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. 2021 Jul;70(7):2049-2057.
doi: 10.1007/s00262-020-02834-y. Epub 2021 Jan 13.

A template to quantify the location and density of CD3 + and CD8 + tumor-infiltrating lymphocytes in colon cancer by digital pathology on whole slides for an objective, standardized immune score assessment

Dordi Lea  1   2   3 , Martin Watson  1   2   4 , Ivar Skaland  3 , Hanne R Hagland  1   5 , Melinda Lillesand  3 , Einar Gudlaugsson  3 , Kjetil Søreide  6   7   8
Affiliations

A template to quantify the location and density of CD3 + and CD8 + tumor-infiltrating lymphocytes in colon cancer by digital pathology on whole slides for an objective, standardized immune score assessment

Dordi Lea et al. Cancer Immunol Immunother. 2021 Jul.

Abstract

Background: In colon cancer, the location and density of tumor-infiltrating lymphocytes (TILs) can classify patients into low and high-risk groups for prognostication. While a commercially available 'Immunoscore®' exists, the incurred expenses and copyrights may prevent universal use. The aim of this study was to develop a robust and objective quantification method of TILs in colon cancer.

Methods: A consecutive, unselected series of specimens from patients with colon cancer were available for immunohistochemistry and assessment of TILs by automated digital pathology. CD3 + and CD8 + cells at the invasive margin and in tumor center were assessed on consecutive sections using automated digital pathology and image analysis software (Visiopharm®). An algorithm template for whole slide assessment, generated cell counts per square millimeters (cells/mm2), from which the immune score was calculated using distribution volumes. Furthermore, immune score was compared with clinical and histopathological characteristics to confirm its relevance.

Results: Based on the quantified TILs numbers by digital image analyses, patients were classified into low (n = 83, 69.7%), intermediate (n = 14, 11.8%) and high (n = 22, 18.5%) immune score groups. High immune score was associated with stage I-II tumors (p = 0.017) and a higher prevalence of microsatellite instable (MSI) tumors (p = 0.030). MSI tumors had a significantly higher numbers of CD3 + TILs in the invasive margin and CD8 + TILs in both tumor center and invasive margin, compared to microsatellite stable (MSS) tumors.

Conclusion: A digital template to quantify an easy-to-use immune score corresponds with clinicopathological features and MSI in colon cancer.

Keywords: Colorectal cancer; Digital image analysis; Immune response; Tumor center; Tumor-infiltrating lymphocytes; Tumor-invasive margin.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1
a Hematoxylin–eosin staining of tumor. b Immunohistochemical staining of CD3 (brown) in the same tumor with marking of tumor center/TC (blue) and invasive margin/IM (green). c Digital image analysis measured the CD3 + tumor-infiltrating lymphocytes (TILs) in TC and IM. The number of positive TILs was calculated pr. mm2. The same tumor area was analyzed for CD3 and CD8 for each patient. d Close-up view that shows positive TILs marked with green. Negative nuclei are marked blue and surrounding stroma is marked red
Fig. 2
Fig. 2
Flowchart for calculating immune response based on mean densities of CD3 + and CD8 + in tumor center (TC) and invasive margin (IM)
Fig. 3
Fig. 3
Distribution of number of CD3 + and CD8 + TILs in tumor center (TC) and invasive margin (IM)
Fig. 4
Fig. 4
Comparison of number of CD3 + and CD8 + tumor-infiltrating lymphocytes (TILs) of MSS and MSI tumors in tumor center (TC) and invasive margin (IM). Extreme values > 4000 cells is not shown in figure
See this image and copyright information in PMC

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