Extracellular vesicles promote migration despite BRAF inhibitor treatment in malignant melanoma cells.

Extracellular vesicles (EVs) constitute a vital component of intercellular communication, exerting significant influence on metastasis formation and drug resistance mechanisms. Malignant melanoma (MM) is one of the deadliest forms of skin cancers, because of its high metastatic potential and often acquired resistance to oncotherapies. The prevalence of BRAF mutations in MM underscores the importance of BRAF-targeted therapies, such as vemurafenib and dabrafenib, alone or in combination with the MEK inhibitor, trametinib. This study aimed to elucidate the involvement of EVs in MM progression and ascertain whether EV-mediated metastasis promotion persists during single agent BRAF (vemurafenib, dabrafenib), or MEK (trametinib) and combined BRAF/MEK (dabrafenib/trametinib) inhibition.Using five pairs of syngeneic melanoma cell lines, we assessed the impact of EVs - isolated from their respective supernatants - on melanoma cell proliferation and migration. Cell viability and spheroid growth assays were employed to evaluate proliferation, while migration was analyzed through mean squared displacement (MSD) and total traveled distance (TTD) measurements derived from video microscopy and single-cell tracking.Our results indicate that while EV treatments had remarkable promoting effect on cell migration, they exerted only a modest effect on cell proliferation and spheroid growth. Notably, EVs demonstrated the ability to mitigate the inhibitory effects of BRAF inhibitors, albeit they were ineffective against a MEK inhibitor and the combination of BRAF/MEK inhibitors. In summary, our findings contribute to the understanding of the intricate role played by EVs in tumor progression, metastasis, and drug resistance in MM.

Predicting immune checkpoint therapy response in three independent metastatic melanoma cohorts.

While Immune checkpoint inhibition (ICI) therapy shows significant efficacy in metastatic melanoma, only about 50% respond, lacking reliable predictive methods. We introduce a panel of six proteins aimed at predicting response to ICI therapy. Evaluating previously reported proteins in two untreated melanoma cohorts, we used a published predictive model (EaSIeR score) to identify potential proteins distinguishing responders and non-responders. Six proteins initially identified in the ICI cohort correlated with predicted response in the untreated cohort. Additionally, three proteins correlated with patient survival, both at the protein, and at the transcript levels, in an independent immunotherapy treated cohort. Our study identifies predictive biomarkers across three melanoma cohorts, suggesting their use in therapeutic decision-making.

Research autopsy programmes in oncology: shared experience from 14 centres across the world.

While there is a great clinical need to understand the biology of metastatic cancer in order to treat it more effectively, research is hampered by limited sample availability. Research autopsy programmes can crucially advance the field through synchronous, extensive, and high-volume sample collection. However, it remains an underused strategy in translational research. Via an extensive questionnaire, we collected information on the study design, enrolment strategy, study conduct, sample and data management, and challenges and opportunities of research autopsy programmes in oncology worldwide. Fourteen programmes participated in this study. Eight programmes operated 24 h/7 days, resulting in a lower median postmortem interval (time between death and start of the autopsy, 4 h) compared with those operating during working hours (9 h). Most programmes (n = 10) succeeded in collecting all samples within a median of 12 h after death. A large number of tumour sites were sampled during each autopsy (median 15.5 per patient). The median number of samples collected per patient was 58, including different processing methods for tumour samples but also non-tumour tissues and liquid biopsies. Unique biological insights derived from these samples included metastatic progression, treatment resistance, disease heterogeneity, tumour dormancy, interactions with the tumour micro-environment, and tumour representation in liquid biopsies. Tumour patient-derived xenograft (PDX) or organoid (PDO) models were additionally established, allowing for drug discovery and treatment sensitivity assays. Apart from the opportunities and achievements, we also present the challenges related with postmortem sample collections and strategies to overcome them, based on the shared experience of these 14 programmes. Through this work, we hope to increase the transparency of postmortem tissue donation, to encourage and aid the creation of new programmes, and to foster collaborations on these unique sample collections. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Identifying Ferroptosis Inducers, HDAC, and RTK Inhibitor Sensitivity in Melanoma Subtypes through Unbiased Drug Target Prediction.

