Malignant T cells induce skin barrier defects through cytokine-mediated JAK/STAT signaling in cutaneous T-cell lymphoma.

Cutaneous T-cell lymphoma (CTCL) is a devastating lymphoid malignancy characterized by the accumulation of malignant T cells in the dermis and epidermis. Skin lesions cause serious symptoms that hamper quality of life and are entry sites for bacterial infection, a major cause of morbidity and mortality in advanced diseases. The mechanism driving the pathological processes that compromise the skin barrier remains unknown. Here, we report increased transepidermal water loss and compromised expression of the skin barrier proteins filaggrin and filaggrin-2 in areas adjacent to TOX-positive T cells in CTCL skin lesions. Malignant T cells secrete mediators (including cytokines such as interleukin 13 [IL-13], IL-22, and oncostatin M) that activate STAT3 signaling and downregulate filaggrin and filaggrin-2 expression in human keratinocytes and reconstructed human epithelium. Consequently, the repression of filaggrins can be counteracted by a cocktail of antibodies targeting these cytokines/receptors, small interfering RNA-mediated knockdown of JAK1/STAT3, and JAK1 inhibitors. Notably, we show that treatment with a clinically approved JAK inhibitor, tofacitinib, increases filaggrin expression in lesional skin from patients with mycosis fungoides. Taken together, these findings indicate that malignant T cells secrete cytokines that induce skin barrier defects via a JAK1/STAT3-dependent mechanism. As clinical grade JAK inhibitors largely abrogate the negative effect of malignant T cells on skin barrier proteins, our findings suggest that such inhibitors provide novel treatment options for patients with CTCL with advanced disease and a compromised skin barrier.

IGFBP7 is not required for B-RAF-induced melanocyte senescence.

Induction of senescence permanently restricts cellular proliferation after oncogenic stimulation thereby acting as a potent barrier to tumor development. The relevant effector proteins may therefore be fundamental to cancer development. A recent study identified IGFBP7 as a secreted factor mediating melanocyte senescence induced by oncogenic B-RAF, which is found commonly in cutaneous nevi. In contrast to the previous report, we demonstrate that B-RAF signaling does not induce IGFBP7 expression, nor the expression of the IGFBP7 targets, BNIP3L, SMARCB1, or PEA15, in human melanocytes or fibroblasts. We also found no correlation between B-RAF mutational status and IGFBP7 protein expression levels in 22 melanoma cell lines, 90 melanomas, and 46 benign nevi. Furthermore, using a lentiviral silencing strategy we show that B-RAF induces senescence in melanocytes and fibroblasts, irrespective of the presence of IGFBP7. Therefore, we conclude that the secreted protein IGFBP7 is dispensable for B-RAF(V600E)-induced senescence in human melanocytes.

Adjuvant immunotherapy with nivolumab versus observation in completely resected Merkel cell carcinoma (ADMEC-O): disease-free survival results from a randomised, open-label, phase 2 trial.

BACKGROUND: Merkel cell carcinoma (MCC) is an immunogenic but aggressive skin cancer. Even after complete resection and radiation, relapse rates are high. PD-1 and PD-L1 checkpoint inhibitors showed clinical benefit in advanced MCC. We aimed to assess efficacy and safety of adjuvant immune checkpoint inhibition in completely resected MCC (ie, a setting without an established systemic standard-of-care treatment). METHODS: In this multicentre phase 2 trial, patients (any stage, Eastern Cooperative Oncology Group performance status 0-1) at 20 academic medical centres in Germany and the Netherlands with completely resected MCC lesions were randomly assigned 2:1 to receive nivolumab 480 mg every 4 weeks for 1 year, or observation, stratified by stage (American Joint Committee on Cancer stages 1-2 vs stages 3-4), age (<65 vs ≥65 years), and sex. Landmark disease-free survival (DFS) at 12 and 24 months was the primary endpoint, assessed in the intention-to-treat populations. Overall survival and safety were secondary endpoints. This planned interim analysis was triggered when the last-patient-in was followed up for more than 1 year. This study is registered with ClinicalTrials.gov (NCT02196961) and with the EU Clinical Trials Register (2013-000043-78). FINDINGS: Between Oct 1, 2014, and Aug 31, 2020, 179 patients were enrolled (116 [65%] stage 3-4, 122 [68%] ≥65 years, 111 [62%] male). Stratification factors (stage, age, sex) were balanced across the nivolumab (n=118) and internal control group (observation, n=61); adjuvant radiotherapy was more common in the control group. At a median follow-up of 24·3 months (IQR 19·2-33·4), median DFS was not reached (between-groups hazard ratio 0·58, 95% CI 0·30-1·12); DFS rates in the nivolumab group were 85% at 12 months and 84% at 24 months, and in the observation group were 77% at 12 months and 73% at 24 months. Overall survival results were not yet mature. Grade 3-4 adverse events occurred in 48 [42%] of 115 patients who received at least one dose of nivolumab and seven [11%] of 61 patients in the observation group. No treatment-related deaths were reported. INTERPRETATION: Adjuvant therapy with nivolumab resulted in an absolute risk reduction of 9% (1-year DFS) and 10% (2-year DFS). The present interim analysis of ADMEC-O might suggest clinical use of nivolumab in this area of unmet medical need. However, overall survival events rates, with ten events in the active treatment group and six events in the half-the-size observation group, are not mature enough to draw conclusions. The explorative data of our trial support the continuation of ongoing, randomised trials in this area. ADMEC-O suggests that adjuvant immunotherapy is clinically feasible in this area of unmet medical need. FUNDING: Bristol Myers Squibb.

