Elucidating the Heterogeneity of Immunotherapy Response and Immune-Related Toxicities by Longitudinal ctDNA and Immune Cell Compartment Tracking in Lung Cancer.

PURPOSE: Although immunotherapy is the mainstay of therapy for advanced non-small cell lung cancer (NSCLC), robust biomarkers of clinical response are lacking. The heterogeneity of clinical responses together with the limited value of radiographic response assessments to timely and accurately predict therapeutic effect-especially in the setting of stable disease-calls for the development of molecularly informed real-time minimally invasive approaches. In addition to capturing tumor regression, liquid biopsies may be informative in capturing immune-related adverse events (irAE). EXPERIMENTAL DESIGN: We investigated longitudinal changes in circulating tumor DNA (ctDNA) in patients with metastatic NSCLC who received immunotherapy-based regimens. Using ctDNA targeted error-correction sequencing together with matched sequencing of white blood cells and tumor tissue, we tracked serial changes in cell-free tumor load (cfTL) and determined molecular response. Peripheral T-cell repertoire dynamics were serially assessed and evaluated together with plasma protein expression profiles. RESULTS: Molecular response, defined as complete clearance of cfTL, was significantly associated with progression-free (log-rank P = 0.0003) and overall survival (log-rank P = 0.01) and was particularly informative in capturing differential survival outcomes among patients with radiographically stable disease. For patients who developed irAEs, on-treatment peripheral blood T-cell repertoire reshaping, assessed by significant T-cell receptor (TCR) clonotypic expansions and regressions, was identified on average 5 months prior to clinical diagnosis of an irAE. CONCLUSIONS: Molecular responses assist with the interpretation of heterogeneous clinical responses, especially for patients with stable disease. Our complementary assessment of the peripheral tumor and immune compartments provides an approach for monitoring of clinical benefits and irAEs during immunotherapy.

Elucidating the heterogeneity of immunotherapy response and immune-related toxicities by longitudinal ctDNA and immune cell compartment tracking in lung cancer.

Purpose: Although immunotherapy is the mainstay of therapy for advanced non-small cell lung cancer (NSCLC), robust biomarkers of clinical response are lacking. The heterogeneity of clinical responses together with the limited value of radiographic response assessments to timely and accurately predict therapeutic effect -especially in the setting of stable disease-call for the development of molecularly-informed real-time minimally invasive predictive biomarkers. In addition to capturing tumor regression, liquid biopsies may be informative in evaluating immune-related adverse events (irAEs). Experimental design: We investigated longitudinal changes in circulating tumor DNA (ctDNA) in patients with metastatic NSCLC who received immunotherapy-based regimens. Using ctDNA targeted error-correction sequencing together with matched sequencing of white blood cells and tumor tissue, we tracked serial changes in cell-free tumor load (cfTL) and determined molecular response for each patient. Peripheral T-cell repertoire dynamics were serially assessed and evaluated together with plasma protein expression profiles. Results: Molecular response, defined as complete clearance of cfTL, was significantly associated with progression-free (log-rank p=0.0003) and overall survival (log-rank p=0.01) and was particularly informative in capturing differential survival outcomes among patients with radiographically stable disease. For patients who developed irAEs, peripheral blood T-cell repertoire reshaping, assessed by significant TCR clonotypic expansions and regressions were noted on-treatment. Conclusions: Molecular responses assist with interpretation of heterogeneous clinical responses especially for patients with stable disease. Our complementary assessment of the tumor and immune compartments by liquid biopsies provides an approach for monitoring of clinical benefit and immune-related toxicities for patients with NSCLC receiving immunotherapy. Statement of translational relevance: Longitudinal dynamic changes in cell-free tumor load and reshaping of the peripheral T-cell repertoire capture clinical outcomes and immune-related toxicities during immunotherapy for patients with non-small cell lung cancer.

