Proteomic Profiling of Advanced Melanoma Patients to Predict Therapeutic Response to Anti-PD-1 Therapy.

PURPOSE: Despite high clinical need, there are no biomarkers that accurately predict the response of patients with metastatic melanoma to anti-PD-1 therapy. EXPERIMENTAL DESIGN: In this multicenter study, we applied protein depletion and enrichment methods prior to various proteomic techniques to analyze a serum discovery cohort (n = 56) and three independent serum validation cohorts (n = 80, n = 12, n = 17). Further validation analyses by literature and survival analysis followed. RESULTS: We identified several significantly regulated proteins as well as biological processes such as neutrophil degranulation, cell-substrate adhesion, and extracellular matrix organization. Analysis of the three independent serum validation cohorts confirmed the significant differences between responders (R) and nonresponders (NR) observed in the initial discovery cohort. In addition, literature-based validation highlighted 30 markers overlapping with previously published signatures. Survival analysis using the TCGA database showed that overexpression of 17 of the markers we identified correlated with lower overall survival in patients with melanoma. CONCLUSIONS: Ultimately, this multilayered serum analysis led to a potential marker signature with 10 key markers significantly altered in at least two independent serum cohorts: CRP, LYVE1, SAA2, C1RL, CFHR3, LBP, LDHB, S100A8, S100A9, and SAA1, which will serve as the basis for further investigation. In addition to patient serum, we analyzed primary melanoma tumor cells from NR and found a potential marker signature with four key markers: LAMC1, PXDN, SERPINE1, and VCAN.

Combinatorial Treatment with PARP and MAPK Inhibitors Overcomes Phenotype Switch-Driven Drug Resistance in Advanced Melanoma.

Metastatic melanoma is either intrinsically resistant or rapidly acquires resistance to targeted therapy treatments, such as MAPK inhibitors (MAPKi). A leading cause of resistance to targeted therapy is a dynamic transition of melanoma cells from a proliferative to a highly invasive state, a phenomenon called phenotype switching. Mechanisms regulating phenotype switching represent potential targets for improving treatment of patients with melanoma. Using a drug screen targeting chromatin regulators in patient-derived three-dimensional MAPKi-resistant melanoma cell cultures, we discovered that PARP inhibitors (PARPi) restore sensitivity to MAPKis, independent of DNA damage repair pathways. Integrated transcriptomic, proteomic, and epigenomic analyses demonstrated that PARPis induce lysosomal autophagic cell death, accompanied by enhanced mitochondrial lipid metabolism that ultimately increases antigen presentation and sensitivity to T-cell cytotoxicity. Moreover, transcriptomic and epigenetic rearrangements induced by PARP inhibition reversed epithelial-mesenchymal transition-like phenotype switching, which redirected melanoma cells toward a proliferative and MAPKi-sensitive state. The combination of PARP and MAPKis synergistically induced cancer cell death both in vitro and in vivo in patient-derived xenograft models. Therefore, this study provides a scientific rationale for treating patients with melanoma with PARPis in combination with MAPKis to abrogate acquired therapy resistance. SIGNIFICANCE: PARP inhibitors can overcome resistance to MAPK inhibitors by activating autophagic cell death and reversing phenotype switching, suggesting that this synergistic combination could help improve the prognosis of patients with melanoma.

Dynamic thresholding and tissue dissociation optimization for CITE-seq identifies differential surface protein abundance in metastatic melanoma.

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.

Tumor-derived GDF-15 blocks LFA-1 dependent T cell recruitment and suppresses responses to anti-PD-1 treatment.

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/ß2-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.

ROS Induction Targets Persister Cancer Cells with Low Metabolic Activity in NRAS-Mutated Melanoma.

