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Abstract Immune checkpoint inhibitors (ICIs) have transformed cancer treatment, providing significant benefit to patients across various tumour types, including melanoma. However, around 40% of melanoma patients do not benefit from ICI treatment, and accurately predicting ICI response remains challenging. We now describe a novel and simple approach that integrates immune-associated transcriptome signatures and tumour volume burden to better predict ICI response in melanoma patients. RNA sequencing was performed on pre-treatment (PRE) tumour specimens derived from 32 patients with advanced melanoma treated with combination PD1 and CTLA4 inhibitors. Of these 32 patients, 11 also had early during treatment (EDT, 5–15 days after treatment start) tumour samples. Tumour volume was assessed at PRE for all 32 patients, and at first computed tomography (CT) imaging for the 11 patients with EDT samples. Analysis of the Hallmark IFNγ gene set revealed no association with ICI response at PRE (AUC ROC curve = 0.6404, p = 0.24, 63% sensitivity, 71% specificity). When IFNg activity was evaluated with tumour volume (ratio of gene set expression to tumour volume) using logistic regression to predict ICI response, we observed high discriminative power in separating ICI responders from non-responders (AUC = 0.7760, p = 0.02, 88% sensitivity, 67% specificity); this approach was reproduced with other immune-associated transcriptomic gene sets. These findings were further replicated in an independent cohort of 23 melanoma patients treated with PD1 inhibitor. Hence, integrating tumour volume with immune-associated transcriptomic signatures improves the prediction of ICI response, and suggest that higher levels of immune activation relative to tumour burden are required for durable ICI response.

Abstract Background Neoadjuvant therapy improves recurrence-free survival (RFS) in resectable stage III cutaneous melanoma. However, accurately predicting individual recurrence risk remains a significant challenge. We investigated circulating tumour DNA (ctDNA) as a biomarker for recurrence in measurable stage IIIB/C melanoma patients undergoing neoadjuvant immunotherapy. Methods Plasma samples were collected pre-neoadjuvant treatment, pre-surgery and/or six weeks post-surgery from 40 patients enrolled in the OpACIN-neo and PRADO clinical trials. Patients received two cycles of ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1) before surgery. Cell free DNA (cfDNA) underwent unbiased pre-amplification followed by tumour-informed mutation detection using droplet digital polymerase chain reaction (ddPCR) with the Bio-Rad QX600 PCR system. Results Pre-treatment ctDNA was detectable in 19/40 (48%) patients. Among these, 17/19 (89%) zero-converted within six weeks of surgery and none recurred. Positive ctDNA post-surgery (N = 4), irrespective of pre-treatment ctDNA status, was 100% predictive of recurrence (sensitivity 44%, specificity 100%). Furthermore, ctDNA cleared prior to surgery in 7/9 (78%) patients who did not recur, warranting further investigation into ctDNA-guided surgical management. Conclusion Post-surgery ctDNA positivity and zero-conversion are highly predictive of recurrence, offering a window for personalised modification of adjuvant therapy.

Background: Anti-Program-Death-1 (PD-1) is a standard adjuvant therapy for patients with resected melanoma. We hypothesized that there are discrepancies in survival, recurrence pattern and toxicity to adjuvant PD-1 between different ethnicities and melanoma subtypes. Objective: We performed a multicenter cohort study incorporating 6 independent institutions in Australia, China, Japan, and the United States. The primary outcomes were recurrence free survival (RFS) and overall survival (OS). Secondary outcomes were disease recurrence patterns and toxicities. Results: In total 534 patients were included. East-Asian/Hispanic/African reported significantly poorer RFS/OS. Nonacral cutaneous or melanoma of unknown primary reported the best RFS/OS, followed by acral, and mucosal was the poorest. Within the nonacral cutaneous or melanoma of unknown primary subtypes, East-Asian/Hispanic/African reported significantly poorer RFS/OS than Caucasian. In the multivariate analysis incorporating ethnicity/melanoma-subtype/age/sex/stage/lactate dehydrogenase/BRAF (v-Raf murine sarcoma viral oncogene homolog B)-mutation/adjuvant radiotherapy, East-Asian/Hispanic/African had independently significantly poorer outcomes (RFS: HR, 1.71; 95% CI, 1.19-2.44 and OS: HR, 2.34; 95% CI, 1.39-3.95), as was mucosal subtype (RFS: HR, 3.25; 95% CI, 2.04-5.17 and OS: HR, 3.20; 95% CI, 1.68-6.08). Mucosal melanoma was an independent risk factor for distant metastasis, especially liver metastasis. East-Asian/Hispanic/African had significantly lower incidence of gastrointestinal/musculoskeletal/respiratory/other-rare-type-toxicities; but higher incidences of liver toxicities. Limitations: A retrospective study. Conclusions: Ethnicity and melanoma subtype are associated with survival and recurrence pattern in melanoma patients treated with adjuvant anti-PD-1. Toxicity profile differs by ethnicity and may require a precision toxicity surveillance strategy.