Porocarcinomas with PAK1/2/3 fusions: a series of 12 cases.

AIMS: Porocarcinoma is a malignant sweat gland tumour differentiated toward the upper part of the sweat duct and may arise from the transformation of a preexisting benign poroma. In 2019, Sekine et al. demonstrated the presence of YAP1::MAML2 and YAP1::NUTM1 fusions in most poromas and porocarcinomas. Recently, our group identified PAK2-fusions in a subset of benign poromas. Herein we report a series of 12 porocarcinoma cases harbouring PAK1/2/3 fusions. METHODS AND RESULTS: Five patients were male and the median age was 79 years (ranges: 59-95). Tumours were located on the trunk (n = 7), on the thigh (n = 3), neck (n = 1), or groin area (n = 1). Four patients developed distant metastases. Microscopically, seven cases harboured a benign poroma component and a malignant invasive part. Ductal formations were observed in all, while infundibular/horn cysts and cells with vacuolated cytoplasm were detected in seven and six tumours, respectively. In three cases, the invasive component consisted of a proliferation of elongated cells, some of which formed pseudovascular spaces, whereas the others harboured a predominant solid or trabecular growth pattern. Immunohistochemical staining for CEA and EMA confirmed the presence of ducts. Focal androgen receptor expression was detected in three specimens. Whole RNA sequencing evidenced LAMTOR1::PAK1 (n = 2), ZDHHC5::PAK1 (n = 2), DLG1::PAK2, CTDSP1::PAK1, CTNND1::PAK1, SSR1::PAK3, CTNNA1::PAK2, RNF13::PAK2, ROBO1::PAK2, and CD47::PAK2. Activating mutation of HRAS (G13V, n = 3, G13R, n = 1, Q61L, n = 2) was present in six cases. CONCLUSION: Our study suggests that PAK1/2/3 fusions is the oncogenic driver of a subset of porocarcinomas lacking YAP1 rearrangement.

S1-Guideline Sebaceous Carcinoma.

Sebaceous gland carcinomas are rare malignant cutaneous adnexal tumors with sebocytic differentiation. The typical predilection area is the head and neck region, where sebaceous gland carcinomas are the most common malignant adnexal tumors of the skin. According to their localization a distinction is made between periocular and extraocular sebaceous gland carcinomas. Muir-Torre syndrome (MTS) should always be ruled out if it is suspected. In terms of prognosis, sebaceous gland carcinomas are potentially aggressive tumors with a clear tendency to recur and metastasize. Only small extraocular sebaceous gland carcinomas that have been completely resected have a very good prognosis. Sebaceous gland carcinomas most frequently metastasize lymphogenously to regional or distant lymph nodes; organ metastasis occurs less frequently. Periocular sebaceous gland carcinomas have a higher metastasis rate (up to 15%) than extraocular sebaceous gland carcinomas (up to 2%). Complete micrographically controlled surgery (MCS) of the primary tumor is the therapy of first choice, regardless of periocular or extraocular localization. Adjuvant or therapeutic radiotherapy may be considered. There is currently no established standard therapy for advanced, inoperable, or metastatic sebaceous gland carcinomas. Local procedures and systemic therapies such as chemotherapy or immunotherapy can be considered. The procedure should be determined individually by an interdisciplinary tumor board. Close follow-up care is recommended for these potentially aggressive carcinomas.

