Sebaceous tumours: more than skin deep.

CLINICAL PRESENTATION: A 77-year-old man presented to our skin cancer centre with various cutaneous tumours occurring in 2006-2017. Histopathology showed a 'hidradenocarcinoma' on the left upper back (2006) and a sebaceous adenoma (figure 1) on the left shoulder (2011). In 2017, he developed a sebaceous carcinoma on the middle upper back, which manifested as a slowly enlarging, asymptomatic nodule. Medical history was significant for curative resection of colorectal cancer in 1988.gutjnl;67/11/1957/F1F1F1Figure 1Clinical appearance of the sebaceous adenoma on the patient's left shoulder in 2011.The most recent lesion was subjected to extensive immunohistochemical assessment. The neoplastic cells were positive for cytokeratin 5/6, cytokeratin 7, cluster of differentiation antigen 10, adipophilin, androgen receptor, epithelial membrane antigen, KI67 antigen, MLH1 and PMS2, but stained negative for gross cystic disease fluid protein 15, prostate-specific antigen, carbohydrate antigen 19/9, CDX2 protein, hepatocyte-specific antigen, carcinoembryonic antigen, cluster of differentiation antigen 117 and cytokeratin 19. Given the variety of histological manifestations of the patient's skin neoplasms, further studies were performed. They revealed positive nuclear expression signals for MLH1, MSH6 and PMS2, whereas MSH2 expression was absent in almost all tumour cells (figure 2). Positron emission tomography (PET)/CT and colonoscopy did not detect any pathological findings. However, molecular genetic analysis of peripheral blood showed a heterozygous deletion of exon 7 of the MSH2 gene. Subsequently, several family members tested positive for MSH2 mutations and underwent genetic counselling.gutjnl;67/11/1957/F2F2F2Figure 2(A-D) Histopathological images of the patient's most recent lesion (diaminobenzidine, original magnification, ×100). The tumour cells demonstrated strong nuclear positivity for MLH1 (A) and PMS2 (B), but were essentially negative for MSH6 (C) and MSH2 (D). QUESTION: What is your diagnosis? DIAGNOSIS: Muir-Torre syndrome (MTS).

SF3B1 and BAP1 mutations in blue nevus-like melanoma.

Blue nevi are melanocytic tumors originating in the cutaneous dermis. Malignant tumors may arise in association with or resembling blue nevi, so called 'blue nevus-like melanoma', which can metastasize and result in patient death. Identifying which tumors will behave in a clinically aggressive manner can be challenging. Identifying genetic alterations in such tumors may assist in their diagnosis and prognostication. Blue nevi are known to be genetically related to uveal melanomas (eg, both harboring GNAQ and GNA11 mutations). In this study, we analyzed a large cohort (n=301) of various morphologic variants of blue nevi and related tumors including tumors diagnosed as atypical blue nevi (n=21), and blue nevus-like melanoma (n=12), screening for all gene mutations known to occur in uveal melanoma. Similar to published reports, we found the majority of blue nevi harbored activating mutations in GNAQ (53%) or GNA11 (15%). In addition, rare CYSLTR2 (1%) and PLCB4 (1%) mutations were identified. EIF1AX, SF3B1, and BAP1 mutations were also detected, with BAP1 and SF3B1 R625 mutations being present only in clearly malignant tumors (17% (n=2) and 25% (n=3) of blue nevus-like melanoma, respectively). In sequencing data from a larger cohort of cutaneous melanomas, this genetic profile was also identified in tumors not originally diagnosed as blue nevus-like melanoma. Our findings suggest that the genetic profile of coexistent GNAQ or GNA11 mutations with BAP1 or SF3B1 mutations can aid the histopathological diagnosis of blue nevus-like melanoma and distinguish blue nevus-like melanoma from conventional epidermal-derived melanomas. Future studies will need to further elucidate the prognostic implications and appropriate clinical management for patients with tumors harboring these mutation profiles.

Angioplasmacellular hyperplasia - a new histopathologic clue for anogenital herpes simplex recidivans in immunocompromised patients?

