PURPOSE Approximately 50% of patients with advanced Merkel cell carcinoma (MCC) have primary or acquired resistance to PD-(L)1 blockade, which may be overcome using combination immune checkpoint inhibition (ICI) with anti–cytotoxic T lymphocyte antigen-4 antibody. We present results from the recurrent/metastatic MCC cohort in CheckMate 358, a nonrandomized, multicohort, phase I/II study of nivolumab (NIVO) with or without ipilimumab (IPI) in virus-associated cancers (ClinicalTrials.gov identifier: NCT02488759 ). METHODS ICI-naïve patients with recurrent/metastatic MCC and 0-2 previous systemic therapies were administered NIVO monotherapy at 240 mg once every 2 weeks or combination therapy with NIVO 3 mg/kg once every 2 weeks + IPI 1 mg/kg once every 6 weeks. The primary end point was objective response. Secondary end points included duration of response (DOR), progression-free survival (PFS), and overall survival (OS). RESULTS Sixty-eight patients received NIVO (n = 25) or NIVO + IPI (n = 43). The objective response rate (95% CI) and median DOR (95% CI), respectively, were 60% (38.7 to 78.9) and 60.6 months (16.7 to not applicable [NA]) with NIVO and 58% (42.1 to 73) and 25.9 months (10.4 to NA) with NIVO + IPI. The median PFS (95% CI) and OS (95% CI), respectively, were 21.3 (9.2 to 62.5) and 80.7 (23.3 to NA) months with NIVO and 8.4 (3.7 to 24.3) and 29.8 (8.5 to 48.3) months with NIVO + IPI. The incidence of grade 3/4 treatment-related adverse events was 28% with NIVO and 47% with the combination. CONCLUSION This nonrandomized study showed frequent and durable responses with both NIVO and NIVO + IPI in patients with ICI-naïve advanced MCC. However, it did not show improvement in efficacy with the combination, thus contradicting previous study reports that had suggested clinical benefit with combination ICI. A randomized trial of NIVO + IPI versus NIVO monotherapy is warranted.

BACKGROUND:While coronavirus disease 2019 (COVID-19) is primarily a respiratory infection, few studies have characterised the immune response to COVID-19 in lung tissue. We sought to understand the pathogenic role of microenvironmental interactions and the extracellular matrix in post-mortem COVID-19 lung using an integrative multi-omic approach. METHODS:Post-mortem formalin-fixed paraffin-embedded lung tissue from fatal COVID-19 and nonrespiratory death control lung underwent multi-omic evaluation by Quantseq Bulk RNA sequencing, Nanostring GeoMx spatial transcriptomics, RNAscope, multiplex immunofluorescence and immunohistochemistry, to evaluate virus distribution, immune composition and the extracellular matrix. Markers of extracellular synthesis and breakdown were measured in the serum of 215 patients with COVID-19 and 54 healthy volunteer controls using ELISA. RESULTS:We found that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection was restricted to the pneumocytes and macrophages of early-stage disease. Spatial analyses revealed an immunosuppressive virus microenvironment, enriched for PDL1+IDO1+ macrophages and depleted of T-cells. Oligoclonal T-cells in COVID-19 lung showed no enrichment of SARS-CoV-2 specific T-cell receptors. Collagen VI was upregulated and contributed to alveolar wall thickening and impaired gas exchange in COVID-19 lung. Serum from COVID-19 patients showed increased levels of PRO-C6, a marker of collagen VI synthesis, predicted mortality in hospitalised patients. CONCLUSIONS:Our data refine the current model of respiratory COVID-19 with regard to virus distribution, immune niches and the role of the noncellular microenvironment in pathogenesis and risk stratification in COVID-19. We show that collagen deposition is an early event in the course of the disease.

