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The treatment of patients with metastatic melanoma with immune checkpoint inhibitors (ICI) leads to impressive response rates but primary and secondary resistance to ICI reduces progression-free survival. Novel strategies that interfere with resistance mechanisms are key to further improve patient outcome during ICI therapy. P53 is often inactivated by mouse-double-minute-2 (MDM2), which may decrease immunogenicity of melanoma cells. We analyzed primary patient-derived melanoma cell lines, performed bulk sequencing analysis of patient-derived melanoma samples, and used melanoma mouse models to investigate the role of MDM2-inhibition for enhanced ICI therapy. We found increased expression of IL15 and MHC-II in murine melanoma cells upon p53 induction by MDM2-inhibition. MDM2-inhibitor induced MHC-II and IL15-production, which was p53 dependent as Tp53 knockdown blocked the effect. Lack of IL15-receptor in hematopoietic cells or IL15 neutralization reduced the MDM2-inhibition/p53-induction–mediated antitumor immunity. P53 induction by MDM2-inhibition caused anti-melanoma immune memory as T cells isolated from MDM2-inhibitor–treated melanoma-bearing mice exhibited anti-melanoma activity in secondary melanoma-bearing mice. In patient-derived melanoma cells p53 induction by MDM2-inhibition increased IL15 and MHC-II. IL15 and CIITA expressions were associated with a more favorable prognosis in patients bearing WT but not TP53-mutated melanoma. Implications: MDM2-inhibition represents a novel strategy to enhance IL15 and MHC-II–production, which disrupts the immunosuppressive tumor microenvironment. On the basis of our findings, a clinical trial combining MDM2-inhibition with anti–PD-1 immunotherapy for metastatic melanoma is planned.

Healing wounds and cancers present remarkable cellular and molecular parallels, but the specific roles of the healing phases are largely unknown. We developed a bioinformatics pipeline to identify genes and pathways that define distinct phases across the time-course of healing. Their comparison to cancer transcriptomes revealed that a resolution phase wound signature is associated with increased severity in skin cancer and enriches for extracellular matrix-related pathways. Comparisons of transcriptomes of early- and late-phase wound fibroblasts vs skin cancer-associated fibroblasts (CAFs) identified an "early wound" CAF subtype, which localizes to the inner tumor stroma and expresses collagen-related genes that are controlled by the RUNX2 transcription factor. A "late wound" CAF subtype localizes to the outer tumor stroma and expresses elastin-related genes. Matrix imaging of primary melanoma tissue microarrays validated these matrix signatures and identified collagen- vs elastin-rich niches within the tumor microenvironment, whose spatial organization predicts survival and recurrence. These results identify wound-regulated genes and matrix patterns with prognostic potential in skin cancer., Matrix Biology, 119, ISSN:0945-053X, ISSN:1569-1802

To determine whether there is a difference in orthopaedic trauma patient medication satisfaction and adherence using an oral versus subcutaneous injectable anticoagulant for venous thromboembolism chemoprophylaxis.Randomized controlled trial.Single academic Level 1 trauma center.One hundred twenty adult orthopaedic trauma patients with operative pelvic or lower extremity fractures were randomized and completed the study.Three weeks of either the service standard 40 mg once daily enoxaparin versus trial medication 10 mg once daily rivaroxaban postoperatively.Patient satisfaction as measured by the Treatment Satisfaction Questionnaire for Medication (TSQM-9). Medication adherence as measured by the Morisky Medication Adherence Scale (MMAS-8).Medication adherence was similar in both groups. Medication satisfaction was significantly higher in the oral rivaroxaban group based on the TSQM-9 and patient-reported data. Secondary outcomes found no significant difference in the incidence of bleeding events or clinically relevant venous thromboembolism. The enoxaparin group experienced more adverse medication-related events. The rivaroxaban medication regimen costs 7.5-10× less out of pocket for uninsured patients.The results of this randomized controlled trial demonstrate that patients with surgical orthopaedic trauma prefer an oral anticoagulant for postoperative venous thromboembolism chemoprophylaxis and suggest that rivaroxaban may be a viable option. Furthermore, large-scale studies are needed to confirm safety and efficacy for rivaroxaban in this population as a potential alternative to enoxaparin and aspirin.Therapeutic Level II. See Instructions for Authors for a complete description of levels of evidence.

