, viral integration and E6/E7 expression are required to establish how discriminatory a marker p16INK4a truly is.