Sequential Colocalization of ERa, PR, and AR Hormone Receptors Using Confocal Microscopy Enables New Insights into Normal Breast and Prostate Tissue and Cancers

Multiplex immunohistochemistry (mIHC) use markers staining different cell populations applying widefield optical microscopy. Resolution is low not resolving subcellular co-localization. We sought to colocalize markers at subcellular level with antibodies validated for clinical diagnosis, including the single secondary antibody (combination of anti-rabbit/mouse-antibodies) used for diagnostic IHC with any primary antibody, and confocal microscopy. We explore colocalization in the nucleus (ColNu) of nuclear hormone receptors (ERa, PR, and AR) along with the baseline marker p63 in paired samples of breast and prostate tissues. We established ColNu mIHCF as a reliable technique easily implemented in a hospital setting. In ERa+ breast cancer, we identified different colocalization patterns (nuclear or cytoplasmatic) with PR and AR on the luminal epithelium. A triple-negative breast-cancer case expressed membrane-only ERa. A PR-only case was double positive PR/p63. In normal prostate, we identified an ERa+/p63+/AR-negative distinct population. All prostate cancer cases characteristically expressed ERa on the apical membrane of the AR+ epithelium. We confirmed this using ERa IHC and needle-core biopsies. ColNu mIHCF is feasible and already revealed a new marker for prostate cancer and identified sub-patterns in breast cancer. It could be useful for pathology as well as for functional studies in normal prostate and breast tissues

Sequential Colocalization of ERa, PR, and AR Hormone Receptors Using Confocal Microscopy Enables New Insights into Normal Breast and Prostate Tissue and Cancers

Multiplex immunohistochemistry (mIHC) use markers staining different cell populations applying widefield optical microscopy. Resolution is low not resolving subcellular co-localization. We sought to colocalize markers at subcellular level with antibodies validated for clinical diagnosis, including the single secondary antibody (combination of anti-rabbit/mouse-antibodies) used for diagnostic IHC with any primary antibody, and confocal microscopy. We explore colocalization in the nucleus (ColNu) of nuclear hormone receptors (ERa, PR, and AR) along with the baseline marker p63 in paired samples of breast and prostate tissues. We established ColNu mIHCF as a reliable technique easily implemented in a hospital setting. In ERa+ breast cancer, we identified different colocalization patterns (nuclear or cytoplasmatic) with PR and AR on the luminal epithelium. A triple-negative breast-cancer case expressed membrane-only ERa. A PR-only case was double positive PR/p63. In normal prostate, we identified an ERa+/p63+/AR-negative distinct population. All prostate cancer cases characteristically expressed ERa on the apical membrane of the AR+ epithelium. We confirmed this using ERa IHC and needle-core biopsies. ColNu mIHCF is feasible and already revealed a new marker for prostate cancer and identified sub-patterns in breast cancer. It could be useful for pathology as well as for functional studies in normal prostate and breast tissuesThis study has been supported by projects from the State Research Agency (AEI), and Ministry of Science and Innovation, Spain, MINECO-FEDER PID2019-110437RB-I00 (to CVA) and Instituto de Salud Carlos III (ISCIII)-FEDER PI19/01316 (to JMCT). Financial support code: ED431G 2019/02 from the Xunta de Galicia and the European Union (European Regional Development Fund - ERDF) to the Centro singular de Investigación de Galicia accreditation 2019-2022 is gratefully acknowledgedS

In search of an evidence-based strategy for quality assessment of human tissue samples: report of the tissue Biospecimen Research Working Group of the Spanish Biobank Network

