Protocol for the EACH trial: a multicentre phase II study evaluating the safety and antitumour activity of the combination of avelumab, an anti-PD-L1 agent, and cetuximab, as any line treatment for patients with recurrent/metastatic head and neck squamous cell cancer (HNSCC) in the UK.

IntroductionHead and neck cancer is the eighth most common cancer in the UK. Current standard of care treatment for patients with recurrent/metastatic squamous cell head and neck carcinoma (HNSCC) is platinum-based chemotherapy combined with the anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibody, cetuximab. However, most patients will have poor median overall survival (OS) of 6-9 months despite treatment. HNSCC tumours exhibit an immune landscape poised to respond to immunotherapeutic approaches, with most tumours expressing the immunosuppressive receptor programmed death-ligand 1 (PD-L1). We undertook the current study to determine the safety and efficacy of avelumab, a monoclonal antibody targeting the interaction between PD-L1 and its receptor on cytotoxic T-cells, in combination with cetuximab.Methods and analysisThis is a multi-centre, single-arm dose de-escalation phase II safety and efficacy study of avelumab combined with cetuximab; the study was to progress to a randomised phase II trial, however, the study will now complete after the safety run-in component. Up to 16 participants with histologically/cytologically recurrent/metastatic squamous cell carcinoma (including HNSCC) who have not received cetuximab previously will be recruited. All patients will receive 10 mg/kg avelumab and cetuximab (500, 400 or 300 mg/m2 depending on the cohort open at time of registration) on days 1 and 15 of 4-week cycles for up to 1 year, (avelumab not given cycle 1 day 1). A modified continual reassessment method will be used to determine dose de-escalation. The primary objective is to establish the safety of the combination and to determine the optimum dose of cetuximab. Secondary objectives include assessing evidence of antitumour activity by evaluating response rates and disease control rates at 6 and 12 months as well as progression-free and OS.Ethics and disseminationApproval granted by City and East REC (18/LO/0021). Findings will be published in peer-reviewed journals and disseminated at conferences.Trial registration numberNCT03494322

Fc Effector Function Contributes to the Activity of Human Anti-CTLA-4 Antibodies.

With the use of a mouse model expressing human Fc-gamma receptors (FcγRs), we demonstrated that antibodies with isotypes equivalent to ipilimumab and tremelimumab mediate intra-tumoral regulatory T (Treg) cell depletion in vivo, increasing the CD8+ to Treg cell ratio and promoting tumor rejection. Antibodies with improved FcγR binding profiles drove superior anti-tumor responses and survival. In patients with advanced melanoma, response to ipilimumab was associated with the CD16a-V158F high affinity polymorphism. Such activity only appeared relevant in the context of inflamed tumors, explaining the modest response rates observed in the clinical setting. Our data suggest that the activity of anti-CTLA-4 in inflamed tumors may be improved through enhancement of FcγR binding, whereas poorly infiltrated tumors will likely require combination approaches

The evolution of lung cancer and impact of subclonal selection in TRACERx

Lung cancer is the leading cause of cancer-associated mortality worldwide. Here we analysed 1,644 tumour regions sampled at surgery or during follow-up from the first 421 patients with non-small cell lung cancer prospectively enrolled into the TRACERx study. This project aims to decipher lung cancer evolution and address the primary study endpoint: determining the relationship between intratumour heterogeneity and clinical outcome. In lung adenocarcinoma, mutations in 22 out of 40 common cancer genes were under significant subclonal selection, including classical tumour initiators such as TP53 and KRAS. We defined evolutionary dependencies between drivers, mutational processes and whole genome doubling (WGD) events. Despite patients having a history of smoking, 8% of lung adenocarcinomas lacked evidence of tobacco-induced mutagenesis. These tumours also had similar detection rates for EGFR mutations and for RET, ROS1, ALK and MET oncogenic isoforms compared with tumours in never-smokers, which suggests that they have a similar aetiology and pathogenesis. Large subclonal expansions were associated with positive subclonal selection. Patients with tumours harbouring recent subclonal expansions, on the terminus of a phylogenetic branch, had significantly shorter disease-free survival. Subclonal WGD was detected in 19% of tumours, and 10% of tumours harboured multiple subclonal WGDs in parallel. Subclonal, but not truncal, WGD was associated with shorter disease-free survival. Copy number heterogeneity was associated with extrathoracic relapse within 1 year after surgery. These data demonstrate the importance of clonal expansion, WGD and copy number instability in determining the timing and patterns of relapse in non-small cell lung cancer and provide a comprehensive clinical cancer evolutionary data resource