Differentiating atypical lipomatous tumors (ALTs) and well-differentiated liposarcomas (WDLs) from benign lipomatous lesions is important for guiding clinical management, though conventional visual analysis of these lesions is challenging due to overlap of imaging features. Radiomics-based approaches may serve as a promising alternative and/or supplementary diagnostic approach to conventional imaging.The purpose of this study is to review the practice of radiomics-based imaging and systematically evaluate the literature available for studies evaluating radiomics applied to differentiating ALTs/WDLs from benign lipomas.A background review of the radiomic workflow is provided, outlining the steps of image acquisition, segmentation, feature extraction, and model development. Subsequently, a systematic review of MEDLINE, EMBASE, Scopus, the Cochrane Library, and the grey literature was performed from inception to June 2022 to identify size studies using radiomics for differentiating ALTs/WDLs from benign lipomas. Radiomic models were shown to outperform conventional analysis in all but one model with a sensitivity ranging from 68 to 100% and a specificity ranging from 84 to 100%. However, current approaches rely on user input and no studies used a fully automated method for segmentation, contributing to interobserver variability and decreasing time efficiency.Radiomic models may show improved performance for differentiating ALTs/WDLs from benign lipomas compared to conventional analysis. However, considerable variability between radiomic approaches exists and future studies evaluating a standardized radiomic model with a multi-institutional study design and preferably fully automated segmentation software are needed before clinical application can be more broadly considered.

The 2018 ovarian-adnexal reporting and data system (O-RADS) guidelines are aimed at providing a system for consistent reports and risk stratification for ovarian lesions found on ultrasound. It provides key characteristics and findings for lesions, a lexicon of descriptors to communicate findings, and risk characterization and associated follow-up recommendation guidelines. However, the O-RADS guidelines have not been validated in North American institutions or amongst less experienced readers.To evaluate the diagnostic accuracy and inter-reader reliability of ultrasound O-RADS risk stratification amongst less experienced readers in a North American institution with and without pre-test training.A single-center retrospective study was performed using 100 ovarian/adnexal lesions of varying O-RADS scores. Of these cases, 50 were allotted to a training cohort and 50 to a testing cohortMean patient age was 40 ± 16 years with lesions ranging from 1.2 to 22.5 cm. Readers demonstrated excellent specificities (85%-100% pre-training and 91%-100% post-training) and NPVs (89%-100% pre-training and 91-100% post-training) across the O-RADS categories. Sensitivities were variable (55%-100% pre-training and 64%-100% post-training) with malignant O-RADS 4 and 5 Lesions pre-training and post-training AUC values of 0.87-0.95 and 0.94-098, respectively (Less experienced readers in North America achieved excellent specificities and AUC values with very good pairwise inter-reader reliability. They may be subject to misclassification of potentially malignant lesions, and specific training around dermoid features and smooth

To describe morphokinetic parameters and ploidy among low-quality blastocysts not meeting the criteria for clinical use.Prospective cohort study.Academic medical center.Two hundred patients undergoing in vitro fertilization between February 2018 and November 2019.All embryos were cultured in a time-lapse incubator. All expanded blastocysts underwent preimplantation genetic testing for aneuploidy using next-generation sequencing.Static blastocyst morphology grading; morphokinetic parameters, including time to each cell division (2-cell formation to 8-cell formation); time to morula formation; time to the start of blastulation; time to blastocyst formation; and preimplantation genetic testing for aneuploidy results.A total of 1,306 embryos progressed to the expanded blastocyst stage; of these, 935 embryos met the criteria for clinical use and were designated as high quality, whereas 371 embryos were graded as low quality and did not meet the criteria for use. In morphokinetic evaluation, low-quality embryos developed more quickly to 5-cell formation (t5) 48.4 [42.4-48.7) vs 50.2 [46.3-50.1] hours, but progressed more slowly thereafter with tM 91.5 [85.9-92.3] vs 88.3 [82.1-88.3] and tB 114.0 [106.4-113.9] vs 106.9 [101.3-107.4] hours. Among the low-quality embryos, 75.5% were aneuploid, 22.4% were euploid, and 2.2% had undetermined chromosome copy number results. Morphokinetic parameters did not differ between the euploid and aneuploid low-quality embryos.Morphokinetic analysis did not distinguish between euploid and aneuploid low-quality embryos.