Adjuvant nivolumab plus ipilimumab or nivolumab alone versus placebo in patients with resected stage IV melanoma with no evidence of disease (IMMUNED): final results of a randomised, double-blind, phase 2 trial.

BACKGROUND: The IMMUNED trial previously showed significant improvements in recurrence-free survival for adjuvant nivolumab plus ipilimumab as well as for adjuvant nivolumab alone in patients with stage IV melanoma with no evidence of disease after resection or radiotherapy. Here, we report the final analysis, including overall survival data. METHODS: IMMUNED was an investigator-sponsored, double-blind, placebo-controlled, three-arm, phase 2 trial conducted in 20 academic medical centres in Germany. Eligible patients were aged 18-80 years with stage IV melanoma with no evidence of disease after surgery or radiotherapy. Patients were randomly assigned (1:1:1) to either nivolumab plus ipilimumab (nivolumab 1 mg/kg plus ipilimumab 3 mg/kg every 3 weeks for four doses followed by nivolumab 3 mg/kg every 2 weeks), nivolumab monotherapy (nivolumab 3 mg/kg every 2 weeks), or matching placebo, for up to 1 year. The primary endpoint was recurrence-free survival in the intention-to-treat population. Secondary endpoints were time-to-recurrence, overall survival, progression-free survival or recurrence-free survival 2 (in patients in the placebo group who crossed over to nivolumab monotherapy after experiencing disease recurrence), and safety endpoints. This trial is registered on ClinicalTrials.gov (NCT02523313), and is complete. FINDINGS: Between Sept 2, 2015, and Nov 20, 2018, 175 patients were enrolled in the study, and 167 were randomly assigned to receive either nivolumab plus ipilimumab (n=56), nivolumab plus ipilimumab-matching placebo (n=59), or double placebo control (n=52). At a median follow-up of 49·2 months (IQR 34·9-58·1), 4-year recurrence-free survival was 64·2% (95% CI 49·2-75·9) in the nivolumab plus ipilimumab group, 31·4% (19·7-43·8) in the nivolumab alone group, and 15·0% (6·7-26·6) in the placebo group. The hazard ratio (HR) for recurrence for the nivolumab plus ipilimumab group versus placebo was 0·25 (97·5% CI 0·13-0·48; p<0·0001), and for the nivolumab group versus placebo was 0·60 (0·36-1·00; p=0·024). Median overall survival was not reached in any treatment group. The HR for overall survival was significantly in favour of the nivolumab plus ipilimumab group versus placebo (HR 0·41; 95% CI 0·17-0·99; p=0·040), but not for the nivolumab group versus placebo (HR 0·75; 0·36-1·56; p=0·44). 4-year overall survival was 83·8% (95% CI 68·8-91·9) in the nivolumab plus ipilimumab group, 72·6% (57·4-83·2) in the nivolumab alone group, and 63·1% (46·9-75·6) in the placebo group. The median progression-free survival or recurrence-free survival 2 of patients in the placebo group who crossed over to nivolumab monotherapy after experiencing disease recurrence was not reached (95% CI 21·2 months to not reached). Rates of grade 3-4 treatment-related adverse events remained largely unchanged compared with our previous report, occurring in 71% (95% CI 57-82) of the nivolumab plus ipilimumab group, and 29% (95% CI 17-42) of patients receiving nivolumab alone. There were no treatment-related deaths. INTERPRETATION: Both active regimens continued to show significantly improved recurrence-free survival compared with placebo in patients with stage IV melanoma with no evidence of disease who were at high risk of recurrence. Overall survival was significantly improved for patients receiving nivolumab plus ipilimumab compared with placebo. Use of subsequent anti-PD-1-based therapy was high in patients in the placebo group after recurrence and most likely impacted the overall survival comparison of nivolumab alone versus placebo. The recurrence-free and overall survival benefit of nivolumab plus ipilimumab over placebo reinforces the change of practice already initiated for the treatment of patients with stage IV melanoma with no evidence of disease. FUNDING: Bristol-Myers Squibb.

Mesenchymal-epithelial transition in lymph node metastases of oral squamous cell carcinoma is accompanied by ZEB1 expression.