The evolution of non-small cell lung cancer metastases in TRACERx

Metastatic disease is responsible for the majority of cancer-related deaths. We report the longitudinal evolutionary analysis of 126 non-small cell lung cancer (NSCLC) tumours from 421 prospectively recruited patients in TRACERx who developed metastatic disease, compared with a control cohort of 144 non-metastatic tumours. In 25% of cases, metastases diverged early, before the last clonal sweep in the primary tumour, and early divergence was enriched for patients who were smokers at the time of initial diagnosis. Simulations suggested that early metastatic divergence more frequently occurred at smaller tumour diameters (less than 8 mm). Single-region primary tumour sampling resulted in 83% of late divergence cases being misclassified as early, highlighting the importance of extensive primary tumour sampling. Polyclonal dissemination, which was associated with extrathoracic disease recurrence, was found in 32% of cases. Primary lymph node disease contributed to metastatic relapse in less than 20% of cases, representing a hallmark of metastatic potential rather than a route to subsequent recurrences/disease progression. Metastasis-seeding subclones exhibited subclonal expansions within primary tumours, probably reflecting positive selection. Our findings highlight the importance of selection in metastatic clone evolution within untreated primary tumours, the distinction between monoclonal versus polyclonal seeding in dictating site of recurrence, the limitations of current radiological screening approaches for early diverging tumours and the need to develop strategies to target metastasis-seeding subclones before relapse

Genomic–transcriptomic evolution in lung cancer and metastasis

Intratumour heterogeneity (ITH) fuels lung cancer evolution, which leads to immune evasion and resistance to therapy. Here, using paired whole-exome and RNA sequencing data, we investigate intratumour transcriptomic diversity in 354 non-small cell lung cancer tumours from 347 out of the first 421 patients prospectively recruited into the TRACERx study. Analyses of 947 tumour regions, representing both primary and metastatic disease, alongside 96 tumour-adjacent normal tissue samples implicate the transcriptome as a major source of phenotypic variation. Gene expression levels and ITH relate to patterns of positive and negative selection during tumour evolution. We observe frequent copy number-independent allele-specific expression that is linked to epigenomic dysfunction. Allele-specific expression can also result in genomic–transcriptomic parallel evolution, which converges on cancer gene disruption. We extract signatures of RNA single-base substitutions and link their aetiology to the activity of the RNA-editing enzymes ADAR and APOBEC3A, thereby revealing otherwise undetected ongoing APOBEC activity in tumours. Characterizing the transcriptomes of primary–metastatic tumour pairs, we combine multiple machine-learning approaches that leverage genomic and transcriptomic variables to link metastasis-seeding potential to the evolutionary context of mutations and increased proliferation within primary tumour regions. These results highlight the interplay between the genome and transcriptome in influencing ITH, lung cancer evolution and metastasis

Antibodies against endogenous retroviruses promote lung cancer immunotherapy

B cells are frequently found in the margins of solid tumours as organized follicles in ectopic lymphoid organs called tertiary lymphoid structures (TLS). Although TLS have been found to correlate with improved patient survival and response to immune checkpoint blockade (ICB), the underlying mechanisms of this association remain elusive. Here we investigate lung-resident B cell responses in patients from the TRACERx 421 (Tracking Non-Small-Cell Lung Cancer Evolution Through Therapy) and other lung cancer cohorts, and in a recently established immunogenic mouse model for lung adenocarcinoma. We find that both human and mouse lung adenocarcinomas elicit local germinal centre responses and tumour-binding antibodies, and further identify endogenous retrovirus (ERV) envelope glycoproteins as a dominant anti-tumour antibody target. ERV-targeting B cell responses are amplified by ICB in both humans and mice, and by targeted inhibition of KRAS(G12C) in the mouse model. ERV-reactive antibodies exert anti-tumour activity that extends survival in the mouse model, and ERV expression predicts the outcome of ICB in human lung adenocarcinoma. Finally, we find that effective immunotherapy in the mouse model requires CXCL13-dependent TLS formation. Conversely, therapeutic CXCL13 treatment potentiates anti-tumour immunity and synergizes with ICB. Our findings provide a possible mechanistic basis for the association of TLS with immunotherapy response

