Automated tumor immunophenotyping predicts clinical benefit from anti-PD-L1 immunotherapy.

Cancer immunotherapy has transformed the clinical approach to patients with malignancies, as profound benefits can be seen in a subset of patients. To identify this subset, biomarker analyses increasingly focus on phenotypic and functional evaluation of the tumor microenvironment to determine if density, spatial distribution, and cellular composition of immune cell infiltrates can provide prognostic and/or predictive information. Attempts have been made to develop standardized methods to evaluate immune infiltrates in the routine assessment of certain tumor types; however, broad adoption of this approach in clinical decision-making is still missing. We developed approaches to categorize solid tumors into 'desert', 'excluded', and 'inflamed' types according to the spatial distribution of CD8+ immune effector cells to determine the prognostic and/or predictive implications of such labels. To overcome the limitations of this subjective approach, we incrementally developed four automated analysis pipelines of increasing granularity and complexity for density and pattern assessment of immune effector cells. We show that categorization based on 'manual' observation is predictive for clinical benefit from anti-programmed death ligand 1 therapy in two large cohorts of patients with non-small cell lung cancer or triple-negative breast cancer. For the automated analysis we demonstrate that a combined approach outperforms individual pipelines and successfully relates spatial features to pathologist-based readouts and the patient's response to therapy. Our findings suggest that tumor immunophenotype generated by automated analysis pipelines should be evaluated further as potential predictive biomarkers for cancer immunotherapy. © 2024 The Pathological Society of Great Britain and Ireland.

Anti-TIGIT antibody improves PD-L1 blockade through myeloid and Treg cells.

Tiragolumab, an anti-TIGIT antibody with an active IgG1κ Fc, demonstrated improved outcomes in the phase 2 CITYSCAPE trial (ClinicalTrials.gov: NCT03563716 ) when combined with atezolizumab (anti-PD-L1) versus atezolizumab alone1. However, there remains little consensus on the mechanism(s) of response with this combination2. Here we find that a high baseline of intratumoural macrophages and regulatory T cells is associated with better outcomes in patients treated with atezolizumab plus tiragolumab but not with atezolizumab alone. Serum sample analysis revealed that macrophage activation is associated with a clinical benefit in patients who received the combination treatment. In mouse tumour models, tiragolumab surrogate antibodies inflamed tumour-associated macrophages, monocytes and dendritic cells through Fcγ receptors (FcγR), in turn driving anti-tumour CD8+ T cells from an exhausted effector-like state to a more memory-like state. These results reveal a mechanism of action through which TIGIT checkpoint inhibitors can remodel immunosuppressive tumour microenvironments, and suggest that FcγR engagement is an important consideration in anti-TIGIT antibody development.

Immune heterogeneity in small-cell lung cancer and vulnerability to immune checkpoint blockade.

Atezolizumab (anti-PD-L1), combined with carboplatin and etoposide (CE), is now a standard of care for extensive-stage small-cell lung cancer (ES-SCLC). A clearer understanding of therapeutically relevant SCLC subsets could identify rational combination strategies and improve outcomes. We conduct transcriptomic analyses and non-negative matrix factorization on 271 pre-treatment patient tumor samples from IMpower133 and identify four subsets with general concordance to previously reported SCLC subtypes (SCLC-A, -N, -P, and -I). Deeper investigation into the immune heterogeneity uncovers two subsets with differing neuroendocrine (NE) versus non-neuroendocrine (non-NE) phenotypes, demonstrating immune cell infiltration hallmarks. The NE tumors with low tumor-associated macrophage (TAM) but high T-effector signals demonstrate longer overall survival with PD-L1 blockade and CE versus CE alone than non-NE tumors with high TAM and high T-effector signal. Our study offers a clinically relevant approach to discriminate SCLC patients likely benefitting most from immunotherapies and highlights the complex mechanisms underlying immunotherapy responses.

Clinical and molecular characterization of long-term survivors with extensive-stage small cell lung cancer treated with first-line atezolizumab plus carboplatin and etoposide.