Dynamics of Sequence and Structural Cell-Free DNA Landscapes in Small-Cell Lung Cancer.

PURPOSE: Patients with small-cell lung cancer (SCLC) have an exceptionally poor prognosis, calling for improved real-time noninvasive biomarkers of therapeutic response. EXPERIMENTAL DESIGN: We performed targeted error-correction sequencing on 171 serial plasmas and matched white blood cell (WBC) DNA from 33 patients with metastatic SCLC who received treatment with chemotherapy (n = 16) or immunotherapy-containing (n = 17) regimens. Tumor-derived sequence alterations and plasma aneuploidy were evaluated serially and combined to assess changes in total cell-free tumor load (cfTL). Longitudinal dynamic changes in cfTL were monitored to determine circulating cell-free tumor DNA (ctDNA) molecular response during therapy. RESULTS: Combined tiered analyses of tumor-derived sequence alterations and plasma aneuploidy allowed for the assessment of ctDNA molecular response in all patients. Patients classified as molecular responders (n = 9) displayed sustained elimination of cfTL to undetectable levels. For 14 patients, we observed initial molecular responses, followed by ctDNA recrudescence. A subset of patients (n = 10) displayed a clear pattern of molecular progression, with persistence of cfTL across all time points. Molecular responses captured the therapeutic effect and long-term clinical outcomes in a more accurate and rapid manner compared with radiographic imaging. Patients with sustained molecular responses had longer overall (log-rank P = 0.0006) and progression-free (log-rank P < 0.0001) survival, with molecular responses detected on average 4 weeks earlier than imaging. CONCLUSIONS: ctDNA analyses provide a precise approach for the assessment of early on-therapy molecular responses and have important implications for the management of patients with SCLC, including the development of improved strategies for real-time tumor burden monitoring. See related commentary by Pellini and Chaudhuri, p. 2176.

Liquid biopsy approaches to capture tumor evolution and clinical outcomes during cancer immunotherapy.

Circulating cell-free tumor DNA (ctDNA) can serve as a real-time biomarker of tumor burden and provide unique insights into the evolving molecular landscape of cancers under the selective pressure of immunotherapy. Tracking the landscape of genomic alterations detected in ctDNA may reveal the clonal architecture of the metastatic cascade and thus improve our understanding of the molecular wiring of therapeutic responses. While liquid biopsies may provide a rapid and accurate evaluation of tumor burden dynamics during immunotherapy, the complexity of antitumor immune responses is not fully captured through single-feature ctDNA analyses. This underscores a need for integrative studies modeling the tumor and the immune compartment to understand the kinetics of tumor clearance in association with the quality of antitumor immune responses. Clinical applications of ctDNA testing in patients treated with immune checkpoint inhibitors have shown both predictive and prognostic value through the detection of genomic biomarkers, such as tumor mutational burden and microsatellite instability, as well as allowing for real-time monitoring of circulating tumor burden and the assessment of early on-therapy responses. These efforts highlight the emerging role of liquid biopsies in selecting patients for cancer immunotherapy, monitoring therapeutic efficacy, determining the optimal duration of treatment and ultimately guiding treatment selection and sequencing. The clinical translation of liquid biopsies is propelled by the increasing number of ctDNA-directed interventional clinical trials in the immuno-oncology space, signifying a critical step towards implementation of liquid biopsies in precision immuno-oncology.

Peripheral blood immune cell dynamics reflect antitumor immune responses and predict clinical response to immunotherapy.