Hospital admission and emergency care attendance risk for SARS-CoV-2 delta (B.1.617.2) compared with alpha (B.1.1.7) variants of concern: a cohort study

Background: The SARS-CoV-2 delta (B.1.617.2) variant was first detected in England in March, 2021. It has since rapidly become the predominant lineage, owing to high transmissibility. It is suspected that the delta variant is associated with more severe disease than the previously dominant alpha (B.1.1.7) variant. We aimed to characterise the severity of the delta variant compared with the alpha variant by determining the relative risk of hospital attendance outcomes. Methods: This cohort study was done among all patients with COVID-19 in England between March 29 and May 23, 2021, who were identified as being infected with either the alpha or delta SARS-CoV-2 variant through whole-genome sequencing. Individual-level data on these patients were linked to routine health-care datasets on vaccination, emergency care attendance, hospital admission, and mortality (data from Public Health England's Second Generation Surveillance System and COVID-19-associated deaths dataset; the National Immunisation Management System; and NHS Digital Secondary Uses Services and Emergency Care Data Set). The risk for hospital admission and emergency care attendance were compared between patients with sequencing-confirmed delta and alpha variants for the whole cohort and by vaccination status subgroups. Stratified Cox regression was used to adjust for age, sex, ethnicity, deprivation, recent international travel, area of residence, calendar week, and vaccination status. Findings: Individual-level data on 43 338 COVID-19-positive patients (8682 with the delta variant, 34 656 with the alpha variant; median age 31 years [IQR 17–43]) were included in our analysis. 196 (2·3%) patients with the delta variant versus 764 (2·2%) patients with the alpha variant were admitted to hospital within 14 days after the specimen was taken (adjusted hazard ratio [HR] 2·26 [95% CI 1·32–3·89]). 498 (5·7%) patients with the delta variant versus 1448 (4·2%) patients with the alpha variant were admitted to hospital or attended emergency care within 14 days (adjusted HR 1·45 [1·08–1·95]). Most patients were unvaccinated (32 078 [74·0%] across both groups). The HRs for vaccinated patients with the delta variant versus the alpha variant (adjusted HR for hospital admission 1·94 [95% CI 0·47–8·05] and for hospital admission or emergency care attendance 1·58 [0·69–3·61]) were similar to the HRs for unvaccinated patients (2·32 [1·29–4·16] and 1·43 [1·04–1·97]; p=0·82 for both) but the precision for the vaccinated subgroup was low. Interpretation: This large national study found a higher hospital admission or emergency care attendance risk for patients with COVID-19 infected with the delta variant compared with the alpha variant. Results suggest that outbreaks of the delta variant in unvaccinated populations might lead to a greater burden on health-care services than the alpha variant. Funding: Medical Research Council; UK Research and Innovation; Department of Health and Social Care; and National Institute for Health Research

Changes in symptomatology, reinfection, and transmissibility associated with the SARS-CoV-2 variant B.1.1.7: an ecological study

