Publications by authors named "Matthew Humphries"

46 Publications

HistoClean: Open-source software for histological image pre-processing and augmentation to improve development of robust convolutional neural networks.

Comput Struct Biotechnol J 2021 26;19:4840-4853. Epub 2021 Aug 26.

Patrick G Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, Northern Ireland.

The growth of digital pathology over the past decade has opened new research pathways and insights in cancer prediction and prognosis. In particular, there has been a surge in deep learning and computer vision techniques to analyse digital images. Common practice in this area is to use image pre-processing and augmentation to prevent bias and overfitting, creating a more robust deep learning model. This generally requires consultation of documentation for multiple coding libraries, as well as trial and error to ensure that the techniques used on the images are appropriate. Herein we introduce HistoClean; a user-friendly, graphical user interface that brings together multiple image processing modules into one easy to use toolkit. HistoClean is an application that aims to help bridge the knowledge gap between pathologists, biomedical scientists and computer scientists by providing transparent image augmentation and pre-processing techniques which can be applied without prior coding knowledge. In this study, we utilise HistoClean to pre-process images for a simple convolutional neural network used to detect stromal maturity, improving the accuracy of the model at a tile, region of interest, and patient level. This study demonstrates how HistoClean can be used to improve a standard deep learning workflow via classical image augmentation and pre-processing techniques, even with a relatively simple convolutional neural network architecture. HistoClean is free and open-source and can be downloaded from the Github repository here: https://github.com/HistoCleanQUB/HistoClean.
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http://dx.doi.org/10.1016/j.csbj.2021.08.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8426467PMC
August 2021

Orthogonal MET analysis in a population-representative stage II-III colon cancer cohort: prognostic and potential therapeutic implications.

Mol Oncol 2021 Aug 24. Epub 2021 Aug 24.

Precision Medicine Centre of Excellence, Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, Northern Ireland.

Clinical trials for MET inhibitors have demonstrated limited success for their use in colon cancer (CC). However, clinical efficacy may be obscured by a lack of standardisation in MET assessment for patient stratification. In this study, we aimed to determine the molecular context in which MET is deregulated in CC using a series of genomic and proteomic tests to define MET expression and identify patient subgroups that should be considered in future studies with MET-targeted agents. To this aim, orthogonal expression analysis of MET was conducted in a population-representative cohort of stage II/III CC patients (n=240) diagnosed in Northern Ireland from 2004 to 2008. Targeted sequencing was used to determine the relative incidence of MET R970C and MET T992I mutations within the cohort. MET amplification was assessed using dual-color dual-hapten brightfield in situ hybridisation (DDISH). Expression of transcribed MET and c-MET protein within the cohort was assessed using digital image analysis on MET RNA in situ hybridisation (ISH) and c-MET immunohistochemistry (IHC) stained slides. We found that less than 2% of the stage II/III CC patient population assessed demonstrated a genetic MET aberration. Determination of a high MET RNA-ISH/low c-MET IHC protein subgroup was found to be associated with poor five-year cancer-specific outcomes compared to patients with concordant MET RNA-ISH and c-MET IHC protein expression (HR 2.12 [95%CI: 1.27-3.68]). The MET RNA-ISH/c-MET IHC protein biomarker paradigm identified in this study demonstrates that subtyping of MET expression may be required to identify MET-addicted malignancies in CC patients who will truly benefit from MET inhibition.
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http://dx.doi.org/10.1002/1878-0261.13089DOI Listing
August 2021

A Means of Assessing Deep Learning-Based Detection of ICOS Protein Expression in Colon Cancer.

Cancers (Basel) 2021 Jul 29;13(15). Epub 2021 Jul 29.

Precision Medicine Centre of Excellence, The Patrick G Johnston Centre for Cancer Research, Queen's University Belfast, Belfast BT9 7AE, UK.

Biomarkers identify patient response to therapy. The potential immune-checkpoint biomarker, Inducible T-cell COStimulator (ICOS), expressed on regulating T-cell activation and involved in adaptive immune responses, is of great interest. We have previously shown that open-source software for digital pathology image analysis can be used to detect and quantify ICOS using cell detection algorithms based on traditional image processing techniques. Currently, artificial intelligence (AI) based on deep learning methods is significantly impacting the domain of digital pathology, including the quantification of biomarkers. In this study, we propose a general AI-based workflow for applying deep learning to the problem of cell segmentation/detection in IHC slides as a basis for quantifying nuclear staining biomarkers, such as ICOS. It consists of two main parts: a simplified but robust annotation process, and cell segmentation/detection models. This results in an optimised annotation process with a new user-friendly tool that can interact with1 other open-source software and assists pathologists and scientists in creating and exporting data for deep learning. We present a set of architectures for cell-based segmentation/detection to quantify and analyse the trade-offs between them, proving to be more accurate and less time consuming than traditional methods. This approach can identify the best tool to deliver the prognostic significance of ICOS protein expression.
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http://dx.doi.org/10.3390/cancers13153825DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8345140PMC
July 2021

The clinical and molecular significance associated with STING signaling in breast cancer.

NPJ Breast Cancer 2021 Jun 25;7(1):81. Epub 2021 Jun 25.

School of Pharmacy, Queen's University Belfast, Belfast, Northern Ireland, UK.

STING signaling in cancer is a crucial component of response to immunotherapy and other anti-cancer treatments. Currently, there is no robust method of measuring STING activation in cancer. Here, we describe an immunohistochemistry-based assay with digital pathology assessment of STING in tumor cells. Using this novel approach in estrogen receptor-positive (ER+) and ER- breast cancer, we identify perinuclear-localized expression of STING (pnSTING) in ER+ cases as an independent predictor of good prognosis, associated with immune cell infiltration and upregulation of immune checkpoints. Tumors with low pnSTING are immunosuppressed with increased infiltration of "M2"-polarized macrophages. In ER- disease, pnSTING does not appear to have a significant prognostic role with STING uncoupled from interferon responses. Importantly, a gene signature defining low pnSTING expression is predictive of poor prognosis in independent ER+ datasets. Low pnSTING is associated with chromosomal instability, MYC amplification and mTOR signaling, suggesting novel therapeutic approaches for this subgroup.
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http://dx.doi.org/10.1038/s41523-021-00283-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8233333PMC
June 2021

Colonic epithelial cathelicidin (LL-37) expression intensity is associated with progression of colorectal cancer and presence of CD8 T cell infiltrate.

J Pathol Clin Res 2021 Sep 14;7(5):495-506. Epub 2021 May 14.

Division of Molecular & Clinical Medicine, School of Medicine, University of Dundee, Dundee, UK.

