P1265: tmem244 gene expression as a potential blood diagnostic marker that can distinguish sézary syndrome from mycosis fungoides and benign erythroderma

Background: Sézary syndrome (SS) is a leukemic form of cutaneous T-cell lymphoma (CTCL) characterized by erythroderma, intractable pruritis, and the presence of circulating malignant CD4+ T cells called Sézary cells. The diagnosis of SS is challenging and requires the integration of clinical presentation, histopathologic evaluation, and molecular studies. The immunophenotype of Sézary cells is heterogeneous and often varies among patients. What’s more, SS can resemble inflammatory dermatoses as well as another CTCL type Mycosis Fungoides (MF). Therefore, there is a need for a relevant diagnostic tool, that will also allow sensitive disease monitoring and accurate assessment of treatment response. Aims: This study aimed to analyze and compare the expression of the TMEM244 gene in peripheral blood mononuclear cells (PBMC), CD4+ T-cells from peripheral blood, and lymphocytes from skin biopsies, from SS, MF, and benign erythroderma (E) patients. Moreover, TMEM244 expression was measured in different subpopulations of PBMC from healthy donors (HD). Methods:TMEM244 expression was evaluated in 18 SS, 5 MF, 8 E, and 44 HD samples. PBMC were separated from the whole blood by density gradient centrifugation and the CD4+ T-cells were separated using negative selection on magnetic beads. The skin biopsies were cut into 1 mm3 pieces, treated overnight with Collagenase P, and passed through nylon strainers (100-40 µm). The following subpopulations of PBMC from 6 HD: CD4, CD8, CD45RO, CD45RA, CD31, CD56, CD14, CD19 were separated by PE-conjugated antibody staining and positive selection on magnetic beads. RNA was extracted by TRI Reagent, reverse transcribed, and TMEM244 expression was analyzed by qRT-PCR. All samples were assayed in triplicates, the relative gene expression was calculated (2^-ΔCT). Statistical significance was determined with the unpaired t-test with Welch’s correction and the Mann–Whitney test. Results: Within the PBMC samples mean TMEM244 expression fold change in SS was 46.9 relative to HD and 52.24 relative to MF/E (p-values of < 0.0001 respectively) (Fig. 1A; 2A). The relative fold TMEM244 expression in CD4+ T cells was even higher than in the whole population of PBMC, in SS it was 359.2 relative to HD and 42.4 relative to MF/E (p-values of < 0.0001 respectively) (Fig. 1B; 2B). The experiment failed to show differential relative TMEM244 expression in SS skin samples compared to MF/E samples (p-values > 0.05). In blood cells, relative TMEM244 expression in T cells, both CD4+ (4.7E-04) and CD8+ (6.9E-04) was higher than in any other population of PBMC, including CD19+ B-cells (2.7E-05), CD56+ NK cells (1.2E-04), and CD14+ monocytes (8.8E-05). Moreover, the analysis of naïve CD45RA+ (5.2E-05) and memory CD45RO+ (3.8E-04) populations, as well as the youngest subset of naïve T cells CD31+ (7.6E-05) showed that the expression of TMEM244 is associated more with the later stages of T cell activation. Image:Summary/Conclusion: The analysis showed that elevated TMEM244 expression is only present in the blood cells of Sezary syndrome patients but not in healthy individuals. What is more, the expression of TMEM244 in the blood can differentiate Sezary syndrome patients from those suffering from benign erythroderma or other types of CTCL. Moreover, both CD4+ and CD45RO+ markers, a characteristic immunophenotype of Sezary cells, represented subpopulations with a higher basic expression of TMEM244, suggesting the involvement of this gene in the disease onset.