Hypothemycin, an epoxide derivative of (5

RNA editing refers to non-transient RNA modifications that occur after transcription and prior to translation by the ribosomes. RNA editing is more widespread in cancer cells than in non-transformed cells and is associated with tumorigenesis of various cancer tissues. However, RNA editing can also generate neo-antigens that expose tumour cells to host immunosurveillance. Global RNA editing in melanoma and its relevance to clinical outcome currently remain poorly characterized. The present study compared RNA editing as well as gene expression in tumour cell lines from melanoma patients of short or long metastasis-free survival, patients relapsing or not after immuno- and targeted therapy and tumours harbouring

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.

Summary Recently, CITE-seq emerged as a multimodal single-cell technology capturing gene expression and surface protein information from the same single-cells, which allows unprecedented insights into disease mechanisms and heterogeneity, as well as immune cell profiling. Multiple single-cell profiling methods exist, but they are typically focussed on either gene expression or antibody analysis, not their combination. Moreover, existing software suites are not easily scalable to a multitude of samples. To this end, we designed gExcite, a start-to-end workflow that provides both gene and antibody expression analysis, as well as hashing deconvolution. Embedded in the Snakemake workflow manager, gExcite facilitates reproducible and scalable analyses. We showcase the output of gExcite on a study of different dissociation protocols on PBMC samples. Availability gExcite is open source available on github at https://github.com/ETH-NEXUS/gExcite_pipeline. The software is distributed under the GNU General Public License 3 (GPL3). Supplementary information Supplementary data are available at Bioinformatics online.

The treatment of patients with metastatic melanoma with immune checkpoint inhibitors (ICI) leads to impressive response rates but primary and secondary resistance to ICI reduces progression-free survival. Novel strategies that interfere with resistance mechanisms are key to further improve patient outcome during ICI therapy. P53 is often inactivated by mouse-double-minute-2 (MDM2), which may decrease immunogenicity of melanoma cells. We analyzed primary patient-derived melanoma cell lines, performed bulk sequencing analysis of patient-derived melanoma samples, and used melanoma mouse models to investigate the role of MDM2-inhibition for enhanced ICI therapy. We found increased expression of IL15 and MHC-II in murine melanoma cells upon p53 induction by MDM2-inhibition. MDM2-inhibitor induced MHC-II and IL15-production, which was p53 dependent as Tp53 knockdown blocked the effect. Lack of IL15-receptor in hematopoietic cells or IL15 neutralization reduced the MDM2-inhibition/p53-induction–mediated antitumor immunity. P53 induction by MDM2-inhibition caused anti-melanoma immune memory as T cells isolated from MDM2-inhibitor–treated melanoma-bearing mice exhibited anti-melanoma activity in secondary melanoma-bearing mice. In patient-derived melanoma cells p53 induction by MDM2-inhibition increased IL15 and MHC-II. IL15 and CIITA expressions were associated with a more favorable prognosis in patients bearing WT but not TP53-mutated melanoma.Implications:MDM2-inhibition represents a novel strategy to enhance IL15 and MHC-II–production, which disrupts the immunosuppressive tumor microenvironment. On the basis of our findings, a clinical trial combining MDM2-inhibition with anti–PD-1 immunotherapy for metastatic melanoma is planned.

Supplementary Figures Legends and Methods

Evaluation of caspae 3 staining after treatment with Aβ(25-35) in mice

Evaluation of the role of CD271 in drug resistance

Evaluation of Aβ(25-35) in 3D spheroids and zebrafish larvae

Mechanism of action Aβ(25-35) treatment in CD271high and CD271low cells

Aβ(25-35) + chemotherapy reduces viability and induces cell death of melanoma cells

