Mass Cytometric Analysis of Early-Stage Mycosis Fungoides.

Mycosis fungoides (MF) is the most common subtype of cutaneous T-cell lymphoma. Early-stage disease is characterized by superficial infiltrates of small- to medium-sized atypical epidermotropic T lymphocytes that are clonal related. Nevertheless, the percentage of atypical T cells is low with many admixed reactive immune cells. Despite earlier studies, the composition and spatial characteristics of the cutaneous lymphocytic infiltrate has been incompletely characterized. Here, we applied mass cytometry to profile the immune system in skin biopsies of patients with early-stage MF and in normal skin from healthy individuals. Single-cell suspensions were prepared and labeled with a 43-antibody panel, and data were acquired on a Helios mass cytometer. Unbiased hierarchical clustering of the data identified the major immune lineages and heterogeneity therein. This revealed patient-unique cell clusters in both the CD4+ and myeloid cell compartments but also phenotypically distinct cell clusters that were shared by most patients. To characterize the immune compartment in the tissue context, we developed a 36-antibody panel and performed imaging mass cytometry on MF skin tissue. This visualized the structure of MF skin and the distribution of CD4+ T cells, regulatory T cells, CD8+ T cells, malignant T cells, and various myeloid cell subsets. We observed clusters of CD4+ T cells and multiple types of dendritic cells (DCs) identified through differential expression of CD11c, CD1a, and CD1c in the dermis. These results indicated substantial heterogeneity in the composition of the local immune infiltrate but suggest a prominent role for clustered CD4-DC interactions in disease pathogenesis. Probably, the local inhibition of such interactions may constitute an efficient treatment modality.

Increased risk of cancer other than melanoma in CDKN2A founder mutation (p16-Leiden)-positive melanoma families.

PURPOSE: We report the largest study to date analyzing the risk of cancers other than melanoma in melanoma families positive for the same CDKN2A mutation. EXPERIMENTAL DESIGN: We studied family members of 22 families positive for the p16-Leiden founder mutation who had attended a surveillance clinic or were their close relatives. Within this cohort, observed and expected rates of cancer were computed by mutation status consisting of 221 (proven plus obligate) carriers, 639 (proven plus obligate) noncarriers, and 668 first-degree relatives whose carrier risk was estimated from the relationship to known carriers and the age and melanoma status of that person and their relatives. RESULTS: Our analysis shows a relative risk (RR) of cancer other than melanoma and nonmelanoma skin cancer of 4.4 [95% confidence interval (95% CI), 3.3-5.6], predominantly attributable to the increased risk for pancreatic cancer (RR, 46.6; 95% CI, 24.7-76.4), but also for other cancers. We provide substantial proof for pancreatic cancer being a key component of the p16-Leiden phenotype. Inclusion of this cancer in a penetrance analysis leads to an estimated RR of pancreatic cancer for mutation carriers of 47.8 (95% CI, 28.4-74.7). CONCLUSIONS: This study shows clear evidence of increased risk of cancers other than melanoma in CDKN2A families carrying the p16-Leiden mutation. Further research is necessary to determine if similar risks apply to families with CDKN2A mutations other than p16-Leiden.

Epigenetic inactivation of RASSF1a in uveal melanoma.

PURPOSE: The RAS association domain family 1 (RASSF1) gene is a tumor-suppressor gene located on chromosome 3p21.3. The alternative transcript (RASSF1a) has been shown to be inactivated by hypermethylation in several human malignancies, including breast, prostate, and lung cancer, and in cutaneous melanoma. The purpose of this study was to evaluate the methylation status of RASSF1a in human uveal melanoma. METHODS: The methylation status of the RASSF1a promoter region was analyzed using PCR in combination with melting curve analysis, sequencing, and restriction enzyme analysis. Eleven human uveal melanoma cell lines, normal melanocytes, 39 archival frozen tumor specimens, and a metastatic lesion of untreated primary uveal melanoma were studied. In addition, whether RASSF1a methylation correlates with patient survival and development of metastatic disease was investigated. RESULTS: RASSF1a promoter methylation was detected in 10 of the 11 (91%) cell lines, in 19 of the 38 (50%) patients with primary uveal melanoma and in the metastatic lesion. A positive correlation was found between RASSF1a promoter methylation and development of metastatic disease (P = 0.041). A correlation with disease-free survival could not be established, but a positive trend was observed (P = 0.063). CONCLUSIONS: These data show that RASSF1a methylation is a common epigenetic event in uveal melanoma development, potentially of clinical relevance. The presence of a methylated RASSF1a promoter region might therefore serve as a tumor marker and as a possible target for therapeutic intervention.

UVB-induced leucocyte trafficking in the epidermis of photosensitive lupus erythematosus patients: normal depletion of Langerhans cells.

BACKGROUND: The pathogenic mechanisms of UV-induced skin lesions of lupus erythematosus (LE) are unknown. In a recent study of pathogenic mechanisms of polymorphic light eruption (PLE), significantly more Langerhans cells (LCs) persisted in the epidermis after UVB overexposure than in healthy individuals. Interestingly, the same phenomenon was observed in one subacute cutaneous lupus erythematosus (SCLE) patient. It could therefore be hypothesized that both photodermatoses share a common pathogenic mechanism of photosensitivity. In the present study, we tested this hypothesis by investigating leucocyte trafficking in the initial phase of cutaneous LE after intense UVB exposure. METHODS: In 22 photosensitive LE patients (12 chronic discoid lupus erythematosus, seven systemic lupus erythematosus and three SCLE) and nine age/sex-matched controls, uninvolved buttock skin was exposed to six minimal erythemal dose (MED) UVB radiation. Subsequently, biopsies were taken after 24, 48 and 72 h, and one control biopsy was taken from unirradiated skin. Skin sections were stained for the presence of LCs, neutrophils and macrophages. Areal percentages of positively stained cells within the epidermis were quantified and compared between the patients and controls. RESULTS: A gradual decrease of epidermal LCs and a gradual increase of epidermal neutrophils and macrophages at several timepoints after six MED irradiation was observed equally in both LE patients and controls. CONCLUSION: Immunohistopathology of irradiated uninvolved skin of photosensitive LE patients did not reveal the same pathologic trafficking of LCs and neutrophils as described for PLE patients. We conclude that different mechanisms are operative in the pathogenesis of PLE and photosensitive LE.