JAK/STAT Inhibition Normalizes Lipid Composition in 3D Human Epidermal Equivalents Challenged with Th2 Cytokines.

Derangement of the epidermal barrier lipids and dysregulated immune responses are key pathogenic features of atopic dermatitis (AD). The Th2-type cytokines interleukin IL-4 and IL-13 play a prominent role in AD by activating the Janus Kinase/Signal Transduction and Activator of Transcription (JAK/STAT) intracellular signaling axis. This study aimed to investigate the role of JAK/STAT in the lipid perturbations induced by Th2 signaling in 3D epidermal equivalents. Tofacitinib, a low-molecular-mass JAK inhibitor, was used to screen for JAK/STAT-mediated deregulation of lipid metabolism. Th2 cytokines decreased the expression of elongases 1, 3, and 4 and serine-palmitoyl-transferase and increased that of sphingolipid delta(4)-desaturase and carbonic anhydrase 2. Th2 cytokines inhibited the synthesis of palmitoleic acid and caused depletion of triglycerides, in association with altered phosphatidylcholine profiles and fatty acid (FA) metabolism. Overall, the ceramide profiles were minimally affected. Except for most sphingolipids and very-long-chain FAs, the effects of Th2 on lipid pathways were reversed by co-treatment with tofacitinib. An increase in the mRNA levels of CPT1A and ACAT1, reduced by tofacitinib, suggests that Th2 cytokines promote FA beta-oxidation. In conclusion, pharmacological inhibition of JAK/STAT activation prevents the lipid disruption caused by the halted homeostasis of FA metabolism.

Stenotrophomonas maltophilia skin infection in an immunocompetent patient: Primary cutaneous CD30+ T-cell lymphoproliferative disorder or pseudolymphoma?

Cutaneous pseudolymphomas are a wide group of diseases mimicking cutaneous lymphoma. They comprise several skin conditions with different etiopathogenesis, clinical-pathological features, and prognosis, which may occur in the absence of an identifiable trigger factor or after administration of medications or vaccinations, tattoos, infections, or arthropod bites. They present with different manifestations: from solitary to regionally clustered lesions, up to generalized distribution and, in rare cases, erythroderma. They persist variably, from weeks to years, and resolve spontaneously or after antibiotics, but may recur in some cases. CD30+ T-cell pseudolymphomas are characterized by the presence of large, activated lymphoid cells, generally in response to viral infections, arthropod assault reactions, and drug eruptions. Stenotrophomonas maltophilia is a ubiquitous Gram-negative bacillus responsible for opportunistic infections in immunocompromised patients. Infection of intact skin in immunocompetent patients is particularly rare. Here, we report a case of a man presenting an isolated nodule histopathologically mimicking a primary cutaneous CD30+ T-cell lymphoproliferative disorder.

Dermoscopic, Histological, Confocal Microscopy Correlation of Atypical-Dysplastic Melanocytic Nevi.

INTRODUCTION: The term "atypical melanocytic nevus" (AMN) is used as a synonym for dysplastic nevus (DN) in clinical practice. Although the criteria for diagnosis of AMN/DN by the Agency for Research on Cancer helps to differentiate AMN/DN from common acquired nevi, they do not have high degrees of specificity, as they are similar to those used for the diagnosis of melanoma. OBJECTIVES: In this retrospective study we evaluated the correlation and diagnostic concordance of dermoscopy, confocal microscopy, and histological examination in 50 AMN. METHODS: A graded scale was used to compare histological examination with dermoscopy and confocal microscopy. Low magnification histological images of only the central part of lesions were examined. This allowed histological diagnoses based almost exclusively on architectural criteria instead of simultaneously architectural and cytological, as in the global histological examination. RESULTS: Our data demonstrate that the diagnostic accuracy of dermoscopy and confocal microscopy diagnosis of the clinical aspects of AMN/DN as nevi or melanomas tends to be equivalent, being fair for nevi and excellent for melanomas. The total percentage of AMN suggested that the accuracy of confocal microscopy in the diagnosis of melanoma (86.7%) is greater than that of dermoscopy (73.3%). CONCLUSIONS: This study demonstrated that diagnostic assessments of AMN/DN by dermoscopy and confocal microscopy are accurate and often coincide with those of histological examination and that their combined use helps to better manage and monitor these patients by facilitating early detection of melanomas and reducing unnecessary excisions of benign melanocytic lesions.