Abstract The biological underpinnings of therapeutic resistance to immune checkpoint inhibitors (ICI) in adolescent and young adult (AYA) melanoma patients are incompletely understood. Here, we characterize the immunogenomic profile and spatial architecture of the tumor microenvironment (TME) in AYA (aged ≤ 30 years) and older adult (aged 31–84 years) patients with melanoma, to determine the AYA-specific features associated with ICI treatment outcomes. We identify two ICI-resistant spatiotypes in AYA patients with melanoma showing stroma-infiltrating lymphocytes (SILs) that are distinct from the adult TME. The SILhigh subtype was enriched in regulatory T cells in the peritumoral space and showed upregulated expression of immune checkpoint molecules, while the SILlow subtype showed a lack of immune activation. We establish a young immunosuppressive melanoma score that can predict ICI responsiveness in AYA patients and propose personalized therapeutic strategies for the ICI-resistant subgroups. These findings highlight the distinct immunogenomic profile of AYA patients, and individualized TME features in ICI-resistant AYA melanoma that require patient-specific treatment strategies.

Abstract Background The combination of nivolumab + relatlimab is superior to nivolumab alone in the treatment of naive patients and has activity in PD-1 refractory melanoma. We had previously observed a reduced expression of LAG3 in melanoma tissue from patients with type 2 diabetes. Method To evaluate the impact of diabetes on oncological outcomes of patients with advanced melanoma treated with nivolumab plus the LAG3 inhibitor relatlimab we performed a retrospective multicenter study. Results Overall, 129 patients were included: 88 without diabetes before the treatment, 37 who were diagnosed with type 2 diabetes before the start of treatment, and 4 without diabetes before treatment who developed immune checkpoint inhibitor-induced diabetes (ICI-DM). PFS was 21.71 months (95% CI: 15.61–27.81) in patients without diabetes, 10.23 months (95% CI: 5.81–14.66) in patients with type 2 diabetes, and 50.85 months (95% CI: 23.04–78.65) in patients who developed ICI-DM. OS was 37.94 months (95% CI: 31.02–44.85) in patients without diabetes, 22.12 months (95% CI: 14.41–29.85) in those with type 2 diabetes and 57.64 months (95% CI: 42.29–72.99) in those who developed ICI-DM. Multivariate analysis showed that the presence of diabetes and LDH was correlated with OS and PFS. The mean OS was 64.63 months in subjects with low levels of glucose ( 1.5) had a worse prognosis than those whose glucose level had not increased. This result was observed also in subgroups treated either in first line or further lines. Patients who developed ICI-DM during the study period had better outcomes than the overall population and patients without diabetes. Conclusions LAG3 inhibition for treating metastatic or unresectable melanoma has a reduced efficacy in patients with type 2 diabetes, possibly due to a low expression of LAG3 in tumor tissue. Higher level evidence should be obtained.

Abstract Outcomes for patients with melanoma have improved over the past decade with the clinical development and approval of immunotherapies targeting immune checkpoint receptors such as programmed death-1 (PD-1), programmed death ligand 1 (PD-L1) or cytotoxic T lymphocyte antigen-4 (CTLA-4). Combinations of these checkpoint therapies with other agents are now being explored to improve outcomes and enhance benefit-risk profiles of treatment. Alternative inhibitory receptors have been identified that may be targeted for anti-tumor immune therapy, such as lymphocyte-activation gene-3 (LAG-3), as have several potential target oncogenes for molecularly targeted therapy, such as tyrosine kinase inhibitors. Unfortunately, many patients still progress and acquire resistance to immunotherapy and molecularly targeted therapies. To bypass resistance, combination treatment with immunotherapies and single or multiple TKIs have been shown to improve prognosis compared to monotherapy. The number of new combinations treatment under development for melanoma provides options for the number of patients to achieve a therapeutic benefit. Many diagnostic and prognostic assays have begun to show clinical applicability providing additional tools to optimize and individualize treatments. However, the question on the optimal algorithm of first- and later-line therapies and the search for biomarkers to guide these decisions are still under investigation. This year, the Melanoma Bridge Congress (Dec 1st–3rd, 2022, Naples, Italy) addressed the latest advances in melanoma research, focusing on themes of paramount importance for melanoma prevention, diagnosis and treatment. This included sessions dedicated to systems biology on immunotherapy, immunogenicity and gene expression profiling, biomarkers, and combination treatment strategies.

Immune-related adverse events (irAEs) are side effects of immune checkpoint inhibitor therapy (ICI). While common irAEs have been well characterized, there are more limited data on rare immune related adverse events (RirAEs) due to low incidence. Lack of characterization of these entities has led to difficulties in accurate diagnosis and management. Here, we conducted a multi-institution analysis of all patients with stage III/IV melanoma who developed RirAEs after being treated with ICIs (anti-PD-1/L1, anti-CTLA-4, and combination PD-1/CTLA-4 blockade) at three institutions (Vanderbilt University Medical Center, Massachusetts General Hospital, and Melanoma Institute of Australia). RirAEs were defined as those occurring in approximately