OBJECTIVES: In the Phase I/III IMpower133 study, first-line atezolizumab plus carboplatin and etoposide (CP/ET) treatment for extensive-stage small cell lung cancer (ES-SCLC) significantly improved overall survival (OS) and progression-free survival versus placebo plus CP/ET. We explored patient and disease characteristics associated with long-term survival in IMpower133, and associations of differential gene expression and SCLC-A (ASCL1-driven), SCLC-N (NEUROD1-driven), SCLC-P (POU2F3-driven), and SCLC-inflamed (SCLC-I) transcriptional subtypes with long-term survival. MATERIALS AND METHODS: Patients with previously untreated ES-SCLC were randomized 1:1 to four 21-day cycles of CP/ET with atezolizumab or placebo. Long-term survivors (LTS) were defined as patients who lived ≥ 18 months post randomization. A generalized linear model was used to evaluate the odds of living ≥ 18 months. Differential gene expression was analyzed using RNA-sequencing data in LTS and non-LTS. OS was assessed by T-effector and B-cell gene signature expression. Distribution of SCLC transcriptional subtypes was assessed in LTS and non-LTS. RESULTS: More LTS were in the atezolizumab arm (34%) than in the placebo arm (20%). The odds ratio for living ≥ 18 months in the atezolizumab arm versus the placebo arm was 2.1 (P < 0.03). Enhanced immune-related signaling was seen in LTS in both arms. Exploratory OS analyses showed atezolizumab treatment benefit versus placebo across T-effector and B-cell gene signature expression subgroups. A higher proportion of LTS than non-LTS in both arms had the SCLC-I subtype; this difference was particularly pronounced in the atezolizumab arm. CONCLUSION: These exploratory analyses suggest that long-term survival is more likely with atezolizumab than placebo in ES-SCLC, confirming the treatment benefit of the IMpower133 regimen. CLINICALTRIAL: gov Identifier: NCT02763579.

The ectonucleotidase CD39 identifies tumor-reactive CD8+ T cells predictive of immune checkpoint blockade efficacy in human lung cancer.

Improved identification of anti-tumor T cells is needed to advance cancer immunotherapies. CD39 expression is a promising surrogate of tumor-reactive CD8+ T cells. Here, we comprehensively profiled CD39 expression in human lung cancer. CD39 expression enriched for CD8+ T cells with features of exhaustion, tumor reactivity, and clonal expansion. Flow cytometry of 440 lung cancer biospecimens revealed weak association between CD39+ CD8+ T cells and tumoral features, such as programmed death-ligand 1 (PD-L1), tumor mutation burden, and driver mutations. Immune checkpoint blockade (ICB), but not cytotoxic chemotherapy, increased intratumoral CD39+ CD8+ T cells. Higher baseline frequency of CD39+ CD8+ T cells conferred improved clinical outcomes from ICB therapy. Furthermore, a gene signature of CD39+ CD8+ T cells predicted benefit from ICB, but not chemotherapy, in a phase III clinical trial of non-small cell lung cancer. These findings highlight CD39 as a proxy of tumor-reactive CD8+ T cells in human lung cancer.

LRRC15+ myofibroblasts dictate the stromal setpoint to suppress tumour immunity.

