Anatomic position determines oncogenic specificity in melanoma.

Oncogenic alterations to DNA are not transforming in all cellular contexts1,2. This may be due to pre-existing transcriptional programmes in the cell of origin. Here we define anatomic position as a major determinant of why cells respond to specific oncogenes. Cutaneous melanoma arises throughout the body, whereas the acral subtype arises on the palms of the hands, soles of the feet or under the nails3. We sequenced the DNA of cutaneous and acral melanomas from a large cohort of human patients and found a specific enrichment for BRAF mutations in cutaneous melanoma and enrichment for CRKL amplifications in acral melanoma. We modelled these changes in transgenic zebrafish models and found that CRKL-driven tumours formed predominantly in the fins of the fish. The fins are the evolutionary precursors to tetrapod limbs, indicating that melanocytes in these acral locations may be uniquely susceptible to CRKL. RNA profiling of these fin and limb melanocytes, when compared with body melanocytes, revealed a positional identity gene programme typified by posterior HOX13 genes. This positional gene programme synergized with CRKL to amplify insulin-like growth factor (IGF) signalling and drive tumours at acral sites. Abrogation of this CRKL-driven programme eliminated the anatomic specificity of acral melanoma. These data suggest that the anatomic position of the cell of origin endows it with a unique transcriptional state that makes it susceptible to only certain oncogenic insults.

Loss of 5-hydroxymethylcytosine is an epigenetic hallmark of melanoma.

DNA methylation at the 5 position of cytosine (5-mC) is a key epigenetic mark that is critical for various biological and pathological processes. 5-mC can be converted to 5-hydroxymethylcytosine (5-hmC) by the ten-eleven translocation (TET) family of DNA hydroxylases. Here, we report that "loss of 5-hmC" is an epigenetic hallmark of melanoma, with diagnostic and prognostic implications. Genome-wide mapping of 5-hmC reveals loss of the 5-hmC landscape in the melanoma epigenome. We show that downregulation of isocitrate dehydrogenase 2 (IDH2) and TET family enzymes is likely one of the mechanisms underlying 5-hmC loss in melanoma. Rebuilding the 5-hmC landscape in melanoma cells by reintroducing active TET2 or IDH2 suppresses melanoma growth and increases tumor-free survival in animal models. Thus, our study reveals a critical function of 5-hmC in melanoma development and directly links the IDH and TET activity-dependent epigenetic pathway to 5-hmC-mediated suppression of melanoma progression, suggesting a new strategy for epigenetic cancer therapy.

Undifferentiated and Dedifferentiated Metastatic Melanomas Masquerading as Soft Tissue Sarcomas: Mutational Signature Analysis and Immunotherapy Response.

The distinction between undifferentiated melanoma (UM) or dedifferentiated melanoma (DM) from undifferentiated or unclassifiable sarcoma can be difficult and requires the careful correlation of clinical, pathologic, and genomic findings. In this study, we examined the utility of mutational signatures to identify patients with UM/DM with particular attention as to whether this distinction matters for treatment because the survival of patients with metastatic melanoma has dramatically improved with immunologic therapy, whereas durable responses are less frequent in sarcomas. We identified 19 cases of UM/DM that were initially reported as unclassified or undifferentiated malignant neoplasm or sarcoma and submitted for targeted next-generation sequencing analysis. These cases were confirmed as UM/DM by harboring melanoma driver mutations, UV signature, and high tumor mutation burden. One case of DM showed melanoma in situ. Meanwhile, 18 cases represented metastatic UM/DM. Eleven patients had a prior history of melanoma. Thirteen of 19 (68%) of the tumors were immunohistochemically completely negative for 4 melanocytic markers (S100, SOX10, HMB45, and MELAN-A). All cases harbored a dominant UV signature. Frequent driver mutations involved BRAF (26%), NRAS (32%), and NF1 (42%). In contrast, the control cohort of undifferentiated pleomorphic sarcomas (UPS) of deep soft tissue exhibited a dominant aging signature in 46.6% (7/15) without evidence of UV signature. The median tumor mutation burden for DM/UM vs UPS was 31.5 vs 7.0 mutations/Mb (P < .001). A favorable response to immune checkpoint inhibitor therapy was observed in 66.6% (12/18) of patients with UM/DM. Eight patients exhibited a complete response and were alive with no evidence of disease at the last follow-up (median 45.5 months). Our findings support the usefulness of the UV signature in discriminating DM/UM vs UPS. Furthermore, we present evidence suggesting that patients with DM/UM and UV signatures can benefit from immune checkpoint inhibitor therapy.