The utilization of PD1 and CTLA4 inhibitors has revolutionized the treatment of malignant melanoma (MM). However, resistance to targeted and immune-checkpoint-based therapies still poses a significant problem. Here we mine large scale MM proteogenomic data integrating it with MM cell line dependency screen, and drug sensitivity data to identify druggable targets and forecast treatment efficacy and resistance. Leveraging protein profiles from established MM subtypes and molecular structures of 82 cancer treatment drugs, we identified nine candidate hub proteins, mTOR, FYN, PIK3CB, EGFR, MAPK3, MAP4K1, MAP2K1, SRC and AKT1, across five distinct MM subtypes. These proteins serve as potential drug targets applicable to one or multiple MM subtypes. By analyzing transcriptomic data from 48 publicly accessible melanoma cell lines sourced from Achilles and CRISPR dependency screens, we forecasted 162 potentially targetable genes. We also identified genetic resistance in 260 genes across at least one melanoma subtype. In addition, we employed publicly available compound sensitivity data (Cancer Therapeutics Response Portal, CTRPv2) on the cell lines to assess the correlation of compound effectiveness within each subtype. We have identified 20 compounds exhibiting potential drug impact in at least one melanoma subtype. Remarkably, employing this unbiased approach, we have uncovered compounds targeting ferroptosis, that demonstrate a striking 30x fold difference in sensitivity among different subtypes. This implies that the proteogenomic classification of melanoma has the potential to predict sensitivity to ferroptosis compounds. Our results suggest innovative and novel therapeutic strategies by stratifying melanoma samples through proteomic profiling, offering a spectrum of novel therapeutic interventions and prospects for combination therapy. Highlights: (1) Proteogenomic subtype classification can define the landscape of genetic dependencies in melanoma (2) Nine proteins from molecular subtypes were identified as potential drug targets for specified MM patients (3) 20 compounds identified that show potential effectiveness in at least one melanoma subtype (4) Proteogenomics can predict specific ferroptosis inducers, HDAC, and RTK Inhibitor sensitivity in melanoma subtypes.

Inter- and intratumoral proteomics and glycosaminoglycan characterization of ALK rearranged lung adenocarcinoma tissues: a pilot study.

Lung cancer is one of the most common types of cancer with limited therapeutic options, therefore a detailed understanding of the underlying molecular changes is of utmost importance. In this pilot study, we investigated the proteomic and glycosaminoglycan (GAG) profile of ALK rearranged lung tumor tissue regions based on the morphological classification, mucin and stromal content. Principal component analysis and hierarchical clustering revealed that both the proteomic and GAG-omic profiles are highly dependent on mucin content and to a lesser extent on morphology. We found that differentially expressed proteins between morphologically different tumor types are primarily involved in the regulation of protein synthesis, whereas those between adjacent normal and different tumor regions take part in several other biological processes (e.g. extracellular matrix organization, oxidation-reduction processes, protein folding) as well. The total amount and the sulfation profile of heparan sulfate and chondroitin sulfate showed small differences based on morphology and larger differences based on mucin content of the tumor, while an increase was observed in both the total amount and the average rate of sulfation in tumors compared to adjacent normal regions.

Corrigendum: A potential bioelectromagnetic method to slow down the progression and prevent the development of ultimate pulmonary fibrosis by COVID-19.

[This corrects the article DOI: 10.3389/fimmu.2020.556335.].

Deep Proteomic Analysis on Biobanked Paraffine-Archived Melanoma with Prognostic/Predictive Biomarker Read-Out.