Molecular Profile of Subungual Melanoma: A MelaNostrum Consortium Study of 68 Cases Reporting BRAF, NRAS, KIT, and TERT Promoter Status.

BACKGROUND: Subungual melanoma (SM) is an unusual type of melanocytic tumor affecting the nail apparatus. The mutational prevalence of the most prominently mutated genes in melanoma has been reported in small cohorts of SM, with unclear conclusions on whether SM is different from the rest of melanomas arising in acral locations or not. Hence, the molecular profile of a large series of SM is yet to be described. OBJECTIVES: The aim of this study was to describe the molecular characteristics of a large series of SM and their association with demographic and histopathological features. METHODS: Patients diagnosed with SM between 2001 and 2021 were identified from six Spanish and Italian healthcare centers. The mutational status for BRAF, NRAS, KIT, and the promoter region of TERT (TERTp) were determined either by Sanger sequencing or next-generation sequencing. Clinical data were retrieved from the hospital databases to elucidate potential associations. RESULTS: A total of 68 SM cases were included. Mutations were most common in BRAF (10.3%) and KIT (10%), followed by NRAS (7.6%), and TERTp (3.8%). Their prevalence was similar to that of non-subungual acral melanoma but higher in SM located on the hand than on the foot. CONCLUSIONS: To date, this study represents the largest cohort of SM patients with data on the known driver gene mutations. The low mutation rate supports a different etiopathogenic mechanism for SM in comparison of non-acral cutaneous melanoma, particularly for SM of the foot.

Targeting Fatty Acid Amide Hydrolase Counteracts the Epithelial-to-Mesenchymal Transition in Keratinocyte-Derived Tumors.

The endocannabinoid system regulates physiological processes, and the modulation of endogenous endocannabinoid (eCB) levels is an attractive tool to contrast the development of pathological skin conditions including cancers. Inhibiting FAAH (fatty acid amide hydrolase), the degradation enzyme of the endocannabinoid anandamide (AEA) leads to the increase in AEA levels, thus enhancing its biological effects. Here, we evaluated the anticancer property of the FAAH inhibitor URB597, investigating its potential to counteract epithelial-to-mesenchymal transition (EMT), a process crucially involved in tumor progression. The effects of the compound were determined in primary human keratinocytes, ex vivo skin explants, and the squamous carcinoma cell line A431. Our results demonstrate that URB597 is able to hinder the EMT process by downregulating mesenchymal markers and reducing migratory potential. These effects are associated with the dampening of the AKT/STAT3 signal pathways and reduced release of pro-inflammatory cytokines and tumorigenic lipid species. The ability of URB597 to contrast the EMT process provides insight into effective approaches that may also include the use of FAAH inhibitors for the treatment of skin cancers.

Bleomycin Electrochemotherapy of Dermal Cylindroma as an Alternative Treatment in a Rare Adnexal Neoplasm: A Case Report and Literature Review.

BACKGROUND: Brooke-Spiegler syndrome is a rare autosomal dominant disorder characterized by the continuous development of multiple benign skin appendage tumors. It is treated usually by repeated standard surgery. Here, we present a case study where electrochemotherapy (ECT) with bleomycin was used as an effective alternative approach in treating advanced dermal cylindromatosis of the head and neck in a patient with Brooke-Spiegler syndrome. PATIENTS AND METHODS: A 45-year-old woman presented with multiple recurrent dermal cylindroma lesions on her scalp. Previous treatment consisted of several surgical excisions that resulted in psychological deterioration due to the formation of numerous scars and extensive alopecic areas. ECT was offered to provide tumor removal and disease control and to improve the patient's quality of life. RESULTS: The treatment was well tolerated, and a significant reduction in neoplastic tissue was achieved. Importantly, scalp skin condition significantly improved, regaining a fair follicular density on the margins. CONCLUSION: This report suggests the feasibility of bleomycin ECT as a less invasive alternative option for controlling multiple scalp cylindroma lesions with cosmetically acceptable results, and improving quality of life.