Lymphocyte-activation gene-3 (LAG-3), an immune checkpoint receptor, negatively regulates T-cell function and facilitates immune escape of tumors. Dual inhibition of LAG-3 and programmed cell death receptor-1 (PD-1) significantly improved progression-free survival (PFS) in metastatic melanoma patients compared to anti-PD-1 therapy alone. Investigating the utility of LAG-3 expression as a biomarker of response to anti-LAG-3 + anti-PD-1 immunotherapy is of great clinical relevance. This study sought to evaluate the association between baseline LAG-3 expression and clinical outcomes following anti-LAG-3 and anti-PD-1-based immunotherapy in metastatic melanoma. LAG-3 immunohistochemistry (clone D2G4O) was performed on pre-treatment formalin-fixed, paraffin-embedded metastatic melanoma specimens from 53 patients treated with combination anti-LAG-3 + anti-PD-1-based therapies. Eleven patients had received prior anti-PD-1-based treatment. Patients were categorized as responders (complete/partial response; n = 36) or non-responders (stable/progressive disease; n = 17) based on the Response Evaluation Criteria in Solid Tumours (RECIST). Tumor-infiltrating lymphocytes (TILs) were scored on hematoxylin and eosin-stained sections. LAG-3 expression was observed in 81% of patients, with staining in TILs and dendritic cells. Responders displayed significantly higher proportions of LAG-3+ cells compared to non-responders (P = .0210). LAG-3 expression positively correlated with TIL score (P .05). Patients with ≥ 1% LAG-3+ cells in their tumors had significantly longer PFS compared to patients with

Abstract Background Gene expression profiling is increasingly being utilised as a diagnostic, prognostic and predictive tool for managing cancer patients. Single-sample scoring approach has been developed to alleviate instability of signature scores due to variations from sample composition. However, it is a challenge to achieve comparable signature scores across different expressional platforms. Methods The pre-treatment biopsies from a total of 158 patients, who have received single-agent anti-PD-1 (n = 84) or anti-PD-1 + anti-CTLA-4 therapy (n = 74), were performed using NanoString PanCancer IO360 Panel. Multiple immune-related signature scores were measured from a single-sample rank-based scoring approach, singscore. We assessed the reproducibility and the performance in reporting immune profile of singscore based on NanoString assay in advance melanoma. To conduct cross-platform analyses, singscores between the immune profiles of NanoString assay and the previous orthogonal whole transcriptome sequencing (WTS) data were compared through linear regression and cross-platform prediction. Results singscore-derived signature scores reported significantly high scores in responders in multiple PD-1, MHC-1-, CD8 T-cell-, antigen presentation-, cytokine- and chemokine-related signatures. We found that singscore provided stable and reproducible signature scores among the repeats in different batches and cross-sample normalisations. The cross-platform comparisons confirmed that singscores derived via NanoString and WTS were comparable. When singscore of WTS generated by the overlapping genes to the NanoString gene set, the signatures generated highly correlated cross-platform scores (Spearman correlation interquartile range (IQR) [0.88, 0.92] and r 2 IQR [0.77, 0.81]) and better prediction on cross-platform response (AUC = 86.3%). The model suggested that Tumour Inflammation Signature (TIS) and Personalised Immunotherapy Platform (PIP) PD-1 are informative signatures for predicting immunotherapy-response outcomes in advanced melanoma patients treated with anti-PD-1-based therapies. Conclusions Overall, the outcome of this study confirms that singscore based on NanoString data is a feasible approach to produce reliable signature scores for determining patients’ immune profiles and the potential clinical utility in biomarker implementation, as well as to conduct cross-platform comparisons, such as WTS.

Immunotherapy resistance is common among melanoma patients. Here, the authors identify three resistance mechanism subtypes across tumor-derived cell lines and matched samples and highlight antigen presentation disruption as a key mediator of resistance.

Hypercalcaemia is a common complication seen in malignancy, frequently due to paraneoplastic parathyroid hormone-related peptide production or osteolytic bony metastases. We present a 58-year-old female with immunotherapy-mediated hypophysitis causing secondary cortisol deficiency resulting in severe glucocorticoid-responsive hypercalcaemia. Whilst hypophysitis is a well recognised adverse event in those receiving immunotherapy for advanced malignancy, it does not typically present with hypercalcaemia. The mechanism responsible for hypercalcaemia due to hypocortisolaemia has not been fully elucidated although hypotheses include the effects of volume depletion and thyroxine’s action on bone. Prompt treatment with glucocorticoids caused an improvement in the patient’s symptoms and corrected her hypercalcaemia which later returned after an attempted glucocorticoid wean. With the increasing uptake of immunotherapy, clinicians should be aware of this unusual presentation of immunotherapy-related hypophysitis and secondary hypocortisolaemia which can be life-threatening if the diagnosis is delayed.