Recent single-cell studies of cancer in both mice and humans have identified the emergence of a myofibroblast population specifically marked by the highly restricted leucine-rich-repeat-containing protein 15 (LRRC15)1-3. However, the molecular signals that underlie the development of LRRC15+ cancer-associated fibroblasts (CAFs) and their direct impact on anti-tumour immunity are uncharacterized. Here in mouse models of pancreatic cancer, we provide in vivo genetic evidence that TGFß receptor type 2 signalling in healthy dermatopontin+ universal fibroblasts is essential for the development of cancer-associated LRRC15+ myofibroblasts. This axis also predominantly drives fibroblast lineage diversity in human cancers. Using newly developed Lrrc15-diphtheria toxin receptor knock-in mice to selectively deplete LRRC15+ CAFs, we show that depletion of this population markedly reduces the total tumour fibroblast content. Moreover, the CAF composition is recalibrated towards universal fibroblasts. This relieves direct suppression of tumour-infiltrating CD8+ T cells to enhance their effector function and augments tumour regression in response to anti-PDL1 immune checkpoint blockade. Collectively, these findings demonstrate that TGFß-dependent LRRC15+ CAFs dictate the tumour-fibroblast setpoint to promote tumour growth. These cells also directly suppress CD8+ T cell function and limit responsiveness to checkpoint blockade. Development of treatments that restore the homeostatic fibroblast setpoint by reducing the population of pro-disease LRRC15+ myofibroblasts may improve patient survival and response to immunotherapy.

Genomic Profiling of Bronchoalveolar Lavage Fluid in Lung Cancer.

Genomic profiling of bronchoalveolar lavage (BAL) samples may be useful for tumor profiling and diagnosis in the clinic. Here, we compared tumor-derived mutations detected in BAL samples from subjects with non-small cell lung cancer (NSCLC) to those detected in matched plasma samples. Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq) was used to genotype DNA purified from BAL, plasma, and tumor samples from patients with NSCLC. The characteristics of cell-free DNA (cfDNA) isolated from BAL fluid were first characterized to optimize the technical approach. Somatic mutations identified in tumor were then compared with those identified in BAL and plasma, and the potential of BAL cfDNA analysis to distinguish lung cancer patients from risk-matched controls was explored. In total, 200 biofluid and tumor samples from 38 cases and 21 controls undergoing BAL for lung cancer evaluation were profiled. More tumor variants were identified in BAL cfDNA than plasma cfDNA in all stages (P < 0.001) and in stage I to II disease only. Four of 21 controls harbored low levels of cancer-associated driver mutations in BAL cfDNA [mean variant allele frequency (VAF) = 0.5%], suggesting the presence of somatic mutations in nonmalignant airway cells. Finally, using a Random Forest model with leave-one-out cross-validation, an exploratory BAL genomic classifier identified lung cancer with 69% sensitivity and 100% specificity in this cohort and detected more cancers than BAL cytology. Detecting tumor-derived mutations by targeted sequencing of BAL cfDNA is technically feasible and appears to be more sensitive than plasma profiling. Further studies are required to define optimal diagnostic applications and clinical utility. SIGNIFICANCE: Hybrid-capture, targeted deep sequencing of lung cancer mutational burden in cell-free BAL fluid identifies more tumor-derived mutations with increased allele frequencies compared with plasma cell-free DNA. See related commentary by Rolfo et al., p. 2826.

Inferring gene expression from cell-free DNA fragmentation profiles.

Profiling of circulating tumor DNA (ctDNA) in the bloodstream shows promise for noninvasive cancer detection. Chromatin fragmentation features have previously been explored to infer gene expression profiles from cell-free DNA (cfDNA), but current fragmentomic methods require high concentrations of tumor-derived DNA and provide limited resolution. Here we describe promoter fragmentation entropy as an epigenomic cfDNA feature that predicts RNA expression levels at individual genes. We developed 'epigenetic expression inference from cell-free DNA-sequencing' (EPIC-seq), a method that uses targeted sequencing of promoters of genes of interest. Profiling 329 blood samples from 201 patients with cancer and 87 healthy adults, we demonstrate classification of subtypes of lung carcinoma and diffuse large B cell lymphoma. Applying EPIC-seq to serial blood samples from patients treated with PD-(L)1 immune-checkpoint inhibitors, we show that gene expression profiles inferred by EPIC-seq are correlated with clinical response. Our results indicate that EPIC-seq could enable noninvasive, high-throughput tissue-of-origin characterization with diagnostic, prognostic and therapeutic potential.

Intratumoral plasma cells predict outcomes to PD-L1 blockade in non-small cell lung cancer.

