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Background: Pemphigoid diseases are a heterogeneous group of autoimmune blistering disorders characterized by predominant deposition of immunoglobulin G or immunoglobulin A autoantibodies against structural proteins of the dermoepidermal junction (DEJ). Sole linear immunoglobulin M (IgM) deposits at the DEJ in pemphigoid diseases have been observed; however, IgM-specific target antigens have not been identified.Objective: Characterization of patients with IgM pemphigoid.Methods: Skin biopsy specimens and sera from IgM-positive patients were assessed using histopathology, direct and indirect immunofluorescence microscopy, enzyme-linked immunosorbent assays, immunoblotting, cryosection assay, complement fixation test, and internalization assays.Results: Tissue-bound linear IgM deposits along the DEJ and circulating IgM autoantibodies against type XVII collagen (Col17) were detected. These circulating IgM autoantibodies showed no complement activating or blister inducing capacity, but the ability of Col17 internalization ex vivo.Limitations: Limited number of patients.Conclusion: This study provides further evidence for the role of IgM autoantibodies in pemphigoid disease and highlights Col17 as a target antigen in IgM pemphigoid. [ABSTRACT FROM AUTHOR]

Besides central tolerance peripheral tolerance is an important mechanism to avoid development of autoimmunity. Naturally occurring thymic-derived regulatory T cells (nTreg) mediate peripheral tolerance by suppressing autoreactive T cells clones having escaped thymic deletional control. This implies that nTreg have therapeutic potential to dampen autoimmune disease. However, one of the main challenges for the therapeutic application of nTreg still remains the scarce amount of nTreg available. Transforming growth factor beta (TGF-β1) plays a critical role in the generation and immunosuppressive function of nTreg thereby contributing to immune homeostasis. TGF-β1 is thought to be essential for the generation and function of nTreg and regulatory T cells with suppressive properties can be induced in vitro by TGF-β1. These so-called TGF-β1-induced regulatory T cells (iTreg) can be induced in vitro from conventional CD4 + T cells by addition of TGF-β1 and this discovery has added new options to use regulatory T cells therapeutically. Here we discuss the generation and in vitro and in vivo functions of murine and human TGF-β1-induced regulatory T cells in light of potential application as treatment for autoimmune diseases including current problems and drawbacks in their therapeutic use. [ABSTRACT FROM AUTHOR]

The coronavirus disease 2019 (COVID-19) pandemic has changed the lives of people around the world. Fortunately, sufficient vaccines are now available. Local reactions with ipsilateral lymphadenopathy are among the most common side effects. We investigated the impact of lymphadenopathy after COVID-19 vaccination on the value of ultrasound in tumour patients. Patients with melanoma or Merkel cell carcinoma were included who underwent lymph node excision and received COVID-19 vaccination within 6 weeks before surgery. The consistency of the preoperative ultrasound findings with the histopathologic findings was investigated. Eight patients were included (two Merkel cell carcinoma and six melanoma patients) who underwent lymph node excision between 16th April 2021 and 19th May 2021 and had previously received COVID-19 vaccination. In three of the eight patients (one Merkel cell carcinoma and two melanoma patients), lymph node metastases were erroneously diagnosed preoperatively during tumour follow-up with physical examination, ultrasound, and or fluorodeoxyglucose (FDG)–positron emission tomography (PET)/computed tomography (CT). In these three patients, the suspected lymph node metastases were located in the left axilla after COVID-19 vaccination in the left upper arm, which resulted in selective lymph node removal in two patients and complete lymphadenectomy in one patient. COVID-19 vaccine–associated lymphadenopathy is expected to be observed much more frequently in the near future because of increasing vaccination rates. This cause of lymphadenopathy, which may in ultrasound as well as in FDG PET/CT resemble lymph node metastases, must be considered, especially in oncologic patients undergoing tumour follow-up. In addition, COVID-19 vaccination should be given as far away as possible from an underlying primary on the contralateral side to avoid oncologic misdiagnosis followed by malpractice. • Lymphadenopathy after coronavirus disease 2019 (COVID-19) vaccination mimics lymph node metastasis in ultrasound. • Lymphadenopathy after COVID-19 vaccination may lead to unnecessary lymph node extirpations. • Vaccination should be administered as far as possible from the site of the primary tumour. [ABSTRACT FROM AUTHOR]

