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Abstract Deep single-cell multi-omic profiling offers a promising approach to understand and overcome drug resistance in relapsed or refractory (rr) acute myeloid leukemia (AML). Here, we combine single-cell ex vivo drug profiling (pharmacoscopy) with single-cell and bulk DNA, RNA, and protein analyses, alongside clinical data from 21 rrAML patients. Unsupervised data integration reveals reduced ex vivo response to the Bcl-2 inhibitor venetoclax (VEN) in patients treated with both a hypomethylating agent (HMA) and VEN, compared to those pre-exposed to chemotherapy or HMA alone. Integrative analysis identifies both known and unreported mechanisms of innate and treatment-related VEN resistance and suggests alternative treatments, like targeting increased proliferation with the PLK inhibitor volasertib. Additionally, high CD36 expression in VEN-resistant blasts associates with sensitivity to CD36-targeted antibody treatment ex vivo. This study demonstrates how single-cell multi-omic profiling can uncover drug resistance mechanisms and treatment vulnerabilities, providing a valuable resource for future AML research.

Abstract Cancer-associated immune dysfunction is a major challenge for effective therapies. The emergence of antibodies targeting tumor cell-surface antigens led to advancements in the treatment of hematopoietic malignancies, particularly blood cancers. Yet their impact is constrained against tumors of hematopoietic origin manifesting in the skin. In this study, we employ a clonality-supervised deep learning methodology to dissect key pathological features implicated in mycosis fungoides, the most common cutaneous T-cell lymphoma. Our investigations unveil the prominence of the IL-32β–major histocompatibility complex (MHC)-I axis as a critical determinant in tumor T-cell immune evasion within the skin microenvironment. In patients’ skin, we find MHC-I to detrimentally impact the functionality of natural killer (NK) cells, diminishing antibody-dependent cellular cytotoxicity and promoting resistance of tumor skin T-cells to cell-surface targeting therapies. Through murine experiments in female mice, we demonstrate that disruption of the MHC-I interaction with NK cell inhibitory Ly49 receptors restores NK cell anti-tumor activity and targeted T-cell lymphoma elimination in vivo. These findings underscore the significance of attenuating the MHC-I-dependent immunosuppressive networks within skin tumors. Overall, our study introduces a strategy to reinvigorate NK cell-mediated anti-tumor responses to overcome treatment resistance to existing cell-surface targeted therapies for skin lymphoma.

Lichen planus (LP) is a highly prevalent inflammatory skin disease. While various clinical subtypes have been defined, detailed comparisons of these variants are lacking. This study aimed to elucidate differences in gene expression and cellular composition across LP subtypes. Lesional skin biopsies from 28 LP patients (classical, oral, genital, and lichen planopilaris) and seven non-diseased skin controls (NDC) were analyzed. Gene expression profiling of 730 inflammation-related genes was conducted using NanoString. Immune cell compositions were assessed by multiplex immunohistochemistry. Gene expression profiles revealed unique inflammatory signatures for each LP subtype. Lichen planopilaris exhibited the most divergence, with downregulated gene expression and upregulation of complement pathway genes (C5-7), along with elevated M2 macrophages. Oral and genital LP demonstrated similar profiles with strong upregulation of TNF-related and Toll-like receptor-associated genes. Oral LP showed the highest upregulation of cytotoxicity-associated genes, as well as high numbers of CD8+ IL-17A+ (Tc17) cells (8.02%). Interferon gene signatures were strongly upregulated in oral and classical LP. The study highlights distinct differences in inflammatory gene expression and cell composition across LP subtypes, emphasizing the need for tailored therapeutic approaches.