Background Colitis was the third most common immune-related adverse effect in melanoma patients treated with immune-checkpoint inhibitor (ICI). With the limitation of real-world data in the UK population, this study was conducted in a UK hospital where a fresh colitis guidance was introduced to evaluate the overall grading (OG) to assess colitis severity and guide the treatment. Aims This study aimed to investigate colitis severity by Common Terminology Criteria for Adverse Events (CTCAE) grade and overall grade at time of presentation. Colitis treatment and outcome were evaluated to compare the impact of these two categories. Methods A single-center, retrospective observational study was performed in melanoma patient developing colitis symptoms. Results A total of 44 advanced melanoma patient with colitis symptoms were included. Median time to colitis onset was 67 days (range 4–890). Majority of patients developed G1/ G2 of CTCAE scale (70.4%) but moderate or severe overall grade (84.1%). There were 65.9% of patients treated with steroids, and 38.2% with infliximab and 4.5% with vedolizumab. The median time of colitis resolution was 28 days (range 0–282). Both treatment modality and time to resolution were associated with severity of colitis assessed by complete OG(p<0.0001) rather than CTCAE grading (p>0.05). Conclusions This study provided a comprehensive description about ICI-induced colitis management in a single center of the UK. The more completed OG was proposed to stratify colitis patient and guide the investigation and treatment decision at presenting time, replacing the old CTCAE grading.

AimsThe MAPK pathway is constitutively activated in uveal melanoma (UM). Selumetinib (AZD6244, ARRY-142886), a MEK inhibitor, has shown limited activity as monotherapy in metastatic UM. Pre-clinical studies support synergistic cytotoxic activity for MEK inhibitors combined with taxanes, and here we sought to assess the clinical efficacy of combining selumetinib and paclitaxel.Patients and MethodsSeventy-seven patients with metastatic UM who had not received prior chemotherapy were randomised to selumetinib alone, or combined with paclitaxel with or without interruption in selumetinib two days before paclitaxel. The primary endpoint was progression free survival (PFS). After amendment, the combination arms were combined for analysis and the sample size adjusted to detect a hazard ratio (HR): 0.55, 80% power at 1-sided 5% significance level.ResultsThe median PFS in the combination arms was 4.8 months (95% CI: 3.8 - 5.6) compared with 3.4 months (2.0 - 3.9) in the selumetinib arm (HR 0.62 [90% CI 0.41 - 0.92], 1-sided p-value = 0.022). ORR was 14% and 4% in the combination and monotherapy arms respectively. Median OS was 9 months for the combination and was not significantly different from selumetinib alone (10 months) with HR of 0.98 [90% CI 0.58 - 1.66], 1-sided p-value = 0.469. Toxicity was in keeping with the known profiles of the agents involved.ConclusionsSelPac met its primary endpoint, demonstrating an improvement in PFS for combination selumetinib and paclitaxel. No improvement in OS was observed, and the modest improvement in PFS is not practice changing.

Introduction Immune-related hepatitis is an adverse effect following treatment with immune-checkpoint inhibitors, such as ipilimumab, nivolumab and pembrolizumab. International guidelines advise on the use of corticosteroids as first-line treatment, although guidance on how to treat cases resistant to corticosteroids is limited. We aimed to evaluate the presentation and management of patients with grade 3-4 immune-related hepatitis, following treatment with immune-checkpoint inhibitors for stage 4 or unresectable or stage 3 melanoma, with a particular focus on steroid-refractory cases. Methods A retrospective observational review of patients developing immune-related hepatitis whilst undergoing treatment with immune checkpoint inhibitors for advanced melanoma from July 2014 to February 2020 at a tertiary oncology centre. Results Forty-one patients developed immune-related hepatitis, of which 83% had been treated with the combination of ipilimumab and nivolumab. The median time to onset of IR-hepatitis was 47 days (range: 4-476), and the median time to peak alanine aminotransferase was 71 days (range: 4-478). Four patients had resolution of grade 3 immune-related hepatitis without the introduction of corticosteroids. A total of 37 patients were treated with corticosteroids. A total of 12 required oral treatment only and 13 were successfully managed as outpatients. Six patients had steroid-refractory immune-related hepatitis; and all received tacrolimus, with one also receiving mycophenolate mofetil and infliximab. Conclusions This study describes the largest UK series of immune-related hepatitis patients in the literature. We present two important deviations from current guidelines. Firstly, there is some evidence that withholding steroids is possible in grade 3-4 immune-related hepatitis. Secondly, tacrolimus can be used successfully to manage patients resistant to corticosteroids, with the early introduction most beneficial to reduce time on steroids.

Additional detail of materials and methods used. Figure 1. Flow chart detailing dose escalation schema. Figure 2. MVA-EBNA1/LMP2 Vaccination does not alter broad subsets of immune cells. Figure 3. EBNA1 antibody response before and after MVA-EL vaccination. Table 1 MHC class I and II restricted epitope peptides in EBNA1 and LMP2 used as targets in ELIspot assays. Table 2 Summary of EBNA1- and LMP2-specific immune responses stimulated by vaccination of UK NPC patients.