Summary: Background: Both dabrafenib/trametinib (D/T) and anti-PD-1 monotherapy (PD-1) are approved adjuvant therapies for patients with stage III BRAF V600-mutant melanoma. However, there is still a lack of head-to-head comparative data. We aimed to describe efficacy and toxicity outcomes for these two standard therapies across melanoma centers. Methods: This multicenter, retrospective cohort study was conducted in 15 melanoma centers in Australia, China, Germany, Italy, Japan, UK, and US. We included adult patients with resected stage III BRAF V600-mutant melanoma who received either adjuvant D/T or PD-1 between Jul 2015 and Oct 2022. The primary endpoint was relapse-free survival (RFS). Secondary endpoints included overall survival (OS), recurrence pattern and toxicity. Findings: We included 598 patients with stage III BRAF V600-mutant melanoma who received either adjuvant D/T (n = 393 [66%]) or PD-1 (n = 205 [34%]) post definitive surgery between Jul 2015 and Oct 2022. At a median follow-up of 33 months (IQR 21–43), the median RFS was 51.0 months (95% CI 41.0-not reached [NR]) in the D/T group, significantly longer than PD-1 (44.8 months [95% CI 28.5-NR]) (univariate: HR 0.66, 95% CI 0.50–0.87, P = 0.003; multivariate: HR 0.58, 95% CI 0.39–0.86, P = 0.007), with comparable OS with PD-1 (multivariate, HR 0.90, 95% CI 0.48–1.70, P = 0.75). Similar findings were observed using a restricted-mean-survival-time model. Among those who experienced recurrence, the proportion of distant metastases was higher in the D/T cohort. D/T had a higher incidence of treatment modification due to adverse events (AEs) than PD-1, but fewer persistent AEs. Interpretation: In patients with stage III BRAF V600-mutant melanoma post definitive surgery, D/T yielded better RFS than PD-1, with higher transient but lower persistent toxicity, and comparable OS. D/T seems to provide a better outcome compared with PD-1, but a longer follow-up and ideally a large prospective trial are needed. Funding: Dr. Xue Bai was supported by the Beijing Hospitals Authority Youth Programme (QMS20211101) for her efforts devoted to this study. Dr. Keith T. Flaherty was funded by Adelson Medical Research Foundation for the efforts devoted to this study.

IntroductionIncreasing implementation of the highly efficacious immune checkpoint inhibitors (ICIs) has raised awareness of their various complications in the form of immune-related adverse events (irAEs). Transverse myelitis following ICIs is thought to be a rare but serious neurologic irAE and knowledge is limited about this distinct clinical entity.CasesWe describe four patients across three tertiary centers in Australia with ICI-induced transverse myelitis. Three patients had a diagnosis of stage III–IV melanoma treated with nivolumab and one patient had stage IV non-small cell lung cancer treated with pembrolizumab. All patients had longitudinally extensive transverse myelitis on magnetic resonance imaging (MRI) spine and clinical presentation was accompanied by inflammatory cerebrospinal fluid (CSF) findings. Half of our cohort had received spinal radiotherapy, with the areas of transverse myelitis extending beyond the level of previous radiation field. Inflammatory changes on neuroimaging did not extend to the brain parenchyma or caudal nerve roots, except for one case involving the conus medullaris. All patients received high dose glucocorticoids as first-line therapy, however the majority relapsed or had a refractory state (3/4) despite this, requiring escalation of their immunomodulation, with either induction intravenous immunoglobulin (IVIg) or plasmapheresis. Patients in our cohort who relapsed had a poorer outcome with more severe disability and reduced functional independence following resolution of their myelitis. Two patients had no progression of their malignancy and two patients had malignancy progression. Of the three patients who survived, two had resolution of their neurological symptoms and one remained symptomatic.ConclusionWe propose that prompt intensive immunomodulation is favored for patients with ICI-transverse myelitis in an attempt to reduce associated significant morbidity and mortality. Furthermore, there is a significant risk of relapse following cessation of immunomodulatory therapy. We suggest one treatment approach of IVMP and induction IVIg for all patients presenting with ICI-induced transverse myelitis based on such findings. With the increasing use of ICIs across oncology, further studies are required to explore this neurological phenomenon in greater detail to help establish management consensus guidelines.

Abstract Background Adjuvant immunotherapy is revolutionising care for patients with resected stage III and IV melanoma. However, immunotherapy may be associated with toxicity, making treatment decisions complicated. This study aimed to identify factors physicians and nurses considered regarding adjuvant immunotherapy for melanoma. Methods In-depth interviews were conducted with physicians (medical oncologists, surgeons and dermatologists) and nurses managing patients with resected stage III melanoma at three Australian tertiary melanoma centres between July 2019 and March 2020. Factors considered regarding adjuvant immunotherapy were explored. Recruitment continued until data saturation and thematic analysis was undertaken. Results Twenty-five physicians and nurses, aged 28–68 years, 60% females, including eleven (44%) medical oncologists, eight (32%) surgeons, five (20%) nurses, and one (4%) dermatologist were interviewed. Over half the sample managed five or more new resected stage III patients per month who could be eligible for adjuvant immunotherapy. Three themes about adjuvant immunotherapy recommendations emerged: [1] clinical and patient factors, [2] treatment information provision, and [3] individual physician/nurse factors. Melanoma sub-stage and an individual patient’s therapy risk/benefit profile were primary considerations. Secondary factors included uncertainty about adjuvant immunotherapy’s effectiveness and their views about treatment burden patients might consider acceptable. Conclusions Patients’ disease sub-stage and their treatment risk versus benefit drove the melanoma health care professionals’ adjuvant immunotherapy endorsement. Findings clarify clinician preferences and values, aiding clinical communication with patients and facilitating clinical decision-making about management options for resected stage III melanoma.

Immune-related adverse events represent a major hurdle to the success of immunotherapy. The immunological mechanisms underlying their development and relation to antitumor responses are poorly understood. By examining both systemic and tissue-specific immune changes induced by combination anti–CTLA-4 and anti–PD-1 immunotherapy, we found distinct repertoire changes in patients who developed moderate-severe colitis, irrespective of their antitumor response to therapy. The proportion of circulating monocytes were significantly increased at baseline in patients who subsequently developed colitis compared with patients who did not develop colitis, and biopsies from patients with colitis showed monocytic infiltration of both endoscopically and histopathologically normal and inflamed regions of colon. The magnitude of systemic expansion of T cells following commencement of immunotherapy was also greater in patients who developed colitis. Importantly, we show expansion of specific T cell subsets within inflamed regions of the colon, including tissue-resident memory CD8+ T cells and Th1 CD4+ T cells in patients who developed colitis. Our data also suggest that CD8+ T cell expansion was locally induced, while Th1 cell expansion was systemic. Together, our data show that exaggerated innate and T cell responses to combination immunotherapy synergize to propel colitis in susceptible patients.