Abstract Melanoma provides a primary benchmark for targeted drug therapy. Most melanomas with BRAFV600 mutations regress in response to BRAF/MEK inhibitors (BRAFi/MEKi). However, nearly all relapse within the first two years, and there is a connection between BRAFi/MEKi-resistance and poor response to immune checkpoint therapy. We reported that androgen receptor (AR) activity is required for melanoma cell proliferation and tumorigenesis. We show here that AR expression is markedly increased in BRAFi-resistant melanoma cells, and in sensitive cells soon after BRAFi exposure. Increased AR expression is sufficient to render melanoma cells BRAFi-resistant, eliciting transcriptional changes of BRAFi-resistant subpopulations, including elevated EGFR and SERPINE1 expression, of likely clinical significance. Inhibition of AR expression or activity blunts changes in gene expression and suppresses proliferation and tumorigenesis of BRAFi-resistant melanoma cells, promoting clusters of CD8+ T cells infiltration and cancer cells killing. Our findings point to targeting AR as possible co-therapeutical approach in melanoma treatment.

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.

Summary: Characterization of extracellular vesicles (EVs) holds great promise for biomarker discovery and understanding of diseases, including melanoma, the deadliest skin cancer type. Here, we describe a size-exclusion chromatography method to isolate and concentrate EVs from patient material including (1) patient-derived melanoma cell line supernatants and (2) plasma and serum biopsies. Additionally, we provide a protocol to analyze EVs by nano-flow cytometry. EV suspensions obtained with the presented protocol can be used for several downstream analyses including RNA sequencing and proteomics. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Fresh vs. slow-frozen tissues from various malignancies are compared for the establishment of 2D, 3D and ex vivo cultures, as well as for scRNAseq analysis, and found to be comparable and suitable for cancer research.

Dendritic cell (DC) migration from peripheral tissues via afferent lymphatic vessels to draining lymph nodes (dLNs) is important for the organism’s immune regulation and immune protection. Several lymphatic endothelial cell (LEC)-expressed adhesion molecules have thus far been found to support transmigration and movement within the lymphatic vasculature. In this study, we investigated the contribution of CD112, an adhesion molecule that we recently found to be highly expressed in murine LECs, to this process. Performing in vitro assays in the murine system, we found that transmigration of bone marrow-derived dendritic cells (BM-DCs) across or adhesion to murine LEC monolayers was reduced when CD112 was absent on LECs, DCs, or both cell types, suggesting the involvement of homophilic CD112–CD112 interactions. While CD112 was highly expressed in murine dermal LECs, CD112 levels were low in endogenous murine dermal DCs and BM-DCs. This might explain why we observed no defect in the in vivo lymphatic migration of adoptively transferred BM-DCs or endogenous DCs from the skin to dLNs. Compared to murine DCs, human monocyte-derived DCs expressed higher CD112 levels, and their migration across human CD112-expressing LECs was significantly reduced upon CD112 blockade. CD112 expression was also readily detected in endogenous human dermal DCs and LECs by flow cytometry and immunofluorescence. Upon incubating human skin punch biopsies in the presence of CD112-blocking antibodies, DC emigration from the tissue into the culture medium was significantly reduced, indicating impaired lymphatic migration. Overall, our data reveal a contribution of CD112 to human DC migration.

Melanomas are highly immunogenic tumors that have been shown to activate the immune response. Nonetheless, a significant portion of melanoma cases are either unresponsive to immunotherapy or relapsed due to acquired resistance. During melanomagenesis, melanoma and immune cells undergo immunomodulatory mechanisms that aid in immune resistance and evasion. The crosstalk within melanoma microenvironment is facilitated through the secretion of soluble factors, growth factors, cytokines, and chemokines. In addition, the release and uptake of secretory vesicles known as extracellular vesicles (EVs) play a key role in shaping the tumor microenvironment (TME). Melanoma-derived EVs have been implicated in immune suppression and escape, promoting tumor progression. In the context of cancer patients, EVs are usually isolated from biofluids such as serum, urine, and saliva. Nonetheless, this approach neglects the fact that biofluid-derived EVs reflect not only the tumor, but also include contributions from different organs and cell types. For that, isolating EVs from tissue samples allows for studying different cell populations resident at the tumor site, such as tumor-infiltrating lymphocytes and their secreted EVs, which play a central anti-tumor role. Herein, we outline the first instance of a method for EV isolation from frozen tissue samples at high purity and sensitivity that can be easily reproduced without the need for complicated isolation methods. Our method of processing the tissue not only circumvents the need for hard-to-acquire freshly isolated tissue samples, but also preserves EV surface proteins which allows for multiplex surface markers profiling. Tissue-derived EVs provide insight into the physiological role of EVs enrichment at tumor sites, which can be overlooked when studying circulating EVs coming from different sources. Tissue-derived EVs could be further characterized in terms of their genomics and proteomics to identify possible mechanisms for regulating the TME. Additionally, identified markers could be correlated to overall patient survival and disease progression for prognostic purposes.