Colorectal cancer (CRC) remains a leading cause of cancer mortality. Here, we define the colonic epithelial expression of cathelicidin (LL-37) in CRC. Cathelicidin exerts pleotropic effects including anti-microbial and immunoregulatory functions. Genetic knockout of cathelicidin led to increased size and number of colorectal tumours in the azoxymethane-induced murine model of CRC. We aimed to translate this to human disease. The expression of LL-37 in a large (n = 650) fully characterised cohort of treatment-naïve primary human colorectal tumours and 50 matched normal mucosa samples with associated clinical and pathological data (patient age, gender, tumour site, tumour stage [UICC], presence or absence of extra-mural vascular invasion, tumour differentiation, mismatch repair protein status, and survival to 18 years) was assessed by immunohistochemistry. The biological consequences of LL-37 expression on the epithelial barrier and immune cell phenotype were assessed using targeted quantitative PCR gene expression of epithelial permeability (CLDN2, CLDN4, OCLN, CDH1, and TJP1) and cytokine (IL-1β, IL-18, IL-33, IL-10, IL-22, and IL-27) genes in a human colon organoid model, and CD3 , CD4 , and CD8 lymphocyte phenotyping by immunohistochemistry, respectively. Our data reveal that loss of cathelicidin is associated with human CRC progression, with a switch in expression intensity an early feature of CRC. LL-37 expression intensity is associated with CD8 T cell infiltrate, influenced by tumour characteristics including mismatch repair protein status. There was no effect on epithelial barrier gene expression. These data offer novel insights into the contribution of LL-37 to the pathogenesis of CRC and as a therapeutic molecule.
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http://dx.doi.org/10.1002/cjp2.222DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8363930PMC
September 2021

Evolutionary genetic algorithm identifies as a potential predictive biomarker for immune-checkpoint therapy in colorectal cancer.

NAR Genom Bioinform 2021 Jun 20;3(2):lqab016. Epub 2021 Apr 20.

Patrick G Johnston Centre for Cancer Research, Queen's University Belfast, BT9 7AE, Northern Ireland.

Identifying robust predictive biomarkers to stratify colorectal cancer (CRC) patients based on their response to immune-checkpoint therapy is an area of unmet clinical need. Our evolutionary algorithm Atlas Correlation Explorer (ACE) represents a novel approach for mining The Cancer Genome Atlas (TCGA) data for clinically relevant associations. We deployed ACE to identify candidate predictive biomarkers of response to immune-checkpoint therapy in CRC. We interrogated the colon adenocarcinoma (COAD) gene expression data across nine immune-checkpoints ( and ). was identified as the most common gene associated with immune-checkpoint genes in CRC. Using human/murine single-cell RNA-seq data, we demonstrated that was expressed predominantly in a subset of T-cells associated with increased immune-checkpoint expression ( < 0.0001). Confirmatory IL2RB immunohistochemistry (IHC) analysis in a large MSI-H colon cancer tissue microarray (TMA;  = 115) revealed sensitive, specific staining of a subset of lymphocytes and a strong association with FOXP3+ lymphocytes ( < 0.0001). mRNA positively correlated with three previously-published gene signatures of response to immune-checkpoint therapy ( < 0.0001). Our evolutionary algorithm has identified to be extensively linked to immune-checkpoints in CRC; its expression should be investigated for clinical utility as a potential predictive biomarker for CRC patients receiving immune-checkpoint blockade.
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http://dx.doi.org/10.1093/nargab/lqab016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8057496PMC
June 2021

The Potential of Digital Image Analysis to Determine Tumor Cell Content in Biobanked Formalin-Fixed, Paraffin-Embedded Tissue Samples.

Biopreserv Biobank 2021 Aug 29;19(4):324-331. Epub 2021 Mar 29.

Northern Ireland Biobank, Center for Cancer Research and Cell Biology, Queen's University, Belfast, United Kingdom.

Best practices dictate that biobanks ensure accurate determination of tumor content before supplying formalin-fixed, paraffin-embedded (FFPE) tissue samples to researchers for nucleic acid extraction and downstream molecular testing. It is advisable that trained and competent individuals, who understand the requirements of the downstream molecular tests, perform the microscopic morphological examination. However, the special skills, time, and costs associated with these assessments can be prohibitive, especially in large case cohorts requiring extensive pathological review. Determination of tumor content reliably by digital image analysis (DIA) could represent a significant advantage if validated, utilized, and deployed by biobanks. Whole slide digital scanned images of colorectal, lung, and breast cancer specimens were created. The scanned images were imported into the DIA software QuPath and digital annotations were completed by biobank technicians, under the direction of trained histopathology senior scientists. Automated cell detection was conducted and tumor epithelial cells were classified and quantified. DIA scores were highly concordant with the manual assessment for 376 of 435 samples (86%). A detailed review of discordant cases indicated digital scores had a higher accuracy than the manual estimation. Automated digital quantification has the potential to replace visual estimations with reduced subjectivity and increased reliability compared with manual tumor estimations. We recommend the use of DIA by biobanks involved in provision of FFPE tissue samples, especially in large research studies requiring high volumes of cases to be analyzed.
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http://dx.doi.org/10.1089/bio.2020.0105DOI Listing
August 2021

PD-L1 Multiplex and Quantitative Image Analysis for Molecular Diagnostics.

Cancers (Basel) 2020 Dec 23;13(1). Epub 2020 Dec 23.

Precision Medicine Centre of Excellence, The Patrick G Johnston Centre for Cancer Research, Queen's University, Belfast BT9 7AE, UK.

Multiplex immunofluorescence (mIF) and digital image analysis (DIA) have transformed the ability to analyse multiple biomarkers. We aimed to validate a clinical workflow for quantifying PD-L1 in non-small cell lung cancer (NSCLC). NSCLC samples were stained with a validated mIF panel. Immunohistochemistry (IHC) was conducted and mIF slides were scanned on an Akoya Vectra Polaris. Scans underwent DIA using QuPath. Single channel immunofluorescence was concordant with single-plex IHC. DIA facilitated quantification of cell types expressing single or multiple phenotypic markers. Considerations for analysis included classifier accuracy, macrophage infiltration, spurious staining, threshold sensitivity by DIA, sensitivity of cell identification in the mIF. Alternative sequential detection of biomarkers by DIA potentially impacted final score. Strong concordance was observed between 3,3'-Diaminobenzidine (DAB) IHC slides and mIF slides (R = 0.7323). Comparatively, DIA on DAB IHC was seen to overestimate the PD-L1 score more frequently than on mIF slides. Overall, concordance between DIA on DAB IHC slides and mIF slides was 95%. DIA of mIF slides is rapid, highly comparable to DIA on DAB IHC slides, and enables comprehensive extraction of phenotypic data and specific microenvironmental detail intrinsic to the sample. Exploration of the clinical relevance of mIF in the context of immunotherapy treated cases is warranted.
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http://dx.doi.org/10.3390/cancers13010029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7796246PMC
December 2020

Metastasis and Immune Evasion from Extracellular cGAMP Hydrolysis.

Cancer Discov 2021 05 28;11(5):1212-1227. Epub 2020 Dec 28.

Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York, New York.

