Differences in the pathological, transcriptomic, and prognostic implications of lymphoid structures between primary and metastatic cutaneous melanomas

Abstract

Background: While the prognostic role of tertiary lymphoid structures (TLS) has been well studied in solid cancers, the prevalence and impact of immature precursor lymphoid structures known as lymphoid aggregates (LA) remain unresolved in relation to the disease process. In this study, we examined characteristics and the prognostic utility of LA and TLS status in histological samples from patients with melanoma.

Methods: We assessed The Cancer Genomic Atlas-skin cutaneous melanoma digital slides and melanoma specimens from the University of Pittsburgh for the presence of LA and TLS using H&E staining, multiplex immunofluorescence (mIF) and transcriptomic analyses. Cox proportional hazard regression models were used to assess the prognostic value associated with the presence of lymphoid structures in melanomas.

Results: A total of 278 evaluable samples were analyzed and split into primary melanomas in skin (N=195) and metastatic melanomas involving skin/subcutaneous/soft tissue sites (N=83). 72% of tumor specimens contained histologically defined LA located in peritumoral (34%), intratumoral (5.6%) or stromal (6.1%) locations, with the remaining samples (54.3%) exhibiting LA in multiple locations. In contrast to LA which tended to form more commonly in primary melanoma samples, TLS with germinal centers predominantly formed in peritumoral (45.2%) or stromal (35.5%) locations in metastatic melanomas (p=0.02), with TLS observed in 11% of all melanoma specimens evaluated. mIF analyses revealed cellular heterogeneity of lymphoid structures, with CD20⁺ (B) cells present in nodule-shaped and stromal locations where they exhibited a high degree of colocalization with CD4⁺ and CD8⁺ T cells. A previously defined 12-chemokine gene expression score was significantly higher in samples with evidence of LA versus none (p<0.001), and samples without LA/TLS were enriched with pigmentation/neural network gene signatures. The presence of LA was significantly associated with tumor-free regional lymph node status (p=0.002). In multivariable analysis, after adjusting for age, sex, sample type, and stage, the presence of LA was associated with improved patient overall survival (OS) (HR=0.52, 95% CI 0.31 to 0.87, p=0.01).

Conclusion: Melanoma frequently contains LA, which tends to form in diverse locations in the tumor microenvironment in association with improved overall survival and tumor-free regional lymph node status in patients with primary disease.

Keywords: B cell; Immunotherapy; Melanoma.

Figure 1. Overview of lymphoid aggregate (LA) and tertiary lymphoid structure (TLS) characteristics based on H&E assessments. Stacked bar plots comparing the percentages of (A) LA and (B) TLS among primary and metastatic samples. (C) Bar plot displaying LA subtypes. Bar plots showing locations of (D) LA and (E) TLS. (F) H&E slide showing subcutaneous nodule of melanoma, which is surrounded by adipose tissue, red arrow indicates TLS forming within adipose tissue.

Figure 2. Prognostic significance of lymphoid structures in relation to overall survival. (A) Kaplan-Meier plot comparing overall survival probability between patients with LA and those without LA. Cox proportional hazard regression models for overall survival adjusting for (B) LA presence, age, sex, stage, and sample type, (C) LA subtype, (D) LA location. (E) Hazard regression plot comparing overall survival between patients with LA with TLS and LA only with adjustment for age, sex, stage, sample type. IT, intratumoral; LA, lymphoid aggregates; PT, peritumoral; TLS, tertiary lymphoid structures.

Figure 3. Assessment of lymphoid aggregates in primary melanomas in relation to regional lymph node (RLN) status and histopathological features. (A) RLN positivity, p=0.02, (B) histological subtype, p=0.01(C) regression, p=0.05 and (D) log-transformed Breslow thickness, p=0.36. RLN, regional lymph node, SS, superficial spreading.

Figure 4. Cellular composition of lymphoid structures and heterogeneity based on subtypes, location evaluated by multiplex immunofluorescence assessment of lymphoid infiltrates. (A) Multiplex immunofluorescence analysis of lymphoid structures. Stack bar plots represent the proportion of each cell. Layer 1 represents the whole slide, and each region of interest was annotated and presented in x axis. (B) Heatmap represents the proportion of cell types for each slide across cluster and nodule from intratumorally (n=17), peritumorally (n=72), and stromal (n=42) annotated regions of interest. (C) Box plots comparing the composition of CD8+T cells, (D) CD20⁺ cells, (E) CD21⁺ cells, (F) PNAd across subtypes and location of lymphoid aggregates. (G) Box plot shows the distribution of CD8 cells at IT, (n=17), PT (n=72), and ST (n=42). (H) CD20 cell distribution (I). PNAd distribution. The p value was calculated between the two groups by using the Wilcoxon rank test. (J) Correlation between CD20 and CD8 counts over the whole slide, Spearman correlation coefficient and p value are depicted over figure showing strong positive correlation. IT, intratumoral; PT, peritumoral; ST, stromal.

Figure 5. Cell colocalization quantified using the cross-K function method for an individual case using CD20 as a reference and measurement of entropy. The black line represents the input image, the red line represents a randomly distributed point pattern. With the increase of radius (x axis), the black line diverges further from the red line, meaning that there is at least one mixed cluster of two types of points, which is also suggested by the positive value of the AUC score for (A) CD20-CD4, (B) CD20-CD8, (C) CD20-PNAD, and (D) CD20-CD21. (E) For CD20-SOX10, the AUC score was close to 0, suggesting the two types of cells do not exhibit a positional relationship. Entropy measurement based on CD20 as reference (F) with CD4 (G), CD21 (H) or PNAD (I). Higher entropies (blue) are obtained when the cell types considered are balanced and suggest high cell colocalization, and lower entropies arise when a particular cell type(s) is rare and indicate low cell colocalization. AUC, area under the curve.

Figure 6. Transcriptomic profiling and functional Gene Set Enrichment Analyses of melanoma samples with and without LA. (A) Heatmap illustrating differentially expressed genes in samples with/without LA. (B) Bar plots showing upregulated and downregulated MSigDB hallmark gene sets, Gene Ontology pathways, and REACTOME pathways in LA samples. (C) Box plot showing 12-chemokine score in samples with LA±TLS which are combined in LA group and without LA (No-LA). Table further shows 12-CK score in LA and TLS groups, separately. P value was calculated using the Wilcoxon rank-sum test. LA, lymphoid aggregates; TLS, tertiary lymphoid structures.