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.

Cutaneous melanoma is one of the most aggressive human malignancies and shows increasing incidence. Mast cells (MCs), long-lived tissue-resident cells that are particularly abundant in human skin where they regulate both innate and adaptive immunity, are associated with melanoma stroma (MAMCs). Thus, MAMCs could impact melanoma development, progression, and metastasis by secreting proteases, pro-angiogenic factors, and both pro-inflammatory and immuno-inhibitory mediators. To interrogate the as-yet poorly characterized role of human MAMCs, we have purified MCs from melanoma skin biopsies and performed RNA-seq analysis. Here, we demonstrate that MAMCs display a unique transcriptome signature defined by the downregulation of the FcεRI signaling pathway, a distinct expression pattern of proteases and pro-angiogenic factors, and a profound upregulation of complement component C3. Furthermore, in melanoma tissue, we observe a significantly increased number of C3+ MCs in stage IV melanoma. Moreover, in patients, C3 expression significantly correlates with the MC-specific marker TPSAB1, and the high expression of both markers is linked with poorer melanoma survival. In vitro, we show that melanoma cell supernatants and tumor microenvironment (TME) mediators such as TGF-β, IL-33, and IL-1β induce some of the changes found in MAMCs and significantly modulate C3 expression and activity in MCs. Taken together, these data suggest that melanoma-secreted cytokines such as TGF-β and IL-1β contribute to the melanoma microenvironment by upregulating C3 expression in MAMCs, thus inducing an MC phenotype switch that negatively impacts melanoma prognosis.

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.

PurposeWe explored imaging and blood bio-markers for survival prediction in a cohort of patients with metastatic melanoma treated with immune checkpoint inhibition.Materials and Methods94 consecutive metastatic melanoma patients treated with immune checkpoint inhibition were included into this study. PET/CT imaging was available at baseline (Tp0), 3 months (Tp1) and 6 months (Tp2) after start of immunotherapy. Radiological response at Tp2 was evaluated using iRECIST. Total tumor burden (TB) at each time-point was measured and relative change of TB compared to baseline was calculated. LDH, CRP and S-100B were also analyzed. Cox proportional hazards model and logistic regression were used for survival analysis.ResultsiRECIST at Tp2 was significantly associated with overall survival (OS) with C-index=0.68. TB at baseline was not associated with OS, whereas TB at Tp1 and Tp2 provided similar predictive power with C-index of 0.67 and 0.71, respectively. Appearance of new metastatic lesions during follow-up was an independent prognostic factor (C-index=0.73). Elevated LDH and S-100B ratios at Tp2 were significantly associated with worse OS: C-index=0.73 for LDH and 0.73 for S-100B. Correlation of LDH with TB was weak (r=0.34). A multivariate model including TB change, S-100B, and appearance of new lesions showed the best predictive performance with C-index=0.83.ConclusionOur analysis shows only a weak correlation between LDH and TB. Additionally, baseline TB was not a prognostic factor in our cohort. A multivariate model combining early blood and imaging biomarkers achieved the best predictive power with regard to survival, outperforming iRECIST.