Efficacy of immunotherapy with combination of cryotherapy and topical imiquimod for treatment of Kaposi sarcoma.

Multiple treatment modalities for Kaposi sarcoma (KS) have been reported, including chemotherapy, radiation therapy, surgical excision, electrochemotherapy, and cryotherapy. Common topical treatments include timolol, imiquimod, and alitretinoin. We searched our institutional database for patients with ICD-9 or 10 codes for KS seen by a dermatologist with experience in KS management from July 1, 2004 to January 1, 2022. We screened patient charts to include patients who received combination therapy of cryotherapy followed by topical imiquimod three times a week for 2 months (n = 9). Patients were followed in the clinic every 3 months. Time to resolution was assessed by photographic evidence of resolution as determined by a dermatologist and corroborated with clinical documentation in patient charts. Median age (IQR) at KS diagnosis was 58 (27.5) years. All patients were male (n = 9, 100%). Majority were white (n = 7, 78%) and non-Hispanic (n = 8, 89%). Five (56%) had classic KS, one (11%) had HIV-associated KS, and three (33%) were HIV-negative men who have sex with men. Median time to resolution was 30.5 weeks, with a median of two treatments. In our study, 93% (n = 42/45) of lesions and 89% (n = 8/9) of patients experienced complete resolution during a median (range) duration of follow-up of 58 (13-209) weeks. Side effects were limited to pain during cryotherapy, occasional blister formation after cryotherapy, and mild inflammation due to imiquimod. No infections were observed. Combination therapy of cryotherapy and topical imiquimod may be an efficacious and comparatively low-risk treatment for limited, cutaneous KS.

Autophagy gene Atg16L1 prevents lethal T cell alloreactivity mediated by dendritic cells.

Atg16L1 mediates the cellular degradative process of autophagy and is considered a critical regulator of inflammation based on its genetic association with inflammatory bowel disease. Here we find that Atg16L1 deficiency leads to an exacerbated graft-versus-host disease (GVHD) in a mouse model of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Atg16L1-deficient allo-HSCT recipients with GVHD displayed increased T cell proliferation due to increased dendritic cell (DC) numbers and costimulatory molecule expression. Reduced autophagy within DCs was associated with lysosomal abnormalities and decreased amounts of A20, a negative regulator of DC activation. These results broaden the function of Atg16L1 and the autophagy pathway to include a role in limiting a DC-mediated response during inflammatory disease, such as GVHD.

Lentigo maligna melanoma mapping using reflectance confocal microscopy correlates with staged excision: A prospective study.

BACKGROUND: Lentigo maligna/lentigo maligna melanoma (LM/LMM) can present with subclinical extension that may be difficult to define preoperatively and lead to incomplete excision and potential recurrence. Preliminarily studies have used reflectance confocal microscopy (RCM) to assess LM/LMM margins. OBJECTIVE: To evaluate the correlation of LM/LMM subclinical extension defined by RCM compared with the gold standard histopathology. METHODS: Prospective study of LM/LMM patients referred for dermatologic surgery. RCM was performed at the clinically defined initial surgical margin followed by margin-controlled staged excision with paraffin-embedded tissue, and histopathology was correlated with RCM results. RESULTS: Seventy-two patients were included. Mean age was 66.8 years (standard deviation, 11.1; range, 38-89); 69.4% were men. Seventy of 72 lesions (97.2%) were located on the head and neck with mean largest clinical diameter of 1.3 cm (range, 0.3-5). Diagnostic accuracy for detection of residual melanoma in the tumor debulk (after biopsy) had a sensitivity of 96.7% and a specificity of 66.7% when compared with histopathology. RCM margin assessment revealed an overall agreement with final histopathology of 85.9% (κ = 0.71; P < .001). LIMITATIONS: No RCM imaging beyond initial planned margins was performed. CONCLUSION: RCM showed moderate to excellent overall agreement between RCM imaging of LM/LMM and histopathology of staged excision margins.

