Meta-analysis of tumor- and T cell-intrinsic mechanisms of sensitization to checkpoint inhibition.

Checkpoint inhibitors (CPIs) augment adaptive immunity. Systematic pan-tumor analyses may reveal the relative importance of tumor-cell-intrinsic and microenvironmental features underpinning CPI sensitization. Here, we collated whole-exome and transcriptomic data for >1,000 CPI-treated patients across seven tumor types, utilizing standardized bioinformatics workflows and clinical outcome criteria to validate multivariable predictors of CPI sensitization. Clonal tumor mutation burden (TMB) was the strongest predictor of CPI response, followed by total TMB and CXCL9 expression. Subclonal TMB, somatic copy alteration burden, and histocompatibility leukocyte antigen (HLA) evolutionary divergence failed to attain pan-cancer significance. Dinucleotide variants were identified as a source of immunogenic epitopes associated with radical amino acid substitutions and enhanced peptide hydrophobicity/immunogenicity. Copy-number analysis revealed two additional determinants of CPI outcome supported by prior functional evidence: 9q34 (TRAF2) loss associated with response and CCND1 amplification associated with resistance. Finally, single-cell RNA sequencing (RNA-seq) of clonal neoantigen-reactive CD8 tumor-infiltrating lymphocytes (TILs), combined with bulk RNA-seq analysis of CPI-responding tumors, identified CCR5 and CXCL13 as T-cell-intrinsic markers of CPI sensitivity.

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

Lung cancer is the leading cause of cancer-associated mortality worldwide1. 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.

Tracking early lung cancer metastatic dissemination in TRACERx using ctDNA.

Circulating tumour DNA (ctDNA) can be used to detect and profile residual tumour cells persisting after curative intent therapy1. The study of large patient cohorts incorporating longitudinal plasma sampling and extended follow-up is required to determine the role of ctDNA as a phylogenetic biomarker of relapse in early-stage non-small-cell lung cancer (NSCLC). Here we developed ctDNA methods tracking a median of 200 mutations identified in resected NSCLC tissue across 1,069 plasma samples collected from 197 patients enrolled in the TRACERx study2. A lack of preoperative ctDNA detection distinguished biologically indolent lung adenocarcinoma with good clinical outcome. Postoperative plasma analyses were interpreted within the context of standard-of-care radiological surveillance and administration of cytotoxic adjuvant therapy. Landmark analyses of plasma samples collected within 120 days after surgery revealed ctDNA detection in 25% of patients, including 49% of all patients who experienced clinical relapse; 3 to 6 monthly ctDNA surveillance identified impending disease relapse in an additional 20% of landmark-negative patients. We developed a bioinformatic tool (ECLIPSE) for non-invasive tracking of subclonal architecture at low ctDNA levels. ECLIPSE identified patients with polyclonal metastatic dissemination, which was associated with a poor clinical outcome. By measuring subclone cancer cell fractions in preoperative plasma, we found that subclones seeding future metastases were significantly more expanded compared with non-metastatic subclones. Our findings will support (neo)adjuvant trial advances and provide insights into the process of metastatic dissemination using low-ctDNA-level liquid biopsy.

Pervasive chromosomal instability and karyotype order in tumour evolution.

Chromosomal instability in cancer consists of dynamic changes to the number and structure of chromosomes1,2. The resulting diversity in somatic copy number alterations (SCNAs) may provide the variation necessary for tumour evolution1,3,4. Here we use multi-sample phasing and SCNA analysis of 1,421 samples from 394 tumours across 22 tumour types to show that continuous chromosomal instability results in pervasive SCNA heterogeneity. Parallel evolutionary events, which cause disruption in the same genes (such as BCL9, MCL1, ARNT (also known as HIF1B), TERT and MYC) within separate subclones, were present in 37% of tumours. Most recurrent losses probably occurred before whole-genome doubling, that was found as a clonal event in 49% of tumours. However, loss of heterozygosity at the human leukocyte antigen (HLA) locus and loss of chromosome 8p to a single haploid copy recurred at substantial subclonal frequencies, even in tumours with whole-genome doubling, indicating ongoing karyotype remodelling. Focal amplifications that affected chromosomes 1q21 (which encompasses BCL9, MCL1 and ARNT), 5p15.33 (TERT), 11q13.3 (CCND1), 19q12 (CCNE1) and 8q24.1 (MYC) were frequently subclonal yet appeared to be clonal within single samples. Analysis of an independent series of 1,024 metastatic samples revealed that 13 focal SCNAs were enriched in metastatic samples, including gains in chromosome 8q24.1 (encompassing MYC) in clear cell renal cell carcinoma and chromosome 11q13.3 (encompassing CCND1) in HER2+ breast cancer. Chromosomal instability may enable the continuous selection of SCNAs, which are established as ordered events that often occur in parallel, throughout tumour evolution.