The purpose of the present work is to underline the importance of obtaining a standardized procedure to ensure and evaluate both clinical and research usability of human tissue samples. The study, which was carried out by the Biospecimen Science Working Group of the Spanish Biobank Network, is based on a general overview of the current situation about quality assurance in human tissue biospecimens. It was conducted an exhaustive review of the analytical techniques used to evaluate the quality of human tissue samples over the past 30 years, as well as their reference values if they were published, and classified them according to the biomolecules evaluated: (i) DNA, (ii) RNA, and (iii) soluble or/and fixed proteins for immunochemistry. More than 130 publications released between 1989 and 2019 were analysed, most of them reporting results focused on the analysis of tumour and biopsy samples. A quality assessment proposal with an algorithm has been developed for both frozen tissue samples and formalin-fixed paraffin-embedded (FFPE) samples, according to the expected quality of sample based on the available pre-analytical information and the experience of the participants in the Working Group. The high heterogeneity of human tissue samples and the wide number of pre-analytic factors associated to quality of samples makes it very difficult to harmonize the quality criteria. However, the proposed method to assess human tissue sample integrity and antigenicity will not only help to evaluate whether stored human tissue samples fit for the purpose of biomarker development, but will also allow to perform further studies, such as assessing the impact of different pre-analytical factors on very well characterized samples or evaluating the readjustment of tissue sample collection, processing and storing procedures. By ensuring the quality of the samples used on research, the reproducibility of scientific results will be guaranteed.This work was funded by the Ministerio de Ciencia, Innovacion y Universidades of Spain and Instituto de Salud Carlos III (PI16/00528, PI16/00946, PI16/01207 and PI16/01276), co-funded by the Spanish Biobank Network (PT13/0010/0030, PT17/0015/0001, PT17/0015/0021, PT17/0015/0049, PT17/0015/0018, PT17/0015/0002, PT17/0015/0016, PT17/0015/0038, PT17/0015/0027, PT17/0015/0004, PT17/0015/0047, PT17/0015/0014, PT17/0015/0041, and PT17/0015/0006), European Regional Development Fund (FEDER) "A way to make Europe" and granted by Conselleria d'Innovacio, Recerca i Turisme del Govern de les Illes Balears (TEC/002/2017).S

Transcriptome-wide association study of breast cancer risk by estrogen-receptor status

Previous transcriptome-wide association studies (TWAS) have identified breast cancer risk genes by integrating data from expression quantitative loci and genome-wide association studies (GWAS), but analyses of breast cancer subtype-specific associations have been limited. In this study, we conducted a TWAS using gene expression data from GTEx and summary statistics from the hitherto largest GWAS meta-analysis conducted for breast cancer overall, and by estrogen receptor subtypes (ER+ and ER-). We further compared associations with ER+ and ER- subtypes, using a case-only TWAS approach. We also conducted multigene conditional analyses in regions with multiple TWAS associations. Two genes, STXBP4 and HIST2H2BA, were specifically associated with ER+ but not with ER- breast cancer. We further identified 30 TWAS-significant genes associated with overall breast cancer risk, including four that were not identified in previous studies. Conditional analyses identified single independent breast-cancer gene in three of six regions harboring multiple TWAS-significant genes. Our study provides new information on breast cancer genetics and biology, particularly about genomic differences between ER+ and ER- breast cancer.Peer reviewe

In search of an evidence-based strategy for quality assessment of human tissue samples: report of the tissue biospecimen research working group of the spanish biobank network

The purpose of the present work is to underline the importance of obtaining a standardized procedure to ensure and evaluate both clinical and research usability of human tissue samples. The study, which was carried out by the Biospecimen Science Working Group of the Spanish Biobank Network, is based on a general overview of the cur‑ rent situation about quality assurance in human tissue biospecimens. It was conducted an exhaustive review of the analytical techniques used to evaluate the quality of human tissue samples over the past 30 years, as well as their reference values if they were published, and classifed them according to the biomolecules evaluated: (i) DNA, (ii) RNA, and (iii) soluble or/and fxed proteins for immunochemistry. More than 130 publications released between 1989 and 2019 were analysed, most of them reporting results focused on the analysis of tumour and biopsy samples. A qual‑ ity assessment proposal with an algorithm has been developed for both frozen tissue samples and formalin-fxed parafn-embedded (FFPE) samples, according to the expected quality of sample based on the available pre-analytical information and the experience of the participants in the Working Group. The high heterogeneity of human tissue samples and the wide number of pre-analytic factors associated to quality of samples makes it very difcult to harmo‑ nize the quality criteria. However, the proposed method to assess human tissue sample integrity and antigenicity will not only help to evaluate whether stored human tissue samples ft for the purpose of biomarker development, but will also allow to perform further studies, such as assessing the impact of diferent pre-analytical factors on very well characterized samples or evaluating the readjustment of tissue sample collection, processing and storing procedur