Cytosolic DNA is characteristic of chromosomally unstable metastatic cancer cells, resulting in constitutive activation of the cGAS-STING innate immune pathway. How tumors co-opt inflammatory signaling while evading immune surveillance remains unknown. Here, we show that the ectonucleotidase ENPP1 promotes metastasis by selectively degrading extracellular cGAMP, an immune-stimulatory metabolite whose breakdown products include the immune suppressor adenosine. ENPP1 loss suppresses metastasis, restores tumor immune infiltration, and potentiates response to immune checkpoint blockade in a manner dependent on tumor cGAS and host STING. Conversely, overexpression of wild-type ENPP1, but not an enzymatically weakened mutant, promotes migration and metastasis, in part through the generation of extracellular adenosine, and renders otherwise sensitive tumors completely resistant to immunotherapy. In human cancers, ENPP1 expression correlates with reduced immune cell infiltration, increased metastasis, and resistance to anti-PD-1/PD-L1 treatment. Thus, cGAMP hydrolysis by ENPP1 enables chromosomally unstable tumors to transmute cGAS activation into an immune-suppressive pathway. SIGNIFICANCE: Chromosomal instability promotes metastasis by generating chronic tumor inflammation. ENPP1 facilitates metastasis and enables tumor cells to tolerate inflammation by hydrolyzing the immunotransmitter cGAMP, preventing its transfer from cancer cells to immune cells..
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http://dx.doi.org/10.1158/2159-8290.CD-20-0387DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8102348PMC
May 2021

In-depth Clinical and Biological Exploration of DNA Damage Immune Response as a Biomarker for Oxaliplatin Use in Colorectal Cancer.

Clin Cancer Res 2021 01 7;27(1):288-300. Epub 2020 Oct 7.

MRC Clinical Trials Unit, University College London, London, United Kingdom.

Purpose: The DNA damage immune response (DDIR) assay was developed in breast cancer based on biology associated with deficiencies in homologous recombination and Fanconi anemia pathways. A positive DDIR call identifies patients likely to respond to platinum-based chemotherapies in breast and esophageal cancers. In colorectal cancer, there is currently no biomarker to predict response to oxaliplatin. We tested the ability of the DDIR assay to predict response to oxaliplatin-based chemotherapy in colorectal cancer and characterized the biology in DDIR-positive colorectal cancer.

Experimental Design: Samples and clinical data were assessed according to DDIR status from patients who received either 5-fluorouracil (5-FU) or 5FUFA (bolus and infusion 5-FU with folinic acid) plus oxaliplatin (FOLFOX) within the FOCUS trial ( = 361, stage IV), or neoadjuvant FOLFOX in the FOxTROT trial ( = 97, stage II/III). Whole transcriptome, mutation, and IHC data of these samples were used to interrogate the biology of DDIR in colorectal cancer.

Results: Contrary to our hypothesis, DDIR-negative patients displayed a trend toward improved outcome for oxaliplatin-based chemotherapy compared with DDIR-positive patients. DDIR positivity was associated with microsatellite instability (MSI) and colorectal molecular subtype 1. Refinement of the DDIR signature, based on overlapping IFN-related chemokine signaling associated with DDIR positivity across colorectal cancer and breast cancer cohorts, further confirmed that the DDIR assay did not have predictive value for oxaliplatin-based chemotherapy in colorectal cancer.

Conclusions: DDIR positivity does not predict improved response following oxaliplatin treatment in colorectal cancer. However, data presented here suggest the potential of the DDIR assay in identifying immune-rich tumors that may benefit from immune checkpoint blockade, beyond current use of MSI status.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-3237DOI Listing
January 2021

Identifying mismatch repair-deficient colon cancer: near-perfect concordance between immunohistochemistry and microsatellite instability testing in a large, population-based series.

Histopathology 2021 Feb 11;78(3):401-413. Epub 2020 Oct 11.

Department of Cellular Pathology, Belfast Health and Social Care Trust, Belfast, UK.

Aims: Establishing the mismatch repair (MMR) status of colorectal cancers is important to enable the detection of underlying Lynch syndrome and inform prognosis and therapy. Current testing typically involves either polymerase chain reaction (PCR)-based microsatellite instability (MSI) testing or MMR protein immunohistochemistry (IHC). The aim of this study was to compare these two approaches in a large, population-based cohort of stage 2 and 3 colon cancer cases in Northern Ireland.

Methods And Results: The study used the Promega pentaplex assay to determine MSI status and a four-antibody MMR IHC panel. IHC was applied to tumour tissue microarrays with triplicate tumour sampling, and assessed manually. Of 593 cases with available MSI and MMR IHC results, 136 (22.9%) were MSI-high (MSI-H) and 135 (22.8%) showed abnormal MMR IHC. Concordance was extremely high, with 97.1% of MSI-H cases showing abnormal MMR IHC, and 97.8% of cases with abnormal IHC showing MSI-H status. Under-representation of tumour epithelial cells in samples from heavily inflamed tumours resulted in misclassification of several cases with abnormal MMR IHC as microsatellite-stable. MMR IHC revealed rare cases with unusual patterns of MMR protein expression, unusual combinations of expression loss, or secondary clonal loss of expression, as further illustrated by repeat immunostaining on whole tissue sections.

Conclusions: MSI PCR testing and MMR IHC can be considered to be equally proficient tests for establishing MMR/MSI status, when there is awareness of the potential pitfalls of either method. The choice of methodology may depend on available services and expertise.
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http://dx.doi.org/10.1111/his.14233DOI Listing
February 2021

Immune status is prognostic for poor survival in colorectal cancer patients and is associated with tumour hypoxia.

Br J Cancer 2020 10 20;123(8):1280-1288. Epub 2020 Jul 20.

Precision Medicine Centre of Excellence, Centre for Cell Research and Cell Biology, Queen's University Belfast, Belfast, Northern Ireland.

Background: Immunohistochemical quantification of the immune response is prognostic for colorectal cancer (CRC). Here, we evaluate the suitability of alternative immune classifiers on prognosis and assess whether they relate to biological features amenable to targeted therapy.

Methods: Overall survival by immune (CD3, CD4, CD8, CD20 and FOXP3) and immune-checkpoint (ICOS, IDO-1 and PD-L1) biomarkers in independent CRC cohorts was evaluated. Matched mutational and transcriptomic data were interrogated to identify associated biology.

Results: Determination of immune-cold tumours by combined low-density cell counts of CD3, CD4 and CD8 immunohistochemistry constituted the best prognosticator across stage II-IV CRC, particularly in patients with stage IV disease (HR 1.98 [95% CI: 1.47-2.67]). These immune-cold CRCs were associated with tumour hypoxia, confirmed using CAIX immunohistochemistry (P = 0.0009), which may mediate disease progression through common biology (KRAS mutations, CRIS-B subtype and SPP1 mRNA overexpression).