The discovery of novel protein biomarkers in melanoma is crucial. Our introduction of formalin-fixed paraffin-embedded (FFPE) tumor protocol provides new opportunities to understand the progression of melanoma and open the possibility to screen thousands of FFPE samples deposited in tumor biobanks and available at hospital pathology departments. In our retrospective biobank pilot study, 90 FFPE samples from 77 patients were processed. Protein quantitation was performed by high-resolution mass spectrometry and validated by histopathologic analysis. The global protein expression formed six sample clusters. Proteins such as TRAF6 and ARMC10 were upregulated in clusters with enrichment for shorter survival, and proteins such as AIFI1 were upregulated in clusters with enrichment for longer survival. The cohort's heterogeneity was addressed by comparing primary and metastasis samples, as well comparing clinical stages. Within immunotherapy and targeted therapy subgroups, the upregulation of the VEGFA-VEGFR2 pathway, RNA splicing, increased activity of immune cells, extracellular matrix, and metabolic pathways were positively associated with patient outcome. To summarize, we were able to (i) link global protein expression profiles to survival, and they proved to be an independent prognostic indicator, as well as (ii) identify proteins that are potential predictors of a patient's response to immunotherapy and targeted therapy, suggesting new opportunities for precision medicine developments.

Mapping the Melanoma Plasma Proteome (MPP) Using Single-Shot Proteomics Interfaced with the WiMT Database.

Plasma analysis by mass spectrometry-based proteomics remains a challenge due to its large dynamic range of 10 orders in magnitude. We created a methodology for protein identification known as Wise MS Transfer (WiMT). Melanoma plasma samples from biobank archives were directly analyzed using simple sample preparation. WiMT is based on MS1 features between several MS runs together with custom protein databases for ID generation. This entails a multi-level dynamic protein database with different immunodepletion strategies by applying single-shot proteomics. The highest number of melanoma plasma proteins from undepleted and unfractionated plasma was reported, mapping >1200 proteins from >10,000 protein sequences with confirmed significance scoring. Of these, more than 660 proteins were annotated by WiMT from the resulting ~5800 protein sequences. We could verify 4000 proteins by MS1t analysis from HeLA extracts. The WiMT platform provided an output in which 12 previously well-known candidate markers were identified. We also identified low-abundant proteins with functions related to (i) cell signaling, (ii) immune system regulators, and (iii) proteins regulating folding, sorting, and degradation, as well as (iv) vesicular transport proteins. WiMT holds the potential for use in large-scale screening studies with simple sample preparation, and can lead to the discovery of novel proteins with key melanoma disease functions.

Evidence based tools to improve efficiency of currently administered oncotherapies for tumors of the hepatopancreatobiliary system.

Hepatopancreatobiliary tumors are challenging to treat, and the advanced or metastatic forms have a very low 5-year survival rate. Several drug combinations have been tested, and new therapeutic approaches have been introduced in the last decades, including radiofrequency and heat based methods. Hyperthermia is the artificial heating of tumors by various biophysical methods that may possess immunostimulant, tumoricidal, and chemoradiotherapy sensitizer effects. Both whole-body and regional hyperthermia studies have been conducted since the 1980s after the introduction of deep-seated tumor hyperthermia techniques. Results of the effects of hyperthermia in hepatocellular and pancreatic cancer are known from several studies. Hyperthermia in biliary cancers is a less investigated area. High local and overall responses to treatment, increased progression-free and overall survival, and improved laboratory and quality-of-life results are associated with hyperthermia in all three tumor types. With the evolution of chemotherapeutic agents and the introduction of newer techniques, the combination of adjuvant hyperthermia with those therapies is advantageous and has not been associated with an increase in alarming adverse effects. However, despite the many positive effects of hyperthermia, its use is still only known at the experimental level, and its concomitant utilization in routine cancer treatment is not certain because of the lack of thorough clinical studies.

The Human Melanoma Proteome Atlas-Complementing the melanoma transcriptome.

