title: Next-generation tissue microarray (ngTMA) increases the quality of biomarker studies: an example using CD3, CD8, and CD45RO in the tumor microenvironment of six different solid tumor types.
abstract: BACKGROUND
  Tissue microarray (TMA) technology revolutionized the investigation of potential biomarkers from paraffin-embedded tissues. However, conventional TMA construction is laborious, time-consuming and imprecise. Next-generation tissue microarrays (ngTMA) combine histological expertise with digital pathology and automated tissue microarraying. The aim of this study was to test the feasibility of ngTMA for the investigation of biomarkers within the tumor microenvironment (tumor center and invasion front) of six tumor types, using CD3, CD8 and CD45RO as an example.
  
  
  METHODS
  Ten cases each of malignant melanoma, lung, breast, gastric, prostate and colorectal cancers were reviewed. The most representative H&E slide was scanned and uploaded onto a digital slide management platform. Slides were viewed and seven TMA annotations of 1 mm in diameter were placed directly onto the digital slide. Different colors were used to identify the exact regions in normal tissue (n=1), tumor center (n=2), tumor front (n=2), and tumor microenvironment at invasion front (n=2) for subsequent punching. Donor blocks were loaded into an automated tissue microarrayer. Images of the donor block were superimposed with annotated digital slides. Exact annotated regions were punched out of each donor block and transferred into a TMA block. 420 tissue cores created two ngTMA blocks. H&E staining and immunohistochemistry for CD3, CD8 and CD45RO were performed.
  
  
  RESULTS
  All 60 slides were scanned automatically (total time<10 hours), uploaded and viewed. Annotation time was 1 hour. The 60 donor blocks were loaded into the tissue microarrayer, simultaneously. Alignment of donor block images and digital slides was possible in less than 2 minutes/case. Automated punching of tissue cores and transfer took 12 seconds/core. Total ngTMA construction time was 1.4 hours. Stains for H&E and CD3, CD8 and CD45RO highlighted the precision with which ngTMA could capture regions of tumor-stroma interaction of each cancer and the T-lymphocytic immune reaction within the tumor microenvironment.
  
  
  CONCLUSIONS
  Based on a manual selection criteria, ngTMA is able to precisely capture histological zones or cell types of interest in a precise and accurate way, aiding the pathological study of the tumor microenvironment. This approach would be advantageous for visualizing proteins, DNA, mRNA and microRNAs in specific cell types using in situ hybridization techniques.
authors.forename: Inti
authors.initials: I
authors.lastname: Zlobec
authors.forename: Viktor H
authors.initials: VH
authors.lastname: Koelzer
authors.forename: Heather
authors.initials: H
authors.lastname: Dawson
authors.forename: Aurel
authors.initials: A
authors.lastname: Perren
authors.forename: Alessandro
authors.initials: A
authors.lastname: Lugli
mesh_terms: D014408:Biomarkers, Tumor / Q000378:metabolism*; D017252:CD3 Complex / Q000378:metabolism*; D016827:CD8 Antigens / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D017493:Leukocyte Common Antigens / Q000378:metabolism*; D009369:Neoplasms / Q000378:metabolism* / Q000473:pathology; D046888:Tissue Array Analysis / Q000379:methods*; D059016:Tumor Microenvironment
chemical_list: D014408:Biomarkers, Tumor; D017252:CD3 Complex; D016827:CD8 Antigens; D017493:Leukocyte Common Antigens