Intron 4-5 hTERT DNA Hypermethylation in Merkel Cell Carcinoma: Frequency, Association with Other Clinico-pathological Features and Prognostic Relevance

Abstract

Merkel cell carcinoma (MCC) is an aggressive skin tumor with neuroendocrine differentiation, mainly affecting elderly population or immunocompromised individuals. As methylation of the human telomerase reverse transcriptase (mhTERT) has been shown to be a prognostic factor in different tumors, we investigated its role in MCC, in particular in intron 4-5 where rs10069690 has been mapped and recognized as a cancer susceptibility locus. DNA methylation analysis of hTERT gene was assessed retrospectively in a cohort of 69 MCC patients from the University of Bologna, University of Turin and University of Insubria. Overall mortality was evaluated with Kaplan-Meier curves and multivariable Royston-Parmar models. High levels of mhTERT (mhTERT_high) (HR = 2.500, p = 0.015) and p63 (HR = 2.659, p = 0.016) were the only two clinico-pathological features significantly associated with a higher overall mortality at the multivariate analysis. We did not find different levels of mhTERT between MCPyV (+) and (-) cases (21 vs 14, p = 0.554); furthermore, mhTERT_high was strongly associated with older age (80.5 vs 72 years, p = 0.026), no angioinvasion (40.7% vs 71.0%, p = 0.015), lower Ki67 (50 vs 70%, p = 0.005), and PD-L1 expressions in both tumor (0 vs 3%, p = 0.021) and immune cells (0 vs 10%, p = 0.002). mhTERT is a frequently involved epigenetic mechanism and a relevant prognostic factor in MCC. In addition, it belongs to the shared oncogenic pathways of MCC (MCPyV and UV-radiations) and it could be crucial, together with other epigenetic and genetic mechanisms as gene amplification, in determining the final levels of hTERT mRNA and telomerase activity in these patients.

Keywords: HTERT; HTERT intron 4–5; Merkel cell carcinoma; Merkel cell polyomavirus; Methylation; Rs10069690; Telomerase.

Fig. 1

The lollipop graph displays the percent methylation per CpG position for 10 randomly selected samples, 5 mhTERT_high cases, and 5 mhTERT_low ones, respectively. The color of the circles represents the percent methylation as shown in the color legend on the right side of the plot. On the lower x-axis, the genomic positions of the CpGs are displayed, the upper x-axis shows the summarized average percent methylation for this CpG position

Fig. 2

Kaplan-Meier survival curve shows a higher mortality in mhTERT_high group (log-rank test: t = 2.76, p = 0.097), with the curves diverging about 12 months after the surgery

Fig. 3

In the final multivariable RP model, mhTERT_high (p = 0.015) and p63 (p = 0.016) strongly influenced overall survival, whit borderline-significant associations for angioinvasion (p = 0.060) and absence of MCPyV (p = 0.056). Blue-dashed line: mhTERT_low or absence of examined parameter (p63, angioinvasion and MCPyV); red line: mhTERT_high or presence of examined parameter (p63, angioinvasion and MCPyV)