The MM500 meta-study aims to establish a knowledge basis of the tumor proteome to serve as a complement to genome and transcriptome studies. Somatic mutations and their effect on the transcriptome have been extensively characterized in melanoma. However, the effects of these genetic changes on the proteomic landscape and the impact on cellular processes in melanoma remain poorly understood. In this study, the quantitative mass-spectrometry-based proteomic analysis is interfaced with pathological tumor characterization, and associated with clinical data. The melanoma proteome landscape, obtained by the analysis of 505 well-annotated melanoma tumor samples, is defined based on almost 16 000 proteins, including mutated proteoforms of driver genes. More than 50 million MS/MS spectra were analyzed, resulting in approximately 13,6 million peptide spectrum matches (PSMs). Altogether 13 176 protein-coding genes, represented by 366 172 peptides, in addition to 52 000 phosphorylation sites, and 4 400 acetylation sites were successfully annotated. This data covers 65% and 74% of the predicted and identified human proteome, respectively. A high degree of correlation (Pearson, up to 0.54) with the melanoma transcriptome of the TCGA repository, with an overlap of 12 751 gene products, was found. Mapping of the expressed proteins with quantitation, spatiotemporal localization, mutations, splice isoforms, and PTM variants was proven not to be predicted by genome sequencing alone. The melanoma tumor molecular map was complemented by analysis of blood protein expression, including data on proteins regulated after immunotherapy. By adding these key proteomic pillars, the MM500 study expands the knowledge on melanoma disease.

A Potential Bioelectromagnetic Method to Slow Down the Progression and Prevent the Development of Ultimate Pulmonary Fibrosis by COVID-19.

Introduction: Right now, we are facing a global pandemic caused by the coronavirus SARS-CoV-2 that causes the highly contagious human disease COVID-19. The number of COVID-19 cases is increasing at an alarming rate, more and more people suffer from it, and the death toll is on the rise since December 2019, when COVID-19 has presumably appeared. We need an urgent solution for the prevention, treatment, and recovery of the involved patients. Methods: Modulated electro-hyperthermia (mEHT) is known as an immuno-supportive therapy in oncology. Our proposal is to apply this method to prevent the progression of the disease after its identification, to provide treatment when necessary, and deliver rehabilitation to diminish the fibrotic-often fatal-consequences of the infection. Hypothesis: The effects of mEHT, which are proven for oncological applications, could be utilized for the inactivation of the virus or for treating the fibrotic consequences. The hypothesized mEHT effects, which could have a role in the antiviral treatment, it could be applied for viral-specific immune-activation and for anti-fibrotic treatments.

DNA Polymerase Alpha Subunit B Is a Binding Protein for Erlotinib Resistance in Non-Small Cell Lung Cancer.

Erlotinib inhibits epithelial growth factor receptor (EGFR) kinase activity and is used to treat non-small cell lung cancer (NSCLC). Despite its high efficacy, recurrence can occur in patients who become resistant to the drug. To address the underlying mechanism of Erlotinib resistance, we investigated additional mechanisms related to mode-of-drug-action, by multiple protein-binding interactions, besides EGFR by using drug affinity responsive target stability (DARTS) and liquid chromatography-mass spectrometry (LC-MS/MS) methods with non-labeled Erlotinib. DNA polymerase alpha subunit B (POLA2) was identified as a new Erlotinib binding protein that was validated by the DARTS platform, complemented with cellular thermal shift assays. Genetic knock-down of POLA2 promoted the anti-proliferative effect of the drug in the Erlotinib-resistant cell line H1299 with high POLA2 expression, whereas the overexpression of POLA2 restored anti-proliferative effects in the Erlotinib-sensitive cell line HCC827 with low POLA2 expression. Importantly, POLA2 expression levels in four NSCLC cell lines were positively correlated with anti-proliferative Erlotinib efficacy (Pearson correlation coefficient, R = 0.9886). These results suggest that POLA2 is a novel complementary target protein of Erlotinib, and could clinically provide validity as a surrogate marker for drug resistance in patients with NSCLC.

Protein Expression in Metastatic Melanoma and the Link to Disease Presentation in a Range of Tumor Phenotypes.