Conclusions: Given the significantly poorer survival of immune-cold CRC patients, these data illustrate that assessment of CD4-expressing cells complements low CD3 and CD8 immunohistochemical quantification in the tumour bulk, potentially facilitating immunophenotyping of patient biopsies to predict prognosis. In addition, we found immune-cold CRCs to associate with a difficult-to-treat, poor prognosis hypoxia signature, indicating that these patients may benefit from hypoxia-targeting clinical trials.
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http://dx.doi.org/10.1038/s41416-020-0985-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7555485PMC
October 2020

A robust multiplex immunofluorescence and digital pathology workflow for the characterisation of the tumour immune microenvironment.

Mol Oncol 2020 10 1;14(10):2384-2402. Epub 2020 Sep 1.

Patrick G Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK.

Multiplex immunofluorescence is a powerful tool for the simultaneous detection of tissue-based biomarkers, revolutionising traditional immunohistochemistry. The Opal methodology allows up to eight biomarkers to be measured concomitantly without cross-reactivity, permitting identification of different cell populations within the tumour microenvironment. In this study, we aimed to validate a multiplex immunofluorescence workflow in two complementary multiplex panels and evaluate the tumour immune microenvironment in colorectal cancer (CRC) formalin-fixed paraffin-embedded tissue. We stained CRC and tonsil samples using Opal multiplex immunofluorescence on a Leica BOND RX immunostainer. We then acquired images on an Akoya Vectra Polaris and performed multispectral unmixing using inform. Antibody panels were validated on tissue microarray sections containing cores from six normal tissue types, using qupath for image analysis. Comparisons between chromogenic immunohistochemistry and multiplex immunofluorescence on consecutive sections from the same tissue microarray showed significant correlation (r  > 0.9, P-value < 0.0001), validating both panels. We identified many factors that influenced the quality of the acquired fluorescent images, including biomarker co-expression, staining order, Opal-antibody pairing, sample thickness, multispectral unmixing and biomarker detection order during image analysis. Overall, we report the optimisation and validation of a multiplex immunofluorescence process, from staining to image analysis, ensuring assay robustness. Our multiplex immunofluorescence protocols permit the accurate detection of multiple immune markers in various tissue types, using a workflow that enables rapid processing of samples, above and beyond previous workflows.
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http://dx.doi.org/10.1002/1878-0261.12764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7530793PMC
October 2020

Comparison of different anti-Ki67 antibody clones and hot-spot sizes for assessing proliferative index and grading in pancreatic neuroendocrine tumours using manual and image analysis.

Histopathology 2020 Oct 2;77(4):646-658. Epub 2020 Sep 2.

Department of Cellular Pathology, Belfast Health and Social Care Trust, Belfast, Northern Ireland, UK.

Aims: Ki67 proliferative index (PI) is essential for grading gastroenteric and pancreatic neuroendocrine tumours (GEP NETs). Analytical and preanalytical variables can affect Ki67 PI. In contrast to counting methodology, until now little attention has focused on the question of clone equivalence and the effect of hot-spot size on Ki67 PI in GEP NETs. Using manual counting and image analysis, this study compared the Ki67 PI achieved using MM1, K2 and 30-9 to MIB1, a clone which has been validated for, and is referenced in, guidelines relating to assessment of Ki67 PI in GEP NETs.

Methods And Results: Forty-two pancreatic NETs were each immunohistochemically stained for the anti-Ki67 clones MIB1, MM1, K2 and 30-9. Ki67 PI was calculated manually and by image analysis, the latter using three different hot-spot sizes. In manual comparisons using single hot-spot high-power fields, non-MIB1 clones overestimated Ki67 PI compared to MIB1, resulting in grading discordances. Image analysis shows good agreement with manual Ki67 PI but a tendency to overestimate absolute Ki67 PI. Increasing the size of tumour hot-spot from 500 to 2000 cells resulted in a decrease in Ki67 PI.

Conclusion: Different anti-Ki67 clones do not produce equivalent PIs in GEP NETs, and clone selection may therefore affect patient care. Increasing the hot-spot size decreases the Ki67 PI. Greater standardisation in terms of antibody clone selection and hot-spot size is required for grading GEP NETs. Image analysis is an effective tool for assisting Ki67 assessment and allows easier standardisation of the size of the tumour hot-spot.
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http://dx.doi.org/10.1111/his.14200DOI Listing
October 2020

Glucocorticoid Receptor Expression Predicts Good Outcome in response to Taxane-Free, Anthracycline-Based Therapy in Triple Negative Breast Cancer.

J Oncol 2020 20;2020:3712825. Epub 2020 May 20.

School of Pharmacy, Queen's University Belfast, Belfast BT9 7BL, UK.

Triple negative breast cancer (TNBC) is a poor outcome subset of breast cancers characterised by the lack of expression of ER , PR, and HER2 amplification. It is a heterogeneous group of cancers which fail to derive benefit from modern, more targeted treatments such as Tamoxifen and Herceptin. Current standard of care (SoC) is cytotoxic chemotherapy, which is effective for some patients, with other patients deriving little/no benefit and lacking alternative treatments. This study has identified the glucocorticoid receptor (GR) as a potential predictive biomarker of response to anthracycline-based chemotherapy in triple negative breast cancer (TNBC). GR gene expression levels in patient samples were analysed through publicly available microarray datasets as well as protein expression through immunohistochemistry (IHC) and correlated with clinical/pathological outcomes, including survival. While the results confirmed previous observations that high GR expression is associated with poor outcome in response to taxane-based chemotherapy, this study shows for the first time that high GR expression is associated with improved outcomes in the context of anthracycline-based chemotherapy. GR therefore has the potential to be used as a predictive biomarker to guide treatment choices and ensure that patients derive the greatest benefit from first line treatment, avoiding unnecessary costs, side effects, and disease progression.
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http://dx.doi.org/10.1155/2020/3712825DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7256765PMC
May 2020

The adaptive immune and immune checkpoint landscape of neoadjuvant treated esophageal adenocarcinoma using digital pathology quantitation.

BMC Cancer 2020 Jun 1;20(1):500. Epub 2020 Jun 1.

Precision Medicine Centre of Excellence, Patrick G Johnston Centre for Cancer Research, Queen's University, Belfast, UK.

Background: Limited studies examine the immune landscape in Esophageal Adenocarcinoma (EAC). We aim to identify novel associations, which may inform immunotherapy treatment stratification.

Methods: Three hundred twenty-nine EAC cases were available in Tissue Microarrays (TMA) format. A discovery cohort of 166 EAC cases were stained immunohistochemically for range of adaptive immune (CD3, CD4, CD8 and CD45RO) and immune checkpoint biomarkers (ICOS, IDO-1, PD-L1, PD-1). A validation cohort of 163 EAC cases was also accessed. A digital pathology analysis approach was used to quantify biomarker density.

