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Understanding the evolutionary pathways to metastasis and resistance to immune-checkpoint inhibitors (ICI) in melanoma is critical for improving outcomes. Here, we present the most comprehensive intrapatient metastatic melanoma dataset assembled to date as part of the Posthumous Evaluation of Advanced Cancer Environment (PEACE) research autopsy program, including 222 exome sequencing, 493 panel-sequenced, 161 RNA sequencing, and 22 single-cell whole-genome sequencing samples from 14 ICI-treated patients. We observed frequent whole-genome doubling and widespread loss of heterozygosity, often involving antigen-presentation machinery. We found KIT extrachromosomal DNA may have contributed to the lack of response to KIT inhibitors of a KIT-driven melanoma. At the lesion-level, MYC amplifications were enriched in ICI nonresponders. Single-cell sequencing revealed polyclonal seeding of metastases originating from clones with different ploidy in one patient. Finally, we observed that brain metastases that diverged early in molecular evolution emerge late in disease. Overall, our study illustrates the diverse evolutionary landscape of advanced melanoma.Significance:Despite treatment advances, melanoma remains a deadly disease at stage IV. Through research autopsy and dense sampling of metastases combined with extensive multiomic profiling, our study elucidates the many mechanisms that melanomas use to evade treatment and the immune system, whether through mutations, widespread copy-number alterations, or extrachromosomal DNA.

Supplementary figure 1: Cohort overview. Number of samples sequenced with whole exome, panel or whole RNA sequencing. Supplementary figure 2: Phylogeny and WGD events in CRUKP1047. Supplementary figure 3: Ploidy and SCNA burden. Supplementary figure 4: Overview of each case. Supplementary figure 5: MEDICC2 copy number sample trees. Supplementary figure 6: MEDICC tree of all exome samples demonstrating that samples cluster together by patient, and not by melanoma subtype. Supplementary figure 7: SCNA frequency of cutaneous (a), acral (b) and melanoma of unknown primary (MUP, c). Supplementary figure 8: Correlation between liver copy number distance to other sites and time of emergence after stage IV diagnosis. Supplementary figure 9: Examination of tumour heterogeneity of alterations to antigen-presentation machinery genes, with site and patient annotation. Supplementary figure 10: Boxplots indicating the proportion of losses in the cohort for each segment. Supplementary figure 11: Balance of expression between nonsynonymous mutations that were not predicted to be neoantigens and clonal predicted neoantigens. Supplementary figure 12: Barplot of TIL infiltration score frequencies, determined by pathologist assessment of histology, across all samples. Supplementary figure 13: Number of samples per patient that are classified as either none-low in terms of TILs or moderate-heavy. Supplementary figure 14: Histogram of purity for samples with RNA-seq data. Supplementary figure 15: TME deconvolution. Supplementary figure 16: The effect of metastatic site on transcriptional profiles. Supplementary figure 17: Association of PHF3 copy number with expression. Supplementary figure 18: Overview of gene fusions identified in RNA-seq data. Supplementary figure 19: Comparison of ploidy estimates from panel sequencing data, exome data and FISH. Supplementary figure 20: Comparison of ploidy estimates in panel, exome, FISH and single cell data. Supplementary figure 21: FACs sort plot for CRUKP2567 diaphragmatic metastasis. Supplementary figure 22: Ploidy and wGII values from single cell sequencing of FACS-sorted tumour cells. Supplementary figure 23: Copy number profiles on chromosome 5 for bulk samples from primary and DI_2_R2, a diaphragmatic metastasis. Supplementary figure 24: Histogram of purity of samples for which RNA-seq was performed faceted by patient. Supplementary figure 25: Histogram of purity of samples for which RNA-seq was performed faceted by tissue site.

S. Table 1: List of PEACE Consortium members. S. Table 2: Sample database used for the study for Panel, Exome and RNASeq samples. S. Table 3: Overview of lines of treatment given to each patient. S. Table 4: Lesions and patients included in the lesion-level response to ICI analysis. S. Table 5: Hallmark genesets significantly upregulated in normal tissue vs tumor tissue. Table shows p-values for tissue type, purity; T value for tissue type, purity; q-value for tissue type, purity (FDR corrected). S. Table 6: Hallmark genesets significantly upregulated in tumor tissue vs normal tissue. Table shows p-values for tissue type, purity; T value for tissue type, purity; q-value for tissue type, purity (FDR corrected). S. Table 7: Genes included in the target panel. S. Table 8: Tree topology comparisons between manually constructed trees and pairtree-constructed trees.