Background Acral melanoma is a rare melanoma subtype with poor prognosis. Importantly, these patients were not identified as a specific subgroup in the landmark melanoma trials involving ipilimumab and the anti-programmed cell death protein-1 (PD-1) agents nivolumab and pembrolizumab. There is therefore an absence of prospective clinical trial evidence regarding the efficacy of checkpoint inhibitors (CPIs) in this population. Acral melanoma has lower tumor mutation burden (TMB) than other cutaneous sites, and primary site is associated with differences in TMB. However the impact of this on the effectiveness of immune CPIs is unknown. We examined the efficacy of CPIs in acral melanoma, including by primary site.Methods Patients with unresectable stage III/IV acral melanoma treated with CPI (anti-PD-1 and/or ipilimumab) were studied. Multivariable logistic and Cox regression analyses were conducted. Primary outcome was objective response rate (ORR); secondary outcomes were progression-free survival (PFS) and overall survival (OS).Results In total, 325 patients were included: 234 (72%) plantar, 69 (21%) subungual and 22 (7%) palmar primary sites. First CPI included: 184 (57%) anti-PD-1, 59 (18%) anti-PD-1/ipilimumab combination and 82 (25%) ipilimumab. ORR was significantly higher with initial anti-PD-1/ipilimumab compared with anti-PD-1 (43% vs 26%, HR 2.14, p=0.0004) and significantly lower with ipilimumab (15% vs 26%, HR 0.49, p=0.0016). Landmark PFS at 1 year was highest for anti-PD-1/ipilimumab at 34% (95% CI 24% to 49%), compared with 26% (95% CI 20% to 33%) with anti-PD-1 and 10% (95% CI 5% to 19%) with ipilimumab. Despite a trend for increased PFS, anti-PD-1/ipilimumab combination did not significantly improve PFS (HR 0.85, p=0.35) or OS over anti-PD-1 (HR 1.30, p=0.16), potentially due to subsequent therapies and high rates of acquired resistance. No outcome differences were found between primary sites.Conclusion While the ORR to anti-PD-1/ipilimumab was significantly higher than anti-PD-1 and PFS numerically higher, in this retrospective cohort this benefit did not translate to improved OS. Future trials should specifically include patients with acral melanoma, to help determine the optimal management of this important melanoma subtype.

Abstract Background Immune checkpoint inhibitors (ICI) have become the standard of care in many oncological conditions but are associated with a spectrum of renal immune-related adverse events (IrAEs). We aimed to describe the spectrum, histology, management and outcomes of renal IrAE in patients with metastatic melanoma undergoing ICI therapy. Methods We conducted a retrospective review of 23 patients with a diagnosis of metastatic melanoma treated with ICI between January 2017 and April 2019 who developed a renal IrAE. Baseline demographic data, biochemical and histopathological results, management and outcomes were analyzed. Results The majority of patients who developed renal irAE were male and received combination immunotherapy. The median time of onset from initiation of ICI therapy to renal IrAE was 4 months. 52% of the treated renal IrAE had histopathologically confirmed renal IrAE. The most common histological pattern of injury was acute tubulo-interstitial nephritis (92%). One patient developed anti-GBM disease with non-dialysis dependent stage 5 CKD. In tubulointerstitial injury, there was no association between peak creatinine, renal recovery and histologically reported inflammation or fibrosis. Patients with renal IrAE demonstrated persisting renal dysfunction at 3, 6 and 12 months with a mean baseline, 3 and 12 month creatinine of 90.0 μmol/L, 127.0 μmol/L and 107.5 μmol/L respectively. Conclusion Renal IrAE is most commonly attributable to steroid responsive acute tubulointerstitial nephritis. The outcome of rarer pathologies such as anti-GBM disease may be adversely affected by a delayed diagnosis. There is persisting renal dysfunction following an episode of renal IrAE that may have impact on future renal and overall survival outcomes.

A significant proportion of patients develop innate or acquired resistance to immune checkpoint inhibitors. Here, the authors show that resistance to anti-PD-1 blockade is associated with TGF-beta driven major histocompatibility complex I (MHCI) down-regulation and a de-differentiated phenotype in melanoma patients.

Immune checkpoints are small molecules present on the cell surface of T-lymphocytes. They maintain self-tolerance and regulate the amplitude and duration of T-cell responses. Antagonism of immune checkpoints with monoclonal antibodies (immune checkpoint inhibitors) is a rapidly evolving field of anti-cancer immunotherapy and has become standard of care in management of many cancer subtypes. Immune checkpoint inhibition is an effective cancer treatment but can precipitate immune related adverse events (irAEs). Thyroid dysfunction is the most common endocrine irAE and can occur in up to 40% of treated patients. Both thyrotoxicosis and hypothyroidism occur. The clinical presentation and demographic associations of thyrotoxicosis compared to hypothyroidism suggest unique entities with different etiologies. Thyroid irAEs, particularly overt thyrotoxicosis, are associated with increased immune toxicity in other organ systems, but also with longer progression-free and overall survival. Polygenic risk scores using susceptibility loci associated with autoimmune thyroiditis predict development of checkpoint inhibitor associated irAEs, suggesting potentially shared mechanisms underpinning their development. Our review will provide an up-to-date summary of knowledge in the field of thyroid irAEs. Major focus will be directed toward pathogenesis (including genetic factors shared with autoimmune thyroid disease), demographic associations, clinical presentation and course, treatment, and the relationship with cancer outcomes.