Background Autoimmune bullous diseases/dermatoses (AIBDs) are severe autoantibody-mediated skin diseases. The pathogenic relevance of autoreactive CD4+ T cells for the induction of autoantibody production remains to be fully evaluated. Scurfy mice lack functional regulatory T (Treg) cells, experience spontaneous activation of autoreactive CD4+ T cells, and display severe erosive skin lesions suggestive of AIBDs. Objective We sought to determine whether AIBDs develop in Treg cell–deficient scurfy mice. Methods Histology, indirect immunofluorescence (IF) microscopy, direct IF, and ELISA were used to prove the presence of AIBDs in scurfy mice. Monoclonal autoantibodies from sera of scurfy mice were screened by using indirect IF on murine skin, and immunoprecipitation and mass spectrometry were used for target antigen identification, followed by confirmation in modified human embryonic kidney cells and murine keratinocytes. Pathogenicity was determined by injecting the autoantibody into neonatal mice and transferring scurfy CD4+ T cells into nu/nu mice. Results Autoantibodies against different known autoantigens of AIBDs spontaneously develop in scurfy mice. Histology reveals subepidermal blisters, and direct IF of skin of scurfy mice shows a predominant linear staining pattern. The mAb 20B12 shows a linear staining pattern in indirect IF, recognizes the murine hemidesmosomal protein bullous pemphigoid antigen 230 (BP230) as the target antigen, and cross-reacts with human BP230. Purified mAb 20B12 induces subepidermal blisters in neonatal mice. Transfer of scurfy CD4+ T cells is sufficient to induce antibodies with reactivity to AIBD autoantigens and subepidermal blisters in the skin of recipient T cell–deficient nu/nu mice. Conclusion We show that the absence of Treg cells leads to AIBDs by pathogenic autoantibodies targeting BP230. [ABSTRACT FROM AUTHOR]

In melanoma patients, surgical excision of the first draining lymph node, the sentinel lymph node (SLN), is a routine procedure to evaluate lymphogenic metastases. Metastasis detection by histopathological analysis assesses multiple tissue levels with hematoxylin and eosin and immunohistochemically stained glass slides. Considering the amount of tissue to analyze, the detection of metastasis can be highly time-consuming for pathologists. The application of artificial intelligence in the clinical routine has constantly increased over the past few years. In this multi-center study, a deep learning method was established on histological tissue sections of sentinel lymph nodes collected from the clinical routine. The algorithm was trained to highlight potential melanoma metastases for further review by pathologists, without relying on supplementary immunohistochemical stainings (e.g. anti-S100, anti-MelanA). The established method was able to detect the existence of metastasis on individual tissue cuts with an area under the curve of 0.9630 and 0.9856 respectively on two test cohorts from different laboratories. The method was able to accurately identify tumour deposits>0.1 mm and, by automatic tumour diameter measurement, classify these into 0.1 mm to −1.0 mm and>1.0 mm groups, thus identifying and classifying metastasis currently relevant for assessing prognosis and stratifying treatment. Our results demonstrate that AI-based SLN melanoma metastasis detection has great potential and could become a routinely applied aid for pathologists. Our current study focused on assessing established parameters; however, larger future AI-based studies could identify novel biomarkers potentially further improving SLN-based prognostic and therapeutic predictions for affected patients. • Deep learning algorithm establishment on histological slides in a multi-center study. • The algorithm highlights melanoma metastases on H&E stained tissue of SLN. • The method could accurately identify melanoma deposits of>0.1 mm in diameter. • Metastases detected with an AUC of 0.96 and 0.99 in two cohorts, respectively. [ABSTRACT FROM AUTHOR]