Results: CD3, CD4, CD8, CD45RO, ICOS and PD-1 were individually predictive of better overall survival (OS) (Log rank p = < 0.001; p = 0.014; p = 0.001; p = < 0.001; p = 0.008 and p = 0.026 respectively). Correlation and multivariate analysis identified high CD45RO/ICOS patients with significantly improved OS which was independently prognostic (HR = 0.445, (0.223-0.886), p = 0.021). Assessment of CD45RO and ICOS high cases in the validation cohort revealed an associated with improved OS (HR = 0.601 (0.363-0.996), p = 0.048). Multiplex IHC identified cellular co-expression of high CD45RO/ICOS. High CD45RO/ICOS patients have significantly improved OS.

Conclusions: Multiplexing identifies true cellular co-expression. These data demonstrate that co-expression of immune biomarkers are associated with better outcome in EAC and may provide evidence for immunotherapy treatment stratification.
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http://dx.doi.org/10.1186/s12885-020-06987-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7268770PMC
June 2020

Improving the Diagnostic Accuracy of the PD-L1 Test with Image Analysis and Multiplex Hybridization.

Cancers (Basel) 2020 Apr 29;12(5). Epub 2020 Apr 29.

Precision Medicine Centre of Excellence, The Patrick G Johnston Centre for Cancer Research, Queen's University, Belfast BT9 7BL, UK.

Targeting of the programmed cell death protein (PD-1)/programmed death-ligand 1 (PD-L1) axis with checkpoint inhibitors has changed clinical practice in non-small cell lung cancer (NSCLC). However, clinical assessment remains complex and ambiguous. We aim to assess whether digital image analysis (DIA) and multiplex immunofluorescence can improve the accuracy of PD-L1 diagnostic testing. A clinical cohort of routine NSCLC patients reflex tested for PD-L1 (SP263) immunohistochemistry (IHC), was assessed using DIA. Samples of varying assessment difficulty were assessed by multiplex immunofluorescence. Sensitivity, specificity, and concordance was evaluated between manual diagnostic evaluation and DIA for chromogenic and multiplex IHC. PD-L1 expression by DIA showed significant concordance (R² = 0.8248) to manual assessment. Sensitivity and specificity was 86.8% and 91.4%, respectively. Evaluation of DIA scores revealed 96.8% concordance to manual assessment. Multiplexing enabled PD-L1+/CD68+ macrophages to be readily identified within PD-L1+/cytokeratin+ or PD-L1-/cytokeratin+ tumor nests. Assessment of multiplex vs. chromogenic IHC had a sensitivity and specificity of 97.8% and 91.8%, respectively. Deployment of DIA for PD-L1 diagnostic assessment is an accurate process of case triage. Multiplex immunofluorescence provided higher confidence in PD-L1 assessment and could be offered for challenging cases by centers with appropriate expertise and specialist equipment.
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http://dx.doi.org/10.3390/cancers12051114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7281311PMC
April 2020

FGFR1 Expression and Role in Migration in Low and High Grade Pediatric Gliomas.

Front Oncol 2019 13;9:103. Epub 2019 Mar 13.

Leeds Institute of Medical Research at St James's, University of Leeds, Leeds, United Kingdom.

The heterogeneous and invasive nature of pediatric gliomas poses significant treatment challenges, highlighting the importance of identifying novel chemotherapeutic targets. Recently, recurrent Fibroblast growth factor receptor 1 (FGFR1) mutations in pediatric gliomas have been reported. Here, we explored the clinical relevance of FGFR1 expression, cell migration in low and high grade pediatric gliomas and the role of FGFR1 in cell migration/invasion as a potential chemotherapeutic target. A high density tissue microarray (TMA) was used to investigate associations between FGFR1 and activated phosphorylated FGFR1 (pFGFR1) expression and various clinicopathologic parameters. Expression of FGFR1 and pFGFR1 were measured by immunofluorescence and by immunohistochemistry (IHC) in 3D spheroids in five rare patient-derived pediatric low-grade glioma (pLGG) and two established high-grade glioma (pHGG) cell lines. Two-dimensional (2D) and three-dimensional (3D) migration assays were performed for migration and inhibitor studies with three FGFR1 inhibitors. High FGFR1 expression was associated with age, malignancy, tumor location and tumor grade among astrocytomas. Membranous pFGFR1 was associated with malignancy and tumor grade. All glioma cell lines exhibited varying levels of FGFR1 and pFGFR1 expression and migratory phenotypes. There were significant anti-migratory effects on the pHGG cell lines with inhibitor treatment and anti-migratory or pro-migratory responses to FGFR1 inhibition in the pLGGs. Our findings support further research to target FGFR1 signaling in pediatric gliomas.
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http://dx.doi.org/10.3389/fonc.2019.00103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6425865PMC
March 2019

Automated Tumour Recognition and Digital Pathology Scoring Unravels New Role for PD-L1 in Predicting Good Outcome in ER-/HER2+ Breast Cancer.

J Oncol 2018 17;2018:2937012. Epub 2018 Dec 17.

Centre for Cancer Research and Cell Biology, Queen's University Belfast, Belfast, UK.

The role of PD-L1 as a prognostic and predictive biomarker is an area of great interest. However, there is a lack of consensus on how to deliver PD-L1 as a clinical biomarker. At the heart of this conundrum is the subjective scoring of PD-L1 IHC in most studies to date. Current standard scoring systems involve separation of epithelial and inflammatory cells and find clinical significance in different percentages of expression, e.g., above or below 1%. Clearly, an objective, reproducible and accurate approach to PD-L1 scoring would bring a degree of necessary consistency to this landscape. Using a systematic comparison of technologies and the application of QuPath, a digital pathology platform, we show that high PD-L1 expression is associated with improved clinical outcome in Triple Negative breast cancer in the context of standard of care (SoC) chemotherapy, consistent with previous findings. In addition, we demonstrate for the first time that high PD-L1 expression is also associated with better outcome in ER- disease as a whole including HER2+ breast cancer. We demonstrate the influence of antibody choice on quantification and clinical impact with the Ventana antibody (SP142) providing the most robust assay in our hands. Through sampling different regions of the tumour, we show that tumour rich regions display the greatest range of PD-L1 expression and this has the most clinical significance compared to stroma and lymphoid rich areas. Furthermore, we observe that both inflammatory and epithelial PD-L1 expression are associated with improved survival in the context of chemotherapy. Moreover, as seen with PD-L1 inhibitor studies, a low threshold of PD-L1 expression stratifies patient outcome. This emphasises the importance of using digital pathology and precise biomarker quantitation to achieve accurate and reproducible scores that can discriminate low PD-L1 expression.
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http://dx.doi.org/10.1155/2018/2937012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6311859PMC
December 2018

Critical Appraisal of Programmed Death Ligand 1 Reflex Diagnostic Testing: Current Standards and Future Opportunities.

J Thorac Oncol 2019 01 6;14(1):45-53. Epub 2018 Oct 6.