BACKGROUND: Despite treatment advancements with immunotherapy, our understanding of response relies on tissue-based, static tumor features such as tumor mutation burden (TMB) and programmed death-ligand 1 (PD-L1) expression. These approaches are limited in capturing the plasticity of tumor-immune system interactions under selective pressure of immune checkpoint blockade and predicting therapeutic response and long-term outcomes. Here, we investigate the relationship between serial assessment of peripheral blood cell counts and tumor burden dynamics in the context of an evolving tumor ecosystem during immune checkpoint blockade. METHODS: Using machine learning, we integrated dynamics in peripheral blood immune cell subsets, including neutrophil-lymphocyte ratio (NLR), from 239 patients with metastatic non-small cell lung cancer (NSCLC) and predicted clinical outcome with immune checkpoint blockade. We then sought to interpret NLR dynamics in the context of transcriptomic and T cell repertoire trajectories for 26 patients with early stage NSCLC who received neoadjuvant immune checkpoint blockade. We further determined the relationship between NLR dynamics, pathologic response and circulating tumor DNA (ctDNA) clearance. RESULTS: Integrated dynamics of peripheral blood cell counts, predominantly NLR dynamics and changes in eosinophil levels, predicted clinical outcome, outperforming both TMB and PD-L1 expression. As early changes in NLR were a key predictor of response, we linked NLR dynamics with serial RNA sequencing deconvolution and T cell receptor sequencing to investigate differential tumor microenvironment reshaping during therapy for patients with reduction in peripheral NLR. Reductions in NLR were associated with induction of interferon-γ responses driving the expression of antigen presentation and proinflammatory gene sets coupled with reshaping of the intratumoral T cell repertoire. In addition, NLR dynamics reflected tumor regression assessed by pathological responses and complemented ctDNA kinetics in predicting long-term outcome. Elevated peripheral eosinophil levels during immune checkpoint blockade were correlated with therapeutic response in both metastatic and early stage cohorts. CONCLUSIONS: Our findings suggest that early dynamics in peripheral blood immune cell subsets reflect changes in the tumor microenvironment and capture antitumor immune responses, ultimately reflecting clinical outcomes with immune checkpoint blockade.

Longitudinal profiling of circulating tumour DNA for tracking tumour dynamics in pancreatic cancer.

BACKGROUND: The utility of circulating tumour DNA (ctDNA) for longitudinal tumour monitoring in pancreatic ductal adenocarcinoma (PDAC) has not been explored beyond mutations in the KRAS proto-oncogene. Here, we aimed to characterise and track patient-specific somatic ctDNA variants, to assess longitudinal changes in disease burden and explore the landscape of actionable alterations. METHODS: We followed 3 patients with resectable disease and 4 patients with unresectable disease, including 4 patients with ≥ 3 serial follow-up samples, of whom 2 were rare long survivors (> 5 years). We performed whole exome sequencing of tumour gDNA and plasma ctDNA (n = 20) collected over a ~ 2-year period from diagnosis through treatment to death or final follow-up. Plasma from 3 chronic pancreatitis cases was used as a comparison for analysis of ctDNA mutations. RESULTS: We detected > 55% concordance between somatic mutations in tumour tissues and matched serial plasma. Mutations in ctDNA were detected within known PDAC driver genes (KRAS, TP53, SMAD4, CDKN2A), in addition to patient-specific variants within alternative cancer drivers (NRAS, HRAS, MTOR, ERBB2, EGFR, PBRM1), with a trend towards higher overall mutation loads in advanced disease. ctDNA alterations with potential for therapeutic actionability were identified in all 7 patients, including DNA damage response (DDR) variants co-occurring with hypermutation signatures predictive of response to platinum chemotherapy. Longitudinal tracking in 4 patients with follow-up > 2 years demonstrated that ctDNA mutant allele fractions and clonal trends were consistent with CA19-9 measurements and/or clinically reported disease burden. The estimated prevalence of 'stem clones' was highest in an unresectable patient where changes in ctDNA dynamics preceded CA19-9 levels. Longitudinal evolutionary trajectories revealed ongoing subclonal evolution following chemotherapy. CONCLUSION: These results provide proof-of-concept for the use of exome sequencing of serial plasma to characterise patient-specific ctDNA profiles, and demonstrate the sensitivity of ctDNA in monitoring disease burden in PDAC even in unresectable cases without matched tumour genotyping. They reveal the value of tracking clonal evolution in serial ctDNA to monitor treatment response, establishing the potential of applied precision medicine to guide stratified care by identifying and evaluating actionable opportunities for intervention aimed at optimising patient outcomes for an otherwise intractable disease.