Impact of cancer evolution on immune surveillance and checkpoint inhibitor response.

Intratumour heterogeneity (ITH) is pervasive across all cancers studied and may provide the evolving tumour multiple routes to escape immune surveillance. Immune checkpoint inhibitors (CPIs) are rapidly becoming standard of care for many cancers. Here, we discuss recent work investigating the influence of ITH on patient response to immune checkpoint inhibitor (CPI) therapy. At its simplest, ITH may confound the diagnostic accuracy of predictive biomarkers used to stratify patients for CPI therapy. Furthermore, ITH is fuelled by mechanisms of genetic instability that can both engage immune surveillance and drive immune evasion. A greater appreciation of the interplay between ITH and the immune system may hold the key to increasing the proportion of patients experiencing durable responses from CPI therapy.

Cancer cell - Fibroblast crosstalk via HB-EGF, EGFR, and MAPK signaling promotes the expression of macrophage chemo-attractants in squamous cell carcinoma.

Interactions between cells in the tumor microenvironment (TME) shape cancer progression and patient prognosis. To gain insights into how the TME influences cancer outcomes, we derive gene expression signatures indicative of signaling between stromal fibroblasts and cancer cells, and demonstrate their prognostic significance in multiple and independent squamous cell carcinoma cohorts. By leveraging information within the signatures, we discover that the HB-EGF/EGFR/MAPK axis represents a hub of tumor-stroma crosstalk, promoting the expression of CSF2 and LIF and favoring the recruitment of macrophages. Together, these analyses demonstrate the utility of our approach for interrogating the extent and consequences of TME crosstalk.

PD-1 defines a distinct, functional, tissue-adapted state in Vδ1+ T cells with implications for cancer immunotherapy.

Checkpoint inhibition (CPI), particularly that targeting the inhibitory coreceptor programmed cell death protein 1 (PD-1), has transformed oncology. Although CPI can derepress cancer (neo)antigen-specific αß T cells that ordinarily show PD-1-dependent exhaustion, it can also be efficacious against cancers evading αß T cell recognition. In such settings, γδ T cells have been implicated, but the functional relevance of PD-1 expression by these cells is unclear. Here we demonstrate that intratumoral TRDV1 transcripts (encoding the TCRδ chain of Vδ1+ γδ T cells) predict anti-PD-1 CPI response in patients with melanoma, particularly those harboring below average neoantigens. Moreover, using a protocol yielding substantial numbers of tissue-derived Vδ1+ cells, we show that PD-1+Vδ1+ cells display a transcriptomic program similar to, but distinct from, the canonical exhaustion program of colocated PD-1+CD8+ αß T cells. In particular, PD-1+Vδ1+ cells retained effector responses to TCR signaling that were inhibitable by PD-1 engagement and derepressed by CPI.

Race- and Ethnicity-Related Differences in Heart Failure With Preserved Ejection Fraction Using Natural Language Processing.

Background: Heart failure with preserved ejection fraction (HFpEF) is the predominant form of HF in older adults. It represents a heterogenous clinical syndrome that is less well understood across different ethnicities. Objectives: This study aimed to compare the clinical presentation and assess the diagnostic performance of existing HFpEF diagnostic tools between ethnic groups. Methods: A validated Natural Language Processing (NLP) algorithm was applied to the electronic health records of a large London hospital to identify patients meeting the European Society of Cardiology criteria for a diagnosis of HFpEF. NLP extracted patient demographics (including self-reported ethnicity and socioeconomic status), comorbidities, investigation results (N-terminal pro-B-type natriuretic peptide, H2FPEF scores, and echocardiogram reports), and mortality. Analyses were stratified by ethnicity and adjusted for socioeconomic status. Results: Our cohort consisted of 1,261 (64%) White, 578 (29%) Black, and 134 (7%) Asian patients meeting the European Society of Cardiology HFpEF diagnostic criteria. Compared to White patients, Black patients were younger at diagnosis and more likely to have metabolic comorbidities (obesity, diabetes, and hypertension) but less likely to have atrial fibrillation (30% vs 13%; P < 0.001). Black patients had lower N-terminal pro-B-type natriuretic peptide levels and a lower frequency of H2FPEF scores ≥6, indicative of likely HFpEF (26% vs 44%; P < 0.0001). Conclusions: Leveraging an NLP-based artificial intelligence approach to quantify health inequities in HFpEF diagnosis, we discovered that established markers systematically underdiagnose HFpEF in Black patients, possibly due to differences in the underlying comorbidity patterns. Clinicians should be aware of these limitations and its implications for treatment and trial recruitment.

Artificial intelligence methods for improved detection of undiagnosed heart failure with preserved ejection fraction.