Chronic herpes infections in immunocompromised patients exhibit uncommon features both on clinical and histopathologic levels, which can make correct diagnosis challenging. Better defining histopathologic criteria to identify chronic herpes infection in immunocompromised patients would be of great diagnostic value. In a single-center study, clinical and pathological data of herpes infections confirmed by biopsy was collected. We identified 42 cases overall, of which 21 were from immunocompromised patients and performed a detailed histopathologic pattern analysis of all lesions. In immunocompromised patients, vasculitis was seen in 2/21 cases (9.5%). Involvement of the sweat duct epithelium and/or sebaceous glands was observed in none of the HIV-infected patients but in 5/11 (45.4%) transplant patients with herpes infection. This feature was solely found in patients with acute herpetic lesions (5/5). In the process of histopathologic review, we identified a previously unrecognized morphological pattern of herpes simplex virus infection in immunocompromised patients. The individual clinical history and morphological pattern identified is described for multiple affected patients. In summary, in immunocompromised patients, histopathologic diagnosis of herpes infection can be challenging, in particular in chronic lesions, which lack the presentation of typical herpetic keratinocytes. In our study, we identify angioplasmacellular hyperplasia as a new histopathologic clue, which may be helpful in recognizing chronic herpes infection in immunocompromised patients. Further studies are warranted to implement this clue into routine diagnostics.

pHH3 immunostaining improves interobserver agreement of mitotic index in thin melanomas.

According to the seventh edition of the American Joint Committee on Cancer guidelines, the TNM staging category in thin cutaneous melanomas depends on the mitotic rate (MR). In this study, we analyze the interobserver agreement of the MR in a series of 92 thin cutaneous melanomas. Serial sections of the tumors were either stained with hematoxylin and eosin or immunohistochemically stained with pHH3, an antibody for phosphohistone H3, and analyzed by 4 observers. Determination of MR with pHH3 immunostaining resulted in higher sensitivity in counting mitosis for all observers. Moreover, interobserver agreement was higher with pHH3. Immunostaining with pHH3 is a sensitive method to detect mitosis in thin cutaneous melanomas, with good reproducibility of MR between independent observers. Further studies are needed to find out if higher sensitivity in the detection of mitosis by pHH3 immunostaining has additional prognostic relevance.

Deep Learning Assisted Diagnosis of Onychomycosis on Whole-Slide Images.

BACKGROUND: Onychomycosis numbers among the most common fungal infections in humans affecting finger- or toenails. Histology remains a frequently applied screening technique to diagnose onychomycosis. Screening slides for fungal elements can be time-consuming for pathologists, and sensitivity in cases with low amounts of fungi remains a concern. Convolutional neural networks (CNNs) have revolutionized image classification in recent years. The goal of our project was to evaluate if a U-NET-based segmentation approach as a subcategory of CNNs can be applied to detect fungal elements on digitized histologic sections of human nail specimens and to compare it with the performance of 11 board-certified dermatopathologists. METHODS: In total, 664 corresponding H&E- and PAS-stained histologic whole-slide images (WSIs) of human nail plates from four different laboratories were digitized. Histologic structures were manually annotated. A U-NET image segmentation model was trained for binary segmentation on the dataset generated by annotated slides. RESULTS: The U-NET algorithm detected 90.5% of WSIs with fungi, demonstrating a comparable sensitivity with that of the 11 board-certified dermatopathologists (sensitivity of 89.2%). CONCLUSIONS: Our results demonstrate that machine-learning-based algorithms applied to real-world clinical cases can produce comparable sensitivities to human pathologists. Our established U-NET may be used as a supportive diagnostic tool to preselect possible slides with fungal elements. Slides where fungal elements are indicated by our U-NET should be reevaluated by the pathologist to confirm or refute the diagnosis of onychomycosis.

High-resolution three-dimensional imaging for precise staging in melanoma.