We present an optimized dissociation protocol for preparing high-quality skin cell suspensions for in-depth single-cell RNA-sequencing (scRNA-seq) analysis of fresh and cultured human skin. Our protocol enabled the isolation of a consistently high number of highly viable skin cells from small freshly dissociated punch skin biopsies, which we use for scRNA-seq studies. We recapitulated not only the main cell populations of existing single-cell skin atlases, but also identified rare cell populations, such as mast cells. Furthermore, we effectively isolated highly viable single cells from ex vivo cultured skin biopsy fragments and generated a global single-cell map of the explanted human skin. The quality metrics of the generated scRNA-seq datasets were comparable between freshly dissociated and cultured skin. Overall, by enabling efficient cell isolation and comprehensive cell mapping, our skin dissociation-scRNA-seq workflow can greatly facilitate scRNA-seq discoveries across diverse human skin pathologies and ex vivo skin explant experimentations.

Acral melanoma occurs on the soles of the feet, palms of the hands and in nail beds. Here, the authors reports the genomic landscape of 87 acral melanomas and find that some tumors harbor a UV signature and that the tumors are diverse at the levels of mutational signatures, structural aberrations and copy number signatures.

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.

Background/Aims: Among the severe immune-related adverse events (irAEs) that occur with immune checkpoint inhibitor (ICI) therapy, colitis is the most frequent one. This study aimed at describing the experience from the largest gastroenterology unit in Switzerland with immune checkpoint inhibitor-associated colitis (ICIAC), its clinical presentation, management, and outcomes. Methods: We performed a retrospective review of patients who were referred for the evaluation of ICIAC between January 2011 and October 2018 to the Division of Gastroenterology and Hepatology, University Hospital Zurich. Results: Thirty-three patients with immune-related colitis grade 3 or 4 met the inclusion criteria and were analyzed in detail: All patients had diarrhea, 64% had abdominal pain, 42% had bloody stool, 27% had emesis, and 18% developed fever. In total, 33% were successfully treated with corticosteroids alone; 66% were steroid-refractory and treated with infliximab or vedolizumab. Two of these patients developed severe complications requiring surgery. All patients reached complete remission of ICIAC and its symptoms. At colonoscopy, ulcerations were seen in 37% of steroid-refractory versus 63% of steroid-responsive cases. Deep histological ulcerations invading the submucosa were only present in steroid-refractory cases. Conclusion: ICIAC is a severe irAE which frequently requires high-dose steroids and a close follow-up due to deleterious complications. The detection of histologically diagnosed deep ulcerations may predict a steroid-refractory course and may warrant early application of infliximab. However, larger studies are required to confirm our findings.

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 [...]

Mucosal melanomas are challenging to treat partly because so little is known about the genetic drivers underpinning them. Here, the authors perform a genomic landscape analysis of samples collected from three continents, revealing a potential role for CDK4/6 or MEK inhibition in the treatment of the disease.

RNF5 is a ubiquitin ligase regulating ER stress response. Here the authors show that Rnf5 deficiency potentiates immune response against melanoma via altered microbiota, and isolate bacterial strains that confer the same phenotype to wild type mice.