Dedifferentiated melanoma (DedM) poses significant diagnostic challenges. We aimed to investigate the clinical, histopathological and molecular features of DedM. Methylation signature (MS) and copy number profiling (CNP) were carried out in a subgroup of cases. A retrospective series of 78 DedM tissue samples from 61 patients retrieved from EORTC (European Organisation for Research and Treatment of Cancer) Melanoma Group centres were centrally reviewed. Clinical and histopathological features were retrieved. In a subgroup of patients, genotyping through Infinium Methylation microarray and CNP analysis was carried out. Most patients (60/61) had a metastatic DedM showing most frequently an unclassified pleomorphic, spindle cell, or small round cell morphology akin to undifferentiated soft tissue sarcoma, rarely associated with heterologous elements. Overall, among 20 successfully analysed tissue samples from 16 patients, we found retained melanoma-like MS in only 7 tissue samples while a non-melanoma-like MS was observed in 13 tissue samples. In two patients from whom multiple specimens were analysed, some of the samples had a preserved cutaneous melanoma MS while other specimens exhibited an epigenetic shift towards a mesenchymal/sarcoma-like profile, matching the histological features. In these two patients, CNP was largely identical across all analysed specimens, in line with their common clonal origin, despite significant modification of their epigenome. Our study further highlights that DedM represents a real diagnostic challenge. While MS and genomic CNP may help pathologists to diagnose DedM, we provide proof-of-concept that dedifferentiation in melanoma is frequently associated with epigenetic modifications. • Dedifferentiated melanomas pose significant diagnostic challenges. • Epigenetic and copy number variation features may be useful for diagnostic purposes. • Dedifferentiated melanoma patients respond to systemic therapy. [ABSTRACT FROM AUTHOR]

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. 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. 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. 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. (DRKS00015737). • Three-dimensional light sheet fluorescence analysis of entire sentinel lymph nodes. • Identification of melanoma metastases undetected by the current European Organisation for Research and Treatment of Cancer (EORTC) protocol. • Clinical potential of light sheet microscopy for the exact staging of melanoma patients. • Light sheet microscopy may increase the sensitivity of lymph node metastasis detection. • New approach suitable for the implementation in clinical routine. [ABSTRACT FROM AUTHOR]

Radiological imaging such as computed tomography (CT) is used frequently for disease staging and therapy monitoring in advanced skin cancer patients. Detected lesions of unclear dignity are a common challenge for treating physicians. The aim of this study was to assess the frequency and outcome of CT-guided biopsy (CTGB) of radiologically unclear, suspicious lesions and to depict its usefulness in different clinical settings. This retrospective monocentric study included advanced skin cancer patients (melanoma, Merkel cell carcinoma, squamous cell carcinoma, angiosarcoma, cutaneous lymphoma) with radiologically unclear lesions who underwent CTGB between 2010 and 2018. Of 59 skin cancer patients who received CTGB, 47 received CTGB to clarify radiologically suspicious lesions of unclear dignity. 32 patients had no systemic therapy (cohort A), while 15 patients received systemic treatment at CTGB (cohort B). In both cohorts, CTGB revealed skin cancer metastasis in a large proportion of patients (37.5%, 40.0%, respectively), but benign tissue showing inflammation, fibrosis or infection in an equally large percentage (37.5%, 46.7%, respectively). Additionally, a significant number of other cancer entities was found (25.0%, 13.3%, respectively). In patients receiving BRAF/MEK inhibitors, CTGB confirmed suspicious lesions as skin cancer metastasis in 83.3%, leading to treatment change. In immune checkpoint inhibitor-treated patients, skin cancer metastasis was confirmed in 11.1% of patients only, whereas benign tissue changes (inflammation/fibrosis) were found in 77.8%. Our results highlight the relevance of clarifying radiologically unclear lesions by CTGB before start or change of an anti-tumour therapy to exclude benign alterations and secondary malignancies. • Lesions of unclear dignity are frequent in imaging of skin cancer patients. • CT-guided biopsy (CTGB) can be helpful for clarification. • Frequency, outcome and clinical impact of CTGB of such lesions were assessed. • CTGB revealed >50% unexpected findings (benign findings and secondary carcinomas). • CTGB can therefore prevent unnecessary treatment initiation or discontinuation. [ABSTRACT FROM AUTHOR]

