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Abstract Multi-omics profiling by CITE-seq bridges the RNA-protein gap in single-cell analysis but has been largely applied to liquid biopsies. Applying CITE-seq to clinically relevant solid biopsies to characterize healthy tissue and the tumor microenvironment is an essential next step in single-cell translational studies. In this study, gating of cell populations based on their transcriptome signatures for use in cell type-specific ridge plots allowed identification of positive antibody signals and setting of manual thresholds. Next, we compare five skin dissociation protocols by taking into account dissociation efficiency, captured cell type heterogeneity and recovered surface proteome. To assess the effect of enzymatic digestion on transcriptome and epitope expression in immune cell populations, we analyze peripheral blood mononuclear cells (PBMCs) with and without dissociation. To further assess the RNA-protein gap, RNA-protein we perform codetection and correlation analyses on thresholded protein values. Finally, in a proof-of-concept study, using protein abundance analysis on selected surface markers in a cohort of healthy skin, primary, and metastatic melanoma we identify CD56 surface marker expression on metastatic melanoma cells, which was further confirmed by multiplex immunohistochemistry. This work provides practical guidelines for processing and analysis of clinically relevant solid tissue biopsies for biomarker discovery.

Abstract Immune checkpoint blockade therapy is beneficial and even curative for some cancer patients. However, the majority don’t respond to immune therapy. Across different tumor types, pre-existing T cell infiltrates predict response to checkpoint-based immunotherapy. Based on in vitro pharmacological studies, mouse models and analyses of human melanoma patients, we show that the cytokine GDF-15 impairs LFA-1/β2-integrin-mediated adhesion of T cells to activated endothelial cells, which is a pre-requisite of T cell extravasation. In melanoma patients, GDF-15 serum levels strongly correlate with failure of PD-1-based immune checkpoint blockade therapy. Neutralization of GDF-15 improves both T cell trafficking and therapy efficiency in murine tumor models. Thus GDF-15, beside its known role in cancer-related anorexia and cachexia, emerges as a regulator of T cell extravasation into the tumor microenvironment, which provides an even stronger rationale for therapeutic anti-GDF-15 antibody development.

Melanoma cells can switch between proliferative and invasive phenotypes. Here the authors show that the embryonic stem cell factor Sall4 is a negative regulator of melanoma phenotype switching where its loss leads to the acquisition of an invasive phenotype, due to derepression of invasiveness genes.

The genetic changes that occur in late stage metastatic melanoma are not well delineated. Here, the authors use rapid autopsy samples from metastatic melanoma patients and show that the late stage in the disease is characterised by whole genome doubling and aneuploidy.

Deregulation of cellular metabolism through metabolic rewiring and translational reprogramming are considered hallmark traits of tumor development and malignant progression. The transcription factor YY1 is a master regulator of metabolism that we have previously shown to orchestrate a metabolic program required for melanoma formation. In this study, we demonstrate that YY1, while being essential for primary melanoma formation, suppresses metastatic spreading. Its downregulation or loss resulted in the induction of an invasiveness gene program and sensitized melanoma cells for pro-invasive signaling molecules, such as TGF-β. In addition, NGFR, a key effector in melanoma invasion and phenotype switching, was among the most upregulated genes after YY1 knockdown. High levels of NGFR were also associated with other metabolic stress inducers, further indicating that YY1 knockdown mimics a metabolic stress program associated with an increased invasion potential in melanoma. Accordingly, while counteracting tumor growth, loss of YY1 strongly promoted melanoma cell invasiveness in vitro and metastasis formation in melanoma mouse models in vivo. Thus, our findings show that the metabolic regulator YY1 controls phenotype switching in melanoma.

The bacterial pathogen Helicobacter pylori is known for its ability to induce DNA double-strand breaks in the genome of its target cells. Here, we show that H. pylori-induced DNA damage and replication stress occurs in S-phase cells as a result of R-loop-mediated transcription/replication conflicts that are triggered by activation of the ALPK1/TIFA/NF-κB signaling axis.

Summary: The two human pathogens Helicobacter pylori and Mycobacterium tuberculosis (Mtb) co-exist in many geographical areas of the world. Here, using a co-infection model of H. pylori and the Mtb relative M. bovis bacillus Calmette-Guérin (BCG), we show that both bacteria affect the colonization and immune control of the respective other pathogen. Co-occurring M. bovis boosts gastric Th1 responses and H. pylori control and aggravates gastric immunopathology. H. pylori in the stomach compromises immune control of M. bovis in the liver and spleen. Prior antibiotic H. pylori eradication or M. bovis-specific immunization reverses the effects of H. pylori. Mechanistically, the mutual effects can be attributed to the redirection of regulatory T cells (Treg cells) to sites of M. bovis infection. Reversal of Treg cell redirection by CXCR3 blockade restores M. bovis control. In conclusion, the simultaneous presence of both pathogens exacerbates the problems associated with each individual infection alone and should possibly be factored into treatment decisions.

