High Expression of Sonic Hedgehog Pathway Mediators in Mycosis Fungoides Skin Samples: A Retrospective Analysis

Description

Mycosis Fungoides (MF) is characterized by clonal proliferation of CD4+ lymphocytes within the skin. Recent studies report dysregulation of the Sonic Hedgehog (Shh) pathway expressed in neoplastic cells of primary T-cell lymphomas. We aim to assess the expression of Ptch-1, Bcl-2, Smo, and Gli-1, mediators in Shh pathway, in cutaneous biopsies of MF. Biopsies were retrospectively collected at the University of Miami Hospital between 01/2018 and 12/2021, including samples of clinically proven MF, healthy controls (HC), and basal cell carcinoma (BCC) as positive control. Immunohistochemistry was performed using commercially available antibodies, and two-way ANOVA and Tukey’s multiple comparisons test were used to assess statistical significance. 35 biopsy samples, including 19 MF, 12 HC, and 4 BCC were analyzed, and revealed significantly higher expression of Smo, Gli-1, and Bcl-2 in MF biopsies compared to HC, as quantified by the number of positive cells/mm2.Due to the elevated expression of Shh mediators found in MF samples, larger studies are necessary to assess the potential role of the Shh pathway in MF pathogenesis. The data contains our supplemental images for Bcl-2 and Ptch-1 immunohistochemistry staining of cutaneous biopsies of MF, HC, and basal cell carcinoma. Figure Legends: Supplemental Figure 1. 1a) Bcl-2 expression was significantly elevated compared to healthy controls. The results were heterogenous among samples, ranging from no expression to as high as 1,500 cells/mm2 1b) Expression was primarily localized to the stratum basale layer of the epidermis in MF samples, leaving the dermis relatively spared. Very few cells stained positive for Bcl-2 among BCC or HC samples, with no statistical significance. Supplemental Fig 2. 2a) Ptch-1 expression in MF biopsies was not statistically significant compared to controls. 2b) Ptch-1 was localized sparingly within the papillary dermis and epidermis in MF biopsies. Ptch-1 expression was seen predominantly within the basaloid cells of the basal cell carcinoma (positive controls).

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We used retrospectively collected human biopsies from clinically validated cases of MF, retrieved from the specimen bank at the University of Miami Hospital/Jackson Memorial Hospital. Specimens were obtained from consenting patients receiving standard care, with protocol approved by the University of Miami Institutional Review Board between January 2018 and December 2021. Biopsy-proven MF was correlated to clinical examinations to improve accuracy. Healthy controls (HC) were identified as free margins of previously excised non-melanoma skin cancers due to their closest resemblance to normal skin architecture. MF specimens were age- and sex- matched to negative controls. Positive controls were designated as basal cell carcinomas due to the known involvement of the Shh pathway in its pathogenesis. Formalin-fixed and paraffin-embedded cutaneous tissue was cut into 5–7μm sections using a microtome. Sections were deparaffinized with xylene (EMD Millipore) and rehydrated in graded ethanol. Endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide in methanol and washed with distilled water. Slides were then incubated in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween-20, pH 6.0) for 30 minutes at 95°C for antigen retrieval, allowed to cool, and then treated with Background Punisher (Biocare Medical). Antibodies used were polyclonal rabbit anti-Gli (1:200, Abcam, number Ab217326), polyclonal rabbit anti-PTCH1 (1:200, Abcam, number Ab53715), polyclonal rabbit anti-Smoothened (1:100, Abcam, number Ab235183), and monoclonal rabbit anti–Bcl2 (1:200, Abcam, number Ab32124). Antibodies were diluted in 2% normal goat serum (Sigma-Aldrich) in PBS with 0.1% Tween-20 and applied to the samples for overnight incubation at 4 °C. Detection and chromogenic reaction were carried out using the Universal anti-rabbit HRP-Polymer Detection system (Biocare Medical), following the manufacturer’s instructions. Slides were counterstained using Harris hematoxylin (Leica Microsystems) and then dehydrated in graded ethanol and xylene. Bioimage analysis was performed using QuPath bioimage analysis software. Images were imported into QuPath, and regions of interest were annotated using polygon features within the software, focusing on dermal cells for Gli-1, Ptch-1, Smo, Bcl-2. Analysis of positive staining was determined using positive cell detection commands within the software quantified as DAB+ cells over hematoxylin-stained nuclei (Figure 1 shows sample quantification). For each biopsy, 3 independent sections were immunostained and quantified, resulting in the analysis of approximately 2,000 to 10,000 cells per section. The number of positive cells and area detected were used to calculate the percentage of positive cells/mm2, which were then exported to GraphPad Prism for statistical analysis. Two-way ANOVA, followed by Tukey’s multiple comparisons test, was then used to assess statistical significance.

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