Abstract Background Long-term survival of stage IV melanoma patients has improved significantly with the development of immune checkpoint inhibitors (CIs). Reliable biomarkers to predict response and clinical outcome are needed. Methods We investigated the role of melanoma-associated antibodies as predictive markers for CI therapy in two independent cohorts. In cohort 1, a prospective study, we measured specific antibodies before treatment, after one week and after six to nine weeks of treatment. Cohort 2 consisted of serum samples prior to CI therapy initiation. ELISA assays were performed to quantify specific IgG directed against melanocyte differentiation antigens tyrosinase-related proteins 1 and 2 (TRP1/TYRP1 and TRP2/TYRP2), glycoprotein 100 (gp100), MelanA/MART1, and the cancer-testis antigen NY-ESO-1. Response was defined as either complete or partial remission on CT scan according to RECIST 1.1. Results In cohort 1, baseline levels of these antibodies were higher in the responder group, although statistical significance was only reached for NY-ESO-1 (p = 0.007). In cohort 2, significantly higher antibody baseline levels for MelanA/MART1 (p = 0.003) and gp100 (p = 0.029) were found. After pooling the results from both cohorts, higher levels of MelanA/MART1 (p = 0.013), TRP1/TYRP1 (p = 0.048), TRP2/TYRP2 (p = 0.047) and NY-ESO-1 (p = 0.005) specific antibodies at baseline were independently associated with response. Conclusions Melanoma-associated antibodies may be candidate biomarkers for response and survival in metastatic melanoma patients being treated with CIs. These markers may be used to complement patient assessment, in combination with PD-L1 status, tumor-infiltrating lymphocytes and tumor mutational burden, with the aim to predict outcome of CI treatment in patients with metastatic melanoma. Trial registration Ethikkommission Ostschweiz, EKOS 16/079 https://ongoingprojects.swissethics.ch/runningProjects_list.php?q=%28BASECID~contains~2016-00998%29&orderby=dBASECID.

Ample evidence pinpoints the phenotypic diversity of blood vessels (BVs) and site-specific functions of their lining endothelial cells (ECs). We harnessed single-cell RNA sequencing (scRNA-seq) to dissect the molecular heterogeneity of blood vascular endothelial cells (BECs) in healthy adult human skin and identified six different subpopulations, signifying arterioles, post-arterial capillaries, pre-venular capillaries, post-capillary venules, venules and collecting venules. Individual BEC subtypes exhibited distinctive transcriptomic landscapes associated with diverse biological pathways. These functionally distinct dermal BV segments were characterized by their unique compositions of conventional and novel markers (e.g., arteriole marker GJA5; arteriole capillary markers ASS1 and S100A4; pre-venular capillary markers SOX17 and PLAUR; venular markers EGR2 and LRG1), many of which have been implicated in vascular remodeling upon inflammatory responses. Immunofluorescence staining of human skin sections and whole-mount skin blocks confirmed the discrete expression of these markers along the blood vascular tree in situ, further corroborating BEC heterogeneity in human skin. Overall, our study molecularly refines individual BV compartments, whilst the identification of novel subtype-specific signatures provides more insights for future studies dissecting the responses of distinct vessel segments under pathological conditions.

Abstract Background Molecular precision oncology is an emerging practice to improve cancer therapy by decreasing the risk of choosing treatments that lack efficacy or cause adverse events. However, the challenges of integrating molecular profiling into routine clinical care are manifold. From a computational perspective these include the importance of a short analysis turnaround time, the interpretation of complex drug-gene and gene-gene interactions, and the necessity of standardized high-quality workflows. In addition, difficulties faced when integrating molecular diagnostics into clinical practice are ethical concerns, legal requirements, and limited availability of treatment options beyond standard of care as well as the overall lack of awareness of their existence. Methods To the best of our knowledge, we are the first group in Switzerland that established a workflow for personalized diagnostics based on comprehensive high-throughput sequencing of tumors at the clinic. Our workflow, named SwissMTB (Swiss Molecular Tumor Board), links genetic tumor alterations and gene expression to therapeutic options and clinical trial opportunities. The resulting treatment recommendations are summarized in a clinical report and discussed in a molecular tumor board at the clinic to support therapy decisions. Results Here we present results from an observational pilot study including 22 late-stage cancer patients. In this study we were able to identify actionable variants and corresponding therapies for 19 patients. Half of the patients were analyzed retrospectively. In two patients we identified resistance-associated variants explaining lack of therapy response. For five out of eleven patients analyzed before treatment the SwissMTB diagnostic influenced treatment decision. Conclusions SwissMTB enables the analysis and clinical interpretation of large numbers of potentially actionable molecular targets. Thus, our workflow paves the way towards a more frequent use of comprehensive molecular diagnostics in Swiss hospitals.