PDF file - 99KB, Supplementary Methods: The supplemental material contains additional detail on the pharmacokinetic and pharmacodynamic methods used during the study as well as a summary table of safety events and a figure detailing patient disposition. Supplemental Table 1: Summary of safety events in all study cohorts.

PDF file - 269K, Additional detail of methodology, adverse events, epitope peptides and epitope-specific T cell responses. Also includes all results obtained using ELIspot assays with overlapping peptides and analysis of regulatory T cells in patients.

BRAF+MEK inhibitors extend life expectancy of BRAF V600 mutant advanced melanoma patients; acquired resistance limits duration of benefit. Preclinical and case studies suggested that intermittent dosing could enable patients to remain on treatment longer, delay onset of disease progression and offer improved quality of life (QoL). INTERIM was a UK randomised multicentre phase II trial testing an intermittent dosing regimen. Patients with BRAFV600 mutant advanced melanoma with ECOG PS 0-1 due to start dabrafenib+trametinib were randomised to receive dabrafenib (150mg bid) and trametinib (2mg od) either continuously (CONT) or intermittently (INT; dabrafenib d1-22+trametinib d1–15) on a 28 day cycle. Prior immunotherapy and brain involvement was allowed. Patient recruitment, treatment compliance, QoL and progression-free survival (PFS) were evaluated for the primary endpoint. Secondary endpoints included response rate (ORR), overall survival (OS) and toxicity. The prognostic and predictive value of mutant BRAFV600E ctDNA was measured by droplet digital PCR (ddPCR), using mutant allele frequency of >1% as the detection threshold. 79 patients (39 INT, 40 CONT) were recruited from Dec ‘17–Feb ’20; median age 67 years, 52% PS 1, 65% AJCC (7th ed) M1c, 29% had brain metastases and 46% had LDH > ULN. With median follow-up of 19 months, INT was inferior in all efficacy measures: median PFS 8.5 vs 10.7mo (HR 1.39, 95% CI 0.79–2.45, p = 0.255); median OS 18.1 mo vs not reached (HR 1.69, 95% CI 0.87–3.28, p = 0.121), ORR 57% vs 77%. INT patients experienced fewer treatment related AEs (76% vs 88%), but a higher proportion of grade >3 AEs (53% vs 42%). QoL at 6 months favoured CONT. 66 patients had baseline plasma collected for ctDNA analysis and 27 (41%) of these had detectable BRAFV600E ctDNA. Detection prior to treatment correlated with worse OS (HR 2.55, 95%CI 1.25-5.21, p=0.01) and higher disease burden (LDH>ULN; p<0.001). A change to undetected during treatment did not significantly predict better OS. The UK INTERIM study is consistent with other national studies suggesting that intermittent dosing does not improve efficacy of BRAF+MEKi.

BACKGROUND:BRAF+MEK inhibitors extend life expectancy of patients with BRAFV600 mutant advanced melanoma. Acquired resistance limits duration of benefit, but preclinical and case studies suggest intermittent dosing could overcome this limitation. INTERIM was a phase 2 trial evaluating an intermittent dosing regimen. METHODS:Patients with BRAFV600 mutant advanced melanoma due to start dabrafenib+trametinib were randomised to receive either continuous (CONT), or intermittent (INT; dabrafenib d1-21, trametinib d1-14 every 28 days) dosing. A composite primary endpoint included progression-free survival (PFS) and quality of life (QoL). Secondary endpoints included response rate (ORR), overall survival (OS) and adverse events (AEs). Mutant BRAFV600E ctDNA was measured by droplet digital PCR (ddPCR), using mutant allele frequency of > 1 % as the detection threshold. RESULTS:79 patients (39 INT, 40 CONT) were recruited; median age 67 years, 65 % AJCC (7th ed) stage IV M1c, 29 % had brain metastases. With 19 months median follow-up, INT was inferior in all efficacy measures: median PFS 8.5 vs 10.7mo (HR 1.39, 95 %CI 0.79-2.45, p = 0.255); median OS 18.1mo vs not reached (HR 1.69, 95 %CI 0.87-3.28, p = 0.121), ORR 57 % vs 77 %. INT patients experienced fewer treatment-related AEs (76 % vs 88 %), but more grade > 3 AEs (53 % vs 42 %). QoL favoured CONT. Detection of BRAFV600E ctDNA prior to treatment correlated with worse OS (HR 2.55, 95 %CI 1.25-5.21, p = 0.01) in both arms. A change to undetected during treatment did not significantly predict better OS. CONCLUSION:INTERIM findings are consistent with other recent clinical trials reporting that intermittent dosing does not improve efficacy of BRAF+MEK inhibitors.