Clinical management of melanomas with NRAS mutations is challenging. Targeting MAPK signaling is only beneficial to a small subset of patients due to resistance that arises through genetic, transcriptional, and metabolic adaptation. Identification of targetable vulnerabilities in NRAS-mutated melanoma could help improve patient treatment. Here, we used multiomics analyses to reveal that NRAS-mutated melanoma cells adopt a mesenchymal phenotype with a quiescent metabolic program to resist cellular stress induced by MEK inhibition. The metabolic alterations elevated baseline reactive oxygen species (ROS) levels, leading these cells to become highly sensitive to ROS induction. In vivo xenograft experiments and single-cell RNA sequencing demonstrated that intratumor heterogeneity necessitates the combination of a ROS inducer and a MEK inhibitor to inhibit both tumor growth and metastasis. Ex vivo pharmacoscopy of 62 human metastatic melanomas confirmed that MEK inhibitor-resistant tumors significantly benefited from the combination therapy. Finally, oxidative stress response and translational suppression corresponded with ROS-inducer sensitivity in 486 cancer cell lines, independent of cancer type. These findings link transcriptional plasticity to a metabolic phenotype that can be inhibited by ROS inducers in melanoma and other cancers. SIGNIFICANCE: Metabolic reprogramming in drug-resistant NRAS-mutated melanoma cells confers sensitivity to ROS induction, which suppresses tumor growth and metastasis in combination with MAPK pathway inhibitors.

The impact of the COVID-19 pandemic on the diagnosis of cutaneous melanomas: A retrospective cohort study from five European skin cancer reference centres.

BACKGROUND: The COVID-19 lockdown had a dramatic impact on primary care access and resulted in postponed skin cancer screenings. This raises concerns for a diagnostic delay on primary cutaneous melanomas, which can subsequently increase morbidity and mortality. OBJECTIVES: The aim of the study was to investigate the impact of the COVID-19-related restrictions on the melanoma diagnosis in five European skin cancer reference centres in Switzerland, Germany, Austria and Italy. METHODS: A total of 7865 cutaneous melanoma cases were collected between 01 September 2018 and 31 August 2021. The time period was stratified into pre-COVID (pre-lockdown) and post-COVID (lockdown and post-lockdown) according to the established restrictions in each country. The data collection included demographic, clinical and histopathological data from histologically confirmed cutaneous melanomas. Personal and family history of melanoma, and presence of immunosuppression were used to assess the diagnosis delay in high-risk individuals. RESULTS: There was an overall increase of the Breslow tumour thickness (mean 1.25 mm vs. 1.02 mm) during the post-COVID period, as well as an increase in the proportion of T3-T4 melanomas, rates of ulceration and the number of mitotic rates ≥2 (all, p < 0.001). Patients with immunosuppression and personal history of melanoma showed a decrease in the mean log10-transformed Breslow during lockdown and post-COVID. In the multivariate analysis, age at melanoma diagnosis (p < 0.01) and personal history of melanoma (p < 0.01) showed significant differences in the mean Breslow thickness. CONCLUSIONS: The study confirms the diagnostic delay in cutaneous melanomas due to the COVID-19 lockdown. High-risk individuals, such as patients with personal history of melanoma and elderly individuals, were more hesitant to restart their regular skin cancer screenings post-COVID. Further studies with longer follow-up are required to evaluate the consequences of this diagnostic delay in long-term outcomes.

Impact of Covid-19 on the management of patients with metastatic melanoma.

The Covid-19 pandemic created new uncertainties in the management of metastatic melanoma patients. In particular, the impact of immunotherapy, targeted therapy, or chemotherapy on the risk of Sars-CoV-2 infection and severity was debated. In this study, we analyzed all patients with metastatic melanoma receiving therapy who developed Covid-19 between February 2020 and February 2022. We retrospectively collected demographic data, cancer-specific parameters, melanoma treatment regimen, comorbidities and Covid-19-specific parameters in these patients. Of the 350 patients with metastatic melanoma, 25 had Covid-19. The median age at the time of Covid-19 diagnosis was 66 years (range 36-86), 10 patients were female, and 15 patients were male. The treatment regimen during infection was immunotherapy in 12 cases, followed by targeted therapy (n = 8), chemotherapy (n = 2), and TVEC injections, follow-up and palliative therapy in 1 case each. The severity was mild in 17 patients and 8 had a moderate to critical course. Patients with a severe Covid-19 course were often older and had more comorbidities than patients with a mild infection. Many of the patients had a mild Covid-19 course despite having metastatic melanoma and systemic therapy. We therefore recommend continuing systemic therapy whenever possible, even in such exceptional situations as the Covid-19 pandemic.

