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Intratumoral spatial heterogeneity of tumor-infiltrating lymphocytes is a significant factor for precisely stratifying prognostic immune subgroups of microsatellite instability-high colorectal carcinomas


Although the density of tumor-infiltrating lymphocytes (TILs) is known to be linked to prognosis in various cancers, the prognostic impact and immunologic significance of the spatial heterogeneity of TILs have been rarely investigated. In this study, CD3+ and CD8+ TILs were quantified in independent cohorts (discovery, n = 73; and external validation, n = 93) of colorectal carcinomas (CRCs) with microsatellite instability-high (MSI-H) utilizing whole-slide image analysis of CD3/CD8 immunohistochemistry. The Shannon and Simpson indices, which measure intratumoral patch-to-patch evenness of TIL densities, were used to quantitatively assess the spatial heterogeneity of TILs in each case. To uncover immune-related gene expression signatures of spatial heterogeneity-based TIL subgroups of MSI-H CRCs, representative cases were subjected to GeoMx digital spatial profiler (DSP) analysis. As expected, a low density of TILs was significantly associated with poor disease-free survival (DFS) in MSI-H CRCs. The TIL-low tumors were further classified into two subgroups based on the spatial heterogeneity of TILs: TIL-low/heterogeneity-high and TIL-low/heterogeneity-low subgroups. In both discovery and validation cohorts, the TIL-low/heterogeneity-high, TIL-low/heterogeneity-low, and TIL-high subgroups were significantly associated with poor, intermediate, and good DFS, respectively. In the DSP analysis, the TIL-low/heterogeneity-high subgroup showed higher spatial diversity in the expression of immune-related genes than that of the TIL-low/heterogeneity-low subgroup and exhibited upregulation of genes related to immune checkpoints, chemokine/cytokine receptors, and myeloid cells. TIL-low/heterogeneity-high tumors were also enriched with gene sets related to good response to immune checkpoint inhibitor therapy. In conclusion, TIL-low MSI-H CRCs are prognostically heterogeneous and can be divided into prognostically and immunologically distinct subgroups by considering the spatial heterogeneity of TILs. Our data suggest that intratumoral spatial heterogeneity of TILs can be used as a key element for clinically relevant immunologic subtyping of tumors.

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Fig. 1: Prognostic significance of the spatial heterogeneity of TILs in MSI-H CRCs.
Fig. 2: Validation of the prognostic impact of TIL spatial heterogeneity in MSI-H CRCs.
Fig. 3: GeoMx DSP analysis to identify TIL density-dependent immune-related gene expression signatures in MSI-H CRCs.
Fig. 4: GeoMx DSP analysis to identify TIL spatial heterogeneity-dependent immune-related gene expression signatures in MSI-H CRCs.
Fig. 5

Data availability

The raw dataset generated from GeoMx DSP analysis during the current study are available in the Mendeley data repository ( The other datasets generated or analyzed in the study are available from the corresponding author upon reasonable request.


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We thank Seoul National University Hospital Medical Research Collaborating Center for partially supporting statistical analysis in the study.


This study was supported by a grant from the SNUH Research Fund (04-2020-0550; to JHK), a faculty research grant of Yonsei University College of Medicine (6-2021-0144; to MJ), a grant from Seoul National University College of Medicine (800-20210387; to JHK), a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute funded by the Korea government (Ministry of Health and Welfare) (HI21C0977; to MJ), and the National Research Foundation of Korea grants funded by the Korea government (Ministry of Science and ICT) (NRF-2016R1C1B2010627; NRF-2019R1F1A1059535; to JHK).

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Authors and Affiliations



MJ and JHK performed study concept and design. MJ, JAL, S-YY, JMB, GHK, and JHK collected tissue samples and clinicopathologic data. MJ, JAL, S-YY, and JHK provided acquisition, analysis, and interpretation of data. MJ and JHK conducted statistical analyses. MJ and JHK wrote and revised the manuscript. All authors read and approved the final version of the manuscript.

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Correspondence to Jung Ho Kim.

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The authors declare no competing interests.

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This study was conducted in compliance with the ethical guidelines of the 2013 Declaration of Helsinki and was approved by the Institutional Review Boards of Seoul National University Hospital (IRB No. 1804-036-935; and 1805-018-944) and Severance Hospital (IRB No. 4-2021-1130). The study samples collected from Seoul National University Hospital were previously registered in the Cancer Tissue Bank of Seoul National University Hospital with informed consent obtained from all patients about the research use of their tissues. The study samples collected from Severance Hospital were exempted from informed consent acquisition from patients by the Institutional Review Board because the study used only retrospective, anonymized tissue samples.

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Jung, M., Lee, J.A., Yoo, SY. et al. Intratumoral spatial heterogeneity of tumor-infiltrating lymphocytes is a significant factor for precisely stratifying prognostic immune subgroups of microsatellite instability-high colorectal carcinomas. Mod Pathol (2022).

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