Accurate classification of melanocytic proliferations has important implications for prognostic prediction, treatment and follow-up. Although most melanocytic proliferations can be accurately classified using clinical and pathological criteria, classification (specifically distinction between nevus and melanoma) can be challenging in a subset of cases, including those with spitzoid morphology. Genetic studies have shown that mutation profiles differ between primary melanoma subtypes and Spitz nevi. These differences may aid in distinguishing benign from malignant in some melanocytic tumours. Here, we present a selection of melanocytic proliferations with equivocal histopathological criteria, wherein genetic analysis was requested to help guide classification. In two of four cases, the genetic results offered valuable insights, allowing a definitive diagnosis, indicating the diagnostic value of mutation profiling in a real-world routine clinical setting. Although histopathological assessment remains decisive in melanocytic proliferation classification, we recommend including genetic profiling in cases of borderline or atypical lesion to support accurate classification. • Mutation analysis can be a diagnostic aid in determining the dignity in some spitzoid tumours. • Where no gene mutations are identified, translocation analysis should be considered. • Molecular analysis aids in identifying malignant tumours to be treated as melanoma. [ABSTRACT FROM AUTHOR]

Anti-[programmed cell death protein 1 (PD-1)] antibodies nivolumab and pembrolizumab were approved for adjuvant treatment of melanoma as they demonstrated improved relapse-free survival. Currently, combined anti-PD-1 plus anti-[cytotoxic T-lymphocyte-associated protein 4 (CTLA4)] blockade is being investigated in adjuvant and neoadjuvant trials. Sarcoidosis-like reactions have been described for immune checkpoint inhibitors and are most likely drug-induced. The reported rate of sarcoidosis/sarcoidosis-like reactions within clinical melanoma trials is <2%. We observed that a remarkably higher number of melanoma patients (10/45 patients, 22%) treated with immune checkpoint inhibitor (ICI) within an adjuvant clinical trial-developed drug induced sarcoidosis-like reaction (DISR) mimicking metastasis. Of 45 stage III melanoma patients who were treated at our institute with adjuvant ICI (either nivolumab alone or in combination with ipilimumab) within a two-armed, blinded clinical trial, ten developed a DISR. Three of the ten patients were men, median age was 52 years (range, 32–70 years). DISRs were asymptomatic and generally detected radiographically at first radiographic imaging after the start of therapy (median time, 2.8 months) and described as a differential diagnosis to tumour progression. In one patient, DISR was only apparent 13.1 months after start of therapy and 4 weeks after the end of ICI treatment. DISR presented as mediastinal/hilar lymphadenopathy in 8/10 patients (as only site or in addition to lung, skin and/or bone involvement), one patient had only lung and cutaneous, one patient only cutaneous DISR. Biopsies from lymph nodes, skin and bone were taken in 8/10 patients, and histology confirmed sarcoidosis-like reactions (SLRs). As patients were asymptomatic, no treatment for DISR was required, and study treatment was stopped for DISR in only one patient due to bone involvement. DISRs have resolved or are in remission in all patients. At a median follow-up time of 15.3 months (range, 12–17.6 months), two patients experienced melanoma relapse. In most cases, sarcoidosis could only be differentiated from melanoma progression on biopsy. Treating physicians as well as radiologists have to be aware of the potentially higher rate of DISR in patients receiving adjuvant ICI. A thorough interdisciplinary workup is required to discriminate from true melanoma progression and to decide on continuation of adjuvant ICI treatment. • Potentially higher rate of sarcoidosis-like reactions (SLR) in patients who receive adjuvant immunotherapy than in patients with metastatic disease. • Differentiation between SLR and progression of disease in most cases only possible with biopsy. • No clinical symptoms of the SLR in our patients, no steroid treatment required. • No progression of the SLR after continuation of treatment. • Patients that developed SLR and patients that did not had an equal relapse rate (20%). [ABSTRACT FROM AUTHOR]