INTRODUCTION: Many cancer guidelines include sentinel lymph node (SLN) staging to identify microscopic metastatic disease. Current SLN analysis of melanoma patients is effective but has the substantial drawback that only a small representative portion of the node is sampled, whereas most of the tissue is discarded. This might explain the high clinical false-negative rate of current SLN diagnosis in melanoma. Furthermore, the quantitative assessment of metastatic load and microanatomical localisation might yield prognosis with higher precision. Thus, methods to analyse entire SLNs with cellular resolution apart from tedious sequential physical sectioning are required. PATIENTS AND METHODS: Eleven melanoma patients eligible to undergo SLN biopsy were included in this prospective study. SLNs were fixed, optically cleared, whole-mount stained and imaged using light sheet fluorescence microscopy (LSFM). Subsequently, compatible and unbiased gold standard histopathological assessment allowed regular patient staging. This enabled intrasample comparison of LSFM and histological findings. In addition, the development of an algorithm, RAYhance, enabled easy-to-handle display of LSFM data in a browsable histologic slide-like fashion. RESULTS: We comprehensively quantify total tumour volume while simultaneously visualising cellular and anatomical hallmarks of the associated SLN architecture. In a first-in-human study of 21 SLN of melanoma patients, LSFM not only confirmed all metastases identified by routine histopathological assessment but also additionally revealed metastases not detected by routine histology alone. This already led to additional therapeutic options for one patient. CONCLUSION: Our three-dimensional digital pathology approach can increase sensitivity and accuracy of SLN metastasis detection and potentially alleviate the need for conventional histopathological assessment in the future. GERMAN CLINICAL TRIALS REGISTER: (DRKS00015737).

Role of Tumor-Infiltrating B Cells in Clinical Outcome of Patients with Melanoma Treated With Dabrafenib Plus Trametinib.

PURPOSE: Although patients with unresectable or metastatic melanoma can experience long-term survival with BRAF- and MEK-targeted agents or immune checkpoint inhibitors over 5 years, resistance develops in most patients. There is a distinct lack of pretherapeutic biomarkers to identify which patients are likely to benefit from each therapy type. Most research has focused on the predictive role of T cells in antitumor responses as opposed to B cells. PATIENTS AND METHODS: We conducted prespecified exploratory biomarker analysis using gene expression profiling and digital pathology in 146 patients with previously untreated BRAF V600-mutant metastatic melanoma from the randomized, phase III COMBI-v trial and treated with dabrafenib plus trametinib who had available tumor specimens from screening. RESULTS: Baseline cell-cycle gene expression signature was associated with progression-free survival (P = 0.007). Patients with high T-cell/low B-cell gene signatures had improved median overall survival (not reached [95% confidence interval (CI), 33.8 months-not reached]) compared with patients with high T-cell/high B-cell signatures (19.1 months; 95% CI, 13.4-38.6 months). Patients with high B-cell signatures had high B-cell infiltration into the tumor compartment, corresponding with decreased MAPK activity and increased expression of immunosuppressive markers. CONCLUSIONS: B cells may serve as a potential biomarker to predict clinical outcome in patients with advanced melanoma treated with dabrafenib plus trametinib. As separate studies have shown an opposite effect for B-cell levels and response to immunotherapy, B cells may serve as a potential biomarker to facilitate treatment selection. Further validation in a larger patient cohort is needed.

Localization of PD-L1 on single cancer cells by iSERS microscopy with Au/Au core/satellite nanoparticles.

Programmed cell death-ligand 1 (PD-L1) is an important predictive biomarker. The detection of PD-L1 can be crucial for patients with advanced cancer where the use of immunotherapy is considered. Here, we demonstrate the use of immuno-SERS microscopy (iSERS) for localizing PD-L1 on single cancer SkBr-3 cells. A central advantage of iSERS is that the disturbing autofluorescence from cells and tissues can be efficiently minimized by red to near-infrared laser excitation. In this study we employed Au/Au core/satellite nanoparticles as SERS nanotags because of their remarkable signal brightness and colloidal stability upon red laser excitation. False-color iSERS images of the positive and negative controls clearly reveal the specific localization of PD-L1 with SERS nanotag-labeled antibodies.

Macrophages/Microglia Represent the Major Source of Indolamine 2,3-Dioxygenase Expression in Melanoma Metastases of the Brain.