Detection of melanoma mutations using circulating tumor DNA (ctDNA) is a potential alternative to using genomic DNA from invasive tissue biopsies. To date, mutations in the GC-rich TERT promoter region, which is commonly mutated in melanoma, have been technically difficult to detect in ctDNA using next-generation sequencing (NGS) panels. In this study, we developed a custom melanoma NGS panel for detection of ctDNA, which encompasses the top 15 gene mutations in melanoma including the TERT promoter. We analyzed 21 stage III and IV melanoma patient samples who were treatment-naïve or on therapy. The overall detection rate of the custom panel, based on BRAF/NRAS/TERT promoter mutations, was 14/21 (67%) patient samples which included a TERT C250T mutation in one BRAF and NRAS mutation negative sample. A BRAF or NRAS mutation was detected in the ctDNA of 13/21 (62%) patients while TERT promoter mutations were detected in 10/21 (48%) patients. Co-occurrence of TERT promoter mutations with BRAF or NRAS mutations was found in 9/10 (90%) patients. The custom ctDNA panel showed a concordance of 16/21 (76%) with tissue based-detection and included 12 BRAF/NRAS mutation positive and 4 BRAF/NRAS mutation negative patients. The ctDNA mutation detection rate for stage IV was 12/16 (75%) and for stage III was 1/5 (20%). Based on BRAF, NRAS and TERT promoter mutations, the custom melanoma panel displayed a limit of detection of ~0.2% mutant allele frequency and showed significant correlation with droplet digital PCR. For one patient, a novel MAP2K1 H119Y mutation was detected in an NRAS/BRAF/TERT promoter mutation negative background. To increase the detection rate to >90% for stage IV melanoma patients, we plan to expand our custom panel to 50 genes. This study represents one of the first to successfully detect TERT promoter mutations in ctDNA from cutaneous melanoma patients using a targeted NGS panel.

Immune checkpoint inhibitors have transformed the landscape of oncological therapy, but at the price of a new array of immune related adverse events. Among these is β-cell failure, leading to checkpoint inhibitor-related autoimmune diabetes (CIADM) which entails substantial long-term morbidity. As our understanding of this novel disease grows, parallels and differences between CIADM and classic type 1 diabetes (T1D) may provide insights into the development of diabetes and identify novel potential therapeutic strategies. In this review, we outline the knowledge across the disciplines of endocrinology, oncology and immunology regarding the pathogenesis of CIADM and identify possible management strategies.

IntroductionImmune checkpoint inhibitors (ICI) are increasingly being used to treat numerous cancer types. Together with improved recognition of toxicities, this has led to more frequent identification of rare immune-related adverse events (irAE), for which specific treatment strategies are needed. Neutropenia is a rare hematological irAE that has a potential for a high mortality rate because of its associated risk of sepsis. Prompt recognition and timely treatment of this life-threatening irAE are therefore critical to the outcome of patients with immune-related neutropenia.MethodsThis multicenter international retrospective study was conducted at 17 melanoma centers to evaluate the clinical characteristics, diagnostics, treatment, and outcomes of melanoma patients with grade 4 neutropenia (

A significant number of patients (pts) with metastatic melanoma do not respond to anti-programmed cell death 1 (PD1) therapies. Identifying predictive biomarkers therefore remains an urgent need. We retrospectively analyzed plasma DNA of pts with advanced melanoma treated with PD-1 antibodies, nivolumab or pembrolizumab, for five PD-1 genotype single nucleotide polymorphisms (SNPs): PD1.1 (rs36084323, G>A), PD1.3 (rs11568821, G>A), PD1.5 (rs2227981, C>T) PD1.6 (rs10204225, G>A) and PD1.9 (rs2227982, C>T). Clinico-pathological and treatment parameters were collected, and presence of SNPs correlated with response, progression free survival (PFS) and overall survival (OS). 115 patients were identified with a median follow up of 18.7 months (range 0.26 – 52.0 months). All were Caucasian; 27% BRAF V600 mutation positive. At PD-1 antibody commencement, 36% were treatment-naïve and 52% had prior ipilimumab. The overall response rate was 43%, 19% achieving a complete response. Overall median PFS was 11.0 months (95% CI 5.4 - 17.3) and median OS was 31.1 months (95% CI 23.2 - NA). Patients with the G/G genotype had more complete responses than with A/G genotype (16.5% vs. 2.6% respectively) and the G allele of PD1.3 rs11568821 was significantly associated with a longer median PFS than the AG allele, 14.1 vs. 7.0 months compared to the A allele (p=0.04; 95% CI 0.14 – 0.94). No significant association between the remaining SNPs and responses, PFS or OS were observed. Despite limitations in sample size, this is the first study to demonstrate an association of a germline PD-1 polymorphism and PFS in response to anti-PD-1 therapy in pts with metastatic melanoma. Extrinsic factors like host germline polymorphisms should be considered with tumor intrinsic factors as predictive biomarkers for immune checkpoint regulators.