Molecular Pathology Programme, Centre for Cancer Research and Cell Biology, Queen's University, Belfast, Ireland, United Kingdom; Cellular Pathology, Belfast Health and Social Care Trust, Belfast City Hospital, Belfast, Ireland, United Kingdom. Electronic address:

Introduction: Patient suitability to anti-programmed death ligand 1 (PD-L1) immune checkpoint inhibition is key to the treatment of NSCLC. We present, applied to PD-L1 testing: a comprehensive cross-validation of two immunohistochemistry (IHC) clones; our descriptive experience in diagnostic reflex testing; the concordance of IHC to in situ RNA (RNA-ISH); and application of digital pathology.

Methods: Eight hundred thirteen NSCLC tumor samples collected from 564 diagnostic samples were analyzed prospectively, and 249 diagnostic samples analyzed retrospectively in tissue microarray format. Validated methods for IHC and RNA-ISH were tested in tissue microarrays and full sections and the QuPath system were used for digital pathology analysis.

Results: Antibody concordance of clones SP263 and 22C3 validation was 97% to 98% in squamous cell carcinoma and adenocarcinomas, respectively. Clinical NSCLC cases were reported as PD-L1-negative (48%), 1% to 49% (23%), and more than 50% (29%), with differences associated to tissue-type and EGFR status. Comparison of IHC and RNA-ISH was highly concordant in both subgroups. Comparison of digital assessment versus manual assessment was highly concordant. Discrepancies were mostly around the 1% clinical threshold. Challenging IHC interpretation included 1) calculating the total tumor cell denominator and the nature of PD-L1 expressing cell aggregates in cytology samples; 2) peritumoral expression of positive immune cells; 3) calculation of positive tumor percentages around clinical thresholds; and 4) relevance of the 100 malignant cell rule.

Conclusions: Sample type and EGFR status dictate differences in the expected percentage of PD-L1 expression. Analysis of PD-L1 is challenging, and interpretative guidelines are discussed. PD-L1 evaluations by RNA-ISH and digital pathology appear reliable, particularly in adenocarcinomas.
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http://dx.doi.org/10.1016/j.jtho.2018.09.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6328626PMC
January 2019

Stanniocalcin 2 expression is associated with a favourable outcome in male breast cancer.

J Pathol Clin Res 2018 10 23;4(4):241-249. Epub 2018 Aug 23.

Leeds Institute of Cancer and Pathology, University of Leeds, Leeds, UK.

Breast cancer can occur in either gender; however, it is rare in men, accounting for <1% of diagnosed cases. In a previous transcriptomic screen of male breast cancer (MBC) and female breast cancer (FBC) occurrences, we observed that Stanniocalcin 2 (STC2) was overexpressed in the former. The aim of this study was to confirm the expression of STC2 in MBC and to investigate whether this had an impact on patient prognosis. Following an earlier transcriptomic screen, STC2 gene expression was confirmed by RT-qPCR in matched MBC and FBC samples as well as in tumour-associated fibroblasts derived from each gender. Subsequently, STC2 protein expression was examined immunohistochemically in tissue microarrays containing 477 MBC cases. Cumulative survival probabilities were calculated using the Kaplan-Meier method and multivariate survival analysis was performed using the Cox hazard model. Gender-specific STC2 gene expression showed a 5.6-fold upregulation of STC2 transcripts in MBC, also supported by data deposited in Oncomine™. STC2 protein expression was a positive prognostic factor for disease-free survival (DFS; Log-rank; total p = 0.035, HR = 0.49; tumour cells p = 0.017, HR = 0.44; stroma p = 0.030, HR = 0.48) but had no significant impact on overall survival (Log-rank; total p = 0.23, HR = 0.71; tumour cells p = 0.069, HR = 0.59; stroma p = 0.650, HR = 0.87). Importantly, multivariate analysis adjusted for patient age at diagnosis, node staging, tumour size, ER, and PR status revealed that total STC2 expression as well as expression in tumour cells was an independent prognostic factor for DFS (Cox regression; p = 0.018, HR = 0.983; p = 0.015, HR = 0.984, respectively). In conclusion, STC2 expression is abundant in MBC where it is an independent prognostic factor for DFS.
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http://dx.doi.org/10.1002/cjp2.106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6174618PMC
October 2018

Characterising the adipose-inflammatory microenvironment in male breast cancer.

Endocr Relat Cancer 2018 07 9;25(7):773-781. Epub 2018 May 9.

Leeds Institute of Cancer & PathologyUniversity of Leeds, St James's University Hospital, Leeds, UK

Male breast cancer (MBC) incidence seems to parallel global increases in obesity. The stromal microenvironment contributes to carcinogenesis; yet, the role of adipocytes in this is understudied in MBC. We identified four cohorts of male breast tissues diagnosed when obesity was rare (archival cohort) and more common (contemporary cohort). We examined the microenvironment of archival and contemporary cohorts of MBC, diagnosed 1940-1970 and 1998-2006, respectively, with two cohorts of, archival and contemporary gynaecomastia, diagnosed 1940-1979 and 1996-2011, respectively, serving as controls. We quantified adipocytes, crown-like structures (CLS) and the presence of CD8, α smooth muscle actin (αSMA) and CD68+ macrophages in both cohorts, and determined how these affected survival, in the contemporary MBC cohort. In both MBC cohorts, mean adipocyte diameter was larger in the distant stroma compared with stroma close to the invading tumour (92.2 µm vs 66.7 µm). This was not seen in gynaecomastia. CLS were more frequent in both MBC cohorts than gynaecomastia (44/55 (80%) vs 11/18 (61%),  < 0.001). No relationship was found between CLS number and adipocyte size, although there were greater numbers of CLS in contemporary MBC > archival MBC > gynaecomastia. CD8 and CD68 expression in the stroma was significantly associated with reduced survival, with no effects seen with αSMA. Changes in the adipose-inflammatory microenvironment may be a contributing factor to the increase seen in MBC diagnosis.
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http://dx.doi.org/10.1530/ERC-17-0407DOI Listing
July 2018

Oestrogen receptor β (ERβ) regulates osteogenic differentiation of human dental pulp cells.

J Steroid Biochem Mol Biol 2017 11 12;174:296-302. Epub 2017 Oct 12.

