[The Cutaneous Squamous Cell Carcinoma - An Update]. / Das kutane Plattenepithelkarzinom ­ ein Update.

BACKGROUND: The cutaneous squamous cell carcinoma (CSCC) is the second most common nonmelanoma skin cancer with an increasing incidence rate. Patients presenting with high-risk lesions associated with locally advanced or metastatic CSCC face high rates of recurrence and mortality. METHODS: Selective literature review based on PubMed and consideration of current guidelines "Aktinische Keratosen und Plattenepithelkarzinom der Haut" and "Prävention von Hautkrebs". FINDINGS: Complete surgical excision with histopathological control of excision margins is the gold standard in the treatment of primary CSCC. Radiotherapy can be used as an alternative treatment of inoperable CSCCs. In 2019, the PD1-antibody cemiplimab, has been approved for the treatment of locally advanced and metastatic CSCC by the European Medicines Agency. After 3 years of follow up, Cemiplimab shows overall response rates of 46 %, the median overall survival and median response rate had not been reached yet. Additional immunotherapeutics, combinations with other agents and oncolytic viruses are all potentially worth study to try, so clinical trial data will be forthcoming over the next few years to guide optimal use of these agents. CONCLUSION: Multidisciplinary board decisions are mandatory for all patients with advanced disease who require more than surgery. Further development of existing therapeutic concepts, identification of new combination therapies and the development of new immunotherapeutics will be the key challenge over the next few years.

Immune checkpoint blockade with concurrent electrochemotherapy in advanced melanoma: a retrospective multicenter analysis.

Growing evidence suggests that concurrent loco-regional and systemic treatment modalities may lead to synergistic anti-tumor effects in advanced melanoma. In this retrospective multicenter study, we evaluate the use of electrochemotherapy (ECT) combined with ipilimumab or PD-1 inhibition. We investigated patients with unresectable or metastatic melanoma who received the combination of ECT and immune checkpoint blockade for distant or cutaneous metastases within 4 weeks. Clinical and laboratory data were collected and analyzed with respect to safety and efficacy. A total of 33 patients from 13 centers were identified with a median follow-up time of 9 months. Twenty-eight patients received ipilimumab, while five patients were treated with a PD-1 inhibitor (pembrolizumab n = 3, nivolumab n = 2). The local overall response rate (ORR) was 66.7 %. The systemic ORR was 19.2 and 40.0 % in the ipilimumab and PD-1 cohort, respectively. The median duration of response was not reached in either group. The median time to disease progression was 2.5 months for the entire population with 2 months for ipilimumab and 5 months for PD-1 blockade. The median overall survival was not reached in patients with ipilimumab and 15 months in the PD-1 group. Severe systemic adverse events were detected in 25.0 % in the ipilimumab group. No treatment-related deaths were observed. This is the first reported evaluation of ECT and simultaneous PD-1 inhibition and the largest published dataset on ECT with concurrent ipilimumab. The local response was lower than reported for ECT only. Ipilimumab combined with ECT was feasible, tolerable and showed a high systemic response rate.

Computer-Assisted Differential Diagnosis of Pyoderma Gangrenosum and Venous Ulcers with Deep Neural Networks.

(1) Background: Pyoderma gangrenosum (PG) is often situated on the lower legs, and the differentiation from conventional leg ulcers (LU) is a challenging task due to the lack of clear clinical diagnostic criteria. Because of the different therapy concepts, misdiagnosis or delayed diagnosis bears a great risk for patients. (2) Objective: to develop a deep convolutional neural network (CNN) capable of analysing wound photographs to facilitate the PG diagnosis for health professionals. (3) Methods: A CNN was trained with 422 expert-selected pictures of PG and LU. In a man vs. machine contest, 33 pictures of PG and 36 pictures of LU were presented for diagnosis to 18 dermatologists at two maximum care hospitals and to the CNN. The results were statistically evaluated in terms of sensitivity, specificity and accuracy for the CNN and for dermatologists with different experience levels. (4) Results: The CNN achieved a sensitivity of 97% (95% confidence interval (CI) 84.2−99.9%) and outperformed dermatologists, with a sensitivity of 72.7% (CI 54.4−86.7%) significantly (p < 0.03). However, dermatologists achieved a slightly higher specificity (88.9% vs. 83.3%). (5) Conclusions: For the first time, a deep neural network was demonstrated to be capable of diagnosing PG, solely on the basis of photographs, and with a greater sensitivity compared to that of dermatologists.