Lymphoid neogenesis gives rise to tertiary lymphoid structures (TLS) in the periphery of multiple cancer types including muscle invasive bladder cancer (MIBC) where it has positive prognostic and predictive associations. Here, we explored molecular, clinical, and histological data of The Cancer Genome Atlas, as well as the IMvigor210 dataset to study factors associated with TLS development and function in the tumor microenvironment (TME) of MIBC. We also analyzed tumor immune composition including TLS in an independent, retrospective MIBC cohort. We found that the combination of TLS density and tumor mutational burden provides a novel independent prognostic biomarker in MIBC. Gene expression profiles obtained from intratumoral regions that rarely contain TLS in MIBC showed poor correlation with the prognostic TLS density measured in tumor periphery. Tumors with high TLS density showed increased gene signatures as well as infiltration of activated lymphocytes. Intratumoral B-cell and CD8+ T-cell co-infiltration was frequent in TLS-high samples, and such regions harbored the highest proportion of PD-1+TCF1+ progenitor-like T cells, naïve T cells, and activated B cells when compared to regions predominantly infiltrated by either B cells or CD8+ T cells alone. We found four TLS maturation subtypes; however, differences in TLS composition appeared to be dictated by the TME and not by the TLS maturation status. Finally, we identified one downregulated and three upregulated non-immune cell-related genes in TME with high TLS density, which may represent candidates for tumor-intrinsic regulation of lymphoid neogenesis. Our study provides novel insights into TLS-associated gene expression and immune contexture of MIBC and indicates towards the relevance of B-cell and CD8+ T-cell interactions in anti-tumor immunity within and outside TLS.

In the original article [...]

Abstract Background MAP kinase inhibitor (MAPKi) therapy for BRAF mutated melanoma is characterized by high response rates but development of drug resistance within a median progression-free survival (PFS) of 9–12 months. Understanding mechanisms of resistance and identifying effective therapeutic alternatives is one of the most important scientific challenges in melanoma. Using proteomics, we want to specifically gain insight into the pathophysiological process of cerebral metastases. Methods Cerebral metastases from melanoma patients were initially analyzed by a LC–MS shotgun approach performed on a QExactive HF hybrid quadrupole-orbitrap mass spectrometer. For further validation steps after bioinformatics analysis, a targeted LC-QQQ-MS approach, as well as Western blot, immunohistochemistry and immunocytochemistry was performed. Results In this pilot study, we were able to identify 5977 proteins by LC–MS analysis (data are available via ProteomeXchange with identifier PXD007592). Based on PFS, samples were classified into good responders (PFS ≥ 6 months) and poor responders (PFS $$\le$$ ≤ 3 months). By evaluating these proteomic profiles according to gene ontology (GO) terms, KEGG pathways and gene set enrichment analysis (GSEA), we could characterize differences between the two distinct groups. We detected an EMT feature (up-regulation of N-cadherin) as classifier between the two groups, V-type proton ATPases, cell adhesion proteins and several transporter and exchanger proteins to be significantly up-regulated in poor responding patients, whereas good responders showed an immune activation, among other features. We identified class-discriminating proteins based on nearest shrunken centroids, validated and quantified this signature by a targeted approach and could correlate parts of this signature with resistance using the CPL/MUW proteome database and survival of patients by TCGA analysis. We further validated an EMT-like signature as a major discriminator between good and poor responders on primary melanoma cells derived from cerebral metastases. Higher immune activity is demonstrated in patients with good response to MAPKi by immunohistochemical staining of biopsy samples of cerebral melanoma metastases. Conclusions Employing proteomic analysis, we confirmed known extra-cerebral resistance mechanisms in the cerebral metastases and further discovered possible brain specific mechanisms of drug efflux, which might serve as treatment targets or as predictive markers for these kinds of metastasis.