Correction: Tusup et al. Evaluation of the Interplay between the ADAR Editome and Immunotherapy in Melanoma. Non-Coding RNA 2021, 7, 5.

In the original article [...].

The Prognostic Value of a Single, Randomly Timed Circulating Tumor DNA Measurement in Patients with Metastatic Melanoma.

Melanoma currently lacks validated blood-based biomarkers for monitoring and predicting treatment efficacy. Circulating tumor DNA (ctDNA), originating from tumor cells and detectable in plasma, has emerged as a possible biomarker in patients with metastatic melanoma. In this retrospective, single-center study, we collected 129 plasma samples from 79 patients with stage IIIB-IV melanoma as determined by the American Joint Committee on Cancer (AJCC, 8th edition). For the determination of ctDNA levels, we used eight different assays of droplet digital polymerase chain reaction (ddPCR) to detect the most common hotspot mutations in the BRAF and NRAS genes. The aim of the study was to investigate the association of the detectability of ctDNA at a non-prespecified time point in a patient's treatment with tumor progression, and to correlate ctDNA with commonly used biomarkers (protein S100, LDH, and CRP). Patients with detectable ctDNA progressed more frequently in PET-CT within 12 months than those without detectable ctDNA. Detectability of ctDNA was associated with shorter OS in univariate and multivariate analyses. ctDNA was detectable in a statistically significantly larger proportion of patients with distant metastases (79%) than in patients with no distant metastases or only intracranial metastases (32%). Elevated protein S100 and CRP correlated better with detectable ctDNA than LDH. This study supports the potential of ctDNA as a prognostic biomarker in patients with metastatic melanoma. However, additional prospective longitudinal studies with quantitative assessments of ctDNA are necessary to investigate the limitations and strengths of ctDNA as a biomarker.

Defining the Molecular Landscape of Cancer-Associated Stroma in Cutaneous Squamous Cell Carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer worldwide. Cancer-associated stroma (CAS) is central to tumor development and strongly influences therapy response. Perineural infiltration (PNI) represents a major risk factor for cSCC and likely influences CAS reprogramming. However, stromal reprogramming in cSCC remains poorly characterized, and it is unknown whether and how PNI influences CAS. To address these questions, we analyzed CAS and matched normal stroma from 20 cSCC cases (11 without PNI and 9 with PNI) by laser-capture microdissection using RNA sequencing. Our analysis reveals extensive stromal reprogramming strongly driven by changes in immune cells, as validated using immunohistochemistry. Furthermore, CAS of cSCC displays markers of immune exhaustion, and multiplex spatial analysis suggests that PD-L1 expression on NK T cells contributes to T-cell exhaustion and immunosuppression. Finally, PNI is characterized by increased IL-17A. In PNI-negative cases, IL-17A derives predominantly from CD3+ cells. However, with PNI, we observe an increased contribution of fibroblasts to high IL-17A, which coincides with a significant increase in FAP+ cells. Our analysis elucidates the molecular landscape of CAS in cSCC and identifies the presence of immunosuppressive mechanisms, supporting further research into immunotherapy and anti‒IL-17A in cSCC, especially for cases with PNI.

Loss of YY1, a Regulator of Metabolism in Melanoma, Drives Melanoma Cell Invasiveness and Metastasis Formation.

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-ß. 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.

Mycobacterial infection aggravates Helicobacter pylori-induced gastric preneoplastic pathology by redirection of de novo induced Treg cells.

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.

Specific Activation of the CD271 Intracellular Domain in Combination with Chemotherapy or Targeted Therapy Inhibits Melanoma Progression.

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 ß-amyloid-derived peptide (Aß(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 Aß(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. Aß(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. Aß(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.

Epigenetic control of melanoma cell invasiveness by the stem cell factor SALL4.