Encorafenib, binimetinib plus pembrolizumab triplet therapy in patients with advanced BRAFV600 mutant melanoma: safety and tolerability results from the phase I IMMU-TARGET trial.

BACKGROUND: Combination of immune checkpoint inhibitors and mitogen-activated protein kinase (MAPK) pathway inhibitors (MAPKi) has been proposed to enhance the durability of anti-tumour responses induced by MAPKi. Here, we present phase I safety results from an open-label, phase I/II study of pembrolizumab (PEM), encorafenib (ENC) and binimetinib (BIN) triplet therapy in advanced, B-Raf proto-oncogene serine/threonine kinase (BRAF)V600-mutated melanoma (IMMU-TARGET, NCT02902042). METHODS: The dose finding phase I part used a 3 + 3 design, starting with the approved doses of PEM (200 mg every three weeks), ENC (450 mg once daily [QD]) and BIN (45 mg twice daily [BID]) as dose level (DL) 0. Reduction of the ENC and BIN doses (300 mg QD and 30 mg BID at DL-1 and 200 mg QD and 30 mg BID at DL-2) was preplanned in case of ≥2 dose-limiting toxicities (DLTs). Primary objectives were to estimate the recommended phase II dose of the triplet combination, DLT and safety. As per the sponsor's decision, the study was terminated after the phase I part, as the clinical efficacy of the combination is currently being investigated in a pivotal, placebo-controlled (PEM mono), double-blinded phase III trial (STARBOARD,NCT04657991). RESULTS: Fifteen patients were enrolled. DLTs of DL0 were creatine phosphokinase (CPK) elevation plus cytokine release syndrome (n = 1) and gamma glutamyl transferase (GGT) increase (n = 1). No DLT was observed in further 3 + 3 patients at DL-1. One (isolated GGT elevations) DLT of DL0 was questionable, as the patient had further episodes of isolated GGT elevations after treatment discontinuation. Hence, further 6 patients were enrolled at DL0: here, no DLT occurred. In total, 13 of 15 patients (87%) experienced a treatment-related adverse event (TRAE) and 8 patients (53%), a grade ≥III TRAE; there were no TRAE-related deaths. Increases in aspartate aminotransferases, GGT (6/15 patients) and CPK elevations (4/15) were the most common grade III-IV TRAE. In median, patients received triplet therapy for 24 weeks (interquartile range [IQR], 12-45). Of the 14 patients evaluable for efficacy, the overall response rate was 64% (95% confidence interval [CI], 35-87). At a median follow-up of 25 months (IQR, 9-28), progression-free survival at 12 months was 41% (95% CI, 13-68). CONCLUSIONS: Triplet therapy with PEM, ENC and BIN as used in the study was feasible and safe and led to clinically meaningful disease control.

Rapid induction of tumor-specific type 1 T helper cells in metastatic melanoma patients by vaccination with mature, cryopreserved, peptide-loaded monocyte-derived dendritic cells.

There is consensus that an optimized cancer vaccine will have to induce not only CD8+ cytotoxic but also CD4+ T helper (Th) cells, particularly interferon (IFN)-gamma-producing, type 1 Th cells. The induction of strong, ex vivo detectable type 1 Th cell responses has not been reported to date. We demonstrate now that the subcutaneous injection of cryopreserved, mature, antigen-loaded, monocyte-derived dendritic cells (DCs) rapidly induces unequivocal Th1 responses (ex vivo detectable IFN-gamma-producing effectors as well as proliferating precursors) both to the control antigen KLH and to major histocompatibility complex (MHC) class II-restricted tumor peptides (melanoma-antigen [Mage]-3.DP4 and Mage-3.DR13) in the majority of 16 evaluable patients with metastatic melanoma. These Th1 cells recognized not only peptides, but also DCs loaded with Mage-3 protein, and in case of Mage-3DP4-specific Th1 cells IFN-gamma was released even after direct recognition of viable, Mage-3-expressing HLA-DP4+ melanoma cells. The capacity of DCs to rapidly induce Th1 cells should be valuable to evaluate whether Th1 cells are instrumental in targeting human cancer and chronic infections.