Inhibitors of the programmed cell death-1 (PD-1/PD-L1) signaling axis are approved to treat non-small cell lung cancer (NSCLC) patients, based on their significant overall survival (OS) benefit. Using transcriptomic analysis of 891 NSCLC tumors from patients treated with either the PD-L1 inhibitor atezolizumab or chemotherapy from two large randomized clinical trials, we find a significant B cell association with extended OS with PD-L1 blockade, independent of CD8+ T cell signals. We then derive gene signatures corresponding to the dominant B cell subsets present in NSCLC from single-cell RNA sequencing (RNA-seq) data. Importantly, we find increased plasma cell signatures to be predictive of OS in patients treated with atezolizumab, but not chemotherapy. B and plasma cells are also associated with the presence of tertiary lymphoid structures and organized lymphoid aggregates. Our results suggest an important contribution of B and plasma cells to the efficacy of PD-L1 blockade in NSCLC.

Single-cell analysis of human non-small cell lung cancer lesions refines tumor classification and patient stratification.

Immunotherapy is a mainstay of non-small cell lung cancer (NSCLC) management. While tumor mutational burden (TMB) correlates with response to immunotherapy, little is known about the relationship between the baseline immune response and tumor genotype. Using single-cell RNA sequencing, we profiled 361,929 cells from 35 early-stage NSCLC lesions. We identified a cellular module consisting of PDCD1+CXCL13+ activated T cells, IgG+ plasma cells, and SPP1+ macrophages, referred to as the lung cancer activation module (LCAMhi). We confirmed LCAMhi enrichment in multiple NSCLC cohorts, and paired CITE-seq established an antibody panel to identify LCAMhi lesions. LCAM presence was found to be independent of overall immune cell content and correlated with TMB, cancer testis antigens, and TP53 mutations. High baseline LCAM scores correlated with enhanced NSCLC response to immunotherapy even in patients with above median TMB, suggesting that immune cell composition, while correlated with TMB, may be a nonredundant biomarker of response to immunotherapy.

Detecting Liquid Remnants of Solid Tumors: Circulating Tumor DNA Minimal Residual Disease.

Growing evidence demonstrates that circulating tumor DNA (ctDNA) minimal residual disease (MRD) following treatment for solid tumors predicts relapse. These results suggest that ctDNA MRD could identify candidates for adjuvant therapy and measure response to such treatment. Importantly, factors such as assay type, amount of ctDNA release, and technical and biological background can affect ctDNA MRD results. Furthermore, the clinical utility of ctDNA MRD for treatment personalization remains to be fully established. Here, we review the evidence supporting the value of ctDNA MRD in solid cancers and highlight key considerations in the application of this potentially transformative biomarker. SIGNIFICANCE: ctDNA analysis enables detection of MRD and predicts relapse after definitive treatment for solid cancers, thereby promising to revolutionize personalization of adjuvant and consolidation therapies.

The landscape of tumor cell states and ecosystems in diffuse large B cell lymphoma.

Biological heterogeneity in diffuse large B cell lymphoma (DLBCL) is partly driven by cell-of-origin subtypes and associated genomic lesions, but also by diverse cell types and cell states in the tumor microenvironment (TME). However, dissecting these cell states and their clinical relevance at scale remains challenging. Here, we implemented EcoTyper, a machine-learning framework integrating transcriptome deconvolution and single-cell RNA sequencing, to characterize clinically relevant DLBCL cell states and ecosystems. Using this approach, we identified five cell states of malignant B cells that vary in prognostic associations and differentiation status. We also identified striking variation in cell states for 12 other lineages comprising the TME and forming cell state interactions in stereotyped ecosystems. While cell-of-origin subtypes have distinct TME composition, DLBCL ecosystems capture clinical heterogeneity within existing subtypes and extend beyond cell-of-origin and genotypic classes. These results resolve the DLBCL microenvironment at systems-level resolution and identify opportunities for therapeutic targeting (https://ecotyper.stanford.edu/lymphoma).