Cutaneous adnexal tumors are a diverse group of tumors arising from structures of the hair appendages. Although often benign, malignant entities occur which can metastasize and lead to patients´ death. Correct diagnosis is critical to ensure optimal treatment and best possible patient outcome. Artificial intelligence (AI) in the form of deep neural networks has recently shown enormous potential in the field of medicine including pathology, where we and others have found common cutaneous tumors can be detected with high sensitivity and specificity. To become a widely applied tool, AI approaches will also need to reliably detect and distinguish less common tumor entities including the diverse group of cutaneous adnexal tumors. To assess the potential of AI to recognize cutaneous adnexal tumors, we selected a diverse set of these entities from five German centers. The algorithm was trained with samples from four centers and then tested on slides from the fifth center. The neural network was able to differentiate 14 different cutaneous adnexal tumors and distinguish them from more common cutaneous tumors (i.e. basal cell carcinoma and seborrheic keratosis). The total accuracy on the test set for classifying 248 samples into these 16 diagnoses was 89.92 %. Our findings support AI can distinguish rare tumors, for morphologically distinct entities even with very limited case numbers (< 50) for training. This study further underlines the enormous potential of AI in pathology which could become a standard tool to aid pathologists in routine diagnostics in the foreseeable future. The final diagnostic responsibility will remain with the pathologist. ● Establishment of a algorithm for histological analysis in a multi-center study. ● The algorithm distinguishes the group of less common cutaneous adnexal tumors (CAT). ● The algorithm distinguishes 14 CAT from basal cell carcinoma and seborrheic keratosis. ● The total accuracy for classifying samples into these 16 diagnoses was 89.92 %. [ABSTRACT FROM AUTHOR]

Melanomas arising in acral sites are associated with a poorer prognosis than other melanoma subtypes. The aim of this study was to evaluate clinical-pathological and genetic characteristics as well as therapeutic responses of a larger cohort of patients with melanomas arising in acral sites. Clinical data of 134 patients with melanomas arising in acral sites from the Dept. of Dermatology Essen were collected and analysed with regard to clinicopathological characteristics and treatment responses. Genetic analysis with targeted next-generation sequencing was done on 50 samples. In our cohort, BRAF (30%) , NRAS (28%) , TERT promoter (26%) , NF1 (14%) and KIT (6%) were frequently identified mutations. Comparing tumours situated on palms and soles with melanomas arising on dorsal acral sites, a higher frequency of NRAS (39.1% versus 25%) and NF1 (17.3% versus 0%) and lower frequencies of BRAF (21.7% versus 75%) and TERT promoter (8.6% versus 50%) mutations were observed. MAPK activating mutations were identified in 64% of tumours. Overall survival was longer in patients treated with immune checkpoint inhibitors as first-line treatment than in patients receiving other systemic therapies (i.e. BRAF/MEK inhibitors and chemotherapy). Our data suggest that the genetics of melanomas arising in acral sites varies by tumour location and may influence biological behaviour. • Mutations in the TERT promoter were observed in 26% of tumour samples. • MAPK activating mutations were identified in 64% of tumours. • Tumours on palms and soles had ore NRAS and less BRAF and TERT promoter mutations in than dorsal site tumours. • Longer overall survival in patients receiving immune checkpoint inhibitors than other first-line systemic therapies. [ABSTRACT FROM AUTHOR]