Malignant melanoma is among the most aggressive skin cancers and it has among the highest metastatic potentials. Although surgery to remove the primary tumor is the gold standard treatment, once melanoma progresses and metastasizes to the lymph nodes and distal organs, i.e., metastatic melanoma (MM), the usual outcome is decreased survival. To improve survival rates and life span, advanced treatments have focused on the success of targeted therapies in the MAPK pathway that are based on BRAF (BRAF V600E) and MEK. The majority of patients with tumors that have higher expression of BRAF V600E show poorer prognosis than patients with a lower level of the mutated protein. Based on the molecular basis of melanoma, these findings are supported by distinct tumor phenotypes determined from differences in tumor heterogeneity and protein expression profiles. With these aspects in mind, continued challenges are to: (1) deconvolute the complexity and heterogeneity of MM; (2) identify the signaling pathways involved; and (3) determine protein expression to develop targeted therapies. Here, we provide an overview of the results from protein expression in MM and the link to disease presentation in a variety of tumor phenotypes and how these will overcome the challenges of clinical problems and suggest new promising approaches in metastatic melanoma and cancer therapy.

Novel functional proteins coded by the human genome discovered in metastases of melanoma patients.

In the advanced stages, malignant melanoma (MM) has a very poor prognosis. Due to tremendous efforts in cancer research over the last 10 years, and the introduction of novel therapies such as targeted therapies and immunomodulators, the rather dark horizon of the median survival has dramatically changed from under 1 year to several years. With the advent of proteomics, deep-mining studies can reach low-abundant expression levels. The complexity of the proteome, however, still surpasses the dynamic range capabilities of current analytical techniques. Consequently, many predicted protein products with potential biological functions have not yet been verified in experimental proteomic data. This category of 'missing proteins' (MP) is comprised of all proteins that have been predicted but are currently unverified. As part of the initiative launched in 2016 in the USA, the European Cancer Moonshot Center has performed numerous deep proteomics analyses on samples from MM patients. In this study, nine MPs were clearly identified by mass spectrometry in MM metastases. Some MPs significantly correlated with proteins that possess identical PFAM structural domains; and other MPs were significantly associated with cancer-related proteins. This is the first study to our knowledge, where unknown and novel proteins have been annotated in metastatic melanoma tumour tissue.

The Hidden Story of Heterogeneous B-raf V600E Mutation Quantitative Protein Expression in Metastatic Melanoma-Association with Clinical Outcome and Tumor Phenotypes.

In comparison to other human cancer types, malignant melanoma exhibits the greatest amount of heterogeneity. After DNA-based detection of the BRAF V600E mutation in melanoma patients, targeted inhibitor treatment is the current recommendation. This approach, however, does not take the abundance of the therapeutic target, i.e., the B-raf V600E protein, into consideration. As shown by immunohistochemistry, the protein expression profiles of metastatic melanomas clearly reveal the existence of inter- and intra-tumor variability. Nevertheless, the technique is only semi-quantitative. To quantitate the mutant protein there is a fundamental need for more precise techniques that are aimed at defining the currently non-existent link between the levels of the target protein and subsequent drug efficacy. Using cutting-edge mass spectrometry combined with DNA and mRNA sequencing, the mutated B-raf protein within metastatic tumors was quantitated for the first time. B-raf V600E protein analysis revealed a subjacent layer of heterogeneity for mutation-positive metastatic melanomas. These were characterized into two distinct groups with different tumor morphologies, protein profiles and patient clinical outcomes. This study provides evidence that a higher level of expression in the mutated protein is associated with a more aggressive tumor progression. Our study design, comprised of surgical isolation of tumors, histopathological characterization, tissue biobanking, and protein analysis, may enable the eventual delineation of patient responders/non-responders and subsequent therapy for malignant melanoma.

Improved survival prognostication of node-positive malignant melanoma patients utilizing shotgun proteomics guided by histopathological characterization and genomic data.

Metastatic melanoma is one of the most common deadly cancers, and robust biomarkers are still needed, e.g. to predict survival and treatment efficiency. Here, protein expression analysis of one hundred eleven melanoma lymph node metastases using high resolution mass spectrometry is coupled with in-depth histopathology analysis, clinical data and genomics profiles. This broad view of protein expression allowed to identify novel candidate protein markers that improved prediction of survival in melanoma patients. Some of the prognostic proteins have not been reported in the context of melanoma before, and few of them exhibit unexpected relationship to survival, which likely reflects the limitations of current knowledge on melanoma and shows the potential of proteomics in clinical cancer research.