BACKGROUND: Oral squamous cell carcinoma (OSCC), an HPV-negative head and neck cancer, frequently metastasizes to the regional lymph nodes but only occasionally beyond. Initial phases of metastasis are associated with an epithelial-mesenchymal transition (EMT), while the consolidation phase is associated with mesenchymal-epithelial transition (MET). This dynamic is referred to as epithelial-mesenchymal plasticity (EMP). While it is known that EMP is essential for cancer cell invasion and metastatic spread, less is known about the heterogeneity of EMP states and even less about the heterogeneity between primary and metastatic lesions. METHODS: To assess both the heterogeneity of EMP states in OSCC cells and their effects on stromal cells, we performed single-cell RNA sequencing (scRNAseq) of 5 primary tumors, 9 matching metastatic and 5 tumor-free lymph nodes and re-analyzed publicly available scRNAseq data of 9 additional primary tumors. For examining the cell type composition, we performed bulk transcriptome sequencing. Protein expression of selected genes were confirmed by immunohistochemistry. RESULTS: From the 23 OSCC lesions, the single cell transcriptomes of a total of 7263 carcinoma cells were available for in-depth analyses. We initially focused on one lesion to avoid confounding inter-patient heterogeneity and identified OSCC cells expressing genes characteristic of different epithelial and partial EMT stages. RNA velocity and the increase in inferred copy number variations indicated a progressive trajectory towards epithelial differentiation in this metastatic lesion, i.e., cells likely underwent MET. Extension to all samples revealed a less stringent but essentially similar pattern. Interestingly, MET cells show increased activity of the EMT-activator ZEB1. Immunohistochemistry confirmed that ZEB1 was co-expressed with the epithelial marker cornifin B in individual tumor cells. The lack of E-cadherin mRNA expression suggests this is a partial MET. Within the tumor microenvironment we found immunomodulating fibroblasts that were maintained in primary and metastatic OSCC. CONCLUSIONS: This study reveals that EMP enables different partial EMT and epithelial phenotypes of OSCC cells, which are endowed with capabilities essential for the different stages of the metastatic process, including maintenance of cellular integrity. During MET, ZEB1 appears to be functionally active, indicating a more complex role of ZEB1 than mere induction of EMT.

B-cell lymphoma-extra large is a promising drug target in Merkel cell carcinoma.

BACKGROUND: Merkel cell carcinoma (MCC) is an aggressive skin tumour with neuroendocrine differentiation. Immunotherapies are effective in the treatment of patients with advanced-stage MCC, but for patients whose tumours cannot be controlled by the immune system, alternative approaches are urgently needed. OBJECTIVES: To identify overexpressed oncogenes as potential drug targets for MCC. METHODS: NanoString platform, digital droplet polymerase chain reaction (ddPCR) and fluorescence in situ hybridization (FISH) assays were used to determine copy number variations (CNVs); BCL2L1 and PARP1 mRNA expression levels were determined by quantitative real-time polymerase chain reaction (qRT-PCR), B-cell lymphoma extra-large (Bcl-xL) and poly (ADP-ribose) polymerase 1 (PARP1) protein by immuno-blot. Specific Bcl-xL inhibitors and a PARP1 inhibitor were used alone or in combination to test their antitumour effect. RESULTS: Screening for CNVs in 13 classic Merkel cell polyomavirus (MCPyV)-positive and MCPyV-negative MCC cell lines revealed BCL2L1 gains and amplifications, confirmed by ddPCR in 10 cell lines. By ddPCR and FISH, we demonstrated that BCL2L1 gains are present in tumour tissue. BCL2L1 copy number gains were associated with increased Bcl-xL mRNA and protein expression. However, high Bcl-xL expression was not restricted to MCC cells harbouring a BCL2L1 gain/amplification, suggesting additional epigenetic means of regulation. The functional relevance of Bcl-xL in MCC cells was demonstrated by the fact that specific Bcl-xL inhibitors (A1331852 and WEHI-539) led to the induction of apoptosis. Owing to the strong expression and activation of PARP1 in MCC cell lines, we next tested the combination of Bcl-xL inhibitors with the PARP1 inhibitor olaparib, which showed synergistic antitumour effects. CONCLUSIONS: Bcl-xL, which is highly expressed in MCC, appears to be an attractive therapeutic target for the treatment of this tumour, especially as the effect of specific Bcl-xL inhibitors is synergistically enhanced by simultaneous PARP inhibition.

S2k Guideline - Merkel cell carcinoma (MCC, neuroendocrine carcinoma of the skin) - Update 2022.