The manifestation of brain metastases in patients with advanced melanoma is a common event that limits patient's survival and quality of life. The immunosuppressive properties of the brain parenchyma are very different compared to the rest of the body, making it plausible that the current success of cancer immunotherapies is specifically limited here. In melanoma brain metastases, the reciprocal interplay between immunosuppressive mediators such as indoleamine 2, 3-dioxygenase (IDO) or programmed cell death-ligand 1 (PD-L1) in the context of neoplastic transformation are far from being understood. Therefore, we analyzed the immunoreactive infiltrate (CD45, CD3, CD8, Forkhead box P3 [FoxP3], CD11c, CD23, CD123, CD68, Allograft Inflammatory factor 1[AIF-1]) and PD-L1 with respect to IDO expression and localization in melanoma brain metastases but also in matched metastases at extracranial sites to correlate intra- and interpatient data with therapy response and survival. Comparative tissue analysis identified macrophages/microglia as the major source of IDO expression in melanoma brain metastases. In contrast to the tumor infiltrating lymphocytes, melanoma cells per se exhibited low IDO expression levels paralleled by cell surface presentation of PD-L1 in intracranial metastases. Absolute numbers and pattern of IDO-expressing cells in metastases of the brain correlated with recruitment and localization of CD8+ T cells, implicating dynamic impact on the regulation of T cell function in the brain parenchyma. However, paired analysis of matched intra- and extracranial metastases identified significantly lower fractions of cytotoxic CD8+ T cells in intracranial metastases while all other immune cell populations remain unchanged. In line with the already established clinical benefit for PD-L1 expression in extracranial melanoma metastases, Kaplan-Meier analyses correlated PD-L1 expression in brain metastases with favorable outcome in advanced melanoma patients undergoing immune checkpoint therapy. In summary, our data provide new insights into the landscape of immunosuppressive factors in melanoma brain metastases that may be useful in the implication of novel therapeutic strategies for patients undergoing cancer immunotherapy.

Association of toll-interacting protein gene polymorphisms with atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a common inflammatory skin disorder, affecting up to 15% of children in industrialized countries. Toll-interacting protein (TOLLIP) is an inhibitory adaptor protein within the toll-like receptor (TLR) pathway, a part of the innate immune system that recognizes structurally conserved molecular patterns of microbial pathogens, leading to an inflammatory immune response. METHODS: In order to detect a possible role of TOLLIP variation in the pathogenesis of AD, we screened the entire coding sequence of the TOLLIP gene by SSCP in 50 AD patients. We identified an amino acid exchange in exon 6 (Ala222Ser) and a synonymous variation in exon 4 (Pro139Pro). Subsequently, these two variations and four additional non-coding polymorphisms (-526 C/G, two polymorphisms in intron 1 and one in the 3'UTR) were genotyped in 317 AD patients and 224 healthy controls. RESULTS: The -526G allele showed borderline association with AD in our cohort (p = 0.012; significance level after correction for multiple testing 0.0102). Haplotype analysis did not yield additional information. Evaluation of mRNA expression by quantitative real-time polymerase chain reaction in six probands with the CC and six with the GG genotype at the -526 C/G locus did not reveal significant differences between genotypes. CONCLUSION: Variation in the TOLLIP gene may play a role in the pathogenesis of AD. Yet, replication studies in other cohorts and populations are warranted to confirm these association results.

Melanoma diagnosed in lesions previously treated by laser therapy.

Laser therapy has become a routine procedure in dermatological practice and is frequently also used for pigmented lesions. Few reports exist of melanomas diagnosed in lesions previously treated by laser therapy. Between 2007 and 2014, we identified 11 patients who presented to our department with a melanoma diagnosed in a region previously treated by laser therapy. The course of events until the diagnosis of melanoma was assessed as well as patient outcome including treatment for disease progression. No histological assessment had been performed prior to laser therapy in nine of 11 (82%) cases. Benign melanocytic lesions had been diagnosed by biopsy prior to laser therapy in the other two cases. Time from laser therapy to diagnosis of melanoma ranged from less than 1 to 10 years. Stage of disease at diagnosis varied from stage IA to IIIC. Four patients progressed to stage IV disease, of whom at least one died of melanoma. We conclude that laser treatment of pigmented lesions can complicate the diagnosis of melanoma and lead to diagnosis delay with potentially fatal consequences. Considering this risk, we believe laser therapy for pigmented lesions should either be avoided entirely or at a minimum performed only after prior histological assessment.