Immunohistochemistry for PRAME in Dermatopathology.

ABSTRACT: Preferentially expressed antigen in melanoma (PRAME) is a tumor-associated antigen first identified in a melanoma patient and found to be expressed in most melanomas as well as in variable levels in other malignant neoplasms of epithelial, mesenchymal, or hematolymphoid lineage. Detection of PRAME expression in formalin-fixed paraffin-embedded tissue is possible by immunohistochemistry (IHC) with commercially available monoclonal antibodies. In situ and invasive melanoma frequently show a diffuse pattern of nuclear PRAME immunoreactivity which contrasts with the infrequent and typically nondiffuse staining seen in nevi. In many challenging melanocytic tumors, results of PRAME IHC and other ancillary tests correlate well, but not always: The tests are not interchangeable. Most metastatic melanomas are positive for PRAME, whereas nodal nevi are not. Numerous studies on PRAME IHC have become available in the past few years with results supporting the value of PRAME IHC as an ancillary tool in the evaluation of melanocytic lesions and providing insights into limitations in sensitivity and specificity as well as possible pitfalls that need to be kept in mind by practicing pathologists.

Treatment of Extramammary Paget Disease and the Role of Reflectance Confocal Microscopy: A Prospective Study.

BACKGROUND: Extramammary Paget disease (EMPD) poses treatment challenges. Invasive and noninvasive treatment modalities exist with variable success reported. Reflectance confocal microscopy (RCM) is emerging as an adjuvant diagnostic tool. OBJECTIVE: To evaluate the treatment of EMPD patients and the role of RCM. METHODS: Prospective study. Demographic and tumor characteristics were recorded. Handheld-RCM was performed and correlated with histology. Treatment, clearance, pathology, and follow-up were all recorded. RESULTS: Thirty-six EMPD lesions in 33 patients were included. Mean age was 71.7 years, and 23 were men. Mean number of surgical stages needed to clear margins was 1.9 (SD, 0.9; 1.0-3.0 stages), and mean margin needed to clear was 1.8 cm. Reflectance confocal microscopy correlated well with scouting punch biopsies (kappa, 0.93; p < .001). Disruption of the dermoepidermal junction was associated with invasive EMPD versus in situ (83.3% vs 25.9%) on histology (p = .01). LIMITATIONS: Relatively small sample size. CONCLUSION: Extramammary Paget disease is challenging, and lesion demarcation is of the utmost importance. Using a staged surgical excision approach, the mean margins needed were 1.8 cm, less than previously reported. Nonsurgical modalities, including radiation therapy, imiquimod, or photodynamic therapy can be considered if surgery is not pursued. Reflectance confocal microscopy is a valuable noninvasive imaging modality for the management of EMPD.

Eosinophilic Fasciitis Following Checkpoint Inhibitor Therapy: Four Cases and a Review of Literature.

BACKGROUND: Checkpoint inhibitor therapy is widely known to cause a number of immune-related adverse events. One rare adverse effect that is emerging is eosinophilic fasciitis, a fibrosing disorder causing inflammatory infiltration of subcutaneous fascia. It is characterized clinically by edema and subsequent induration and tightening of the skin and subcutaneous tissues. The condition is rare, yet at our institutions we have seen four cases in the past 3 years. We describe our 4 cases and review 11 other cases reported in the literature. CASE PRESENTATION: We present four cases of eosinophilic fasciitis following treatment with programmed cell death protein 1 or programmed cell death-ligand 1 blockade. All patients had extremity involvement with characteristic skin changes ranging from peripheral edema to induration, tightening, and joint limitation. The patients had varying degrees of peripheral eosinophilia. In two of our patients, the diagnosis was made by full-thickness skin biopsy showing lymphocytic infiltration of the subcutaneous fascia, with CD4+ T cells predominating in one case and CD8+ T cells in the other. In the other two cases, the diagnosis was made on the basis of characteristic imaging findings in the context of clinical features consistent with the diagnosis. All four patients were treated with glucocorticoids with varying degrees of success; immunotherapy had to be discontinued in all four. Patients with advanced melanoma who experienced this adverse effect had either a partial response or a complete response to therapy. CONCLUSION: Eosinophilic fasciitis can occur as a result of checkpoint inhibitor therapy. Although a tissue diagnosis is the gold standard, imaging studies may facilitate the diagnosis in the presence of consistent clinical features, but a degree of suspicion is key to recognizing the condition early. Therapy requires a collaborative approach by oncology, rheumatology, and dermatology; physical therapy is an important adjunct in treatment. For advanced melanoma, it may be a good prognostic indicator. IMPLICATIONS FOR PRACTICE: It is important for clinicians to recognize that eosinophilic fasciitis is a potential immune-related adverse event (irAE) as a consequence of immune checkpoint inhibitor therapy. The presentation is quite stereotypical; the diagnosis can be made by imaging in the absence of a full-thickness skin biopsy. Early intervention is important to limit morbidity. This irAE may be a good prognostic sign among patients with melanoma.

