Transcriptome-wide studies of merkel cell carcinoma and validation of intratumoral CD8+ lymphocyte invasion as an independent predictor of survival

Abstract

Purpose: Merkel cell carcinoma (MCC) is a polyomavirus-associated skin cancer that is frequently lethal and lacks established prognostic biomarkers. This study sought to identify biomarkers that improve prognostic accuracy and provide insight into MCC biology.

Patients and methods: Gene expression profiles of 35 MCC tumors were clustered based on prognosis. The cluster of genes overexpressed in good-prognosis tumors was tested for biologic process enrichment. Relevant mRNA expression differences were confirmed by quantitative polymerase chain reaction and immunohistochemistry. An independent set of 146 nonoverlapping MCC tumors (median follow-up, 25 months among 116 living patients) was employed for biomarker validation. Univariate and multivariate Cox regression analyses were performed.

Results: Immune response gene signatures were prominent in patients with good prognoses. In particular, genes associated with cytotoxic CD8+ lymphocytes were overexpressed in tumors from patients with favorable prognoses. In the independent validation set, cases with robust intratumoral CD8+ lymphocyte infiltration had improved outcomes (100% MCC-specific survival, n = 26) compared with instances characterized by sparse infiltration (60% survival, n = 120). Only stage and intratumoral CD8 infiltration (but not age, sex, or CD8+ lymphocytes localized to the tumor-stroma interface) were significant in both univariate and multivariate Cox regression analyses. Notably, traditional histologic identification of tumor-infiltrating lymphocytes was not a significant independent predictor of survival.

Conclusion: Intratumoral CD8+ lymphocyte infiltration can be readily assessed on paraffin-embedded tissue, is independently associated with improved MCC-specific survival, and therefore, may provide prognostic information that enhances established MCC staging protocols.

Fig 1.

Unbiased gene expression analysis reveals association between immune response and Merkel cell carcinoma (MCC) prognosis. Thirty-five tumors were analyzed from 34 patients who had been categorized by prognosis as described in Patients and Methods. Probes were grouped into six bins by the K means algorithm based on expression pattern. Cluster bin 3 displayed the expression pattern of interest (relatively highly expressed in good prognosis patients) and was further investigated through gene set enrichment analysis as indicated.

Fig 2.

CD8+ lymphocytic infiltration correlates with mRNA expression of CD8a. (A) Top row: hematoxylin and eosin (H/E) –stained sections; arrows indicate tumor-infiltrating lymphocytes. Bottom row: immunohistochemistry (IHC) for CD8 on corresponding serial sections. Peritumoral and intratumoral CD8+ lymphocytic infiltrates were each scored on a 0 to 5 scale (Data Supplement). (B) Correlation between CD8a mRNA expression and immunohistochemistry. Twenty samples had available archival materials.

Fig 3.

T-cell infiltration and Merkel cell carcinoma (MCC)–specific survival in an independent set of 146 patients. (A) Tumor infiltrating lymphocytes (TILs) analysis by routine histology among 129 patients. (*) TILs were prognostically significant on univariate (P = .03) but not multivariate (P = .12) analysis. (B) Intratumoral (IT) CD8+ lymphocyte infiltration. Brisk CD8s were defined as an intratumoral CD8 score of 3 to 5 (corresponding to approximately 60 or more CD8s per typical 40× high power field), sparse as 0 to 2. (†) IT CD8 infiltration was a statistically significant predictor of outcome on univariate (P < .01) and multivariate (P = .01) regression analyses (Table 3). (C) Subgroup breakdown of (B), by extent of disease at presentation (as indicated). Extent of disease at presentation was not known for two patients. Statistical analysis was not performed on subgroups; instead, multivariate Cox regression is listed in Table 3.