Diacylglycerol O-acyltransferase 2 (DGAT2) inhibitors have been shown to lower liver triglyceride content and are being explored clinically as a treatment for non-alcoholic steatohepatitis (NASH). This work details efforts to find an extended-half-life DGAT2 inhibitor. A basic moiety was added to a known inhibitor template, and the basicity and lipophilicity were fine-tuned by the addition of electrophilic fluorines. A weakly basic profile was required to find an appropriate balance of potency, clearance, and permeability. This work culminated in the discovery of PF-07202954 (12), a weakly basic DGAT2 inhibitor that has advanced to clinical studies. This molecule displays a higher volume of distribution and longer half-life in preclinical species, in keeping with its physicochemical profile, and lowers liver triglyceride content in a Western-diet-fed rat model.

PDF file - 25KB, Supplementary Figure S1. Patient disposition: AE, adverse event. KRAS, Kirsten rat sarcoma-2. PD, progressive disease. q2w, every 2 weeks. q3w, every 3 weeks.

BACKGROUND:Merkel cell carcinoma (MCC) is a rare tumour with neuroendocrine differentiation and high associated mortality. Studies that describe the epidemiology of MCC are often limited by small sample size, short duration of follow-up, absence of nationwide data and paucity of data on different risk factors.OBJECTIVES:To determine the incidence, demographics and survival for MCC in England between 2004 and 2018.METHODS:This national retrospective cohort study identified all cases of MCC in England from 2004 to 2018 using national population-based data from the National Disease Registration Service. Crude counts, European age-standardized incidence rates (EASRs) and joinpoint analysis were conducted. Patient demographics and treatments received were described. Multivariable Cox regression analysis was used to study risk factors for MCC-specific mortality, by including a priori defined demographic factors, tumour characteristics and immunosuppression. Treatment data were not included in the Cox regression analysis.RESULTS:A total of 3775 MCC tumours were registered. The median age at diagnosis was 81 years (interquartile range 74-87). Overall, 96·6% of patients identified as White ethnicity, and 8·3% of patients were immunosuppressed. The most common site was the face (27·4%). Patients most often presented with stage one disease (22·8%); however, stage was unknown in 31·0%. In total, 80·7% of patients underwent surgical excision, 43·5% radiotherapy and 9·2% systemic therapy. The EASR increased from 0·43 per 100 000 person-years (PYs) to 0·65 per 100 000 person-years between 2004 and 2018, representing a significant annual percentage change of 3·9%. The EASR was greater in men than in women for all years, with an overall male-to-female ratio of 1·41 : 1. The highest EASR was in South West England. Five-year disease-specific survival was 65·6% [95% confidence interval (CI) 63·8-67·4], with a median follow-up of 767 days. MCC-specific mortality increased with age [hazard ratio (HR) 1·02, 95% CI 1·02-1·03], deprivation (HR 1·43, 95% CI 1·16-1·76), immunosuppression (HR 2·80, 95% CI 2·34-3·34) and stage at diagnosis (HR 8·24, 95% CI 5·84-11·6).CONCLUSIONS:This study presents the largest national MCC dataset in Europe, and the most complete reporting of MCC incidence and survival ever published. With the EASR of MCC increasing and high associated mortality, this study encourages further research into the pathology, diagnosis and therapeutic options for MCC to support management guidelines.