High-dimensional mass and flow cytometry (HDCyto) experiments have become a method of choice for high-throughput interrogation and characterization of cell populations. Here, we present an updated R-based pipeline for differential analyses of HDCyto data, largely based on Bioconductor packages. We computationally define cell populations using FlowSOM clustering, and facilitate an optional but reproducible strategy for manual merging of algorithm-generated clusters. Our workflow offers different analysis paths, including association of cell type abundance with a phenotype or changes in signalling markers within specific subpopulations, or differential analyses of aggregated signals. Importantly, the differential analyses we show are based on regression frameworks where the HDCyto data is the response; thus, we are able to model arbitrary experimental designs, such as those with batch effects, paired designs and so on. In particular, we apply generalized linear mixed models or linear mixed models to analyses of cell population abundance or cell-population-specific analyses of signaling markers, allowing overdispersion in cell count or aggregated signals across samples to be appropriately modeled. To support the formal statistical analyses, we encourage exploratory data analysis at every step, including quality control (e.g., multi-dimensional scaling plots), reporting of clustering results (dimensionality reduction, heatmaps with dendrograms) and differential analyses (e.g., plots of aggregated signals).

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.

The anti-proliferative/pro-oxidant efficacy of green pea, soybean, radish, Red Rambo radish, and rocket microgreens, cultivated under either fluorescent lighting (predominant spectral peaks in green and orange) or combination light-emitting diode (LED, predominant spectral peak in blue) was investigated using Ewing sarcoma lines, RD-ES and A673, respectively. All aqueous microgreen extracts significantly reduced cell proliferation (cancer prevention effect) to varying extents in two-dimensional sarcoma cell cultures. The effect of the polyphenol fraction in the aqueous food matrix was unrelated to total polyphenol content, which differed between species and light treatment. Only Pisum sativum (LED-grown) extracts exercised anti-proliferative and pro-apoptotic effects in both three-dimensional RD-ES and A673 spheroids (early tumor progression prevention), without cytotoxic effects on healthy L929 fibroblasts. A similar anti-tumor effect of Red Rambo radish (LED and fluorescent-grown) was evident only in the RD-ES spheroids. Aside from the promising anti-tumor potential of the polyphenol fraction of green pea microgreens, the latter also displayed favorable growth quality parameters, along with radish, under both light treatments over the 10 day cultivation period.

High-dimensional mass and flow cytometry (HDCyto) experiments have become a method of choice for high-throughput interrogation and characterization of cell populations. Here, we present an updated R-based pipeline for differential analyses of HDCyto data, largely based on Bioconductor packages. We computationally define cell populations using FlowSOM clustering, and facilitate an optional but reproducible strategy for manual merging of algorithm-generated clusters. Our workflow offers different analysis paths, including association of cell type abundance with a phenotype or changes in signalling markers within specific subpopulations, or differential analyses of aggregated signals. Importantly, the differential analyses we show are based on regression frameworks where the HDCyto data is the response; thus, we are able to model arbitrary experimental designs, such as those with batch effects, paired designs and so on. In particular, we apply generalized linear mixed models or linear mixed models to analyses of cell population abundance or cell-population-specific analyses of signaling markers, allowing overdispersion in cell count or aggregated signals across samples to be appropriately modeled. To support the formal statistical analyses, we encourage exploratory data analysis at every step, including quality control (e.g., multi-dimensional scaling plots), reporting of clustering results (dimensionality reduction, heatmaps with dendrograms) and differential analyses (e.g., plots of aggregated signals).

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.

