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Multiplex immunohistochemistry accurately defines the immune context of metastatic melanoma.

ABSTRACT: A prospective study explored the heterogeneous nature of metastatic melanoma using Multiplex immunohistochemistry (IHC) and flow cytometry (FACS). Multiplex IHC data quantitated immune subset number present intra-tumoral (IT) vs the tumor stroma, plus distance of immune subsets from the tumor margin (TM). In addition, mIHC showed a close association between the presence of IT CD8⁺ T cells and PDL1 expression in melanoma, which was more prevalent on macrophages than on melanoma cells. In contrast, FACS provided more detailed information regarding the T cell subset differentiation, their activation status and expression of immune checkpoint molecules. Interestingly, mIHC detected significantly higher Treg numbers than FACS and showed preferential CD4⁺ T cell distribution in the tumor stroma. Based on the mIHC and FACS data, we provide a model which defines metastatic melanoma immune context into four categories using the presence or absence of PDL1⁺ melanoma cells and/or macrophages, and their location within the tumor or on the periphery, combined with the presence or absence of IT CD8⁺ T cells. This model interprets melanoma immune context as a spectrum of tumor escape from immune control, and provides a snapshot upon which interpretation of checkpoint blockade inhibitor (CBI) therapy responses can be built.

SUBMITTER: Halse H

PROVIDER: S-EPMC6057961 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Multiplex immunohistochemistry accurately defines the immune context of metastatic melanoma.

Halse H H Colebatch A J AJ Petrone P P Henderson M A MA Mills J K JK Snow H H Westwood J A JA Sandhu S S Raleigh J M JM Behren A A Cebon J J Darcy P K PK Kershaw M H MH McArthur G A GA Gyorki D E DE Neeson P J PJ

Scientific reports 20180724 1

A prospective study explored the heterogeneous nature of metastatic melanoma using Multiplex immunohistochemistry (IHC) and flow cytometry (FACS). Multiplex IHC data quantitated immune subset number present intra-tumoral (IT) vs the tumor stroma, plus distance of immune subsets from the tumor margin (TM). In addition, mIHC showed a close association between the presence of IT CD8<sup>+</sup> T cells and PDL1 expression in melanoma, which was more prevalent on macrophages than on melanoma cells. ...[more]

PMID: 30042403

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Project description:BackgroundThe FAST study identified claudin-18 (CLDN18.2) as a promising novel therapeutic target for gastric cancer (GC). However, the tumor immune microenvironment and clinicopathological features of CLDN18.2-positive GC are unclear, making it difficult to develop and optimize CLDN18.2-targeted treatments.MethodsThis study included 80 GC patients, 60 of whom received anti-PD-1/PD-L1 treatment. CD4/CD8/CD20/CD66b/CD68/CD163/PD-1/PD-L1/TIM-3/LAG-3/FoxP3/CTLA-4/HLA-DR/STING, and CLDN18.2 were labeled using multiplex immunohistochemistry (m-IHC) to decipher the rate and spatial distribution of T cells, B cells, macrophages, and neutrophils in formalin-fixed, paraffin-embedded tumor tissues isolated from these patients. Tumor immune-microenvironmental features and patient survival stratified by CLDN18.2 expression were analyzed using two independent-sample t-tests and log-rank tests, respectively.ResultsWe considered moderate-to-strong CLDN18.2 expression ≥ 40% of tumor cells as the cut-off for positivity. The proportion of CD8+PD-1-, CD8+LAG-3-, and CD8+TIM-3- T cells was significantly higher in CLDN18.2-positive tumors than in negative tumors (0.039 vs. 0.026, P = 0.009; 0.050 vs.0.035, P = 0.024; 0.045 vs. 0.032, P = 0.038, respectively). In addition, the number of neutrophils (CD66b+) was higher in the CLDN18.2-positive group than in the negative group (0.081 vs. 0.055, P = 0.031, respectively), while the rates of M1 (CD68+CD163-HLA-DR+), M2 macrophages (CD68+CD163+HLA-DR-), and B cells (CD20+) were comparable between the CLDN18.2-positive and negative groups. The average numbers of CD8+PD-1-, CD8+LAG-3-, and CD8+TIM-3-T cells surrounding tumor cells within a 20-μm range were higher in CLDN18.2-positive tumors than in the CLDN18.2-negative tumors (0.16 vs. 0.09, P = 0.011; 0.20 vs. 0.12, P = 0.029; 0.18 vs. 0.12, P = 0.047, respectively). In addition, in the CLDN18.2-positive group, tumor cells surrounded by CD8+PD-1-, CD8+LAG-3- T cells, or M1 macrophages within a 20-μm range accounted for a higher proportion of all tumor cells than those in the CLDN18.2-negative group (10.79% vs. 6.60%, P = 0.015; 12.68% vs. 8.70%, P = 0.049; 9.08% vs. 6.56%, P = 0.033, respectively). These findings suggest that CLDN18.2-positive GC harbors complex immune-microenvironmental features. Additionally, CLDN18.2-positive group had shorter OS and irOS than CLDN18.2-negative group (median OS: 23.33 vs.36.6 months, P < 0.001; median irOS: 10.03 vs. 20.13 months, P = 0.044, respectively).ConclusionsCLDN18.2-positive GC displayed unique immune-microenvironmental characteristics, which is of great significance for the development of CLDN18.2-targeted therapies. However, the impact of CLDN18.2-related microenvironmental features on prognosis requires further investigation.