A Microenvironment-Related Nine-Gene Signature May Predict Survival in Mycosis Fungoides Patients at Diagnosis.

Mycosis fungoides (MF) is the most common cutaneous lymphoma characterized by an indolent course. Prognosis is stage-based but this approach does not reflect the different outcomes within stages. Considering that tumor microenvironment is known to be involved in MF pathogenesis and progression, we decided to investigate 99 MF cases by using the PanCancer Immune Profiling Panel. We identified and validated a signature of 9 genes able to predict MF survival and distinguish a high-risk group with a worse outcome from a low-risk group of cases with a better outcome. At the molecular level, low-risk vs. high-risk cases reported a global upregulation of immune genes, enriched in cytokines, and a higher density of dendritic cells and mast cells, possibly associated with a more favorable clinical course.

Clinical and Pathological Spectrum of Prurigo Pigmentosa in Central European Individuals.

Importance: Based on early studies, prurigo pigmentosa (PP) was considered a rare inflammatory dermatosis affecting primarily Asian individuals. However, several case reports subsequently showed that the disease is not restricted to those of Asian origin. Large studies on PP in central European individuals, on the other hand, are missing. Objective: To increase awareness of PP by describing the clinical, histopathological, and immunohistochemical features in central European individuals. Design, Setting, and Participants: This observational, retrospective case series analyzed clinicopathological features of 20 central European patients diagnosed with PP. Data collection was performed by means of archive material, including physician's letters, clinical photographs, and histopathological records, at the Department of Dermatology at the Medical University of Graz in Austria from January 1998 to January 2022. Main Outcomes and Measures: Demographic, clinical, histopathological, and immunohistochemical characteristics for patients diagnosed with PP were recorded. Results: Of the 20 patients included, 15 (75%) were female, and the mean (range) age was 24.1 (15-51) years. The study cohort consisted entirely of European patients. The most common site of involvement of PP was the breast, followed by the neck and back. Other involved clinical sites were the abdomen, shoulders, face, head, axillae, arms, and genital region and groin. Clinically, lesions were characterized by a symmetric pattern in 90% (n = 18) of all cases. Marked hyperpigmentation was observed only in 25% (n = 5) of patients. In some cases, triggers such as malnutrition, long-term pressure, and friction were noted. Histologic findings revealed presence of neutrophils in all cases and necrotic keratinocytes in 67% (n = 16) of cases. Immunohistochemistry results showed predominance of CD8+ lymphocytes in the epidermis, as well as the presence of plasmacytoid dendritic cells and myeloid cell nuclear differentiation antigen-positive neutrophil precursors. Conclusions and Relevance: This case series found that most clinical features observed in Asian patients were also observed in central European patients, but hyperpigmentation was primarily mild to moderate. Histopathological features were similar to those reported in the literature with the additional presence of myeloid cell nuclear differentiation antigen-positive precursor neutrophils. These results expand previous knowledge about PP in central European individuals.

The Activation of PPARγ by (2Z,4E,6E)-2-methoxyocta-2,4,6-trienoic Acid Counteracts the Epithelial-Mesenchymal Transition Process in Skin Carcinogenesis.

Cutaneous squamous cell carcinoma (cSCC) is the most common UV-induced keratinocyte-derived cancer, and its progression is characterized by the epithelial-mesenchymal transition (EMT) process. We previously demonstrated that PPARγ activation by 2,4,6-octatrienoic acid (Octa) prevents cutaneous UV damage. We investigated the possible role of the PPARγ activators Octa and the new compound (2Z,4E,6E)-2-methoxyocta-2,4,6-trienoic acid (A02) in targeting keratinocyte-derived skin cancer. Like Octa, A02 exerted a protective effect against UVB-induced oxidative stress and DNA damage in NHKs. In the squamous cell carcinoma A431 cells, A02 inhibited cell proliferation and increased differentiation markers' expression. Moreover, Octa and even more A02 counteracted the TGF-ß1-dependent increase in mesenchymal markers, intracellular ROS, the activation of EMT-related signal transduction pathways, and cells' migratory capacity. Both compounds, especially A02, counterbalanced the TGF-ß1-induced cell membrane lipid remodeling and the release of bioactive lipids involved in EMT. In vivo experiments on a murine model useful to study cell proliferation in adult animals showed the reduction of areas characterized by active cell proliferation in response to A02 topical treatment. In conclusion, targeting PPARγ may be useful for the prevention and treatment of keratinocyte-derived skin cancer.

