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Sézary syndrome (SS) is an aggressive leukemic variant of cutaneous T-cell lymphoma (CTCL) with a median life expectancy of less than 4 years. Although initial treatment responses are often good, the vast majority of patients with SS fail to respond to ongoing therapy. We hypothesize that malignant T cells are highly heterogeneous and harbor subpopulations of SS cells that are both sensitive and resistant to treatment. Here, we investigate the presence of single-cell heterogeneity and resistance to histone deacetylase inhibitors (HDACi) within primary malignant T cells from patients with SS. Using single-cell RNA sequencing and flow cytometry, we find that malignant T cells from all investigated patients with SS display a high degree of single-cell heterogeneity at both the mRNA and protein levels. We show that this heterogeneity divides the malignant cells into distinct subpopulations that can be isolated by their expression of different surface antigens. Finally, we show that treatment with HDACi (suberanilohydroxamic acid and romidepsin) selectively eliminates some subpopulations while leaving other subpopulations largely unaffected. In conclusion, we show that patients with SS display a high degree of single-cell heterogeneity within the malignant T-cell population, and that distinct subpopulations of malignant T cells carry HDACi resistance. Our data point to the importance of understanding the heterogeneous nature of malignant SS cells in each individual patient to design combinational and new therapies to counter drug resistance and treatment failure.

Purpose: Mycosis fungoides is one of the most common types of extranodal T-cell lymphomas, considered to be caused by malignant transformation of the mature T cells residing in the skin. However, some clinical observations such as the multifocal distribution of mycosis fungoides lesions or patterns of relapse after radiotherapy are not readily explainable by the mature T-cell origin theory.Experimental Design: We have performed a detailed analysis of T-cell receptor (TCR) rearrangements in single malignant cells and in biopsies from mycosis fungoides tumors composed of >80% of malignant cells using next-generation sequencing (NGS) to pinpoint the relationship between neoplastic cells in mycosis fungoides. We have also aimed to detect malignant, circulating T-cell by whole blood TCR sequencing.Results: We found a substantial clonal heterogeneity in the mycosis fungoides samples with regards to TCR, and we demonstrated that lymphoma cells harboring identical TCRg sequences may harbor different TCRa and b sequences. Lack of absolute TCRa, -b, -g monoclonality was further confirmed by TCR amplification and sequencing from microdissected lymphoma cells. We have also found the TCR rearrangements characteristic for lymphoma cells in patients' peripheral blood despite the lack of leukemic blood involvement; however, the circulating TCRg clonotype did not always represent the dominant cutaneous clonotype.Conclusions: These findings can be explained by a model where malignant transformation takes place during early T-cell development giving rise to circulating premalignant clones, which home to the skin producing clinically apparent lesions of cutaneous lymphoma. Therapeutic strategies in T-cell lymphoma should therefore target those early lymphoma precursor cells.

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Background:The pulsed dye laser is the treatment of choice for children with port-wine stains (PWSs). Evaluation of treatment outcome and adverse effects is traditionally based on subjective clinical scoring systems. We intend to objectify treatment results and adverse reactions after 1 treatment with the pulsed dye laser.Design: A before-and-after trial using skin reflectance to detect changes in skin redness and pigmentation, ultrasonography to evaluate changes in echostructure and skin thickness, and 3-dimensional surface contour analysis to detect changes in surface texture.Patients: Twelve children with PWSs.Setting: A university dermatological department. Results:The skin reflectance-determined change in skin redness correlated with the clinical response (r=0.46, PϽ.002). The percentage of reflectance-determined lightening was equal for pink, red, and dark red PWSs (median, 42.9%). Skin pigmentation increased after laser treat-ment (PϽ.007). Ultrasonography revealed lower dermal echogenicity of preoperative PWSs than of postoperative PWSs (PϽ.007) and healthy skin (PϽ.001). An increase in echogenicity reflected a decrease in the dermal water (blood) content. Variations were found in the dermal localization of the PWS. Skin thickness was significantly higher in the PWS before treatment than after (PϽ.001). The preoperative lesional thickness correlated inversely with the ultrasound-assessed treatment response (r=0.35, PϽ.04). The surface contour parameters decreased significantly after laser treatment, indicating a flattening of the skin surface. The contour changes correlated positively with treatment response. By clinical evaluation, no hypopigmentation or texture changes were detected. Conclusion:The evaluation of treatment outcome and adverse effects is refined by the use of skin reflectance, ultrasonographic, and surface contour analysis.

Brink Buus & Niels Ødum (2020) Staphylococcus aureus alpha-toxin inhibits CD8 + T cell-mediated killing of cancer cells in cutaneous T-cell

Clinical and histopathological data on the characteristics of patients with mycosis fungoides and Sézary syndrome in Denmark are limited. This retrospective study describes the epidemiological, clinical and histopathological features of 43 patients with mycosis fungoides and Sézary syndrome in the eastern part of Denmark during 1990 to 2016. Mean age and clinical stage at the time of diagnosis are in line with similar studies, but, surprisingly, 43% of the patients progressed to a higher disease stage. The risk of disease progression was higher for stage IB than IA. Diagnosis of mycosis fungoides andSézary syndrome can be very challenging. Clinical and histopathological data for patients with mycosis fungoides and Sézary syndrome in Denmark are limited. A retrospective study was performed in Region Zealand, Denmark from 1990 to 2016. A total of 43 patients with mycosis fungoides or Sézary syndrome were identified during the period. At the time of diagnosis the patients' mean age was 64.3 years and 74.5% had early-stage (≤IIA) disease. The mean time from onset of skin disease to diagnosis was 4.4 years. Surprisingly, 43% progressed to a higher disease stage, and risk of disease progression was higher for stage IB than IA (p = 0.01). All cases displayed some degree of epidermotropism and the infiltrates consisted of pleomorphic lymphocytes with a T-helper (CD4 + /CD8 -) phenotype. This study describes, for the first time, all aspects of clinical and histopathological findings in patients with mycosis fungoides and Sézary syndrome in a well-characterized Danish cohort.

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