Melanoma is one of the most lethal cutaneous malignancies, characterized by chemoresistance and a striking propensity to metastasize. The transcription factor ATF2 elicits oncogenic activities in melanoma, and its inhibition attenuates melanoma development. Here, we show that expression of a transcriptionally inactive form of Atf2 (Atf2Δ8,9) promotes development of melanoma in mouse models. Atf2Δ8,9-driven tumors show enhanced pigmentation, immune infiltration, and metastatic propensity. Similar to mouse Atf2Δ8,9, we have identified a transcriptionally inactive human ATF2 splice variant 5 (ATF2SV5) that enhances the growth and migration capacity of cultured melanoma cells and immortalized melanocytes. ATF2SV5 expression is elevated in human melanoma specimens and is associated with poor prognosis. These findings point to an oncogenic function for ATF2 in melanoma development that appears to be independent of its transcriptional activity.

Despite recent progress in melanoma therapy via inhibition of activated oncogenes or immune stimulation, most stage IV melanoma patients still have limited survival times. Existing therapeutic approaches eventually fail to prevent further invasion and metastasis, which is driven by a morphological process termed epithelial-mesenchymal transition (EMT). We previously demonstrated that inhibition of EMT in melanoma cells via antagonizing the bone morphogenetic protein (BMP)-pathway abrogated EMT and neural crest migration of melanoma cells in chick embryos. Here, we show that BMP-2 is highly expressed in invasive melanoma cells and is elevated in the serum of stage IV melanoma patients compared to stage IB-IIC patients and healthy controls. Highly BMP-2-expressing melanoma cells display enhanced invasion in the rhombencephalon of the chick embryo. In addition to driving neural crest migration in the zebrafish embryo, the agonists BMP-2, BMP-7 and nodal induce EMT/invasion in radial growth phase melanoma cells and in human melanocytes in skin reconstructs. Blocking either BMP or nodal signaling by antagonists (noggin, lefty), or the Alk4/5/7-receptor inhibitor SB431542, decreases EMT and invasion of melanoma cells in human epidermal skin reconstructs. Together, our data suggest that inhibition of EMT-inducing pathways in melanoma might be a therapeutic approach to attenuate melanoma cell invasiveness.

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).

Floor-plate-derived extracellular signaling molecules, including canonical axon guidance cues of the Netrin family, control neuronal circuit organization. Despite the importance of the floor plate as an essential signaling center in the developing vertebrate central nervous system, no systematic approach to identify binding partners for floor-plate-expressed cell-surface and secreted proteins has been carried out. Here, we used a high-throughput assay to discover extracellular protein-protein interactions, which likely take place in the zebrafish floor-plate microenvironment. The assembled floor-plate network contains 47 interactions including the hitherto-not-reported interaction between Netrin-1 and Draxin. We further characterized this interaction, narrowed down the binding interface, and demonstrated that Draxin competes with Netrin receptors for binding to Netrin-1. Our results suggest that Draxin functions as an extracellular Netrin signaling modulator in vertebrates. A reciprocal gradient of Draxin might shape or sharpen the active Netrin gradient, thereby critically modulating its effect.

Despite the remarkable clinical response of melanoma harboring BRAF mutations to BRAF inhibitors (BRAFi), most tumors become resistant. Here, we identified the downregulation of the ubiquitin ligase RNF125 in BRAFi-resistant melanomas and demonstrated its role in intrinsic and adaptive resistance to BRAFi in cultures as well as its association with resistance in tumor specimens. Sox10/MITF expression correlated with and contributed to RNF125 transcription. Reduced RNF125 was associated with elevated expression of receptor tyrosine kinases (RTKs), including EGFR. Notably, RNF125 altered RTK expression through JAK1, which we identified as an RNF125 substrate. RNF125 bound to and ubiquitinated JAK1, prompting its degradation and suppressing RTK expression. Inhibition of JAK1 and EGFR signaling overcame BRAFi resistance in melanoma with reduced RNF125 expression, as shown in culture and in in vivo xenografts. Our findings suggest that combination therapies targeting both JAK1 and EGFR could be effective against BRAFi-resistant tumors with de novo low RNF125 expression.

phase II and III clinical trials demonstrated modest anti- tumor activity of Binimetinib (MEK162) - a potent allosteric inhibitor of MEK1 and MEK2- in patients with advanced NRAS mutant melanoma. The analysis of the NEMO study in NRAS mutated melanoma, has shown that pre-treatment with immunotherapy improved the outcome of binimetinib therapy. We discuss this finding in the context of in vitro and in vivo effects of MEK inhibition on immuno-critical pathways and interactions.