Merkel cell carcinoma (MCC, ICD-O M8247/3) is a rare, malignant, primary skin tumor with epithelial and neuroendocrine differentiation. The tumor cells share many morphologic, immunohistochemical, and ultrastructural features with cutaneous Merkel cells. Nevertheless, the cell of origin of MCC is unclear. MCC appears clinically as a reddish to purple spherical tumor with a smooth, shiny surface and a soft to turgid, elastic consistency, usually showing rapid growth. Spontaneous and often complete regressions of the tumor are observed. These likely immunologically-mediated regressions explain the cases in which only lymph node or distant metastases are found at the time of initial diagnosis and why the tumor responds very well to immunomodulatory therapies even at advanced stages. Due to its aggressiveness, the usually given indication for sentinel lymph node biopsy, the indication of adjuvant therapies to be evaluated, as well as the complexity of the necessary diagnostics, clinical management should already be determined by an interdisciplinary tumor board at the time of initial diagnosis.

S3 guideline "actinic keratosis and cutaneous squamous cell carcinoma" - update 2023, part 2: epidemiology and etiology, diagnostics, surgical and systemic treatment of cutaneous squamous cell carcinoma (cSCC), surveillance and prevention.

Actinic keratosis (AK) are common lesions in light-skinned individuals that can potentially progress to cutaneous squamous cell carcinoma (cSCC). Both conditions may be associated with significant morbidity and constitute a major disease burden, especially among the elderly. To establish an evidence-based framework for clinical decision making, the guideline "actinic keratosis and cutaneous squamous cell carcinoma" was updated and expanded by the topics cutaneous squamous cell carcinoma in situ (Bowen's disease) and actinic cheilitis. The guideline is aimed at dermatologists, general practitioners, ear nose and throat specialists, surgeons, oncologists, radiologists and radiation oncologists in hospitals and office-based settings, as well as other medical specialties, policy makers and insurance funds involved in the diagnosis and treatment of patients with AK and cSCC. A separate guideline exists for patients and their relatives. In this part, we will address aspects relating to epidemiology and etiology, diagnostics, surgical and systemic treatment of cutaneous squamous cell carcinoma (cSCC), surveillance and prevention.

High-resolution analysis of Merkel Cell Polyomavirus in Merkel Cell Carcinoma reveals distinct integration patterns and suggests NHEJ and MMBIR as underlying mechanisms.

Merkel Cell Polyomavirus (MCPyV) is the etiological agent of the majority of Merkel Cell Carcinomas (MCC). MCPyV positive MCCs harbor integrated, defective viral genomes that constitutively express viral oncogenes. Which molecular mechanisms promote viral integration, if distinct integration patterns exist, and if integration occurs preferentially at loci with specific chromatin states is unknown. We here combined short and long-read (nanopore) next-generation sequencing and present the first high-resolution analysis of integration site structure in MCC cell lines as well as primary tumor material. We find two main types of integration site structure: Linear patterns with chromosomal breakpoints that map closely together, and complex integration loci that exhibit local amplification of genomic sequences flanking the viral DNA. Sequence analysis suggests that linear patterns are produced during viral replication by integration of defective/linear genomes into host DNA double strand breaks via non-homologous end joining, NHEJ. In contrast, our data strongly suggest that complex integration patterns are mediated by microhomology-mediated break-induced replication, MMBIR. Furthermore, we show by ChIP-Seq and RNA-Seq analysis that MCPyV preferably integrates in open chromatin and provide evidence that viral oncogene expression is driven by the viral promoter region, rather than transcription from juxtaposed host promoters. Taken together, our data explain the characteristics of MCPyV integration and may also provide a model for integration of other oncogenic DNA viruses such as papillomaviruses.

Conversion of Sox2-dependent Merkel cell carcinoma to a differentiated neuron-like phenotype by T antigen inhibition.

Viral cancers show oncogene addiction to viral oncoproteins, which are required for survival and proliferation of the dedifferentiated cancer cell. Human Merkel cell carcinomas (MCCs) that harbor a clonally integrated Merkel cell polyomavirus (MCV) genome have low mutation burden and require viral T antigen expression for tumor growth. Here, we showed that MCV+ MCC cells cocultured with keratinocytes undergo neuron-like differentiation with neurite outgrowth, secretory vesicle accumulation, and the generation of sodium-dependent action potentials, hallmarks of a neuronal cell lineage. Cocultured keratinocytes are essential for induction of the neuronal phenotype. Keratinocyte-conditioned medium was insufficient to induce this phenotype. Single-cell RNA sequencing revealed that T antigen knockdown inhibited cell cycle gene expression and reduced expression of key Merkel cell lineage/MCC marker genes, including HES6, SOX2, ATOH1, and KRT20 Of these, T antigen knockdown directly inhibited Sox2 and Atoh1 expression. MCV large T up-regulated Sox2 through its retinoblastoma protein-inhibition domain, which in turn activated Atoh1 expression. The knockdown of Sox2 in MCV+ MCCs mimicked T antigen knockdown by inducing MCC cell growth arrest and neuron-like differentiation. These results show Sox2-dependent conversion of an undifferentiated, aggressive cancer cell to a differentiated neuron-like phenotype and suggest that the ontology of MCC arises from a neuronal cell precursor.