Evaluation of Aβ (25-35) in ex vivo cultures

CD271 (NGFR) is a neurotrophin receptor that belongs to the tumor necrosis receptor (TNFR) family. Upon ligand binding, CD271 can mediate either survival or cell death. Although the role of CD271 as a marker of tumor-initiating cells is still a matter of debate, its role in melanoma progression has been well documented. Moreover, CD271 has been shown to be upregulated after exposure to both chemotherapy and targeted therapy. In this study, we demonstrate that activation of CD271 by a short β-amyloid–derived peptide (Aβ(25–35)) in combination with either chemotherapy or MAPK inhibitors induces apoptosis in 2D and 3D cultures of eight melanoma cell lines. This combinatorial treatment significantly reduced metastasis in a zebrafish xenograft model and led to significantly decreased tumor volume in mice. Administration of Aβ(25–35) in ex vivo tumors from immunotherapy- and targeted therapy–resistant patients significantly reduced proliferation of melanoma cells, showing that activation of CD271 can overcome drug resistance. Aβ(25–35) was specific to CD271-expressing cells and induced CD271 cleavage and phosphorylation of JNK (pJNK). The direct protein–protein interaction of pJNK with CD271 led to PARP1 cleavage, p53 and caspase activation, and pJNK-dependent cell death. Aβ(25–35) also mediated mitochondrial reactive oxygen species (mROS) accumulation, which induced CD271 overexpression. Finally, CD271 upregulation inhibited mROS production, revealing the presence of a negative feedback loop in mROS regulation. These results indicate that targeting CD271 can activate cell death pathways to inhibit melanoma progression and potentially overcome resistance to targeted therapy.Significance:The discovery of a means to specifically activate the CD271 death domain reveals unknown pathways mediated by the receptor and highlights new treatment possibilities for melanoma.

CD271 overexpression strongly enhances Aβ(25-35) killing effect.

CD271 silencing reduces Aβ(25-35) killing effect.

Tumor Profiler Consortium author list

Table ST5: Table of the 15 most informative proteins upregulated in MEK inhibitor-resistant cell culture (ranked by log2 fold-change).

S13: Immunohistochemical analysis of CyTof biomarker panel.

S12: Analysis of CyTof data from fresh melanoma tissue

S15: Tumor response curves in mice for ionophoric copper-chelators in combination with binimetinib.

Table ST7: Clinical data of the 62 melanoma patient which tumor material was used for pharmacoscopy

S16: scRNA-sequencing analysis of xenograft tumors M130227

Table ST8: Data output of the CCLE cell line screening provided by Prof. Schreiber at Broad Institute.

S7: Pathway enrichment (metabolomics)

S5: Elevated cytoplasmic ROS induces apoptosis after the treatment with neocuproine

S10: Clinical data of orthologic slice cultures

S9: Treatment of binimetinib-sensitive melanoma cell lines with PPP inhibitor

Metabolomics

Figure Legends for Supplemental Figures 1-4

Figure legend for supplementary figures.

Conformation of knock-down for siRNA experiments.

Many metastatic melanoma patients experience durable responses to anti-PD1 and/or anti-CTLA4; however, a significant proportion (over 50%) do not benefit from the therapies. In this study, we sought to assess pretreatment liquid biopsies for biomarkers that may correlate with response to checkpoint blockade. We measured the combinatorial diversity evenness of the T-cell receptor (TCR) repertoire (the DE50, with low values corresponding to more clonality and lack of TCR diversity) in pretreatment peripheral blood mononuclear cells from melanoma patients treated with anti-CTLA4 (n = 42) or anti-PD1 (n = 38) using a multi-N-plex PCR assay on genomic DNA (gDNA). A receiver operating characteristic curve determined the optimal threshold for a dichotomized analysis according to objective responses as defined by RECIST1.1. Correlations between treatment outcome, clinical variables, and DE50 were assessed in multivariate regression models and confirmed with Fisher exact tests. In samples obtained prior to treatment initiation, we showed that low DE50 values were predictive of a longer progression-free survival and good responses to PD-1 blockade, but, on the other hand, predicted a poor response to CTLA4 inhibition. Multivariate logistic regression models identified DE50 as the only independent predictive factor for response to anti-CTLA4 therapy (P = 0.03) and anti-PD1 therapy (P = 0.001). Fisher exact tests confirmed the association of low DE50 with response in the anti-CTLA4 (P = 0.041) and the anti-PD1 cohort (P = 0.0016). Thus, the evaluation of basal TCR repertoire diversity in peripheral blood, using a PCR-based method, could help predict responses to anti-PD1 and anti-CTLA4 therapies.

Supplementary Table S1: Clinical information related to the PDX used in Fig1A

Supplemental Methods, Figures, Figure legends, Table and Movie Legends

Supplementary Movie S1 - 3D representation around the Y axis of A375 cell nucleus

Time course experiment for gene expression analysis of IL13RA2 and IL1R2.

Effect of IM on fibroblasts and melanoma cells and effect of TPA on SCC13 cells.

Chemical structure of ingenol mebutate. Confirmation of inhibition by MEK and ERK inhibitor

Selection of further investigated targets.

Supplementary Table S3: List of the genes that were identified by RNAseq to be significantly deregulated upon SPANX KD.