Alpha, Beta, and Gamma Human Papillomaviruses in Genital Lichen Sclerosus: A Retrospective Cross-Sectional Study.

BACKGROUND: Lichen sclerosus (LS) is an inflammatory disease mostly arising at the genital level. It is unclear whether human papillomaviruses (HPVs) have an etiological significance in LS, and data on their prevalence in patients with LS are controversial. OBJECTIVES: The authors assessed alpha, beta, and gamma HPV prevalence in patients with genital LS. The association of HPV positivity with demographic and clinical factors was also investigated. METHODS: One hundred thirty-two formalin-fixed, paraffin-embedded LS samples (2016-2020) were retrieved from the archives of a pathology department. Alpha HPVs were genotyped with the INNO-LiPA HPV Genotyping Extra II kit. Beta and gamma HPVs were searched by multiplex Polymerase Chain Reaction. Immunostaining for p16 INK4a was performed on high-risk HPV-positive samples. RESULTS: Patients had a median age of 61 years, were mostly women ( n = 73, 55.3%), and with an early disease stage ( n = 79, 59.8%). Alpha HPVs were detected in 12/132 cases (9.1%). Among the 5 high-risk HPV-positive cases, only 2 displayed a strong and diffuse p16 INK4a staining. Beta genus was the most prevalent (35/132, 26.5%) and HPV5 was the most frequent beta genotype (25/132, 18.9%). There were 3 gamma HPV-positive cases among those with a valid result (3/131, 2.3%). Multiple infections with genotypes belonging to different genera were infrequent (3/131, 2.3%). No significant differences in the prevalence of the individual genera were observed according to sex and disease stage. CONCLUSIONS: Of the 3 HPV genera, beta genus showed the highest prevalence. Further research is needed to clarify whether the presence of beta HPVs in genital LS has a clinical significance.

Nonvisualized sentinel node on preoperative lymphoscintigraphy in primary cutaneous melanoma: an 11-year retrospective survey.

BACKGROUND: Sentinel lymph node (SLN) biopsy in cutaneous melanoma patients evaluates the regional draining basin for occult micrometastatic disease. Occasionally, nonidentification of SLN impairs the acquisition of this important prognostic factor. OBJECTIVES: To investigate the outcomes of melanoma patients with negative lymphoscintigraphic findings and patients who underwent SLN biopsy from 2004 to 2015 ( n = 1200) were retrospectively reviewed for tumor characteristics and clinical outcomes. METHODS: Patients with nonvisualized lymph nodes (NV group) who underwent only preoperative lymphoscintigraphy were separated and compared with a cohort drawn from all melanoma patients who completed the surgical procedure within the same period (V group). RESULTS: A negative lymphoscintigraphic scan was observed in 38 cases (3.2% of all patients). The NV group showed a significantly older age (median 66.0 vs. 48.3 years; P < 0.0001). Head and neck melanomas were more frequent in the NV group compared to the control group (25.1 vs. 7.8%; P = 0.009). Tumor characteristics such as ulceration and Breslow thickness do not influence the lymphoscintigraphy result. No differences were found in overall survival (OS) and disease-free survival (DFS) between the groups. CONCLUSIONS: The nonvisualization of regional lymph nodes by lymphoscintigraphy is more frequent in older patients with head and neck melanomas. From the clinical point of view, no specific recommendation emerged for patients' management because the nonvisualization of the SLN did not show a significant influence on DFS and OS rates. However, lack of knowledge of lymph node status suggests performing a tighter follow-up eventually by ultrasound evaluation of all potential lymph node drainage basins.

Prevalence of 13 polyomaviruses in actinic keratosis and matched healthy skin samples of immunocompetent individuals.