Immune checkpoint inhibitors (ICI) predispose patients to immune-related adverse events (irAEs). Although hepatitis is a potentially lethal toxicity, the timing and outcomes have not been well described. In this retrospective study, patients from six international institutions were included if they were treated with ICIs and developed immune-related hepatitis. Patient and tumor characteristics, and hepatitis management and outcomes were evaluated. Of the 164 patients included, most were male (53.7%) with a median age of 63.0 years. Most patients had melanoma (83.5%) and stage IV disease (86.0%). Median follow-up was 585 days; median OS and PFS were not reached. The initial grade of hepatitis was most often grade 2 (30.5%) or 3 (45.7%) with a median time to onset of 61 days. Patients were most commonly asymptomatic (46.2%), but flu-like symptoms, including fatigue/anorexia (17.1%), nausea/emesis (14.0%), abdominal/back pain (11.6%), and arthralgias/myalgias (8.5%) occurred. Most patients received glucocorticoids (92.1%); the median time to improvement by one grade was 13.0 days, and the median time to complete resolution was 52.0 days. Second-line immunosuppression was required in 37 patients (22.6%), and steroid-dose re-escalation in 45 patients (27.4%). Five patients (3%) died of ICI-hepatitis or complications of hepatitis treatment. Ninety-one patients (58.6%) did not resume ICI; of 66 patients (40 grade 1/2, 26 grade 3/4) that were rechallenged, only 25.8% (n = 17) had recurrence. In this multi-institutional cohort, immune-related hepatitis was associated with excellent outcomes but frequently required therapy discontinuation, high-dose steroids, and second-line immunosuppression. Rechallenge was associated with a modest rate of hepatitis recurrence.

Background Patients with cancer who are infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are more likely to develop severe illness and die compared with those without cancer. The impact of immune checkpoint inhibition (ICI) on the severity of COVID-19 illness is unknown. The aim of this study was to investigate whether ICI confers an additional risk for severe COVID-19 in patients with cancer.Methods We analyzed data from 110 patients with laboratory-confirmed SARS-CoV-2 while on treatment with ICI without chemotherapy in 19 hospitals in North America, Europe and Australia. The primary objective was to describe the clinical course and to identify factors associated with hospital and intensive care (ICU) admission and mortality.Findings Thirty-five (32%) patients were admitted to hospital and 18 (16%) died. All patients who died had advanced cancer, and only four were admitted to ICU. COVID-19 was the primary cause of death in 8 (7%) patients. Factors independently associated with an increased risk for hospital admission were ECOG ≥2 (OR 39.25, 95% CI 4.17 to 369.2, p=0.0013), treatment with combination ICI (OR 5.68, 95% CI 1.58 to 20.36, p=0.0273) and presence of COVID-19 symptoms (OR 5.30, 95% CI 1.57 to 17.89, p=0.0073). Seventy-six (73%) patients interrupted ICI due to SARS-CoV-2 infection, 43 (57%) of whom had resumed at data cut-off.Interpretation COVID-19–related mortality in the ICI-treated population does not appear to be higher than previously published mortality rates for patients with cancer. Inpatient mortality of patients with cancer treated with ICI was high in comparison with previously reported rates for hospitalized patients with cancer and was due to COVID-19 in almost half of the cases. We identified factors associated with adverse outcomes in ICI-treated patients with COVID-19.

Background Colitis is one of the common immune-related adverse events that leads to morbidity and treatment discontinuation of immunotherapy. The clinical presentation, endoscopic and histopathological features and best management of this toxicity are not well defined.Patients and methods Patients with metastatic melanoma who received immunotherapy (programmed cell death protein 1 (PD1) antibodies, alone or in combination with a cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) antibody (PD1 +CTLA-4)) and who developed clinically significant colitis (requiring systemic corticosteroids) were identified retrospectively from two academic centers. Clinical data were collected for all patients; endoscopic and histopathological data were examined in a subset.Results From May 2013 to May 2019, 118/1507 (7.8%) patients developed significant colitis; 80/553 (14.5%) after PD1+CTLA-4, 35/1000 (3.5%) PD1 alone, and three patients after Ipilimumab (IPI) alone. Combination therapy-induced colitis was more frequent (14.5% vs 3.5% in PD1 alone, p=

Metastatic melanoma is one of the most immunogenic malignancies due to its high rate of mutations and neoantigen formation. Response to BRAF inhibitors (BRAFi) may be determined by intratumoral immune activation within melanoma metastases. To evaluate whether CD8+ T cell infiltration and distribution within melanoma metastases can predict clinical response to BRAFi, we developed a methodology to integrate immunohistochemistry with automated image analysis of CD8+ T cell position. CD8+ distribution patterns were correlated with gene expression data to identify and quantify “hot” areas within a tumor. Furthermore, the relative activation of CD8+cells, based on transcriptomic analysis, and their relationship to other CD8+ T cells and non-CD8+ cells within the tumor suggested a less crowded distribution of cells around activated CD8+ T cells. Furthermore, the relative activation of these CD8+ T cells was associated with improved clinical outcomes and decreased tumor cell proliferation. This study demonstrates the potential of digital pathomics to incorporate immune cell spatial distribution within metastases and RNAseq analysis to predict clinical response to BRAF inhibition in metastatic melanoma.