Genetic variation in antigen presentation and cancer immunotherapy.

Human leukocyte antigen (HLA) molecules are critical mediators of anti-tumor immune responses. In this issue of Immunity,Chhibber et al. (2021) challenge previous associations between germline HLA zygosity and immunotherapy outcomes, demonstrating that germline HLA genotypes and diversity alone are not independent biomarkers of anti-PD1 clinical efficacy.

COVID-19 in patients with hepatobiliary and pancreatic diseases: a single-centre cross-sectional study in East London.

OBJECTIVE: To explore risk factors associated with COVID-19 susceptibility and survival in patients with pre-existing hepato-pancreato-biliary (HPB) conditions. DESIGN: Cross-sectional study. SETTING: East London Pancreatic Cancer Epidemiology (EL-PaC-Epidem) Study at Barts Health National Health Service Trust, UK. Linked electronic health records were interrogated on a cohort of participants (age ≥18 years), reported with HPB conditions between 1 April 2008 and 6 March 2020. PARTICIPANTS: EL-PaC-Epidem Study participants, alive on 12 February 2020, and living in East London within the previous 6 months (n=15 440). The cohort represents a multi-ethnic population with 51.7% belonging to the non-White background. MAIN OUTCOME MEASURE: COVID-19 incidence and mortality. RESULTS: Some 226 (1.5%) participants had confirmed COVID-19 diagnosis between 12 February and 12 June 2020, with increased odds for men (OR 1.56; 95% CI 1.2 to 2.04) and Black ethnicity (2.04; 1.39 to 2.95) as well as patients with moderate to severe liver disease (2.2; 1.35 to 3.59). Each additional comorbidity increased the odds of infection by 62%. Substance misusers were at more risk of infection, so were patients on vitamin D treatment. The higher ORs in patients with chronic pancreatic or mild liver conditions, age >70, and a history of smoking or obesity were due to coexisting comorbidities. Increased odds of death were observed for men (3.54; 1.68 to 7.85) and Black ethnicity (3.77; 1.38 to 10.7). Patients having respiratory complications from COVID-19 without a history of chronic respiratory disease also had higher odds of death (5.77; 1.75 to 19). CONCLUSIONS: In this large population-based study of patients with HPB conditions, men, Black ethnicity, pre-existing moderate to severe liver conditions, six common medical multimorbidities, substance misuse and a history of vitamin D treatment independently posed higher odds of acquiring COVID-19 compared with their respective counterparts. The odds of death were significantly high for men and Black people.

SNPnexus: a web server for functional annotation of human genome sequence variation (2020 update).

SNPnexus is a web-based annotation tool for the analysis and interpretation of both known and novel sequencing variations. Since its last release, SNPnexus has received continual updates to expand the range and depth of annotations provided. SNPnexus has undergone a complete overhaul of the underlying infrastructure to accommodate faster computational times. The scope for data annotation has been substantially expanded to enhance biological interpretations of queried variants. This includes the addition of pathway analysis for the identification of enriched biological pathways and molecular processes. We have further expanded the range of user directed annotation fields available for the study of cancer sequencing data. These new additions facilitate investigations into cancer driver variants and targetable molecular alterations within input datasets. New user directed filtering options have been coupled with the addition of interactive graphical and visualization tools. These improvements streamline the analysis of variants derived from large sequencing datasets for the identification of biologically and clinically significant subsets in the data. SNPnexus is the most comprehensible web-based application currently available and these new set of updates ensures that it remains a state-of-the-art tool for researchers. SNPnexus is freely available at https://www.snp-nexus.org.