S1 Guidelines for the Kaposi Sarcoma.

Kaposi's sarcoma (KS) is a rare, malignant, multilocular vascular disease originating from lymphatic endothelial cells that can primarily affect the skin and mucous membranes, but also the lymphatic system and internal organs such as the gastrointestinal tract, lungs or liver. Five epidemiological subtypes of KS with variable clinical course and prognosis are distinguished, with increased incidence in specific populations: (1) Classical KS, (2) Iatrogenic KS in immunosuppression, (3) Endemic (African) lymphadenopathic KS, (4) Epidemic, HIV-associated KS and KS associated with immune reconstitution inflammatory syndrome (IRIS), and (5) KS in men who have sex with men (MSM) without HIV infection. This interdisciplinary guideline summarizes current practice-relevant recommendations on diangostics and therapy of the different forms of KS. The recommendations mentioned in this short guideline are elaborated in more detail in the extended version of the guideline (online format of the JDDG).

S1-Leitlinie: Kaposi-Sarkom.

Das Kaposi-Sarkom (KS) ist eine seltene, maligne, von lymphatischen Endothelzellen ausgehende, multilokuläre Gefäßerkrankung, die vor allem Haut und Schleimhäute, aber auch das lymphatische System und innere Organe wie den Gastrointestinaltrakt, die Lunge oder die Leber befallen kann. Fünf epidemiologische Subtypen des KS mit variablem klinischem Verlauf und unterschiedlicher Prognose werden unterschieden, die in spezifischen Populationen vermehrt auftreten: (1) klassisches KS, (2) iatrogenes KS bei Immunsuppression, (3) endemisches (afrikanisches) lymphadenopathisches KS, (4) epidemisches, HIV-assoziiertes KS und mit einem Immunrekonstitutions-Inflammations-Syndrom (IRIS) assoziiertes KS und (5) KS bei Männern, die Sex mit Männern haben (MSM) ohne HIV-Infektion. Diese interdisziplinäre Leitlinie fasst aktuelle praxisrelevante Empfehlungen zu Diagnostik und Therapie der verschiedenen Formen des KS zusammen. Die in dieser Kurzleitlinie genannten Empfehlungen werden in der Langfassung der Leitlinie (Online-Version des JDDG) detaillierter ausgeführt.

Adjuvant nivolumab plus ipilimumab or nivolumab monotherapy versus placebo in patients with resected stage IV melanoma with no evidence of disease (IMMUNED): a randomised, double-blind, placebo-controlled, phase 2 trial.