Abstract Background During embryonic development Wnt family members and bone morphogenetic proteins (BMPs) cooperatively induce epithelial-mesenchymal transition (EMT) in the neural crest. Wnt and BMPs are reactivated during malignant transformation in melanoma. We previously demonstrated that the BMP-antagonist noggin blocked the EMT phenotype of melanoma cells in the neural crest and malignant invasion of melanoma cells in the chick embryo; vice-versa, malignant invasion was induced in human melanocytes in vivo by pre-treatment with BMP-2. Results Although there are conflicting results in the literature about the role of β-catenin for invasion of melanoma cells, we found Wnt/β-catenin signaling to be analogously important for the EMT-like phenotype of human metastatic melanoma cells in the neural crest and during invasion: β-catenin was frequently expressed at the invasive front of human primary melanomas and Wnt3a expression was inversely correlated with survival of melanoma patients. Accordingly, cytoplasmic β-catenin levels were increased during invasion of melanoma cells in the rhombencephalon of the chick embryo. Fibroblast derived Wnt3a reduced melanoma cell adhesion and enhanced migration, while the β-catenin inhibitor PKF115–584 increased adhesion and reduced migration in vitro and in the chick embryonic neural crest environment in vivo. Similarly, knockdown of β-catenin impaired intradermal melanoma cell invasion and PKF115–584 efficiently reduced liver metastasis in a chick chorioallantoic membrane model. Our observations were accompanied by specific alterations in gene expression which are linked to overall survival of melanoma patients. Conclusion We present a novel role for Wnt-signaling in neural crest like melanoma cell invasion and metastasis, stressing the crucial role of embryonic EMT-inducing neural crest signaling for the spreading of malignant melanoma.

The aggressive nature of melanoma cells relies on their ability to switch from a high-proliferative/low-invasive to a low-proliferative/high-invasive state; however, the mechanisms governing this switch are unclear. Here, using in vivo models of human melanoma, the authors show that CD271 is a key regulator of phenotype switching and metastasis formation.

Background: RNA editing is a highly conserved posttranscriptional mechanism that contributes to transcriptome diversity. In mammals, it includes nucleobase deaminations that convert cytidine (C) into uridine (U) and adenosine (A) into inosine (I). Evidence from cancer studies indicates that RNA-editing enzymes promote certain mechanisms of tumorigenesis. On the other hand, recoding editing in mRNA can generate mutations in proteins that can participate in the Major Histocompatibility Complex (MHC) ligandome and can therefore be recognized by the adaptive immune system. Anti-cancer treatment based on the administration of immune checkpoint inhibitors enhance these natural anti-cancer immune responses. Results: Based on RNA-Seq datasets, we evaluated the editome of melanoma cell lines generated from patients pre- and post-immunotherapy with immune checkpoint inhibitors. Our results reveal a differential editing in Arthrobacter luteus (Alu) sequences between samples pre-therapy and relapses during therapy with immune checkpoint inhibitors. Conclusion: These data pave the way towards the development of new diagnostics and therapies targeted to editing that could help in preventing relapses during immunotherapies.

The recent emergence of cancer immunotherapies initiated a significant shift in the clinical management of metastatic melanoma. Prior to 2011, melanoma patients only had palliative treatment solutions which offered little to no survival benefit. In 2018, with immunotherapy, melanoma patients can now contemplate durable or even complete remission. Treatment with novel immune checkpoint inhibitors, anti-cytotoxic T-lymphocyte protein 4 and anti-programmed cell death protein 1, clearly result in superior median and long-term survivals compared to standard chemotherapy; however, more than half of the patients do not respond to immune checkpoint blockade. Currently, clinicians do not have any effective way to stratify melanoma patients for immunotherapies. Research is now focusing on identifying biomarkers which could predict a patient’s response prior treatment initiation (or very early during treatment course), in order to maximize therapeutic efficacy, avoid unnecessary costs, and undesirable heavy side effects for the patient. Given the rapid developments in this field and the translational potential for some of the biomarkers, we will summarize the current state of biomarker research for immunotherapy in melanoma, with an emphasis on omics technologies such as next-generation sequencing and mass cytometry (CyTOF).

The originally published version of this Article was updated shortly after publication to add the words ‘The’ and ‘affinity’ to the title, following their inadvertent removal during the production process. This has now been corrected in both the PDF and HTML versions of the Article.

The Cancer Genome Atlas (TCGA) has given researchers and clinicians unprecedented access to many different cancers through multiple platforms that include exome sequencing, comparative genomic hybridisation (CGH) arrays, DNA methylation arrays, RNA sequencing, reverse protein phase arrays (RPPA), and clinical features. Most data are available to the public in their raw and processed forms; however, analysis and interpretation of these data require specialised training and software. To address this problem, online tools such as cBioportal, canEvolve, GDAC firehose, PROGgeneV2, and UCSC Cancer browser have been developed by various groups to explore and perform analyses on the datasets that are easily understandable by basic researchers and clinicians. In this mini-review, we give an overview of the datasets available from TCGA and the public tools available for integrative analysis of survival with the genomic and transcriptomic datasets, and introduce a tool being developed by our group to analyse the datasets within TCGA.