The American Joint Committee on Cancer (AJCC) 8th staging system introduced several revisions. To assess the impact of the 8th edition American Joint Committee on Cancer (AJCC8) staging system on subgrouping and survival, patients with melanoma from two tertiary skin cancer centres were classified according to both the 7th edition American Joint Committee on Cancer (AJCC7) and AJCC8. A total of 1948 patients aged ≥18 years with cutaneous melanoma stage II-IV were included. The impact of sex and age on reclassification was assessed by log binomial models. The inverse probability of censoring weighting method was used to compute ROC curves from time-to-event data to assess the discriminatory ability of AJCC7 and AJCC8. Melanoma-specific survival (MSS) and overall survival (OS) were calculated, and age- and sex-adjusted MSS hazard ratios were estimated using Cox proportional hazards models. Of all, 23.5% of patients were assigned a different subgroup when classified according to AJCC8. Owing to upshifting to stage IIIC (AJCC7 24.8% vs. AJCC8 50.8%), patient numbers of stages IIIA and IIIB decreased from 28.7% to 16.2% and 46.5% to 28.3%. The prediction accuracy for AJCC7 and AJCC8 was comparable (integrated time-dependent area under the curve [AUC] of 0.75 and 0.74, respectively). Five-year MSS of IIB and IIC AJCC8 was poor and lower than that of IIIA AJCC8 (80%, 67% and 89%, respectively). Compared to results of the International Melanoma Database and Discovery Platform, 5-year MSS was 10–15% points lower for stages IIC, IIIB and IIIC. Upshifting affects primarily stage III subgroups, while effects in stage II are minor. Stage IIB/C (AJCC8) patients have 67–80% MSS and should be considered for adjuvant treatment, while in stage IIIA, the indication of adjuvant treatment is questionable. • Melanoma classification according to the 8th edition American Joint Committee on Cancer (AJCC8) does not improve discriminatory ability compared with the 7th edition American Joint Committee on Cancer (AJCC7). • Five-year melanoma-specific survival (MSS) of stages IIB/IIC is lower than that of IIIA. • MSS is worse in all substages than results of the International Melanoma Database and Discovery Platform cohort. [ABSTRACT FROM AUTHOR]

Background Adalimumab and Etanercept are TNF-α antagonists commonly used for treatment of moderate-to-severe psoriasis and psoriatic-arthritis. Reliable instruments to assist the selection of patients for a specific treatment in a real-world scenario are unavailable. Objective To identify patient characteristics and baseline laboratory parameters predicting response to Adalimumab- and Etanercept-treatment. Methods We report a retrospective observational study including 116 and 64 psoriasis-patients treated with Adalimumab and Etanercept, respectively, at a dermatological outpatient clinic of a university hospital. Thirty four patients contributed data to both biologics. First occurrence of either loss-of-response or serious-side-effects (LOR/SSE) was chosen as clinical endpoint and predictors were identified using Cox-regression. Results Baseline anti-double-stranded DNA (anti-dsDNA) concentrations, number of previous treatments with TNF-α antagonists in general and previous treatment with Etanercept in particular significantly predicted LOR/SSE to Adalimumab. The predictive effect of baseline anti-dsDNA was conserved in TNF-α antagonist naïve patients. Number of previous systemic treatments other than TNF-α antagonists significantly predicted LOR/SSE to Etanercept. Age and baseline psoriasis area and severity index (PASI) did not predict response to either biologic in a clinically significant manner. Conclusion Our data suggests that treatment with Adalimumab may promise best results in psoriasis-patients with (A) low baseline anti-dsDNA concentrations, and (B) no previous TNF-α antagonist treatment. A clinically significant predictive effect of age and baseline PASI on response to Adalimumab and Etanercept is unlikely. [ABSTRACT FROM AUTHOR]