The production of a new MAGE-3 peptide presented to cytolytic T lymphocytes by HLA-B40 requires the immunoproteasome.

By stimulating human CD8(+) T lymphocytes with autologous dendritic cells infected with an adenovirus encoding MAGE-3, we obtained a cytotoxic T lymphocyte (CTL) clone that recognized a new MAGE-3 antigenic peptide, AELVHFLLL, which is presented by HLA-B40. This peptide is also encoded by MAGE-12. The CTL clone recognized MAGE-3--expressing tumor cells only when they were first treated with IFN-gamma. Since this treatment is known to induce the exchange of the three catalytic subunits of the proteasome to form the immunoproteasome, this result suggested that the processing of this MAGE-3 peptide required the immunoproteasome. Transfection experiments showed that the substitution of beta5i (LMP7) for beta5 is necessary and sufficient for producing the peptide, whereas a mutated form of beta5i (LMP7) lacking the catalytically active site was ineffective. Mass spectrometric analyses of in vitro digestions of a long precursor peptide with either proteasome type showed that the immunoproteasome produced the antigenic peptide more efficiently, whereas the standard proteasome more efficiently introduced cleavages destroying the antigenic peptide. This is the first example of a tumor-specific antigen exclusively presented by tumor cells expressing the immunoproteasome.

Melanoma-associated chondroitin sulphate proteoglycan as a new target antigen for CD4+ T cells in melanoma patients.

Melanoma-associated chondroitin sulfate proteoglycan (MCSP) (also known as high molecular weight-melanoma-associated antigen) represents an interesting target antigen for cancer immunotherapy which is expressed on human melanomas and other tumors such as breast carcinomas, gliomas, neuroblastomas and acute leukemias. MCSP seems to play an important functional role in melanoma as it is involved in tumor cell migration, invasion and angiogenesis. In this study, we isolated CD4(+) T helper cells from the blood of a healthy donor, recognizing a peptide from the MCSP core protein presented by HLA-DBR1*1101 molecules. T cell reactivity against the identified peptide could be detected in the blood of healthy donors and melanoma patients. MCSP specific T cells from the blood of a patient could be readily expanded by repeated peptide stimulation and recognized MCSP and HLA-DR expressing tumor cells. Our findings suggest that vaccination against MCSP helper T cell epitopes might be a promising approach to fight melanoma.

A case of multiple familial trichoepitheliomas responding to treatment with the Hedgehog signaling pathway inhibitor vismodegib.

Multiple familial trichoepitheliomas (MFT) is an autosomal dominantly inherited disease characterized by multiple skin appendage tumors. We describe a patient showing a continuous spectrum of follicular differentiated neoplasms including classical trichoepitheliomas but also infiltrative growing and finally metastasizing malignant follicular differentiated tumors. Germline mutation analysis revealed a nonsense mutation in the cylindromatosis (CYLD) gene. Gene expression analysis by real-time PCR of tumor tissue showed overexpression of glioma-associated oncogene Gli1 mRNA. Treatment with the Hedgehog pathway inhibitor vismodegib resulted in a significant regression of the highly differentiated trichoepitheliomas. Gli upregulation is indicative of an active Hedgehog signaling pathway. We hypothesize that its upregulation is indirectly caused by CYLD mutation which promotes tumor development. Vismodegib treatment could thus provide a new treatment option for patients with this debilitating disorder.

Late syphilis mimicking a pseudolymphoma of the skin.