BACKGROUND: Actinic keratosis (AK) is a precursor of cutaneous squamous cell carcinoma (cSCC). UV radiation is the major risk factor for AK, but certain human papillomaviruses (HPVs) of the beta genus are also involved in its development. Differently, the role of polyomaviruses (PyVs) in skin carcinogenesis is still debated. Fiftheen PyVs have been isolated from human tissues so far, including Merkel cell polyomavirus (MCPyV), the aetiological agent of Merkel cell carcinoma. METHODS: The presence of 13 PyVs was assessed in skin samples from AK patients (n = 342). Matched fresh-frozen scrapings from healthy skin (HS) and AK lesions from 242 patients, and formalin-fixed paraffin-embedded AK biopsies from a different cohort of 100 patients were analyzed by multiplex PyVs genotyping assay. RESULTS: The most frequent lesion site was the scalp in men (27.3%), and the cheek area in women (29.0%). Differences between men and women were significant for the scalp, the cheek area and the lips. Almost all the scrapings were PyV-positive (HS: 89.7%, AK: 94.6%; p = 0.04). The three most frequent PyVs were MCPyV, HPyV6 and JCPyV (HS: 87.2%, 58.7%, 6.6%, respectively; AK: 88.8%, 51.2%, 9.9%, respectively). HPyV9, TSPyV, BKPyV, HPyV7, LIPyV and SV40 were detected in < 2% of the scrapings. In most cases, matched HS and AK scrapings were both positive (MCPyV: 78.1%, HPyV6: 41.7%), or both negative for the individual genotypes (for the remaining PyVs). PyV prevalence in AK biopsies was 22.0%. Only MCPyV (21.0%) and HPyV6 (3.0%) were detected in these samples. CONCLUSIONS: PyV prevalence in HS and AK scrapings was high, but detection of PyVs exclusively in AK scrapings was rare. PyV positivity rate in AK biopsies was modest. Further research is need to reach firm conclusions regarding the role of these viruses in AK development.

Bcl-2-like protein-10 increases aggressive features of melanoma cells.

Aim: B-cell lymphoma-2 (Bcl-2)-like protein-10 (Bcl2L10) is the less studied member of Bcl-2 family proteins, with the controversial role in different cancer histotypes. Very recently, Bcl2L10 expression in melanoma tumor specimens and its role in melanoma response to therapy have been demonstrated. Here, the involvement of Bcl2L10 on the in vitro and in vivo properties associated with melanoma aggressive features has been investigated. Methods: Endogenous Bcl2L10 protein expression was detected by western blotting analysis in a panel of patient-derived and commercially available human melanoma cells. In vitro assays to evaluate clonogenicity, cell proliferation, cell migration, cell invasion, and in vitro capillary-like structure formation [vasculogenic mimicry (VM)] have been performed by using human melanoma cells stably overexpressing Bcl2L10 or transiently transfected for loss/gain function of Bcl2L10, grown under two- or three-dimensional (3D) conditions Xenograft melanoma model was employed to evaluate in vivo tumor growth and angiogenesis. Results: Results demonstrated that Bcl2L10 acts as an inducer of in vitro cell migration, invasion, and VM, while in vitro cell proliferation, in vivo tumor growth, as well as colony formation properties were not affected. Dissecting different signaling pathways, it was found that Bcl2L10 positively affects the phosphorylation of extracellular-signal-regulated kinase (ERK) and the expression of markers of cell invasion, such as urokinase plasminogen activator receptor (uPAR) and matrix metalloproteinases (MMPs). Of note, Bcl2L10-dependent in vitro migration, invasion, and VM are linked to uPAR. Bcl2L10 also negatively regulates the intracellular calcium level. Finally, reduced invasion capability in 3D spheroid invasion assay of melanoma cells transiently overexpressing Bcl2L10 was observed after treatment with inhibitors of MMPs and uPAR. Conclusions: Overall, data reported in this paper provide evidence supporting a positive role of Bcl2L10 in melanoma aggressive features.

Altered epidermal proliferation, differentiation, and lipid composition: Novel key elements in the vitiligo puzzle.