Frequent GNAQ, GNA11, and EIF1AX Mutations in Iris Melanoma.

Purpose: The most common malignant intraocular tumors with a high mortality in adults are uveal melanomas. Uveal melanomas arise most frequently in the choroid or ciliary body (97%) and rarely in the iris (3%). Whereas conjunctival and posterior uveal (ciliary body and choroidal) melanomas have been studied in more detail genetically, little data exist regarding iris melanomas. Methods: In our study, we genetically analyzed 19 iris melanomas, 8 ciliary body melanomas, 3 ring melanomas, and 4 iris nevi. A targeted next-generation sequencing approach was applied, covering the mutational hotspot regions of nine genes known to be mutated in conjunctival and uveal melanoma (BRAF, NRAS, KIT, GNAQ, GNA11, CYSLTR2, SF3B1, EIF1AX, and BAP1). Results: Activating GNAQ or GNA11 hotspot mutations were detected in a mutually exclusive fashion in 84% (16/19) of iris melanomas. EIF1AX gene mutations also were frequent, detected in 42% (8/19) of iris melanomas. In 4 iris nevi, one GNAQ mutation was identified. GNAQ, GNA11, EIF1AX, and BAP1 mutations were identified at varying frequencies in ciliary body and ring melanomas. Conclusions: In this most comprehensive genetic analysis of iris melanomas published to date, we find iris melanomas to be related genetically to choroidal and ciliary body melanomas, frequently harboring GNAQ, GNA11, and EIF1AX mutations. Future studies will need to assess if screening mutation profiles in iris melanomas may be of diagnostic or prognostic value.

Corticosteroids augment BRAF inhibitor vemurafenib induced lymphopenia and risk of infection.

We have previously demonstrated an impact of the BRAF inhibitor vemurafenib on patient lymphocyte counts. In the current study, the extent to which concomitant use of corticosteroids in BRAF inhibitor treated patients affects lymphocyte counts and predisposes to infection was investigated. A cohort of 102 patients receiving either the selective BRAF inhibitor vemurafenib or dabrafenib was analyzed. The amount of patients receiving either medication with or without systemic corticosteroids (dexamethasone) was determined and lymphocyte counts before and under therapy assessed. Additionally, the number and severity of infections occurring in these groups was analyzed. Vemurafenib treatment led to a considerable decrease in lymphocyte cell counts, with 62.3% of patients having lymphopenia. Dabrafenib treated patients only rarely demonstrated lymphopenia (12.5%). Dexamethasone co-administration further diminished lymphocyte counts. Lymphopenias were observed in 84.6% of patients receiving vemurafenib and dexamethasone. In our cohort, infections were noted in 9 patients, 4 of these were severe and 2 eventually fatal. All 9 cases with infections demonstrated lymphopenia, 8 of these had received dexamethasone and 7 of these a therapy with vemurafenib. Our findings demonstrate a significant lymphopenia in patients treated with the BRAF inhibitor vemurafenib, which is further augmented by dexamethasone and predisposes to infection. If validated in other studies, risk of infection should be considered when applying corticosteroids in combination with BRAF inhibitors, in particular vemurafenib.

Targeted massively parallel sequencing of angiosarcomas reveals frequent activation of the mitogen activated protein kinase pathway.