Clinical protein science in translational medicine targeting malignant melanoma.

Melanoma of the skin is the sixth most common type of cancer in Europe and accounts for 3.4% of all diagnosed cancers. More alarming is the degree of recurrence that occurs with approximately 20% of patients lethally relapsing following treatment. Malignant melanoma is a highly aggressive skin cancer and metastases rapidly extend to the regional lymph nodes (stage 3) and to distal organs (stage 4). Targeted oncotherapy is one of the standard treatment for progressive stage 4 melanoma, and BRAF inhibitors (e.g. vemurafenib, dabrafenib) combined with MEK inhibitor (e.g. trametinib) can effectively counter BRAFV600E-mutated melanomas. Compared to conventional chemotherapy, targeted BRAFV600E inhibition achieves a significantly higher response rate. After a period of cancer control, however, most responsive patients develop resistance to the therapy and lethal progression. The many underlying factors potentially causing resistance to BRAF inhibitors have been extensively studied. Nevertheless, the remaining unsolved clinical questions necessitate alternative research approaches to address the molecular mechanisms underlying metastatic and treatment-resistant melanoma. In broader terms, proteomics can address clinical questions far beyond the reach of genomics, by measuring, i.e. the relative abundance of protein products, post-translational modifications (PTMs), protein localisation, turnover, protein interactions and protein function. More specifically, proteomic analysis of body fluids and tissues in a given medical and clinical setting can aid in the identification of cancer biomarkers and novel therapeutic targets. Achieving this goal requires the development of a robust and reproducible clinical proteomic platform that encompasses automated biobanking of patient samples, tissue sectioning and histological examination, efficient protein extraction, enzymatic digestion, mass spectrometry-based quantitative protein analysis by label-free or labelling technologies and/or enrichment of peptides with specific PTMs. By combining data from, e.g. phosphoproteomics and acetylomics, the protein expression profiles of different melanoma stages can provide a solid framework for understanding the biology and progression of the disease. When complemented by proteogenomics, customised protein sequence databases generated from patient-specific genomic and transcriptomic data aid in interpreting clinical proteomic biomarker data to provide a deeper and more comprehensive molecular characterisation of cellular functions underlying disease progression. In parallel to a streamlined, patient-centric, clinical proteomic pipeline, mass spectrometry-based imaging can aid in interrogating the spatial distribution of drugs and drug metabolites within tissues at single-cell resolution. These developments are an important advancement in studying drug action and efficacy in vivo and will aid in the development of more effective and safer strategies for the treatment of melanoma. A collaborative effort of gargantuan proportions between academia and healthcare professionals has led to the initiation, establishment and development of a cutting-edge cancer research centre with a specialisation in melanoma and lung cancer. The primary research focus of the European Cancer Moonshot Lund Center is to understand the impact that drugs have on cancer at an individualised and personalised level. Simultaneously, the centre increases awareness of the relentless battle against cancer and attracts global interest in the exceptional research performed at the centre.

Complete Clinical Remission of Stage IV Triple-Negative Breast Cancer Lung Metastasis Administering Low-Dose Immune Checkpoint Blockade in Combination With Hyperthermia and Interleukin-2.

The prognosis of triple-negative breast cancer with metastases after chemotherapy remains dismal. We report the case of a 50-year-old female with first disease recurrence at the axillary lymph node and, later on, bilateral pulmonary metastases with severe shortness of breath. The patient received low-dose immune checkpoint blockade (concurrent nivolumab and ipilimumab) weekly over 3 weeks with regional hyperthermia 3 times a week, followed by systemic fever-range hyperthermia induced by interleukin-2 for 5 days. She went into complete remission of her pulmonary metastases with transient WHO I-II diarrhea and skin rash. The patient remained alive for 27 months after the start of treatment, with recurrence of metastases as a sternal mass, and up to 3 cm pleural metastases. This exceptional response should instigate further research efforts with this protocol, which consists only of approved drugs and treatments.