After a period of declining incidence of syphilis in most of Western Europe until the late 1990s, reports about an increasing trend have been published recently. In contrast to the rising incidence of early syphilis, cases of late (tertiary) syphilis are rarely seen in developed countries. In this report, we describe a 54-year-old patient with infiltrated erythematous plaques on his forehead and neck that histologically revealed a dense lymphocytic cell infiltrate with numerous plasma cells. The serologic examination was characteristic of syphilis and in conjunction with the clinical presentation and the patient's history led to the diagnosis of tertiary syphilis. The diagnosis of this late stage of syphilis can be difficult as clinical pictures can be misleading. Peculiar skin lesions should always remind clinicians of this infectious disease which has still to be considered in differential diagnoses in dermatology.

Mycosis fungoides palmaris et plantaris--an unusual variant of cutaneous T-cell lymphoma.

Mycosis fungoides (MF) represents a low-risk, cutaneous, non-Hodgkin, T-cell lymphoma with a wide spectrum of clinicopathological manifestations and therefore may mimic a number of other dermatoses. Sometimes the clinical diversity makes the diagnosis of MF, and especially its atypical forms, challenging. We report on an 18-year old male patient, who had been previously diagnosed with palmoplantar eczema. Clinical, histopathological, immunohistochemical and molecular findings revealed an atypical case of MF.

Afatinib-associated Stevens-Johnson syndrome in an EGFR-mutated lung cancer patient.

INTRODUCTION: Afatinib is a tyrosine kinase inhibitor (TKI), that has been approved for treating patients with epidermal growth factor receptor (EGFR) mutated advanced non-small-cell lung cancer (NSCLC). Stevens-Johnson syndrome (SJS) related to EGFR directed TKIs is a rare adverse event. CASE PRESENTATION: We report a case of a 79-year-old white female with EGFR-mutated, metastatic non-small-cell lung cancer treated with afatinib as first-line palliative treatment, who developed a SJS after two months of treatment. Discontinuation of the TKI and systemic glucocorticoid treatment led to improvement of symptoms and recovery. CONCLUSION: Severe adverse cutaneous drug reactions that predominantly involve the skin and mucous membranes during treatment with afatinib should alert clinicians to suspect SJS and react appropriately.

Artesunate in the treatment of metastatic uveal melanoma--first experiences.

Artesunate (ART) is a derivative of artemisinin, the active principle of the Chinese herb Artemisia annua L. Artesunate is approved for the treatment of multidrug-resistant malaria and has an excellent safety profile. It has been shown that Artesunate, apart from its anti-malarial activity, has cytotoxic effects on a number of human cancer cell lines, including leukemia, colon cancer and melanoma. We report on the first long-term treatment of two cancer patients with ART in combination with standard chemotherapy. These patients with metastatic uveal melanoma were treated on a compassionate-use basis, after standard chemotherapy alone was ineffective in stopping tumor growth. The therapy-regimen was well tolerated with no additional side effects other than those caused by standard chemotherapy alone. One patient experienced a temporary response after the addition of ART to Fotemustine while the disease was progressing under therapy with Fotemustine alone. The second patient first experienced a stabilization of the disease after the addition of ART to Dacarbazine, followed by objective regressions of splenic and lung metastases. This patient is still alive 47 months after first diagnosis of stage IV uveal melanoma, a situation with a median survival of 2-5 months. Despite the small number of treated patients, ART might be a promising adjuvant drug for the treatment of melanoma and possibly other tumors in combination with standard chemotherapy. Its good tolerability and lack of serious side effects will facilitate prospective randomized trials in the near future.

Antigen loading of dendritic cells with whole tumor cell preparations.