Background The SARS-CoV-2 variant B.1.1.7 was first identified in December, 2020, in England. We aimed to investigate whether increases in the proportion of infections with this variant are associated with differences in symptoms or disease course, reinfection rates, or transmissibility. Methods We did an ecological study to examine the association between the regional proportion of infections with the SARS-CoV-2 B.1.1.7 variant and reported symptoms, disease course, rates of reinfection, and transmissibility. Data on types and duration of symptoms were obtained from longitudinal reports from users of the COVID Symptom Study app who reported a positive test for COVID-19 between Sept 28 and Dec 27, 2020 (during which the prevalence of B.1.1.7 increased most notably in parts of the UK). From this dataset, we also estimated the frequency of possible reinfection, defined as the presence of two reported positive tests separated by more than 90 days with a period of reporting no symptoms for more than 7 days before the second positive test. The proportion of SARS-CoV-2 infections with the B.1.1.7 variant across the UK was estimated with use of genomic data from the COVID-19 Genomics UK Consortium and data from Public Health England on spike-gene target failure (a non-specific indicator of the B.1.1.7 variant) in community cases in England. We used linear regression to examine the association between reported symptoms and proportion of B.1.1.7. We assessed the Spearman correlation between the proportion of B.1.1.7 cases and number of reinfections over time, and between the number of positive tests and reinfections. We estimated incidence for B.1.1.7 and previous variants, and compared the effective reproduction number, Rt, for the two incidence estimates. Findings From Sept 28 to Dec 27, 2020, positive COVID-19 tests were reported by 36 920 COVID Symptom Study app users whose region was known and who reported as healthy on app sign-up. We found no changes in reported symptoms or disease duration associated with B.1.1.7. For the same period, possible reinfections were identified in 249 (0·7% [95% CI 0·6–0·8]) of 36 509 app users who reported a positive swab test before Oct 1, 2020, but there was no evidence that the frequency of reinfections was higher for the B.1.1.7 variant than for pre-existing variants. Reinfection occurrences were more positively correlated with the overall regional rise in cases (Spearman correlation 0·56–0·69 for South East, London, and East of England) than with the regional increase in the proportion of infections with the B.1.1.7 variant (Spearman correlation 0·38–0·56 in the same regions), suggesting B.1.1.7 does not substantially alter the risk of reinfection. We found a multiplicative increase in the Rt of B.1.1.7 by a factor of 1·35 (95% CI 1·02–1·69) relative to pre-existing variants. However, Rt fell below 1 during regional and national lockdowns, even in regions with high proportions of infections with the B.1.1.7 variant. Interpretation The lack of change in symptoms identified in this study indicates that existing testing and surveillance infrastructure do not need to change specifically for the B.1.1.7 variant. In addition, given that there was no apparent increase in the reinfection rate, vaccines are likely to remain effective against the B.1.1.7 variant. Funding Zoe Global, Department of Health (UK), Wellcome Trust, Engineering and Physical Sciences Research Council (UK), National Institute for Health Research (UK), Medical Research Council (UK), Alzheimer's Society

Investigation of hospital discharge cases and SARS-CoV-2 introduction into Lothian care homes

Background The first epidemic wave of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in Scotland resulted in high case numbers and mortality in care homes. In Lothian, over one-third of care homes reported an outbreak, while there was limited testing of hospital patients discharged to care homes. Aim To investigate patients discharged from hospitals as a source of SARS-CoV-2 introduction into care homes during the first epidemic wave. Methods A clinical review was performed for all patients discharges from hospitals to care homes from 1st March 2020 to 31st May 2020. Episodes were ruled out based on coronavirus disease 2019 (COVID-19) test history, clinical assessment at discharge, whole-genome sequencing (WGS) data and an infectious period of 14 days. Clinical samples were processed for WGS, and consensus genomes generated were used for analysis using Cluster Investigation and Virus Epidemiological Tool software. Patient timelines were obtained using electronic hospital records. Findings In total, 787 patients discharged from hospitals to care homes were identified. Of these, 776 (99%) were ruled out for subsequent introduction of SARS-CoV-2 into care homes. However, for 10 episodes, the results were inconclusive as there was low genomic diversity in consensus genomes or no sequencing data were available. Only one discharge episode had a genomic, time and location link to positive cases during hospital admission, leading to 10 positive cases in their care home. Conclusion The majority of patients discharged from hospitals were ruled out for introduction of SARS-CoV-2 into care homes, highlighting the importance of screening all new admissions when faced with a novel emerging virus and no available vaccine

Genomic assessment of quarantine measures to prevent SARS-CoV-2 importation and transmission

Mitigation of SARS-CoV-2 transmission from international travel is a priority. We evaluated the effectiveness of travellers being required to quarantine for 14-days on return to England in Summer 2020. We identified 4,207 travel-related SARS-CoV-2 cases and their contacts, and identified 827 associated SARS-CoV-2 genomes. Overall, quarantine was associated with a lower rate of contacts, and the impact of quarantine was greatest in the 16–20 age-group. 186 SARS-CoV-2 genomes were sufficiently unique to identify travel-related clusters. Fewer genomically-linked cases were observed for index cases who returned from countries with quarantine requirement compared to countries with no quarantine requirement. This difference was explained by fewer importation events per identified genome for these cases, as opposed to fewer onward contacts per case. Overall, our study demonstrates that a 14-day quarantine period reduces, but does not completely eliminate, the onward transmission of imported cases, mainly by dissuading travel to countries with a quarantine requirement