Melanoma cells rely on developmental programs during tumor initiation and progression. Here we show that the embryonic stem cell (ESC) factor Sall4 is re-expressed in the Tyr::NrasQ61K; Cdkn2a-/- melanoma model and that its expression is necessary for primary melanoma formation. Surprisingly, while Sall4 loss prevents tumor formation, it promotes micrometastases to distant organs in this melanoma-prone mouse model. Transcriptional profiling and in vitro assays using human melanoma cells demonstrate that SALL4 loss induces a phenotype switch and the acquisition of an invasive phenotype. We show that SALL4 negatively regulates invasiveness through interaction with the histone deacetylase (HDAC) 2 and direct co-binding to a set of invasiveness genes. Consequently, SALL4 knock down, as well as HDAC inhibition, promote the expression of an invasive signature, while inhibition of histone acetylation partially reverts the invasiveness program induced by SALL4 loss. Thus, SALL4 appears to regulate phenotype switching in melanoma through an HDAC2-mediated mechanism.

Real-life data for first-line combination immune-checkpoint inhibition and targeted therapy in patients with melanoma brain metastases.

BACKGROUND: Melanoma brain metastases (MBM) have a poor prognosis. Systemic treatments that have improved outcomes in advanced melanoma have been shown to have an intracranial (IC) effect. We studied the efficacy and outcomes of combined immune checkpoint inhibitor ipilimumab/nivolumab (Combi-ICI) or targeted therapy (Combi-TT) as first-line treatment in MBM. METHODS: MBM patients treated with Combi-ICI or Combi-TT within 3 months after MBM diagnosis. Endpoints were progression-free survival (PFS) and overall survival (OS). RESULTS: 53 patients received Combi-ICI, 32% had symptomatic MBM and 33.9% elevated LDH. 71.7% required local treatment. The disease control rate was 60.3%. IC response rate (RR) was 43.8% at 3-months with durable responses at 6- (46.5%) and 12-months (53.1%). Extracranial (EC) RR was 44.7% at 3-months and 50% at 12-months. Median PFS was 9.6 months (95% CI 3.6-NR) and median overall survival (mOS) 44.8 months (95% CI; 26.2-NR). 63 patients received Combi-TT, 55.6% of patients had symptomatic MBM, 57.2% of patients had elevated LDH and 68.3% of patients required local treatment. The disease control rate was 60.4%. ICRR was 50% at 3-months, but dropped at 6-months (20.9%). ECRR was 69.2% at 3-months and 17.6% at 12-months. Median PFS was 5.8 months (95% CI 4.2-7.6) and mOS 14.2 months (95% CI 8.99-26.8). In BRAFV600 patients, 26.7% of patients received Combi-ICI and 73.3% Combi-TT with OS (p = 0.0053) and mPFS (p = 0.03) in favour to Combi-ICI. CONCLUSION: Combi-ICI showed prolonged mOS with sustainable IC and EC responses. Despite the initially increased efficacy, Combi-TT responses at 12 months were low. Combi-ICI appeared superior to Combi-TT for OS and PFS in BRAFV600 patients. Other clinical factors are determinants for first-line treatment choice.

Frequency, Treatment and Outcome of Immune-Related Toxicities in Patients with Immune-Checkpoint Inhibitors for Advanced Melanoma: Results from an Institutional Database Analysis.

Immune checkpoint inhibitors (ICIs) can induce immune-related adverse events (irAEs), which may result in treatment discontinuation. We sought to describe the onset, frequency, and kinetics of irAEs in melanoma patients in a real-life setting and to further investigate the prognostic role of irAEs in treatment outcomes. In this retrospective single-center cohort study, we included 249 melanoma patients. Onset, grade, and resolution of irAEs and their treatment were analyzed. A total of 191 (74.6%) patients in the non-adjuvant and 65 (25.3%) in the adjuvant treatment setting were identified. In the non-adjuvant setting, 29 patients (59.2%) with anti-CTLA4, 43 (58.1%) with anti-PD1, and 54 (79.4%) with anti-PD1/anti-CTLA4 experienced some grade of irAE and these had an improved outcome. In the adjuvant setting, the frequency of irAEs was 84.6% in anti-CTLA4 and 63.5% in anti-PD1, but no correlation with disease relapse was observed. Patients with underlying autoimmune conditions have a risk of disease exacerbation. Immunomodulatory agents had no impact on treatment efficacy. IrAEs are correlated with increased treatment efficacy in the non-adjuvant setting. Application of steroids and immunomodulatory agents, such as anti-TNF-alpha or anti-IL6, did not affect ICI efficacy. These data support irAEs as possible prognostic markers for ICI treatment.