Nrf2 regulates CD4+ T cell-induced acute graft-versus-host disease in mice.

Nuclear factor erythroid-derived 2-like 2 (Nrf2) is a ubiquitously expressed transcription factor that is well known for its role in regulating the cellular redox pathway. Although there is mounting evidence suggesting a critical role for Nrf2 in hematopoietic stem cells and innate leukocytes, little is known about its involvement in T-cell biology. In this study, we identified a novel role for Nrf2 in regulating alloreactive T-cell function during allogeneic hematopoietic cell transplantation (allo-HCT). We observed increased expression and nuclear translocation of Nrf2 upon T-cell activation in vitro, especially in CD4+ donor T cells after allo-HCT. Allo-HCT recipients of Nrf2 -/- donor T cells had significantly less acute graft-versus-host disease (GVHD)-induced mortality, morbidity, and pathology. This reduction in GVHD was associated with the persistence of Helios+ donor regulatory T cells in the allograft, as well as defective upregulation of the gut-homing receptor LPAM-1 on alloreactive CD8+ T cells. Additionally, Nrf2 -/- donor CD8+ T cells demonstrated intact cytotoxicity against allogeneic target cells. Tumor-bearing allo-HCT recipients of Nrf2 -/- donor T cells had overall improved survival as a result of preserved graft-versus-tumor activity and reduced GVHD activity. Our findings characterized a previously unrecognized role for Nrf2 in T-cell function, as well as revealed a novel therapeutic target to improve the outcomes of allo-HCT.

Cutaneous Legionella infections in allogeneic hematopoietic cell transplantation recipients.

To date, only twenty cases of cutaneous legionellosis have been reported. Cutaneous legionellosis has heterogeneous manifestations including abscesses, nodules, and cellulitis. The detection of most cutaneous Legionella species requires specific diagnostic cultures and assays. Herein, we report a case of cutaneous legionella in a hematopoietic cell transplantation recipient with culture-negative nodules unresponsive to empiric antibiotics. We also discuss the varied morphology of cutaneous legionellosis and important diagnostic considerations.

Cabozantinib in Patients with Advanced Merkel Cell Carcinoma.

BACKGROUND: This study sought to determine the efficacy and safety profile of cabozantinib in patients with advanced Merkel cell carcinoma (MCC). EXPERIMENTAL DESIGN: This prospective, phase II, single-institution trial enrolled patients with platinum-failure, recurrent/metastatic MCC to receive cabozantinib 60 mg orally daily until disease progression, withdrawal from study, or severe toxicity. The primary endpoint was disease control rate. Secondary endpoints included overall survival (OS), progression-free survival (PFS), and toxicity. Immunohistochemistry for VEGFR-2, MET, and HGF expression and next-generation sequencing of tumor tissue were performed and correlated with outcome. RESULTS: Eight patients were accrued from January 24, 2014, to June 8, 2016. The study was closed prematurely because of toxicity and lack of responses. The most frequent adverse events were grades 1 and 2 and included anorexia, fatigue, nausea, hypothyroidism, and dysgeusia. Two patients developed nonhealing, painful ulcers and tumor-skin fistula. One patient had stable disease for 8 months. One patient withdrew from the study after 2 weeks of therapy because of adverse events. Three patients required dose reduction because of toxicity. Median PFS and OS were 2.1 and 11.2 months, respectively. No expression of MET, HGF, or VEGFR-2 was identified in tumor cells by immunohistochemistry of patients' tissue samples. CONCLUSION: Cabozantinib was poorly tolerated and did not demonstrate activity in patients with recurrent/metastatic, platinum-failure MCC. It is unclear whether preselection of patients with the specific upregulation or genetic alteration in the targets for cabozantinib would have changed the results of this study. (Clinical trial identification number: NCT02036476) IMPLICATIONS FOR PRACTICE: This phase II study demonstrated poor tolerability and lack of activity of cabozantinib in an unselected group of patients with advanced Merkel cell carcinoma. Although it is unclear whether preselection of patients with the specific upregulation and genetic alterations in targets for cabozantinib would have changed the results of this study, this would have likely led to an extremely rare patient population that would take many years to accrue.