Background:Adjuvant immune checkpoint inhibitors are a new standard of care in melanoma. However, the immune related toxicity associated with these agents can be serious, and the long-term implications are yet to be defined especially in the adjuvant setting. We report, to our knowledge, the first case of anti-PD-1-induced eosinophilic asthma in a melanoma patient treated with adjuvant pembrolizumab. Case Presentation. A 72-year-old man commenced pembrolizumab in the adjuvant setting after resection of a stage IIIB cutaneous melanoma. The patient experienced episodes of breathlessness 4 weeks after cycle 1. These episodes were nocturnal and caused acute respiratory distress and cough, occasionally waking him up. The episodes progressed, and he was admitted after cycle 2 with a productive cough, wheeze, and breathlessness. Observations showed saturations on air of 94% and a respiratory rate of 19/min. The only laboratory abnormality was a raised eosinophil count of 1.1 × 109. Spirometry showed a FEV1 of 2.57 (91% predicted), FVC of 4.04 (108% predicted), and ratio of 64%. Peak expiratory flow rate was 94% predicted, and corrected gas transfer was 6.29 (78% predicted) with KCO 1.18 (93% predicted). FeNO was raised at 129 indicating inflammation of his airways, and peak flow was 422 l/min. CT of the chest did not show pneumonitis or other lung pathology. A diagnosis of acute eosinophilic asthma was made. Treatment with steroids and beclometasone dipropionate and formoterol inhaler produced rapid resolution of symptoms and normalisation of the eosinophil count. Pembrolizumab was safely recommenced once steroids had discontinued and symptoms had resolved.Conclusions:Specialist respiratory input was needed for optimal patient management and is ongoing. Although a safe rechallenge with pembrolizumab was possible, treatment in the adjuvant setting is curative in intent and long-term safety follow-up is required to assess for delayed toxicity and long-term health implications. This is likely to require large regional/national/international databases to detect, monitor, and educate the wider medical community as these patients are followed up in primary care following initial specialist follow-up.

An emerging clinical issue associated with immune-oncology agents is the collateral effects on the tolerability of concomitant medications. One report of this phenomenon was the increased incidence of hypersensitivity reactions observed in patients receiving concurrent immune checkpoint inhibitors (ICIs) and sulfasalazine (SLZ). Thus, the aim of this study was to characterize the T cells involved in the pathogenesis of such reactions, and recapitulate the effects of inhibitory checkpoint blockade on de-novo priming responses to compounds within in vitro platforms. A regulatory competent human dendritic cell/T-cell coculture assay was used to model the effects of ICIs on de novo nitroso sulfamethoxazole- and sulfapyridine (SP) (the sulfonamide component of SLZ) hydroxylamine-specific priming responses. The role of T cells in the pathogenesis of the observed reactions was explored in 3 patients through phenotypic characterization of SP/sulfapyridine hydroxylamine (SPHA)-responsive T-cell clones (TCC), and assessment of cross-reactivity and pathways of T-cell activation. Augmentation of the frequency of responding drug-specific T cells and intensity of the T-cell response was observed with PD-1/PD-L1 blockade. Monoclonal populations of SP- and SPHA-responsive T cells were isolated from all 3 patients. A core secretory effector molecule profile (IFN-γ, IL-13, granzyme B, and perforin) was identified for SP and SPHA-responsive TCC, which proceeded through Pi and hapten mechanisms, respectively. Data presented herein provides evidence that drug-responsive T cells are effectors of hypersensitivity reactions observed in oncology patients administered ICIs and SLZ. Perturbation of drug-specific T-cell priming is a plausible explanation for clinical observations of how an increased incidence of these adverse events is occurring.

Spirocyclic ring systems are useful intermediates in the design and synthesis of medicinally active agents and commonly found as cores in natural products. Recently, syntheses of a key intermediate Boc-protected-1-oxa-8-azaspiro[4.5]decan-3-amine 1 were examined. While multigram quantities of the racemic material could be made from the reduction of an energic azide intermediate, larger scale reactions and a chiral synthesis required further investigations. Herein, we describe the use of a continuous three-step flow process to scale the formation and reduction of an azide intermediate, and the use of a transaminase to prepare the desired enantiomer in high yield and enantiomeric excess.

SummaryCOVID-19 placentitis, a rare complication of maternal SARS-CoV-2 infection, only shows detectable virus in the placenta of a subset of cases. We provide a deep multi-omic spatial characterisation of placentitis from obstetrically complicated maternal COVID-19 infection. We found that SARS-CoV-2 infected placentas have a distinct transcriptional and immunopathological signature. This signature overlaps with virus-negative cases supporting a common viral aetiology. An inverse correlation between viral load and disease duration suggests viral clearance over time. Quantitative spatial analyses revealed a unique microenvironment surrounding virus-infected trophoblasts characterised by PDL1-expressing macrophages, T-cell exclusion, and interferon blunting. In contrast to uninfected mothers, ACE2 was localised to the maternal side of the placental trophoblast layer of almost all mothers with placental SARS-CoV-2 infection, which may explain variable susceptibility to placental infection. Our results demonstrate a pivotal role for direct placental SARS-CoV-2 infection in driving the unique immunopathology of COVID-19 placentitis.Graphical Abstract