Sustainable responses in metastatic melanoma patients with and without brain metastases after elective discontinuation of anti-PD1-based immunotherapy due to complete response.

BACKGROUND: Anti-PD1-based immunotherapy is currently used in most patients with advanced melanoma. Despite the remarkable data regarding overall survival, the optimal treatment duration is still unknown. METHODS: We evaluated the outcome of 125 patients with advanced melanoma with and without brain metastases (MBM), treated either with anti-PD1 monotherapy (N = 97) or combined with anti-CTLA4 (N = 28) after elective treatment discontinuation due to complete response (CR) (group A, N = 86), or treatment-limiting toxicity (N = 33) and investigator's decision (ID, N = 6) (group B) with subsequent CR. RESULTS: For group A, median duration of treatment (mDoT) was 22 months (range 5-49) and median time to CR 9 months (range 2-47). Accordingly, mDoT for group B was 3 months (range 0-36) and median time to CR 7 months (range 1-32). Seven patients from group A and three from group B experienced disease recurrence. Off-treatment survival was not reached. Median off-treatment response time (mOTRt) was 19 months (range 0-42) and 25 months (range 0-66), respectively. For MBM, mOTRt was 17 months (range 7-41) and 28 months (range 9-39), respectively. After a median follow-up of 38 months (range 9-70), seven (5.6%) patients had deceased, one (0.8%) due to melanoma. CONCLUSIONS: Treatment discontinuation is feasible also in patients with MBM. Efficacy outcomes seemed to be similar in both groups of patients who achieved CR, regardless of reason for discontinuation. In patients who experienced disease relapse, treatment re-challenge with anti-PD1 resulted in subsequent renewed response.

Prolonged Unfrozen Storage and Repeated Freeze-Thawing of SARS-CoV-2 Patient Samples Have Minor Effects on SARS-CoV-2 Detectability by RT-PCR.

Reliable transportation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) patient samples from a swabbing station to a diagnostics facility is essential for accurate results. Therefore, cooling or freezing the samples is recommended in case of longer transportation times. In this study, SARS-CoV-2 detectability by RT-PCR was assessed after prolonged unfrozen storage or repetitive freeze-thawing of SARS-CoV-2 samples. SARS-CoV-2-positive patient swabs stored in viral transport medium were exposed to different temperatures (4°C, 25°C, and 35°C) and to repetitive freeze-thawing, to assess the effect of storage conditions on RT-PCR detection. SARS-CoV-2 RNA was still reliably detected by RT-PCR after 21 days of storage in viral transport medium, even when the samples had been stored at 35°C. The maximum observed change in cycle threshold value per day was 0.046 (±0.019) at 35°C, and the maximum observed change in cycle threshold value per freeze-thaw cycle per day was 0.197 (±0.06). Compared with storage at 4°C, viral RNA levels deviated little but significantly when stored at 25°C or 35°C, or after repeated freeze-thawing. The results of this study indicate that viral RNA levels are relatively stable at higher temperatures and repetitive freeze-thawing.

Evolution of late-stage metastatic melanoma is dominated by aneuploidy and whole genome doubling.

Although melanoma is initiated by acquisition of point mutations and limited focal copy number alterations in melanocytes-of-origin, the nature of genetic changes that characterise lethal metastatic disease is poorly understood. Here, we analyze the evolution of human melanoma progressing from early to late disease in 13 patients by sampling their tumours at multiple sites and times. Whole exome and genome sequencing data from 88 tumour samples reveals only limited gain of point mutations generally, with net mutational loss in some metastases. In contrast, melanoma evolution is dominated by whole genome doubling and large-scale aneuploidy, in which widespread loss of heterozygosity sculpts the burden of point mutations, neoantigens and structural variants even in treatment-naïve and primary cutaneous melanomas in some patients. These results imply that dysregulation of genomic integrity is a key driver of selective clonal advantage during melanoma progression.