Department of Oral Biology, Wellcome Trust Brenner Building, St James University Hospital, University of Leeds, UK. Electronic address:

Estradiol (E) has many important actions in the tissues of the oral cavity. Disruption of E metabolism or alterations in systemic E concentrations have been associated with compromised periodontal health. In many instances such changes occur secondarily to the well characterised effects of E on bone physiology -especially maintenance of bone mineral density (BMD). Despite these important epidemiological findings, little is known about the mechanism of action of E in oral tissues or the expression and function of oestrogen receptor (ER) isoforms in these tissues. We have isolated human dental pulp cells (hDPCs), which are able to differentiate towards an osteogenic lineage under appropriate culture conditions. We show that hDPCs express ERα, ERβ1, ERβ2 and the cell membrane associated G protein-coupled ER (GPR30). Following osteogenic differentiation of hDPCs, ERβ1 and ERβ2 were up regulated approximately 50-fold while ERα and GPR30 were down regulated, but to a much lesser degree (approximately 2-fold). ERβ was characterised as a 59kDa protein following Western blot analysis with validated antibodies and ERβ was detected in both nuclear and cytoplasmic cell compartments following immunofluorescence (IF) and immunohistochemical (IHC) analysis of cultured cells. Furthermore isoform specific antibodies detected both ERβ1 and ERβ2 in DPC cultures and in situ analysis of ERβ expression in decalcified tooth/pulp sections identified the odontoblast layer of pulp cells juxtaposed to the tooth enamel as strongly reactive for both ERβ isoforms. Finally the use of isoform specific agonists identified ERβ as the main receptor responsible for the pro-osteogenic effect of oestrogenic hormones in this tissue. Our data suggest that oestrogens stimulated osteogenic differentiation in hDPCs and that this action is mediated principally through the ERβ isoform. These findings may have important consequences for the investigation and treatment of oral and periodontal pathologies which are associated with imbalances in oestrogen concentrations and action.
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http://dx.doi.org/10.1016/j.jsbmb.2017.10.012DOI Listing
November 2017

Analysis of the ATR-Chk1 and ATM-Chk2 pathways in male breast cancer revealed the prognostic significance of ATR expression.

Sci Rep 2017 08 14;7(1):8078. Epub 2017 Aug 14.

Division of Medical Oncology 2, "Regina Elena" National Cancer Institute, Via Elio Chianesi 53, 00144, Rome, Italy.

The ATR-Chk1 and ATM-Chk2 pathways are central in DNA damage repair (DDR) and their over-activation may confer aggressive molecular features, being an adaptive response to endogenous DNA damage and oncogene-induced replication stress. Herein we investigated the ATR-Chk1 and ATM-Chk2 signalings in male breast cancer (MBC). The expression of DDR kinases (pATR, pATM, pChk1, pChk2, and pWee1) and DNA damage markers (pRPA32 and γ-H2AX) was evaluated by immunohistochemistry in 289 MBC samples to assess their association. Survival analyses were carried out in 112 patients. Survival curves were estimated with the Kaplan-Meier method and compared by log-rank test. Cox proportional regression models were generated to identify variables impacting survival outcomes. The expression of pATR conferred poorer survival outcomes (log rank p = 0.013, p = 0.007 and p = 0.010 for overall, 15- and 10-year survival, respectively). Multivariate Cox models of 10-year survival and overall indicated that pATR expression, alone or combined with pChk2, was an independent predictor of adverse outcomes (10-year survival: pATR: HR 2.74, 95% CI: 1.23-6.10; pATR/pChk2: HR 2.92, 95% CI: 1.35-6.33; overall survival: pATR: HR 2.58, 95% CI: 1.20-5.53; pATR/pChk2: HR 2.89, 95% CI: 1.37-6.12). Overall, the ATR/ATM-initiated molecular cascade seems to be active in a fraction of MBC patients and may represent a negative prognostic factor.
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http://dx.doi.org/10.1038/s41598-017-07366-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5556084PMC
August 2017

Characterisation of male breast cancer: a descriptive biomarker study from a large patient series.

Sci Rep 2017 03 28;7:45293. Epub 2017 Mar 28.

Leeds Institute of Cancer and Pathology, University of Leeds, Leeds, LS9 7TF, UK.

Male breast cancer (MBC) is rare. We assembled 446 MBCs on tissue microarrays and assessed clinicopathological information, together with data from 15 published studies, totalling 1984 cases. By immunohistochemistry we investigated 14 biomarkers (ERα, ERβ1, ERβ2, ERβ5, PR, AR, Bcl-2, HER2, p53, E-cadherin, Ki67, survivin, prolactin, FOXA1) for survival impact. The main histological subtype in our cohort and combined analyses was ductal (81%, 83%), grade 2; (40%, 44%), respectively. Cases were predominantly ERα (84%, 82%) and PR positive (74%, 71%), respectively, with HER2 expression being infrequent (2%, 10%), respectively. In our cohort, advanced age (>67) was the strongest predictor of overall (OS) and disease free survival (DFS) (p = 0.00001; p = 0.01, respectively). Node positivity negatively impacted DFS (p = 0.04). FOXA1 p = 0.005) and AR p = 0.009) were both positively prognostic for DFS, remaining upon multivariate analysis. Network analysis showed ERα, AR and FOXA1 significantly correlated. In summary, the principle phenotype of MBC was luminal A, ductal, grade 2. In ERα+ MBC, only AR had prognostic significance, suggesting AR blockade could be employed therapeutically.
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http://dx.doi.org/10.1038/srep45293DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5368596PMC
March 2017

Association between AXL, Hippo Transducers, and Survival Outcomes in Male Breast Cancer.

J Cell Physiol 2017 Aug 3;232(8):2246-2252. Epub 2017 Mar 3.

Division of Medical Oncology 2, "Regina Elena" National Cancer Institute, Rome, Italy.

Male breast cancer (MBC) is an uncommon malignancy. We have previously reported that the expression of the Hippo transducers TAZ/YAP and their target CTGF was associated with inferior survival in MBC patients. Preclinical evidence demonstrated that Axl is a transcriptional target of TAZ/YAP. Thus, we herein assessed AXL expression to further investigate the significance of active TAZ/YAP-driven transcription in MBC. For this study, 255 MBC samples represented in tissue microarrays were screened for AXL expression, and 116 patients were included. The association between categorical variables was verified by the Pearson's Chi-squared test of independence (2-tailed) or the Fisher Exact test. The relationship between continuous variables was tested with the Pearson's correlation coefficient. The Kaplan-Meier method was used for estimating survival curves, which were compared by log-rank test. Factors potentially impacting 10-year and overall survival were verified in Cox proportional regression models. AXL was positively associated with the TAZ/CTGF and YAP/CTGF phenotypes (P = 0.001 and P = 0.002, respectively). Patients with TAZ/CTGF/AXL- or YAP/CTGF/AXL-expressing tumors had inferior survival compared with non-triple-positive patients (log rank P = 0.042 and P = 0.048, respectively). The variables TAZ/CTGF/AXL and YAP/CTGF/AXL were adverse factors for 10-year survival in the multivariate Cox models (HR 2.31, 95%CI:1.02-5.22, P = 0.045, and HR 2.27, 95%CI:1.00-5.13, P = 0.050). Nearly comparable results were obtained from multivariate analyses of overall survival. The expression pattern of AXL corroborates the idea of the detrimental role of TAZ/YAP activation in MBC. Overall, Hippo-linked biomarkers deserve increased attention in this rare disease. J. Cell. Physiol. 232: 2246-2252, 2017. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/jcp.25745DOI Listing
August 2017

A Case-Matched Gender Comparison Transcriptomic Screen Identifies eIF4E and eIF5 as Potential Prognostic Markers in Male Breast Cancer.

