An integrative histopathologic clustering model based on immuno-matrix elements to predict the risk of death in malignant mesothelioma

Abstract

Objective: Previous studies have reported a close relationship between malignant mesothelioma (MM) and the immune matricial microenvironment (IMM). One of the major problems in these studies is the lack of adequate adjustment for potential confounders. Therefore, the aim of this study was to identify and quantify risk factors such as IMM and various tumor characteristics and their association with the subtype of MM and survival.

Methods: We examined IMM and other tumor markers in tumor tissues from 82 patients with MM. These markers were evaluated by histochemistry, immunohistochemistry, immunofluorescence, and morphometry. Logistic regression analysis, cluster analysis, and Cox regression analysis were performed.

Results: Hierarchical cluster analysis revealed two clusters of MM that were independent of clinicopathologic features. The high-risk cluster included MM with high tumor cellularity, high type V collagen (Col V) fiber density, and low CD8⁺ T lymphocyte density in the IMM. Our results showed that the risk of death was increased for patients with MM with high tumor cellularity (OR = 1.63, 95% CI = 1.29-2.89, P = .02), overexpression of Col V (OR = 2.60, 95% CI = 0.98-6.84, P = .04), and decreased CD8 T lymphocytes (OR = 1.001, 95% CI = 0.995-1.007, P = .008). The hazard ratio for the high-risk cluster was 2.19 (95% CI = 0.54-3.03, P < .01) for mortality from MM at 40 months.

Conclusion: Morphometric analysis of Col V, CD8⁺ T lymphocytes, and tumor cellularity can be used to identify patients with high risk of death from MM.

Keywords: biomarkers; cluster analysis; collagen type V; computational pathology; extracellular matrix; immunomodulation; mesothelioma.

Figure 1

A representative epithelioid MM section showing blocks of epithelioid cells infiltrating a stromal element (A), correlated with Movat histochemical staining highlighting the matrix constituents surrounding neoplastic cells (C). A representative sarcomatoid MM section composed of malignant spindle cells contrasted by cellular densities and matrix‐forming elements, as explored with Movat staining (D)

Figure 2

Immunofluorescence for collagen type I and type V in epithelioid and sarcomatoid subtypes of MM. Type I collagen shows a fibrillar pattern for both MM subtypes (A, B), while type V collagen surrounds the malignant cellular component (C, D). Double D2‐40 (red) and type V collagen (green) staining (E, F) show overlap of both stainings highly suggestive of deposition of collagen V in the membranes of tumor cells. A computer‐assisted collagen I and V image reconstruction is shown in G, highlighting the fibrillar (collagen I) vs the surrounding pattern of collagen V

Figure 3

CD8⁺ T lymphocytes (A, B), CD2⁺ B lymphocytes (C, D), P53 immunoexpression (E, F), and PMS2 positivity (G, H) are compared between the epithelioid and sarcomatoid subtypes

Figure 4

PD‐1 (A, B), PD‐L1 (C, D), CD30 (E, F), WT1 (G, H), and BAP1 (I, J) immunoexpression in epithelioid and sarcomatoid types

Figure 5

A, Dendrogram illustrating the arrangement of the clusters produced by the corresponding analyses of hierarchical clustering analysis of the studied 82 MM patients. Patients split into two clusters, which are separated by vertical lines. For markers, vertical lines on the left indicate low (below cutoff) expression or cellularity < 80.6 cells/mm² CD4/CD8, collagen V < 7.75 fibers/mm³, and CD8 > 99.77 cells/mm², whereas vertical lines on the right indicate high (above cutoff) expression or cellularity > 80.6 cells/mm² CD4/CD8, collagen V > 7.75 fibers/mm³, and CD8 < 99.77 cells/mm². Only significant (P < .05) discriminating markers or marker groups are annotated. B, Dispersion graph illustrates the grouping distribution of cases by the clustering model showing two risk groups: a distinct low‐risk cluster (grouping 1, blue circle) and a high‐risk cluster (grouping 2). C, Survival analysis of MM clusters. Note the poorer outcome of MM cluster 2 (linked to high expression of cellularity > 80.6 cells/mm² CD4/CD8, higher expression of collagen V > 7.75 fibers/mm³, and lower expression of CD8 < 99.77 cells/mm² compared with cluster 1; P = .039 (for comparison between all two clusters). MM indicates malignant mesothelioma; CD8 indicates cytotoxic lymphocytes