Background Immune-related hepatitis (irHepatitis) is a relatively common immune-related adverse event (irAE) of checkpoint inhibitors. Often, it responds well to steroids; however, in refractory cases, further therapy is needed. Anti-tumor necrosis factor (TNF) antibodies are used for management of multiple irAEs, but there are little data in irHepatitis. Here, we report on safety and efficacy of infliximab in 10 cases of steroid-refractory irHepatitis.Methods We retrospectively reviewed patients treated with infliximab for steroid-refractory grade ≥3 irHepatitis at the Department of Dermatology, University Hospital Zurich. The positive response to infliximab was defined as no further increase in alanine aminotransferase (ALT)/aspartate aminotransferase (AST) above 50% than at the time of first infliximab infusion and control of irHepatitis without therapies other than steroids and infliximab.Results 10 patients with steroid-resistant irHepatitis grade ≥3 were treated with infliximab 5 mg/kg, of whom 7 (70%) responded positively. In two cases, the liver values increased over 50% before the irHepatitis could be controlled. In another case, therapies other than infliximab and steroids were given. At the median follow-up of 487 days, 90% of the patients demonstrated resolved irHepatitis without AST/ALT elevation following infliximab infusions.Conclusions Treatment of irHepatitis with infliximab did not result in hepatotoxicity and led to long-lasting positive response in 9 of 10 of the cases. Further research is needed to evaluate the role of anti-TNF antibodies in management of irHepatitis.

The microphthalmia-associated transcription factor (MITF) is a critical regulator of melanocyte development and differentiation. It also plays an important role in melanoma where it has been described as a molecular rheostat that, depending on activity levels, allows reversible switching between different cellular states. Here, we show that MITF directly represses the expression of genes associated with the extracellular matrix (ECM) and focal adhesion pathways in human melanoma cells as well as of regulators of epithelial-to-mesenchymal transition (EMT) such as CDH2, thus affecting cell morphology and cell-matrix interactions. Importantly, we show that these effects of MITF are reversible, as expected from the rheostat model. The number of focal adhesion points increased upon MITF knockdown, a feature observed in drug-resistant melanomas. Cells lacking MITF are similar to the cells of minimal residual disease observed in both human and zebrafish melanomas. Our results suggest that MITF plays a critical role as a repressor of gene expression and is actively involved in shaping the microenvironment of melanoma cells in a cell-autonomous manner.

A deepened understanding of the cellular and molecular processes in the tumor microenvironment is necessary for the development of precision immunotherapy (IT). We simultaneously investigated CD3, PDL1, and IDO by immunohistochemistry in paired biopsies from various organs of 43 metastatic melanoma patients treated with IT and targeted therapy (TT). Intraindividual biopsies taken after a period of weeks to months demonstrate discordant results in 30% of the cases. Overlap of IDO and PDL1 increased after therapy. IT only marginally impacted PDL1 expression over time in contrast to TT. Standardized repeated assessments of multiple immune markers in repeated biopsies will generate detailed insights in melanoma's immune evolution and adaption during therapies and might be used to support treatment decisions.

Ipilimumab and tremelimumab are human monoclonal antibodies (Abs) against cytotoxic T-lymphocyte antigen-4 (CTLA-4). Ipilimumab was the first agent to show a statistically significant benefit in overall survival in advanced melanoma patients. Currently, there is no proven association between the BRAFV600 mutation and the disease control rate in response to ipilimumab. This analysis was carried out to assess if BRAFV600 and NRAS mutation status affects the clinical outcome of anti-CTLA-4-treated melanoma patients. This is a retrospective multi-center analysis of 101 patients, with confirmed BRAF and NRAS mutation status, treated with anti-CTLA-4 antibodies from December 2006 until August 2012. The median overall survival, defined from the treatment start date with the anti-CTLA-4. Abs-treatment to death or till last follow up, of BRAFV600 or NRAS mutant patients (n = 62) was 10.12 months (95% CI 6.78-13.2) compared to 8.26 months (95% CI 6.02-19.9) in BRAFV600/NRASwt subpopulation (n = 39) (p = 0.67). The median OS of NRAS mutated patients (n = 24) was 12.1 months and although was prolonged compared to the median OS of BRAF mutated patients (n = 38, mOS = 8.03 months) or BRAFV600/NRASwt patients (n = 39, mOS = 8.26 months) the difference didn't reach statistical significance (p = 0.56). 69 patients were able to complete 4 cycles of anti-CTLA-4 treatment. Of the 24 patients treated with selected BRAF- or MEK-inhibitors, 16 patients received anti-CTLA 4 Abs following either a BRAF or MEK inhibitor with only 8 of them being able to finish 4 cycles of treatment. Based on our results, there is no difference in the median OS in patients treated with anti-CTLA-4 Abs implying that the BRAF/NRAS mutation status alone is not sufficient to predict the outcome of patients treated with anti-CTLA-4 Abs.