BACKGROUND: Nivolumab and ipilimumab, alone or in combination, are widely used immunotherapeutic treatment options for patients with advanced-ie, unresectable or metastatic-melanoma. This criterion, however, excludes patients with stage IV melanoma with no evidence of disease. We therefore aimed to evaluate the safety and efficacy of adjuvant nivolumab plus ipilimumab or nivolumab monotherapy versus a placebo in this patient population. METHODS: We did a randomised, double-blind, placebo-controlled, phase 2 trial in 20 German academic medical centres. Eligible patients were aged 18-80 years with stage IV melanoma with no evidence of disease after surgery or radiotherapy. Key exclusion criteria included uveal or mucosal melanoma, previous therapy with checkpoint inhibitors, and any previous immunosuppressive therapy within the 30 days before study drug administration. Eligible patients were randomly assigned (1:1:1), using a central, interactive, online system, to the nivolumab plus ipilimumab group (1 mg/kg of intravenous nivolumab every 3 weeks plus 3 mg/kg of intravenous ipilimumab every 3 weeks for four doses, followed by 3 mg/kg of nivolumab every 2 weeks), nivolumab monotherapy group (3 mg/kg of intravenous nivolumab every 2 weeks plus ipilimumab-matching placebo during weeks 1-12), or double-matching placebo group. The primary endpoint was the recurrence-free survival in the intention-to-treat population. The results presented in this report reflect the prespecified interim analysis of recurrence-free survival after 90 events had been reported. This study is registered with ClinicalTrials.gov, NCT02523313, and is ongoing. FINDINGS: Between Sept 2, 2015, and Nov 20, 2018, 167 patients were randomly assigned to receive nivolumab plus ipilimumab (n=56), nivolumab (n=59), or placebo (n=52). As of July 2, 2019, at a median follow-up of 28·4 months (IQR 17·7-36·8), median recurrence-free survival was not reached in the nivolumab plus ipilimumab group, whereas median recurrence-free survival was 12·4 months (95% CI 5·3-33·3) in the nivolumab group and 6·4 months (3·3-9·6) in the placebo group. The hazard ratio for recurrence for the nivolumab plus ipilimumab group versus placebo group was 0·23 (97·5% CI 0·12-0·45; p<0·0001), and for the nivolumab group versus placebo group was 0·56 (0·33-0·94; p=0·011). In the nivolumab plus ipilimumab group, recurrence-free survival at 1 year was 75% (95% CI 61·0-84·9) and at 2 years was 70% (55·1-81·0); in the nivolumab group, 1-year recurrence-free survival was 52% (38·1-63·9) and at 2 years was 42% (28·6-54·5); and in the placebo group, this rate was 32% (19·8-45·3) at 1 year and 14% (5·9-25·7) at 2 years. Treatment-related grade 3-4 adverse events were reported in 71% (95% CI 57-82) of patients in the nivolumab plus ipilimumab group and in 27% (16-40) of those in the nivolumab group. Treatment-related adverse events of any grade led to treatment discontinuation in 34 (62%) of 55 patients in the nivolumab plus ipilimumab group and seven (13%) of 56 in the nivolumab group. Three deaths from adverse events were reported but were considered unrelated to the study treatment. INTERPRETATION: Adjuvant therapy with nivolumab alone or in combination with ipilimumab increased recurrence-free survival significantly compared with placebo in patients with stage IV melanoma with no evidence of disease. The rates of grade 3-4 treatment-related adverse events in both active treatment groups were higher than the rates reported in previous pivotal trials done in advanced melanoma with measurable disease. FUNDING: Bristol-Myers Squibb.

Activity of ipilimumab plus nivolumab in avelumab-refractory Merkel cell carcinoma.

BACKGROUND: Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine cutaneous malignancy with poor prognosis. In Europe, approved systemic therapies are limited to the PD-L1 inhibitor avelumab. For avelumab-refractory patients, efficient and safe treatment options are lacking. METHODS: At three different sites in Germany, clinical and molecular data of patients with metastatic MCC being refractory to the PD-L1 inhibitor avelumab and who were later on treated with combined IPI/NIVO were retrospectively collected and evaluated. RESULTS: Five patients treated at three different academic sites in Germany were enrolled. Three out of five patients investigated for this report responded to combined IPI/NIVO according to RECIST 1.1. Combined immunotherapy was well tolerated without any grade II or III immune-related adverse events. Two out of three responders to IPI/NIVO received platinum-based chemotherapy in between avelumab and combined immunotherapy. CONCLUSION: In this small retrospective study, we observed a high response rate and durable responses to subsequent combined immunotherapy with IPI/NIVO in avelumab-refractory metastatic MCC patients. In conclusion, our data suggest a promising activity of second- or third-line PD-1- plus CTLA-4-blockade in patients with anti-PD-L1-refractory MCC.

BRAF and MEK inhibition in melanoma patients enables reprogramming of tumor infiltrating lymphocytes.

BACKGROUND: Combined inhibition of BRAF/MEK is an established therapy for melanoma. In addition to its canonical mode of action, effects of BRAF/MEK inhibitors on antitumor immune responses are emerging. Thus, we investigated the effect of these on adaptive immune responses. PATIENTS, METHODS AND RESULTS: Sequential tumor biopsies obtained before and during BRAF/MEK inhibitor treatment of four (n = 4) melanoma patients were analyzed. Multiplexed immunofluorescence staining of tumor tissue revealed an increased infiltration of CD4+ and CD8+ T cells upon therapy. Determination of the T-cell receptor repertoire usage demonstrated a therapy induced increase in T-cell clonotype richness and diversity. Application of the Grouping of Lymphocyte Interactions by Paratope Hotspots algorithm revealed a pre-existing immune response against melanoma differentiation and cancer testis antigens that expanded preferentially upon therapy. Indeed, most of the T-cell clonotypes found under BRAF/MEK inhibition were already present in lower numbers before therapy. This expansion appears to be facilitated by induction of T-bet and TCF7 in T cells, two transcription factors required for self-renewal and persistence of CD8+ memory T cells. CONCLUSIONS: Our results suggest that BRAF/MEK inhibition in melanoma patients allows an increased expansion of pre-existing melanoma-specific T cells by induction of T-bet and TCF7 in these.