AIM: Heart failure with preserved ejection fraction (HFpEF) remains under-diagnosed in clinical practice despite accounting for nearly half of all heart failure (HF) cases. Accurate and timely diagnosis of HFpEF is crucial for proper patient management and treatment. In this study, we explored the potential of natural language processing (NLP) to improve the detection and diagnosis of HFpEF according to the European Society of Cardiology (ESC) diagnostic criteria. METHODS AND RESULTS: In a retrospective cohort study, we used an NLP pipeline applied to the electronic health record (EHR) to identify patients with a clinical diagnosis of HF between 2010 and 2022. We collected demographic, clinical, echocardiographic and outcome data from the EHR. Patients were categorized according to the left ventricular ejection fraction (LVEF). Those with LVEF ≥50% were further categorized based on whether they had a clinician-assigned diagnosis of HFpEF and if not, whether they met the ESC diagnostic criteria. Results were validated in a second, independent centre. We identified 8606 patients with HF. Of 3727 consecutive patients with HF and LVEF ≥50% on echocardiogram, only 8.3% had a clinician-assigned diagnosis of HFpEF, while 75.4% met ESC criteria but did not have a formal diagnosis of HFpEF. Patients with confirmed HFpEF were hospitalized more frequently; however the ESC criteria group had a higher 5-year mortality, despite being less comorbid and experiencing fewer acute cardiovascular events. CONCLUSIONS: This study demonstrates that patients with undiagnosed HFpEF are an at-risk group with high mortality. It is possible to use NLP methods to identify likely HFpEF patients from EHR data who would likely then benefit from expert clinical review and complement the use of diagnostic algorithms.

Quantifying the impact of immunotherapy on RNA dynamics in cancer.

BACKGROUND: Checkpoint inhibitor (CPI) immunotherapies have provided durable clinical responses across a range of solid tumor types for some patients with cancer. Nonetheless, response rates to CPI vary greatly between cancer types. Resolving intratumor transcriptomic changes induced by CPI may improve our understanding of the mechanisms of sensitivity and resistance. METHODS: We assembled a cohort of longitudinal pre-therapy and on-therapy samples from 174 patients treated with CPI across six cancer types by leveraging transcriptomic sequencing data from five studies. RESULTS: Meta-analyses of published RNA markers revealed an on-therapy pattern of immune reinvigoration in patients with breast cancer, which was not discernible pre-therapy, providing biological insight into the impact of CPI on the breast cancer immune microenvironment. We identified 98 breast cancer-specific correlates of CPI response, including 13 genes which are known IO targets, such as toll-like receptors TLR1, TLR4, and TLR8, that could hold potential as combination targets for patients with breast cancer receiving CPI treatment. Furthermore, we demonstrate that a subset of response genes identified in breast cancer are already highly expressed pre-therapy in melanoma, and additionally we establish divergent RNA dynamics between breast cancer and melanoma following CPI treatment, which may suggest distinct immune microenvironments between the two cancer types. CONCLUSIONS: Overall, delineating longitudinal RNA dynamics following CPI therapy sheds light on the mechanisms underlying diverging response trajectories, and identifies putative targets for combination therapy.

Evolutionary characterization of lung adenocarcinoma morphology in TRACERx.

Lung adenocarcinomas (LUADs) display a broad histological spectrum from low-grade lepidic tumors through to mid-grade acinar and papillary and high-grade solid, cribriform and micropapillary tumors. How morphology reflects tumor evolution and disease progression is poorly understood. Whole-exome sequencing data generated from 805 primary tumor regions and 121 paired metastatic samples across 248 LUADs from the TRACERx 421 cohort, together with RNA-sequencing data from 463 primary tumor regions, were integrated with detailed whole-tumor and regional histopathological analysis. Tumors with predominantly high-grade patterns showed increased chromosomal complexity, with higher burden of loss of heterozygosity and subclonal somatic copy number alterations. Individual regions in predominantly high-grade pattern tumors exhibited higher proliferation and lower clonal diversity, potentially reflecting large recent subclonal expansions. Co-occurrence of truncal loss of chromosomes 3p and 3q was enriched in predominantly low-/mid-grade tumors, while purely undifferentiated solid-pattern tumors had a higher frequency of truncal arm or focal 3q gains and SMARCA4 gene alterations compared with mixed-pattern tumors with a solid component, suggesting distinct evolutionary trajectories. Clonal evolution analysis revealed that tumors tend to evolve toward higher-grade patterns. The presence of micropapillary pattern and 'tumor spread through air spaces' were associated with intrathoracic recurrence, in contrast to the presence of solid/cribriform patterns, necrosis and preoperative circulating tumor DNA detection, which were associated with extra-thoracic recurrence. These data provide insights into the relationship between LUAD morphology, the underlying evolutionary genomic landscape, and clinical and anatomical relapse risk.

RAS oncogenic activity predicts response to chemotherapy and outcome in lung adenocarcinoma.