High dimensional mass and flow cytometry (HDCyto) experiments have become a method of choice for high throughput interrogation and characterization of cell populations.Here, we present an R-based pipeline for differential analyses of HDCyto data, largely based on Bioconductor packages. We computationally define cell populations using FlowSOM clustering, and facilitate an optional but reproducible strategy for manual merging of algorithm-generated clusters. Our workflow offers different analysis paths, including association of cell type abundance with a phenotype or changes in signaling markers within specific subpopulations, or differential analyses of aggregated signals. Importantly, the differential analyses we show are based on regression frameworks where the HDCyto data is the response; thus, we are able to model arbitrary experimental designs, such as those with batch effects, paired designs and so on. In particular, we apply generalized linear mixed models to analyses of cell population abundance or cell-population-specific analyses of signaling markers, allowing overdispersion in cell count or aggregated signals across samples to be appropriately modeled. To support the formal statistical analyses, we encourage exploratory data analysis at every step, including quality control (e.g. multi-dimensional scaling plots), reporting of clustering results (dimensionality reduction, heatmaps with dendrograms) and differential analyses (e.g. plots of aggregated signals).

Background: Congenital melanocytic nevi (CMNs) are melanocytic neoplasms that can transform into melanoma. However, this development is impeded in the majority of cases and mostly affects patients with large or giant CMNs. Methods: To elucidate mechanisms that keep CMNs from malignant transformation, CMN tissue biopsies were investigated for p-ERK and senescence markers by immunohistochemistry and for SOX10/CD271 (p75NTR) by immunofluorescence. CMN cells were cultivated, and MTT assays were performed for evaluating cell viability. Mutation status for NRAS and BRAF was performed by real-time PCR. Results: 13 CMNs (from patients aged 0.5-11.8 years, mean: 2.7) showed immunoreactivity for SOX10/CD271 (p75NTR) in 34.2%. p-ERK was immunoreactive in 80% (4/5); β-galactosidase was significantly stronger expressed in CMNs compared to melanocytic nevi of patients over 70 years (p = 0.0085). The 5 CMN cultures were immunoreactive for SOX10/CD271 (p75NTR) in 36.7%. By silencing SOX10 by siRNA in 2 CMN cell cultures, cell viability decreased significantly. NRASQ61K mutation was found in 91.7% (11/12) and BRAFV600E in 6.3% of all analyzable CMNs (1/16). Conclusions: Oncogene-induced senescence might prevent malignant transformation through activation of the mitogen-activated protein kinase pathway. SOX10 is necessary for the viability of human CMN cell cultures and may be responsible for clinical changes during aging. © 2014 S. Karger AG, Basel

Summary Centrosome defects are a common feature of many cancers, and they can predispose fly brain cells to form tumours. In flies, centrosome defects perturb the asymmetric division of the neural stem cells, but it is unclear how this might lead to malignant transformation. One possibility is that centrosome defects might also perturb cellular homeostasis: for example, stress pathways are often activated in response to centrosome defects in cultured cells, and stress contributes to tumourigenesis in some fly models. Here we attempt to assess whether centrosome loss or centrosome amplification perturbs cell physiology in vivo by profiling the global transcriptome of Drosophila larval brains and imaginal discs that either lack centrosomes or have too many centrosomes. Surprisingly, we find that centrosome loss or amplification leads to few changes in the transcriptional profile of these cells, indicating that centrosome defects are surprisingly well tolerated by these cells. These observations indicate that centrosome defects can predispose fly brain cells to form tumours without, at least initially, dramatically altering their physiology.

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.