Antibiotics inhibit tumor and disease activity in cutaneous T-cell lymphoma.

It has been proposed that CD4 T-cell responses to Staphylococcus aureus (SA) can inadvertently enhance neoplastic progression in models of skin cancer and cutaneous T-cell lymphoma (CTCL). In this prospective study, we explored the effect of transient antibiotic treatment on tumor cells and disease activity in 8 patients with advanced-stage CTCL. All patients experienced significant decrease in clinical symptoms in response to aggressive, transient antibiotic treatment. In some patients, clinical improvements lasted for more than 8 months. In 6 of 8 patients, a malignant T-cell clone could be identified in lesional skin, and a significant decrease in the fraction of malignant T cells was observed following antibiotics but an otherwise unchanged treatment regimen. Immunohistochemistry, global messenger RNA expression, and cell-signaling pathway analysis indicated that transient aggressive antibiotic therapy was associated with decreased expression of interleukin-2 high-affinity receptors (CD25), STAT3 signaling, and cell proliferation in lesional skin. In conclusion, this study provides novel evidence suggesting that aggressive antibiotic treatment inhibits malignant T cells in lesional skin. Thus, we provide a novel rationale for treatment of SA in advanced CTCL.

Differences in site-specific incidence and relative survival of cutaneous and mucocutaneous genital squamous cell carcinoma in Germany, 2007-2015.

Direct comparisons of the incidence and survival of cutaneous vs mucocutaneous genital squamous cell carcinomas (SCCs) are lacking even though they may bring important insights. We aimed to compare incidence rates and survival of cutaneous and mucocutaneous genital SCCs head-to-head, using the same source population, cancer registry methodology and statistical methods in a population of predominantly white Caucasian descent. Using data (2007-2015) from the population-based cancer registry of North Rhine-Westphalia, (population of 18 million people), we estimated age-specific and age-standardized (old European standard) incidence rates and age-standardized relative 5-year survival of SCC with the period approach for the period 2012 to 2015. Overall, 83 650 SCC cases were registered. The age-standardized incidence rates (per 100 000 person-years) of cutaneous SCCs were 36.5 (SE 0.17) and 17.0 (SE 0.11) among men and women, respectively, with corresponding rates for mucocutaneous genital skin, 1.3 (SE 0.03) and 4.5 (SE 0.06) for men and women, respectively. In all age groups, incidence rates of mucocutaneous genital SCCs were higher in women than men. Men had higher cutaneous SCC incidence at all nongenital subsites than women, with the exception of the lower extremities. Five-year relative survival was considerably lower for mucocutaneous genital SCCs (men: 71%, women: 75%), especially of the scrotal skin (67%) and labia majora (62%) than for SCC of nongenital skin (men: 93%, women: 97%). Given their relatively high incidence together with a lower survival probability, future studies are warranted to establish therapies for advanced mucocutaneous genital SCC, such as immune checkpoint inhibition.

S3 guideline for actinic keratosis and cutaneous squamous cell carcinoma - short version, part 1: diagnosis, interventions for actinic keratoses, care structures and quality-of-care indicators.

Actinic keratoses (AK) are common lesions in light-skinned individuals that can potentially progress to cutaneous squamous cell carcinoma (cSCC). Both conditions may be associated with significant morbidity and constitute a major disease burden, especially among the elderly. To establish an evidence-based framework for clinical decision making, the guideline "actinic keratosis and cutaneous squamous cell carcinoma" was developed using the highest level of methodology (S3) according to regulations issued by the Association of Scientific Medical Societies in Germany (AWMF). The guideline is aimed at dermatologists, general practitioners, ENT specialists, surgeons, oncologists, radiologists and radiation oncologists in hospitals and office-based settings as well as other medical specialties involved in the diagnosis and treatment of patients with AK and cSCC. The guideline is also aimed at affected patients, their relatives, policy makers and insurance funds. In the first part, we will address aspects relating to diagnosis, interventions for AK, care structures and quality-of-care indicators.