Dendritic cells (DC) based vaccinations have been widely used for the induction of anti-tumoral immunity in clinical studies. Antigen loading of DC with whole tumor cell preparations is an attractive method whenever tumor cell material is available. In order to determine parameters for the loading procedure, we performed dose finding and timing experiments. We found that apoptotic and necrotic melanoma cells up to a ratio of one-to-one, equivalent to 1mg/ml protein per 1 x 10(6) DC, can be added to monocyte derived DC without effecting DC recovery extensively. Using the isolated protein content of tumor cells (lysate) as a parameter, up to 5 mg/ml protein per 1 x 10(6) DC can be added. To achieve significant protein uptake at least 1 mg/ml of protein have to be added for more than 24 h as tested with FITC-labelled ovalbumin. Maturation inducing cytokines can be added simultaneously with the tumor cell preparations to immature DC without affecting the uptake. Furthermore, we tested the feasibility of cryopreservation of loaded and matured DC to facilitate the generation of ready to use aliquots. DC were cryopreserved in a mix of human serum albumin, DMSO and 5% glucose. After thawing, surface expression of molecules indicating the mature status (CD83, costimulatory and MHC molecules), was found to be unaltered. Furthermore, cryopreserved DC kept the capability to stimulate allogenic T-cell proliferation in mixed leukocyte reactions at full level. Loaded and matured DC pulsed with influenza matrix peptide (IMP) retained the capacity to induce the generation of IMP-specific cytotoxic T-lymphocytes after cryopreservation as measured by ELISPOT and tetramer staining. The expression of the chemokine receptor CXCR-4 and CCR-7 remained unaltered during cryopreservation and the migratory responsiveness towards MIP-3beta was unaltered as measured in a migration assay. Thus we conclude that the large scale loading and maturation of DC with whole tumor cell preparations can be performed in a single session. These data will facilitate the clinical application of DC loaded with whole tumor cell preparations.

Tumor-reactive CD4+ T cell responses to the melanoma-associated chondroitin sulphate proteoglycan in melanoma patients and healthy individuals in the absence of autoimmunity.

To avoid immune escape by down-regulation or loss of Ag by the tumor cells, target Ags are needed, which are important for the malignant phenotype and survival of the tumor. We could identify a CD4(+) T cell epitope derived from the human melanoma-associated chondroitin sulfate proteoglycan (MCSP) (also known as high m.w.-melanoma-associated Ag), which is strongly expressed on >90% of human melanoma lesions and is important for the motility and invasion of melanoma cells. However, MCSP is not strictly tumor specific, because it is also expressed in a variety of normal tissues. Therefore, self tolerance should prevent the induction of strong T cell responses against these Ags by vaccination strategies. In contrast, breaking self tolerance to this Ag by effectively manipulating the immune system might mediate antitumor responses, although it would bear the risk of autoimmunity. Surprisingly, we could readily isolate CD4(+) Th cells from the blood of a healthy donor-recognizing peptide MCSP(693-709) on HLA-DR11-expressing melanoma cells. Broad T cell reactivity against this Ag could be detected in the peripheral blood of both healthy donors and melanoma patients, without any apparent signs of autoimmune disease. In some patients, a decline of T cell reactivity was observed upon tumor progression. Our data indicate that CD4(+) T cells are capable of recognizing a membrane glycoprotein that is important in melanoma cell function, and it may be possible that the sizable reactivity to this Ag in most normal individuals contributes to immune surveillance against cancer.

Optimizing the exogenous antigen loading of monocyte-derived dendritic cells.

Dendritic cell (DC) vaccination, i.e. the adoptive transfer of antigen-loaded DC, is still at an early stage and requires standardization. In this study, we investigated the exogenous loading of monocyte-derived DCs with HLA class I- and II-restricted peptides, as despite widespread use, little effort has been put into its pre-clinical validation. We found that only mature DCs (m-DC) but not immature DCs (im-DC) could be sufficiently loaded with exogenous class I-restricted peptides and were by far superior in expanding CD8(+) primary (Melan-A.A2 peptide-specific) and recall [Influenza matrix peptide (IMP) A2-specific] T cell responses. Primary stimulation with peptide-loaded im-DCs even down-regulated antigen-specific T cell responses. Our results indicate that stimulation with m-DCs is superior in terms of quantity and quality compared with im-DCs, supporting their preferred use in clinical DC trials. Loading of m-DCs with high (10 microM) concentrations generated clearly more Melan-A effectors than loading with 1 or 0.1 microM without any negative effect on the quality (affinity) of the resulting T cells. In contrast to the findings with the Melan-A peptide loading with 10 microM IMP was counter-productive, induced apoptosis and yielded fewer specific T cells of inferior affinity as compared with loading with 1 or 0.1 microM. In sharp contrast to the situation for HLA class I, much higher levels and longer half-lives of peptide-HLA class II complexes were obtainable upon loading of im-DCs with exogenous peptide, but m-DCs were functionally preferable to induce T(h)1 responses in vitro. Another surprising finding was that, while presentation to T cells upon simultaneous loading of several peptides with highly varying affinities and competing for the same class I or II molecule was possible, in priming experiments peptide competition clearly inhibited T cell induction. Although peptides will obviously vary in their individual properties, our study clearly points to some important principles that should be taken into account.