The development of changes in T cells, referred to as T cell exhaustion, has been suggested as a cause of primary or acquired resistance to immunotherapy by immune checkpoint blockade (ICB). A limited number of studies, largely performed on tumor infiltrating lymphocytes (TILs), has provided evidence in support of this hypothesis, but whether similar changes occur in circulating blood lymphocytes has received little attention. In the present study, a comprehensive analysis of peripheral blood leukocytes from 42 patients taken over the course of treatment with anti-PD-1 was undertaken. The patients included those grouped as responders (who did not progress), primary non-responders (primary resistance) and those with acquired resistance (who initially responded then subsequently progressed). Analysis included surface markers of exhaustion, production of cytokines following in vitro stimulation, and assessment of transcription factor levels associated with T cell exhaustion. There were differences in innate cell populations between responders and non-responders at baseline and maintained throughout therapy. Frequencies of total and classical CD14+CD16− monocytes were higher and the major subset of NK cells (CD16hiCD56+) was significantly smaller in the primary resistance group compared with responders. However, differences in peripheral blood expression of exhaustion markers were not evident between the treatment groups. T cell exhaustion markers were expressed in practically all patients and the major observation was an increase in CD39 on CD4 T cells during treatment. The results confirm the association of Eomes transcription factor with T cell exhaustion but levels of expression and the ratio with T-bet over Eomes did not differ between the patient groups. Thus, peripheral blood expression of T cell exhaustion markers does not distinguish between responders and non-responders to anti-PD-1 therapy. CD4 T cell expression of IFNγ also differed in pre-treatment samples, indicating that predictors of response unrelated to exhaustion may be present in peripheral blood. The association of response with innate cell populations and CD4 T cell responses requires further study.

Abstract Background Selective kinase and immune checkpoint inhibitors, and their combinations, have significantly improved the survival of patients with advanced metastatic melanoma. Not all patients will respond to treatment however, and some patients will present with significant toxicities. Hence, the identification of biomarkers is critical for the selection and management of patients receiving treatment. Biomarker discovery often involves proteomic techniques that simultaneously profile multiple proteins but few studies have compared these platforms. Methods In this study, we used the multiplex bead-based Eve Technologies Discovery assay and the aptamer-based SomaLogic SOMAscan assay to identify circulating proteins predictive of response to immunotherapy in melanoma patients treated with combination immune checkpoint inhibitors. Expression of four plasma proteins were further validated using the bead-based Millipore Milliplex assay. Results Both the Discovery and the SOMAscan assays detected circulating plasma proteins in immunotherapy-treated melanoma patients. However, these widely used assays showed limited correlation in relative protein quantification, due to differences in specificity and the dynamic range of protein detection. Protein data derived from the Discovery and Milliplex bead-based assays were highly correlated. Conclusions Our study highlights significant limitations imposed by inconsistent sensitivity and specificity due to differences in the detection antibodies or aptamers of these widespread biomarker discovery approaches. Our findings emphasize the need to improve these technologies for the accurate identification of biomarkers.

Abstract Alternate BRAF splicing is the most common mechanism of acquired resistance to BRAF inhibitor treatment in melanoma. Recently, alternate BRAF exon 4–8 splicing was shown to involve an intronic mutation, located 51 nucleotides upstream of BRAF exon 9 within a predicted splicing branch point. This intronic mutation was identified in a single cell line but has not been examined in vivo. Herein we demonstrate that in three melanomas biopsied from patients with acquired resistance to BRAF inhibitors, alternate BRAF exon 4–8 splicing is not associated with this intronic branch point mutation. We also confirm that melanoma cells expressing BRAF splicing variants retain exquisite sensitivity to existing FDA-approved MEK inhibitors.

Immune checkpoint blockade has greatly improved the clinical outcomes of many patients with metastatic melanoma, however, almost half do not respond. Whether the interspatial distribution of immune and tumor cells predicts response to anti-PD-1-based therapies and patient outcomes in any cancer, including melanoma, is currently unknown. Here, we examined the spatial distribution of immune and tumor cells via multiplex immunofluorescence. Pre-treatment melanoma specimens from 27 patients (n = 18 responders; n = 9 non-responders) treated with anti-PD-1 monotherapy and 34 patients (n = 22 responders; n = 12 non-responders) treated with combined ipilimumab and anti-PD-1 immunotherapy were studied. Responders displayed significantly higher densities of CD8+ tumor-infiltrating lymphocytes within a 20 µM distance from a melanoma cell compared to non-responders in both anti-PD-1 alone (p = .0024) and combination-treated patients (p = .0096), that were associated with improved progression-free survival for both therapies (anti-PD-1 p = .0158; combination therapy p = .0088). In multivariate analysis, the best model for 12-month progression-free survival for anti-PD-1 monotherapy included PD-L1+ cells within proximity to tumor cells and intratumoral CD8+ density (AUC = 0.80), and for combination therapy included CD8+ cells in proximity to tumor cells, intratumoral PD-L1+ density and LDH (AUC = 0.85). Assessment of the spatial distribution of immune cells in relation to tumor cells provides insight into their role in modulating immune response and highlights their potential role as predictors of response to anti-PD-1 based therapies.