Short Diagnosis-to-Treatment Interval Is Associated With Higher Circulating Tumor DNA Levels in Diffuse Large B-Cell Lymphoma.

PURPOSE: Patients with Diffuse Large B-cell Lymphoma (DLBCL) in need of immediate therapy are largely under-represented in clinical trials. The diagnosis-to-treatment interval (DTI) has recently been described as a metric to quantify such patient selection bias, with short DTI being associated with adverse risk factors and inferior outcomes. Here, we characterized the relationships between DTI, circulating tumor DNA (ctDNA), conventional risk factors, and clinical outcomes, with the goal of defining objective disease metrics contributing to selection bias. PATIENTS AND METHODS: We evaluated pretreatment ctDNA levels in 267 patients with DLBCL treated across multiple centers in Europe and the United States using Cancer Personalized Profiling by Deep Sequencing. Pretreatment ctDNA levels were correlated with DTI, total metabolic tumor volumes (TMTVs), the International Prognostic Index (IPI), and outcome. RESULTS: Short DTI was associated with advanced-stage disease (P < .001) and higher IPI (P < .001). We also found an inverse correlation between DTI and TMTV (RS = -0.37; P < .001). Similarly, pretreatment ctDNA levels were significantly associated with stage, IPI, and TMTV (all P < .001), demonstrating that both DTI and ctDNA reflect disease burden. Notably, patients with shorter DTI had higher pretreatment ctDNA levels (P < .001). Pretreatment ctDNA levels predicted short DTI independent of the IPI (P < .001). Although each risk factor was significantly associated with event-free survival in univariable analysis, ctDNA level was prognostic of event-free survival independent of DTI and IPI in multivariable Cox regression (ctDNA: hazard ratio, 1.5; 95% CI [1.2 to 2.0]; IPI: 1.1 [0.9 to 1.3]; -DTI: 1.1 [1.0 to 1.2]). CONCLUSION: Short DTI largely reflects baseline tumor burden, which can be objectively measured using pretreatment ctDNA levels. Pretreatment ctDNA levels therefore have utility for quantifying and guarding against selection biases in prospective DLBCL clinical trials.

Global analysis of shared T cell specificities in human non-small cell lung cancer enables HLA inference and antigen discovery.

To identify disease-relevant T cell receptors (TCRs) with shared antigen specificity, we analyzed 778,938 TCRß chain sequences from 178 non-small cell lung cancer patients using the GLIPH2 (grouping of lymphocyte interactions with paratope hotspots 2) algorithm. We identified over 66,000 shared specificity groups, of which 435 were clonally expanded and enriched in tumors compared to adjacent lung. The antigenic epitopes of one such tumor-enriched specificity group were identified using a yeast peptide-HLA A∗02:01 display library. These included a peptide from the epithelial protein TMEM161A, which is overexpressed in tumors and cross-reactive epitopes from Epstein-Barr virus and E. coli. Our findings suggest that this cross-reactivity may underlie the presence of virus-specific T cells in tumor infiltrates and that pathogen cross-reactivity may be a feature of multiple cancers. The approach and analytical pipelines generated in this work, as well as the specificity groups defined here, present a resource for understanding the T cell response in cancer.

Patterns of transcription factor programs and immune pathway activation define four major subtypes of SCLC with distinct therapeutic vulnerabilities.

Despite molecular and clinical heterogeneity, small cell lung cancer (SCLC) is treated as a single entity with predictably poor results. Using tumor expression data and non-negative matrix factorization, we identify four SCLC subtypes defined largely by differential expression of transcription factors ASCL1, NEUROD1, and POU2F3 or low expression of all three transcription factor signatures accompanied by an Inflamed gene signature (SCLC-A, N, P, and I, respectively). SCLC-I experiences the greatest benefit from the addition of immunotherapy to chemotherapy, while the other subtypes each have distinct vulnerabilities, including to inhibitors of PARP, Aurora kinases, or BCL-2. Cisplatin treatment of SCLC-A patient-derived xenografts induces intratumoral shifts toward SCLC-I, supporting subtype switching as a mechanism of acquired platinum resistance. We propose that matching baseline tumor subtype to therapy, as well as manipulating subtype switching on therapy, may enhance depth and duration of response for SCLC patients.