Angiosarcomas are rare malignant mesenchymal tumors of endothelial differentiation. The clinical behavior is usually aggressive and the prognosis for patients with advanced disease is poor with no effective therapies. The genetic bases of these tumors have been partially revealed in recent studies reporting genetic alterations such as amplifications of MYC (primarily in radiation-associated angiosarcomas), inactivating mutations in PTPRB and R707Q hotspot mutations of PLCG1. Here, we performed a comprehensive genomic analysis of 34 angiosarcomas using a clinically-approved, hybridization-based targeted next-generation sequencing assay for 341 well-established oncogenes and tumor suppressor genes. Over half of the angiosarcomas (n = 18, 53%) harbored genetic alterations affecting the MAPK pathway, involving mutations in KRAS, HRAS, NRAS, BRAF, MAPK1 and NF1, or amplifications in MAPK1/CRKL, CRAF or BRAF. The most frequently detected genetic aberrations were mutations in TP53 in 12 tumors(35%) and losses of CDKN2A in9 tumors (26%). MYC amplifications were generally mutually exclusive of TP53 alterations and CDKN2A loss and were identified in 8 tumors (24%), most of which (n = 7, 88%) arose post-irradiation. Previously reported mutations in PTPRB (n = 10, 29%) and one (3%) PLCG1 R707Q mutation were also identified. Our results demonstrate that angiosarcomas are a genetically heterogeneous group of tumors, harboring a wide range of genetic alterations. The high frequency of genetic events affecting the MAPK pathway suggests that targeted therapies inhibiting MAPK signaling may be promising therapeutic avenues in patients with advanced angiosarcomas.

TERT promoter mutation status as an independent prognostic factor in cutaneous melanoma.

BACKGROUND: Recently, TERT promoter mutations were identified at high frequencies in cutaneous melanoma tumor samples and cell lines. The mutations were found to have a UV-signature and to lead to increased TERT gene expression. We analyzed a large cohort of melanoma patients for the presence and distribution of TERT promoter mutations and their association with clinico-pathological characteristics. METHODS: 410 melanoma tumor samples were analyzed by Sanger sequencing for the presence of TERT promoter mutations. An analysis of associations between mutation status and various clinical and pathologic variables was performed. RESULTS: TERT promoter mutations were identified in 154 (43%) of 362 successfully sequenced melanomas. Mutation frequencies varied between melanoma subtype, being most frequent in melanomas arising in nonacral skin (48%) and melanomas with occult primary (50%), and less frequent in mucosal (23%), and acral (19%) melanomas. Mutations carried a UV signature (C>T or CC>TT). The presence of TERT promoter mutations was associated with factors such as BRAF or NRAS mutation (P < .001), histologic type (P = .002), and Breslow thickness (P < .001). TERT promoter mutation was independently associated with poorer overall survival in patients with nonacral cutaneous melanomas (median survival 80 months vs 291 months for wild-type; hazard ratio corrected for other covariates 2.47; 95% confidence interval [CI] = 1.29 to 4.74; P = .006). CONCLUSIONS: UV-induced TERT promoter mutations are one of the most frequent genetic alterations in melanoma, with frequencies varying depending on melanoma subtype. In nonacral cutaneous melanomas, presence of TERT promoter mutations is independently associated with poor prognosis.

TERT promoter mutations are frequent in cutaneous basal cell carcinoma and squamous cell carcinoma.

Activating mutations in the TERT promoter were recently identified in up to 71% of cutaneous melanoma. Subsequent studies found TERT promoter mutations in a wide array of other major human cancers. TERT promoter mutations lead to increased expression of telomerase, which maintains telomere length and genomic stability, thereby allowing cancer cells to continuously divide, avoiding senescence or apoptosis. TERT promoter mutations in cutaneous melanoma often show UV-signatures. Non-melanoma skin cancer, including basal cell carcinoma and squamous cell carcinoma, are very frequent malignancies in individuals of European descent. We investigated the presence of TERT promoter mutations in 32 basal cell carcinomas and 34 cutaneous squamous cell carcinomas using conventional Sanger sequencing. TERT promoter mutations were identified in 18 (56%) basal cell carcinomas and in 17 (50%) cutaneous squamous cell carcinomas. The recurrent mutations identified in our cohort were identical to those previously described in cutaneous melanoma, and showed a UV-signature (C>T or CC>TT) in line with a causative role for UV exposure in these common cutaneous malignancies. Our study shows that TERT promoter mutations with UV-signatures are frequent in non-melanoma skin cancer, being present in around 50% of basal and squamous cell carcinomas and suggests that increased expression of telomerase plays an important role in the pathogenesis of these tumors.