Sox10 and DOG1 Expression in Primary Adnexal Tumors of the Skin.

Primary skin adnexal tumors can be challenging to classify and must be discerned from cutaneous adenocarcinoma metastases from various sites. We evaluated expression of Sox10 and DOG1 in normal cutaneous adnexa and in 194 primary skin adnexal tumors, and compared their performance in discriminating primary skin adnexal tumors from cutaneous metastatic adenocarcinomas with that of p40 and p63. In normal skin adnexa, we noted Sox10 expression in both the secretory and myoepithelial cells in eccrine glands, but only in myoepithelial cells in apocrine glands. DOG1 demonstrated canalicular expression in eccrine glands, and weak expression in myoepithelial cells of apocrine glands, germinative cells of sebaceous glands, and outer root sheath of follicular infundibulum. Sox10 was expressed in 100% of cylindromas and spiradenomas, and in variable frequency in other benign and malignant tumors of sweat glands. DOG1 was positive in most cylindromas (87.5%), in only 10.5% of spiradenomas, and was variably expressed in other benign and malignant tumors of sweat glands. All syringomas (n = 20) were negative for Sox10 and DOG1. One out of the 33 follicular neoplasms was positive for Sox10 and DOG1 (3%). All sebaceous neoplasms were negative for Sox10, and 28.1% of them were positive for DOG1. Sox10 was specific (91.9%) but not sensitive (28.4%) for primary skin origin, and was far less accurate (38.5%) than p63 or p40 (95.5% accuracy). Combining Sox10 with p63 or p40 showed only very minimal gain in accuracy (96%). DOG1 expression in tumors showed low sensitivity and specificity for skin adnexal origin.

Mouse mast cell proteases 4 and 5 mediate epidermal injury through disruption of tight junctions.

We previously established a mast cell (MC)-dependent thermal injury model in mice with ulceration and scar formation that depended on nonredundant functions of mouse MC protease (mMCP)4 and mMCP5. We hypothesized that MC activation is an early event and now find by histology that exocytosis of granule contents occurred by 2 min after thermal injury in wild-type (WT) C57BL/6 mice and in the mMCP4- or mMCP5-deficient mice. The degranulation was equivalent for MCs in the dermis and hypodermis of all three strains, but only the WT mice showed an appreciable increase in epidermal thickness. There was no loss of total MCs, partially degranulated plus intact, during the 4 h of observation. By electron microscopy, MCs in all strains showed early zonal degranulation at 30 s with marked progression in magnitude by 120 s and no mitochondrial injury or cellular necrosis. Concomitantly there was an increase in intercellular spaces indicative of tight junction (TJ) disruption in WT mice but not in the mMCP4- or mMCP5-deficient strains. The desmosomes were intact in all strains. Immunodetection of the TJ protein claudin 4 in WT and mMCP5-deficient mice indicated a significant reduction after scald injury whereas mMCP4(-/-) mice showed no significant changes. Taken together, these findings reveal that a second-degree burn injury can initiate an immediate novel zonal degranulation of MCs throughout all skin layers and a disruption of the epidermal TJs dependent on the nonredundant presence of mMCP4 and mMCP5.

Melanoma spheroid formation involves laminin-associated vasculogenic mimicry.

