Background: Skin cancer, specifically cutaneous squamous cell carcinoma (cSCC), occurs 65-200 times more frequent in organ transplant recipients than in the general population. T cells, which are targeted by immunosuppressive drugs, are important in anti-tumor immune surveillance and it is known that their function is regulated by DNA methylation. Our previous study demonstrated higher DNA methylation of an intragenic region of SERPINB9 when comparing T cells from kidney transplant recipients before the occurrence of cSCC with T cells from recipients without cSCC. SERPINB9 codes for a serine protease inhibitor that inhibits Granzyme B and is actively transcribed in T cells. We hypothesized that high methylation of SERPINB9 is a stable trait and may affect the cytotoxic activity of T cells towards cSCC cells, thereby facilitating the development of cSCC.
Methods: We studied a cohort of kidney transplant recipients (n=30) with recurrent cSCC and recipients without cSCC (n=26) of whom PBMCs were available, general patient characteristics were not different between the two groups. The mean time after transplantation was 13 years (±9 years). The cSCC patients developed their first biopsy-proven cSCC 7 years (±6 years) after transplantation. Peripheral T cells were characterized by the following markers: CD3, CD4, CD8, CD45RO, CCR7, CD25 and CD127. DNA methylation of two regions within SERPINB9, both containing 6 CpG sites, was measured using pyrosequencing analysis.
Results: No significant differences were found for the distribution of naive and memory T cell subsets and T regulatory cells between the cSCC and non-cSCC patients after cell sorting. We analyzed DNA methylation of the isolated T cells per region, which is an average of the 6 CpG sites. Percentage DNA methylation of the first region within SERPINB9 was 59%(±14%) for the cSCC patients and 51%(±13%) for the non-cSCC patients (p=0.03). The second region showed DNA methylation percentages of 55%(±14%) for the cSCC patients and 48%(±13%) for the non-cSCC patients (p=0.03).
Conclusion: Our results demonstrate that high intragenic methylation of SERPINB9 in T cells, previously found before the clinical onset of cSCC, can be validated in a different cohort of kidney transplant recipients during recurrent cSCC. This suggests a functional role for SERPINB9 methylation in cSCC development, possibly impairing T cells in exerting their cytotoxic activity towards cSCC cells. We conclude that high SERPINB9 methylation is stable throughout cSCC development which is a novel insight in the pathogenesis of cSCC in kidney transplant recipients.