Project description:IntroductionQuantitative, multiplexed imaging is revealing complex spatial relationships between phenotypically diverse tumor infiltrating leukocyte populations and their prognostic implications. The underlying mechanisms and tissue structures that determine leukocyte distribution within and around tumor nests, however, remain poorly understood. While presumed players in metastatic dissemination, new preclinical data demonstrates that blood and lymphatic vessels (lymphovasculature) also dictate leukocyte trafficking within tumor microenvironments and thereby impact anti-tumor immunity. Here we interrogate these relationships in primary human cutaneous melanoma.MethodsWe established a quantitative, multiplexed imaging platform to simultaneously detect immune infiltrates and tumor-associated vessels in formalin-fixed paraffin embedded patient samples. We performed a discovery, retrospective analysis of 28 treatment-naïve, primary cutaneous melanomas.ResultsHere we find that the lymphvasculature and immune infiltrate is heterogenous across patients in treatment naïve, primary melanoma. We categorized five lymphovascular subtypes that differ by functionality and morphology and mapped their localization in and around primary tumors. Interestingly, the localization of specific vessel subtypes, but not overall vessel density, significantly associated with the presence of lymphoid aggregates, regional progression, and intratumoral T cell infiltrates.DiscussionWe describe a quantitative platform to enable simultaneous lymphovascular and immune infiltrate analysis and map their spatial relationships in primary melanoma. Our data indicate that tumor-associated vessels exist in different states and that their localization may determine potential for metastasis or immune infiltration. This platform will support future efforts to map tumor-associated lymphovascular evolution across stage, assess its prognostic value, and stratify patients for adjuvant therapy.

Project description:ObjectivesGPC3 has been recognized as a promising target for immunotherapy in hepatocellular carcinoma (HCC). However, the GPC3-targeted immunotherapies have shown limited therapeutic efficacy. The use of anti-PD-1/PD-L1 monoclonal antibodies in HCC treatment is considerably constrained. Furthermore, there is still a notable lack of understanding concerning the immune landscape in HCC, especially regarding varied GPC3 expression levels. Therefore, thorough exploration of the intricate tumor immune microenvironment at different GPC3 expression levels is essential for guiding and improving HCC treatment strategies.MethodsSixty patients with HCC were enrolled in this study, receiving a first-line treatment that combined anti-angiogenesis targeted drugs and immunotherapy. Immunohistochemistry was used to assess the levels of GPC3 expression. Multiple immunohistochemical markers, such as CD8, PD-1, LAG3, TIGIT, TIM-3, CD103, Claudin18.2, PD-L1, CD4, Foxp3, CD68, CD163, GPC3, CD11C, CD14, CD66b, and HLA-DR, were used to characterize the immune microenvironment and spatial distribution of immune cells in HCC tumors with different levels of GPC3 expression. Cell expression levels and spatial distribution were determined by fluorescence staining and subsequent analysis of fluorescence intensity using the Panoramic Pathology Workstation (Pano ATLAS). This approach facilitated a detailed examination of cell characteristics and spatial information within the samples.ResultsBased on the result of GPC3 immunohistochemical analysis, patients with strong positive GPC3 expression were classified as high GPC3 expression, while the others were classified as low GPC3 expression. Patients in the low GPC3 expression group had longer overall survival (OS) than in the high group (P = 0.003, HR = 2.9240). Further exploration of the immune microenvironment based on different GPC3 expression levels revealed that in high GPC3 expression group, the proportions of CD8+ T cells(P = 0.0435), TIM-3+ T cells(P = 0.0447), CD103+CD8+ tissue-resident T cells(P = 0.0410), CD11C+CD14-DC cells(P = 0.0497), CD11C+HLA-DR-DC cells(P = 0.0309), CD11C+CD14-HLA-DR-DC cells(P = 0.0233), and CD11C+CD14-CD66b-DC cells(P = 0.0474) were all higher compared to low expression group. At spatial distances of 10 μm, 20 μm, and 30 μm, the levels of CD8+ T cells were higher in the high expression group compared to the low expression group (high vs. low: P = 0.0281, P = 0.0236, P = 0.0220).ConclusionsMultiple immunohistochemistry is a powerful technique for exploring the intricate immune microenvironment of hepatocellular carcinoma, enabling the precise identification of diverse cell subsets and their spatial distribution within the tumor microenvironment. This methodology provides valuable insights into the complex interactions and spatial organization of immune cells in the context of hepatocellular carcinoma progression. Low GPC3 expression in HCC patients indicates potential benefits from combined targeted and immunotherapy. Different levels of GPC3 expression levels can predict the effectiveness of targeted combination immunotherapy in HCC patients. Additionally, different GPC3 expression patterns in HCC patients correspond to unique tumor immune microenvironments, which have implications for guiding HCC treatment approaches.

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Multiplex immunohistochemistry accurately defines the immune context of metastatic melanoma.

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Multiplex immunohistochemistry accurately defines the immune context of metastatic melanoma.

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