title: Multispectral imaging for highly accurate analysis of tumour-infiltrating lymphocytes in primary melanoma.
abstract: OBJECTIVE
  The quality and quantity of the infiltration of immune cells into tumour tissues have substantial impacts on patients' clinical outcomes, and are associated with response to immunotherapy. Therefore, the precise analysis of tumour-infiltrating lymphocytes (TILs) is becoming an important additional pathological biomarker. Analysis of TILs is usually performed semiquantitatively by pathologists on haematoxylin and eosin-stained or immunostained tissue sections. However, automated quantification outperforms semiquantitative approaches, and is becoming the standard. Owing to the presence of melanin pigment, this approach is seriously hampered in melanoma, because the spectrum of melanin lies close to that of commonly used immunohistochemical stains. Aim of this study is to overcome the technical issues due to the presence of melanin for an automated and accurate quantification of TILs in melanoma.
  
  
  RESULTS
  Here, we successfully applied a novel multispectral imaging (MSI) technique to enumerate T cells in human primary melanomas. This microscopy technique combines imaging with spectroscopy to obtain both quantitative expression data and the tissue distributions of different cellular markers. We demonstrate that MSI allows complete and accurate analysis of TILs, successfully avoiding the blurring of images by melanin pigments, in whole tissue slide primary melanoma lesions, which could otherwise not be accurately detected by conventional digital image methodologies.
  
  
  CONCLUSIONS
  Our study highlights the potential of MSI for accurate assessment of immune cell infiltrates, including those in notoriously difficult tissues, such as pigmented melanomas. Quantification of tumour infiltration by different immune cell types is crucial in the search for new biomarkers to predict patient responses to immunotherapies. Our findings show that this innovative microscopy technique is an important extension of the armamentarium of pathologists.
authors: Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Nijmegen, the Netherlands.
authors: Angela
authors: A
authors: Vasaturo
authors: Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Nijmegen, the Netherlands.
authors: Stefania
authors: S
authors: Di Blasio
authors: Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Nijmegen, the Netherlands.|Department of Pathology, Radboud University Medical Centre, Nijmegen, the Netherlands.
authors: Dagmar
authors: D
authors: Verweij
authors: Department of Pathology, Radboud University Medical Centre, Nijmegen, the Netherlands.
authors: Willeke A M
authors: WA
authors: Blokx
authors: Department of Pathology, Radboud University Medical Centre, Nijmegen, the Netherlands.
authors: J Han
authors: JH
authors: van Krieken
authors: Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Nijmegen, the Netherlands.|Department of Medical Oncology, Radboud University Medical Center, Nijmegen, the Netherlands.
authors: I Jolanda M
authors: IJ
authors: de Vries
authors: Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Nijmegen, the Netherlands.
authors: Carl G
authors: CG
authors: Figdor
mesh_terms: D000465:Algorithms; D003952:Diagnostic Imaging / Q000379:methods*; D006801:Humans; D007090:Image Interpretation, Computer-Assisted / Q000379:methods; D016246:Lymphocytes, Tumor-Infiltrating / Q000473:pathology*; D008545:Melanoma / Q000276:immunology / Q000473:pathology*; D008853:Microscopy / Q000379:methods; D012878:Skin Neoplasms / Q000276:immunology / Q000473:pathology*; D013057:Spectrum Analysis / Q000379:methods
keywords: T-cell infiltration; digital pathology; melanin unmixing; melanoma; multispectral imaging