Clin Cancer Res 2017 May 16;23(10):2575-2583. Epub 2016 Dec 16.

Leeds Institute of Cancer and Pathology, University of Leeds, Leeds, United Kingdom.

Breast cancer affects both genders, but is understudied in men. Although still rare, male breast cancer (MBC) is being diagnosed more frequently. Treatments are wholly informed by clinical studies conducted in women, based on assumptions that underlying biology is similar. A transcriptomic investigation of male and female breast cancer was performed, confirming transcriptomic data Biomarkers were immunohistochemically assessed in 697 MBCs ( = 477, training; = 220, validation set) and quantified in pre- and posttreatment samples from an MBC patient receiving everolimus and PI3K/mTOR inhibitor. Gender-specific gene expression patterns were identified. eIF transcripts were upregulated in MBC. eIF4E and eIF5 were negatively prognostic for overall survival alone (log-rank = 0.013; HR = 1.77, 1.12-2.8 and = 0.035; HR = 1.68, 1.03-2.74, respectively), or when coexpressed ( = 0.01; HR = 2.66, 1.26-5.63), confirmed in the validation set. This remained upon multivariate Cox regression analysis [eIF4E = 0.016; HR = 2.38 (1.18-4.8), eIF5 = 0.022; HR = 2.55 (1.14-5.7); coexpression = 0.001; HR = 7.04 (2.22-22.26)]. Marked reduction in eIF4E and eIF5 expression was seen post BEZ235/everolimus, with extended survival. Translational initiation pathway inhibition could be of clinical utility in MBC patients overexpressing eIF4E and eIF5. With mTOR inhibitors that target this pathway now in the clinic, these biomarkers may represent new targets for therapeutic intervention, although further independent validation is required. .
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http://dx.doi.org/10.1158/1078-0432.CCR-16-1952DOI Listing
May 2017

HMG-CoAR expression in male breast cancer: relationship with hormone receptors, Hippo transducers and survival outcomes.

Sci Rep 2016 10 7;6:35121. Epub 2016 Oct 7.

Division of Medical Oncology B, "Regina Elena" National Cancer Institute, Via Elio Chianesi 53, 00144, Rome, Italy.

Male breast cancer (MBC) is a rare hormone-driven disease often associated with obesity. HMG-CoAR is the central enzyme of the mevalonate pathway, a molecular route deputed to produce cholesterol and steroid-based hormones. HMG-CoAR regulates the oncogenic Hippo transducers TAZ/YAP whose expression was previously associated with shorter survival in MBC. 225 MBC samples were immunostained for HMG-CoAR and 124 were considered eligible for exploring its relationship with hormone receptors (ER, PgR, AR), Hippo transducers and survival outcomes. HMG-CoAR was positively associated with the expression of hormone receptors (ER, PgR, AR) and Hippo transducers. Overall survival was longer in patients with HMG-CoAR-positive tumors compared with their negative counterparts (p = 0.031). Five- and 10-year survival outcomes were better in patients whose tumors expressed HMG-CoAR (p = 0.044 and p = 0.043). Uni- and multivariate analyses for 10-year survival suggested that HMG-CoAR expression is a protective factor (HR 0.50, 95% CI: 0.25-0.99, p = 0.048 and HR 0.53, 95% CI: 0.26-1.07, p = 0.078). Results were confirmed in a sensitivity analysis by excluding uncommon histotypes (multivariate Cox: HR 0.45, 95% CI: 0.21-0.97, p = 0.043). A positive relationship emerged between HMG-CoAR, hormone receptors and TAZ/YAP, suggesting a connection between the mevalonate pathway, the hormonal milieu and Hippo in MBC. Moreover, HMG-CoAR expression may be a favorable prognostic indicator.
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http://dx.doi.org/10.1038/srep35121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5054365PMC
October 2016

Deregulation of IGF-binding proteins -2 and -5 contributes to the development of endocrine resistant breast cancer in vitro.

Oncotarget 2016 May;7(22):32129-43

Department of Oral Biology, St James's University Hospital, Leeds, UK.

Tamoxifen (TAM) remains the adjuvant therapy of choice for pre-menopausal women with ERα-positive breast cancer. Resistance and recurrence remain, however, a major challenge with many women relapsing and subsequently dying. The insulin-like growth factor (IGF) axis is involved in breast cancer pathogenesis and progression to endocrine resistant disease, but there is very little data on the expression and potential role of IGF-binding proteins (IGFBP) during acquisition of the resistant phenotype. The aim of this study was to determine the expression and functional role of IGFBP-2 and -5 in the development of TAM resistance (TamR) in vitro and to test retrospectively whether they were predictive of resistance in a tissue microarray of 77 women with primary breast cancers who relapsed on/after endocrine therapy and 193 who did not with long term follow up. Reciprocal expression of IGFBP-2 and IGFBP-5 was observed at both mRNA and protein level in TamR cells. IGFBP-2 expression was increased by 10-fold while IGFBP-5 was decreased by 100-fold, compared to TAM-sensitive control cells. shRNA-mediated silencing of IGFBP-2 in TamR cells restored TAM sensitivity suggesting a causal role for this gene in TamR. While silencing of IGFBP-5 in control cells had no effect on TAM sensitivity, it significantly increased the migratory capacity of these cells. Quantitative image analysis of immunohistochemical data failed, however, to demonstrate an effect of IGFBP2 expression in endocrine-relapsed patients. Likewise, IGFBP-2 and IGFBP-5 expression failed to show any significant associations with survival either in patients relapsing or those not relapsing on/after endocrine therapy. By contrast, in silico mining of a separate published dataset showed that in patients who received endocrine treatment, loss of expression of IGBP-5 was significantly associated with worse survival. Overall these data suggest that co-ordinated and reciprocal alteration in IGFBP-2 and -5 expression may play a role in the acquisition of endocrine resistance.
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http://dx.doi.org/10.18632/oncotarget.8534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5078002PMC
May 2016

Obesity and male breast cancer: provocative parallels?

BMC Med 2015 Jun 4;13:134. Epub 2015 Jun 4.

Leeds Institute of Cancer and Pathology, University of Leeds, St James's University Hospital, Leeds, LS9 7TF, UK.

While rare compared to female breast cancer the incidence of male breast cancer (MBC) has increased in the last few decades. Without comprehensive epidemiological studies, the explanation for the increased incidence of MBC can only be speculated. Nevertheless, one of the most worrying global public health issues is the exponential rise in the number of overweight and obese people, especially in the developed world. Although obesity is not considered an established risk factor for MBC, studies have shown increased incidence among obese individuals. With this observation in mind, this article highlights the correlation between the increased incidence of MBC and the current trends in obesity as a growing problem in the 21(st) century, including how this may impact treatment. With MBC becoming more prominent we put forward the notion that, not only is obesity a risk factor for MBC, but that increasing obesity trends are a contributing factor to its increased incidence.
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http://dx.doi.org/10.1186/s12916-015-0380-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4457166PMC
June 2015
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