Vitiligo is an acquired skin depigmentation disease involving multiple pathogenetic mechanisms, which ultimately direct cytotoxic CD8+ cells to destroy melanocytes. Abnormalities have been described in several cells even in pigmented skin as an expression of a functional inherited defect. Keratinocytes regulate skin homeostasis by the assembly of a proper skin barrier and releasing and responding to cytokines and growth factors. Alterations in epidermal proliferation, differentiation, and lipid composition as triggers for immune response activation in vitiligo have not yet been investigated. By applying cellular and lipidomic approaches, we revealed a deregulated keratinocyte differentiation with altered lipid composition, associated with impaired energy metabolism and increased glycolytic enzyme expression. Vitiligo keratinocytes secreted inflammatory mediators, which further increased following mild mechanical stress, thus evidencing immune activation. These findings identify intrinsic alterations of the nonlesional epidermis, which can be the prime instigator of the local inflammatory milieu that stimulates immune responses targeting melanocytes.

NTRK Gene Fusion Detection in Atypical Spitz Tumors.

Atypical Spitz tumors (AST) deviate from stereotypical Spitz nevi for one or more atypical features and are now regarded as an intermediate category of melanocytic tumors with uncertain malignant potential. Activating NTRK1/NTRK3 fusions elicit oncogenic events in Spitz lesions and are targetable with kinase inhibitors. However, their prevalence among ASTs and the optimal approach for their detection is yet to be determined. A series of 180 ASTs were screened with pan-TRK immunohistochemistry and the presence of NTRK fusions was confirmed using FISH, two different RNA-based NGS panels for solid tumors, and a specific real time RT-PCR panel. Overall, 26 ASTs showed pan-TRK immunostaining. NTRK1 fusions were detected in 15 of these cases showing cytoplasmic immunoreaction, whereas NTRK3 was detected in one case showing nuclear immunoreaction. Molecular tests resulted all positive in only two ASTs (included the NTRK3 translocated), RNA-based NGS and real time RT-PCR were both positive in three cases, and FISH and real time RT-PCR in another two cases. In seven ASTs NTRK1 fusions were detected only by FISH and in two cases only by real time RT-PCR. The frequency of NTRK fusions in ASTs is 9%, with a clear prevalence of NTRK1 compared to NTRK3 alterations. Pan-TRK immunohistochemistry is an excellent screening test. Confirmation of NTRK fusions may require the use of different molecular techniques.

Evaluation of Hedgehog Pathway Inhibition on Nevoid Basal Cell Carcinoma Syndrome Fibroblasts and Basal Cell Carcinoma-Associated Fibroblasts: Are Vismodegib and Sonidegib Useful to Target Cancer-Prone Fibroblasts?

Activating mutations in the Hh pathway underlies the development of sporadic and familial skin BCC. For these oncogenic proliferations displaying ligand-independent activation of the intracellular pathway, two molecules have been approved for therapeutic purposes: vismodegib and sonidegib. Improper Hh signalling occurs in many human tumours also via a paracrine mechanism (ligand-dependent) in which the secretion of Hh ligands by stromal cells support tumour growth. On the other hand, the mobilization of neoplastic stroma by cancer cells is sustained by the activation of Hh signalling in surrounding fibroblasts suggesting a central role of this bidirectional crosstalk in carcinogenesis. Additionally, loss-of-function mutations in the PTCH1 gene in the context of NBCCS, an autosomal dominant disorder predisposing to multiple BCCs, determine tumour permissive phenotypes in dermal fibroblasts. Here, profiling syndromic and BCC-associated fibroblasts unveiled an extraordinary similarity characterized by overexpression of several Hh target genes and a marked pro-inflammatory outline. Both cell types exposed to Hh inhibitors displayed reversion of the tumour-prone phenotype. Under vismodegib and sonidegib treatment, the Wnt/ß-catenin pathway, frequently over-active in tumour stroma, resulted down-regulated by pAKT-GSK3ß axis and consequent increase of ß-catenin turnover. Overall, this study demonstrated that vismodegib and sonidegib impacting on fibroblast tumour supportive functions might be considered in therapy for BCC independently to the mutation status of Hh components in neoplastic cells.