Melanoma is a tumor where virulence is conferred on transition from flat (radial) to three-dimensional (tumorigenic) growth. Virulence of tumorigenic growth is governed by numerous attributes, including presence of self-renewing stem-like cells and related formation of patterned networks associated with the melanoma mitogen, laminin, a phenomenon known as vasculogenic mimicry. Vasculogenic mimicry is posited to contribute to melanoma perfusion and nutrition in vivo; we hypothesized that it may also play a role in stem cell-driven spheroid formation in vitro. Using a model of melanoma in vitro tumorigenesis, laminin-associated networks developed in association with three-dimensional melanoma spheroids. Real-time PCR analysis of laminin subunits showed that spheroids formed from anchorage-independent melanoma cells expressed increased α4 and ß1 laminin chains and α4 laminin expression was confirmed by in situ hybridization. Association of laminin networks with melanoma stem cell-associated nestin and vascular endothelial growth factor receptor-1 also was documented. Moreover, knockdown of nestin gene expression impaired laminin expression and network formation within spheroids. Laminin networks were remarkably similar to those observed in melanoma xenografts in mice and to those seen in patient melanomas. These data indicate that vasculogenic mimicry-like laminin networks, in addition to their genesis in vivo, are integral to the extracellular architecture of melanoma spheroids in vitro, where they may serve as stimulatory scaffolds to support three-dimensional growth.

Clinicopathological features and histogenesis of penile cysts.

Cysts arising in the penis are uncommon and can be found anywhere from the urethral meatus to the root of the penis involving glans, foreskin, or shaft. Median raphe cysts account for the majority of penile cystic lesions reported in the literature. As their name suggests, they arise on the ventral midline of the penis that extends from the urethral meatus to the scrotum and perineum. Proposed hypotheses for their origin as well as their diverse morphology are discussed.

HPV- and non-HPV-related subtypes of penile squamous cell carcinoma (SCC): Morphological features and differential diagnosis according to the new WHO classification (2015).

The majority of penile carcinomas are squamous cell carcinomas originating in the squamous mucosa covering the glans, coronal sulcus, or inner surface of the foreskin, the 3 latter sites comprising the penile anatomical compartments. There is a variegated spectrum of subtypes of penile squamous cell carcinomas according to recent classification schemes. Currently, because of etiological and prognostic considerations, 2 morphologically and molecularly distinctive groups of subtypes of penile SCCs based on the presence of HPV were delineated. The predominant cell composition of tumors associated with HPV is the basaloid cell, which is the hallmark and best tissue marker for the virus. Tumors negative for the virus, however, are preferentially of lower grade and keratinizing maturing neoplasms with the exception of sarcomatoid carcinoma. HPV is detected in research studies by PCR or in situ hybridization (ISH) technologies, but p16 immunohistochemical stain is an adequate and less-expensive surrogate that is useful in the routine practice of pathology. The aim of this review is to demonstrate the variable morphological phenotypic expression of penile tumors separating non-HPV- and HPV-related neoplasms and to add morphological information that will justify subclassifying squamous cell carcinomas in a number of special subtypes. A brief discussion of the differential diagnosis in each category is also provided.

Merkel cell carcinoma expresses vasculogenic mimicry: demonstration in patients and experimental manipulation in xenografts.

Merkel cell carcinoma (MCC) is a highly virulent cutaneous neoplasm that, like melanoma, is a frequent cause of patient morbidity and mortality. The cellular mechanisms responsible for the aggressive behavior of MCC remain unknown. Vasculogenic mimicry (VM) is a phenomenon associated with cancer virulence, including in melanoma, whereby anastomosing laminin networks form in association with tumor cells that express certain endothelial genes. To determine whether VM is a factor in MCC, we employed a relevant xenograft model using two independent human MCC lines. Experimentally induced tumors were remarkably similar histologically to patient MCC, and both contained laminin networks associated with vascular endothelial-cadherin (CD144) and vascular endothelial growth factor receptor 1, as well as Nodal expression typical of VM in melanoma. Moreover, two established chemotherapeutic agents utilized for human MCC, etoposide and carboplatin, induced necrosis in xenografts on systemic administration while enriching for laminin networks in apparently resistant viable tumor regions that persisted. These findings for the first time establish VM-like laminin networks as a biomarker in MCC, demonstrate the experimental utility of the MCC xenograft model, and suggest that VM-rich regions of MCC may be refractory to conventional chemotherapeutic agents.