Activating mutations in KRAS occur in 32% of lung adenocarcinomas (LUAD). Despite leading to aggressive disease and resistance to therapy in preclinical studies, the KRAS mutation does not predict patient outcome or response to treatment, presumably due to additional events modulating RAS pathways. To obtain a broader measure of RAS pathway activation, we developed RAS84, a transcriptional signature optimised to capture RAS oncogenic activity in LUAD. We report evidence of RAS pathway oncogenic activation in 84% of LUAD, including 65% KRAS wild-type tumours, falling into four groups characterised by coincident alteration of STK11/LKB1, TP53 or CDKN2A, suggesting that the classifications developed when considering only KRAS mutant tumours have significance in a broader cohort of patients. Critically, high RAS activity patient groups show adverse clinical outcome and reduced response to chemotherapy. Patient stratification using oncogenic RAS transcriptional activity instead of genetic alterations could ultimately assist in clinical decision-making.

A local human Vδ1 T cell population is associated with survival in nonsmall-cell lung cancer.

Murine tissues harbor signature γδ T cell compartments with profound yet differential impacts on carcinogenesis. Conversely, human tissue-resident γδ cells are less well defined. In the present study, we show that human lung tissues harbor a resident Vδ1 γδ T cell population. Moreover, we demonstrate that Vδ1 T cells with resident memory and effector memory phenotypes were enriched in lung tumors compared with nontumor lung tissues. Intratumoral Vδ1 T cells possessed stem-like features and were skewed toward cytolysis and helper T cell type 1 function, akin to intratumoral natural killer and CD8+ T cells considered beneficial to the patient. Indeed, ongoing remission post-surgery was significantly associated with the numbers of CD45RA-CD27- effector memory Vδ1 T cells in tumors and, most strikingly, with the numbers of CD103+ tissue-resident Vδ1 T cells in nonmalignant lung tissues. Our findings offer basic insights into human body surface immunology that collectively support integrating Vδ1 T cell biology into immunotherapeutic strategies for nonsmall cell lung cancer.

Metastasis and Immune Evasion from Extracellular cGAMP Hydrolysis.

Cytosolic DNA is characteristic of chromosomally unstable metastatic cancer cells, resulting in constitutive activation of the cGAS-STING innate immune pathway. How tumors co-opt inflammatory signaling while evading immune surveillance remains unknown. Here, we show that the ectonucleotidase ENPP1 promotes metastasis by selectively degrading extracellular cGAMP, an immune-stimulatory metabolite whose breakdown products include the immune suppressor adenosine. ENPP1 loss suppresses metastasis, restores tumor immune infiltration, and potentiates response to immune checkpoint blockade in a manner dependent on tumor cGAS and host STING. Conversely, overexpression of wild-type ENPP1, but not an enzymatically weakened mutant, promotes migration and metastasis, in part through the generation of extracellular adenosine, and renders otherwise sensitive tumors completely resistant to immunotherapy. In human cancers, ENPP1 expression correlates with reduced immune cell infiltration, increased metastasis, and resistance to anti-PD-1/PD-L1 treatment. Thus, cGAMP hydrolysis by ENPP1 enables chromosomally unstable tumors to transmute cGAS activation into an immune-suppressive pathway. SIGNIFICANCE: Chromosomal instability promotes metastasis by generating chronic tumor inflammation. ENPP1 facilitates metastasis and enables tumor cells to tolerate inflammation by hydrolyzing the immunotransmitter cGAMP, preventing its transfer from cancer cells to immune cells.This article is highlighted in the In This Issue feature, p. 995.

Publisher Correction: Pulmonary venous circulating tumor cell dissemination before tumor resection and disease relapse.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Publisher Correction: A clonal expression biomarker associates with lung cancer mortality.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: Scalable and robust SARS-CoV-2 testing in an academic center.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The T cell differentiation landscape is shaped by tumour mutations in lung cancer.

Tumour mutational burden (TMB) predicts immunotherapy outcome in non-small cell lung cancer (NSCLC), consistent with immune recognition of tumour neoantigens. However, persistent antigen exposure is detrimental for T cell function. How TMB affects CD4 and CD8 T cell differentiation in untreated tumours, and whether this affects patient outcomes is unknown. Here we paired high-dimensional flow cytometry, exome, single-cell and bulk RNA sequencing from patients with resected, untreated NSCLC to examine these relationships. TMB was associated with compartment-wide T cell differentiation skewing, characterized by loss of TCF7-expressing progenitor-like CD4 T cells, and an increased abundance of dysfunctional CD8 and CD4 T cell subsets, with significant phenotypic and transcriptional similarity to neoantigen-reactive CD8 T cells. A gene signature of redistribution from progenitor-like to dysfunctional states associated with poor survival in lung and other cancer cohorts. Single-cell characterization of these populations informs potential strategies for therapeutic manipulation in NSCLC.