Differences in phenotype and function between spontaneously occurring melan-A-, tyrosinase- and influenza matrix peptide-specific CTL in HLA-A*0201 melanoma patients.

Melanoma-specific T cells can occur spontaneously or in response to vaccination or other therapies, but the frequency is much lower than observed in viral infections. The presence of tumor-specific T cells does not necessarily translate into clinical regressions for a variety of reasons such as an insufficient frequency, activation state or homing capacity of the T cells or escape strategies of the tumor. Having screened melanoma patients prior to inclusion in vaccination trials for spontaneous tumor-specific T cells either by Elispot or tetramer-staining, we have identified 3 patients with sufficient numbers of tumor-reactive T cells to more than 1 TAA and at least 1 virus-antigen to perform phenotypic and functional analysis directly ex vivo. These stage IV melanoma patients showed specific CTL against melan-A.A2, tyrosinase.A2 and influenza matrix peptide (IMP).A2 readily detectable in peripheral blood. T-cell receptor (TCR) staining using the tetramer technology was combined with phenotypic characterization and functional assays. In contrast to IMP-specific CTL, melanoma-specific CTL were predominantly terminally differentiated effector cells. However, analysis of melan-A- and tyrosinase-specific T-cell lines showed that only a part of the melanoma-specific CTL were able to lyse peptide-loaded target cells. Interestingly, the described phenotypic and functional differences of melan-A- and tyrosinase-specific CTL appeared not only between patients but were also evident within patients, suggesting that the immune response against various tumor antigens is regulated independently.

Generation of an optimized polyvalent monocyte-derived dendritic cell vaccine by transfecting defined RNAs after rather than before maturation.

Transfection with RNA is an attractive method of Ag delivery to dendritic cells (DCs), but has not yet been standardized. We describe in this study the methods to efficiently generate an optimized mature monocyte-derived DC vaccine at clinical scale based on the electroporation of several RNAs either into immature DC followed by maturation or, alternatively, directly into mature DCs, which has not been possible so far with such high efficiency. Electroporation of DCs resulted in high yield, high transfection efficiency (>90%), and high migration capacity. Intracellular staining allowed the study of the expression kinetics of Ags encoded by the transfected RNAs (MelanA, MAGE-3, and survivin) and a validation of the vaccine (>/=90% transfection efficiency). Expression of all three Ags peaked 3-4 h after electroporation in DC transfected either before or after maturation, but decreased differently. The DC vaccine can also be cryopreserved and nevertheless retains its viability, stimulatory capacity as well as migratory activity. In addition, we uncover that DC transfected after rather than before maturation appear to be preferable vaccines not only from a production point of view but also because they appear to be immunologically superior for CTL induction in sharp contrast to common belief. DCs transfected after maturation not only more effectively generate and present the Mage-3.A1 and MelanA.A2.1 epitopes to T cell clones, but they even are superior in priming to the standard proteasome-dependent MelanA.A2.1 wild-type prototype tumor epitope, both in terms of T cell expansion and effector function on a per cell basis.

Segmental neurofibromatosis.

Segmental neurofibromatosis is characterised by a limited, segmental distribution of cutaneous neurofibromatosis type 1 (NF1) lesions. It has been suggested that segmental NF results from a postzygotic NF1 gene mutation, and, recently, this hypothesis has been proven in a patient with regionally distributed café-au-lait (CAL) spots and freckles by demonstrating an NF1 microdeletion restricted to fibroblasts cultured from CAL spots. We describe here a patient with segmental NF in which we could not demonstrate any NF1 gene mutation in fibroblasts cultured from neurofibromas by use of the protein truncation test, enzymatic mutation detection and fluorescence in situ hybridisation. These data are in line with the concept that NF1 mutations in Schwann cells, but not in fibroblasts, correlate with neurofibroma formation.