Background & Aims: Checkpoint inhibitor-related hepatitis (CPI-Hep) is an emerging clinical challenge. We aimed to gain insights into the immunopathology of CPI-Hep by comprehensively characterising myeloid and lymphoid subsets. Methods: CPI-treated patients with or without related hepatitis (CPI-Hep; n = 22 and CPI-noHep; n = 7) were recruited. Phenotypic and transcriptional profiling of peripheral immune subsets was performed and compared with 19 healthy controls (HCs). In vitro monocyte-derived macrophages (MoMFs) were assessed for activation and cytokine production. CD163, CCR2, CD68, CD3, CD8 and granzyme B expression was assessed using immunohistochemistry/immunofluorescence (n = 4). Results: A significant total monocyte depletion was observed in CPI-Hep compared with HCs (p = 0.04), along with a proportionate increase in the classical monocyte population (p = 0.0002) and significant upregulation of CCR2, CD163 and downregulation of CCR7. Soluble CD163 levels were significantly elevated in CPI-Hep compared with HCs (p

Patients with blood cancer continue to have a greater risk of inadequate immune responses following three COVID-19 vaccine doses and risk of severe COVID-19 disease. In the context of the CAPTURE study (NCT03226886), we report immune responses in 80 patients with blood cancer who received a fourth dose of BNT162b2. We measured neutralizing antibody titers (NAbTs) using a live virus microneutralization assay against wild-type (WT), Delta, and Omicron BA.1 and BA.2 and T cell responses against WT and Omicron BA.1 using an activation-induced marker (AIM) assay. The proportion of patients with detectable NAb titers and T cell responses after the fourth vaccine dose increased compared with that after the third vaccine dose. Patients who received B cell-depleting therapies within the 12 months before vaccination have the greatest risk of not having detectable NAbT. In addition, we report immune responses in 57 patients with breakthrough infections after vaccination.

Patients with cancer have higher COVID-19 morbidity and mortality. Here we present the prospective CAPTURE study, integrating longitudinal immune profiling with clinical annotation. Of 357 patients with cancer, 118 were SARS-CoV-2 positive, 94 were symptomatic and 2 died of COVID-19. In this cohort, 83% patients had S1-reactive antibodies and 82% had neutralizing antibodies against wild type SARS-CoV-2, whereas neutralizing antibody titers against the Alpha, Beta and Delta variants were substantially reduced. S1-reactive antibody levels decreased in 13% of patients, whereas neutralizing antibody titers remained stable for up to 329 days. Patients also had detectable SARS-CoV-2-specific T cells and CD4+ responses correlating with S1-reactive antibody levels, although patients with hematological malignancies had impaired immune responses that were disease and treatment specific, but presented compensatory cellular responses, further supported by clinical recovery in all but one patient. Overall, these findings advance the understanding of the nature and duration of the immune response to SARS-CoV-2 in patients with cancer.

Not all patients with cancer, in particular those with hematogic malignancies, develop functional immunity against SARS-CoV-2 variants of concern (VOC) following COVID-19 vaccines. Durability of vaccine-induced immunity after two doses and the impact of a third dose were evaluated in CAPTURE (NCT03226886), a longitudinal prospective cohort study of vaccine responses in patients with cancer. In evaluating 316 patients, at a median of 111 days following two doses of either BNT16b2 or ChadOX, we observed a time-dependant decline in neutralising antibody titres (NAbT) in a proportion of patients, where NAbTs became undetectable against Delta and Beta in 17% and 15% of patients, respectively. Vaccine-induced T cell responses declined in 44% of patients. Patients with breakthrough infections following two vaccines doses were characterised by absent/low NAbT to Delta prior to infection. Administration of the third vaccine dose boosted NAb responses against VOC in the majority of patients with cancer, especially those with solid cancer. In patients with hematologic malignancies who had undetectable NAbT against Delta after two vaccine doses, 54% did not develop NAb against both Beta and Delta following the third dose. Third vaccine dose boosted T cell responses were boosted in patients with both solid and hematologic malignancies. These results provide critical information on vaccine responses in patients with cancer, especially against VOCs and support widespread access to a third COVID-19 vaccination in this patient group.

Coronavirus disease 2019 (COVID-19) antiviral response in a pan-tumor immune monitoring (CAPTURE) (NCT03226886) is a prospective cohort study of COVID-19 immunity in patients with cancer. Here we evaluated 585 patients following administration of two doses of BNT162b2 or AZD1222 vaccines, administered 12 weeks apart. Seroconversion rates after two doses were 85% and 59% in patients with solid and hematological malignancies, respectively. A lower proportion of patients had detectable titers of neutralizing antibodies (NAbT) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC) versus wild-type (WT) SARS-CoV-2. Patients with hematological malignancies were more likely to have undetectable NAbT and had lower median NAbT than those with solid cancers against both SARS-CoV-2 WT and VOC. By comparison with individuals without cancer, patients with hematological, but not solid, malignancies had reduced neutralizing antibody (NAb) responses. Seroconversion showed poor concordance with NAbT against VOC. Previous SARS-CoV-2 infection boosted the NAb response including against VOC, and anti-CD20 treatment was associated with undetectable NAbT. Vaccine-induced T cell responses were detected in 80% of patients and were comparable between vaccines or cancer types. Our results have implications for the management of patients with cancer during the ongoing COVID-19 pandemic.

Immune checkpoint inhibitors have transformed the prognosis and treatment paradigm of many cancer types, through the potential for durable responses. However, the majority of patients still do not benefit. Response to checkpoint inhibition is determined by dynamic host, tumour and tumour microenvironment factors that display spatial and temporal variability, but our understanding of these interactions is incomplete. Through investigating biomarkers of resistance and response, opportunities arise to discover new therapeutic targets and shape personalised treatment strategies. Here we review approved and emerging biomarkers of response to immune checkpoint inhibitors, in particular the recent rapid progress in host and tumour genomics. It is unlikely that a single biomarker will precisely predict response, but multivariate multiomic markers may provide a balanced assessment of these factors and more accurately identify patients who will benefit. Further efforts are required to translate these groundbreaking discoveries into novel therapeutics and biomarker driven clinical trials, to provide durable treatment response to a greater population of patients.

Background: Patients with cancer are at increased risk of severe outcomes from COVID-19. Understanding the impact of SARS-CoV-2 infection and vaccination induced-immunity is an area of unmet need. Methods: CAPTURE (NCT03226886) is a prospective longitudinal cohort study of COVID-19 vaccine or SARS-CoV-2 infection-induced immunity. SARS-CoV-2 infections were confirmed by RT-PCR and ELISA. Neutralising antibody titres (NAbT) against wild-type (WT) SARS-CoV-2 and variants of concern (VOC;Alpha, Beta, Delta) and SARS-CoV-2 specific T-cells (SsT-cells) were quantified. Results: 118 patients (89% solid malignancy, [SM]) were SARS-CoV-2-positive (median follow-up: 154 days). 85% patients were symptomatic;2 died of COVID-19. 82% had S1-reactive antibodies, of whom 89% had neutralising antibodies (NAbs);NAbT were lower against all VOCs. While S1-reactive antibody levels declined over time, NAbT remained stable up to 329 days. Most patients had detectable SsT-cells (76% CD4+, 52% CD8+). Haematological malignancy (HM) patients had impaired immune responses that were disease and treatment-specific (anti-CD20), but with evidence suggestive of compensation from T-cells. 585 patients were evaluated following 2 doses of BNT162b2 or AZD1222 vaccines, administered 12 weeks apart. Seroconversion rates after 2 doses were 85% and 54% in patients with SM and HM, respectively. A lower proportion of patients had detectable NAbs against SARS-CoV-2 VOC (Alpha 62%, Beta 54%, Delta 49%) vs WT (84%), with corresponding significantly lower NAbT. Patients with HM were more likely to have an undetectable NAb and had lower NAbT vs solid malignancies to both WT and VOCs. Seroconversion showed poor concordance with NAbTs against VOCs. Prior SARS-CoV-2 infection boosted NAbT including against VOCs. Anti-CD20 treatment was associated with severely diminished NAbTs. Vaccine-induced T-cell responses were detected in 80% of patients, with no differences between vaccines or cancer types. Conclusions: Patients with HM had blunted humoural responses to infection and vaccination, particularly against VOCs, but preserved cellular responses might contribute to protection. Our results lend support to prioritisation of all cancer patients for further booster vaccination. Clinical trial identification: NCT03226886. Legal entity responsible for the study: The Royal Marsden NHS Foundation Trust. Funding: The Royal Marsden Charity;The National Institute for Health Research (NIHR) Biomedical Research Centre (BRC) at the Royal Marsden Hospital and Institute for Cancer Research (ICR). Disclosure: All authors have declared no conflicts of interest.

Background Targeted therapy and immunotherapy agents for advanced melanoma are associated with novel toxicities. Melanoma clinical nurse consultants (CNCs) provide multifaceted clinical care. . Objectives The objective was to evaluate the type of support, excluding clinic and inpatient care, provided by CNCs for patients not enrolled in a clinical trial. . Methods A prospective review of CNC support provided during a 12-week period was conducted. . Findings From May to August 2015, 105 patients attended clinic, and 72 received CNC support. Initial patient encounters with CNCs were documented (n = 150), as well as additional interactions (n = 291). The most common problem identified per initial encounter was symptom/drug toxicity. The most common therapy-related concern was related to anti-programmed cell death protein 1 immunotherapy and BRAF plus MEK inhibition. CNC interventions commonly involved clinical advice and counseling and care coordination.