Single cell analysis reveals distinct immune landscapes in transplant and primary sarcomas that determine response or resistance to immunotherapy.

Immunotherapy fails to cure most cancer patients. Preclinical studies indicate that radiotherapy synergizes with immunotherapy, promoting radiation-induced antitumor immunity. Most preclinical immunotherapy studies utilize transplant tumor models, which overestimate patient responses. Here, we show that transplant sarcomas are cured by PD-1 blockade and radiotherapy, but identical treatment fails in autochthonous sarcomas, which demonstrate immunoediting, decreased neoantigen expression, and tumor-specific immune tolerance. We characterize tumor-infiltrating immune cells from transplant and primary tumors, revealing striking differences in their immune landscapes. Although radiotherapy remodels myeloid cells in both models, only transplant tumors are enriched for activated CD8+ T cells. The immune microenvironment of primary murine sarcomas resembles most human sarcomas, while transplant sarcomas resemble the most inflamed human sarcomas. These results identify distinct microenvironments in murine sarcomas that coevolve with the immune system and suggest that patients with a sarcoma immune phenotype similar to transplant tumors may benefit most from PD-1 blockade and radiotherapy.

Noninvasive Early Identification of Therapeutic Benefit from Immune Checkpoint Inhibition.

Although treatment of non-small cell lung cancer (NSCLC) with immune checkpoint inhibitors (ICIs) can produce remarkably durable responses, most patients develop early disease progression. Furthermore, initial response assessment by conventional imaging is often unable to identify which patients will achieve durable clinical benefit (DCB). Here, we demonstrate that pre-treatment circulating tumor DNA (ctDNA) and peripheral CD8 T cell levels are independently associated with DCB. We further show that ctDNA dynamics after a single infusion can aid in identification of patients who will achieve DCB. Integrating these determinants, we developed and validated an entirely noninvasive multiparameter assay (DIREct-On, Durable Immunotherapy Response Estimation by immune profiling and ctDNA-On-treatment) that robustly predicts which patients will achieve DCB with higher accuracy than any individual feature. Taken together, these results demonstrate that integrated ctDNA and circulating immune cell profiling can provide accurate, noninvasive, and early forecasting of ultimate outcomes for NSCLC patients receiving ICIs.

KEAP1/NFE2L2 Mutations Predict Lung Cancer Radiation Resistance That Can Be Targeted by Glutaminase Inhibition.

Tumor genotyping is not routinely performed in localized non-small cell lung cancer (NSCLC) due to lack of associations of mutations with outcome. Here, we analyze 232 consecutive patients with localized NSCLC and demonstrate that KEAP1 and NFE2L2 mutations are predictive of high rates of local recurrence (LR) after radiotherapy but not surgery. Half of LRs occurred in tumors with KEAP1/NFE2L2 mutations, indicating that they are major molecular drivers of clinical radioresistance. Next, we functionally evaluate KEAP1/NFE2L2 mutations in our radiotherapy cohort and demonstrate that only pathogenic mutations are associated with radioresistance. Furthermore, expression of NFE2L2 target genes does not predict LR, underscoring the utility of tumor genotyping. Finally, we show that glutaminase inhibition preferentially radiosensitizes KEAP1-mutant cells via depletion of glutathione and increased radiation-induced DNA damage. Our findings suggest that genotyping for KEAP1/NFE2L2 mutations could facilitate treatment personalization and provide a potential strategy for overcoming radioresistance conferred by these mutations. SIGNIFICANCE: This study shows that mutations in KEAP1 and NFE2L2 predict for LR after radiotherapy but not surgery in patients with NSCLC. Approximately half of all LRs are associated with these mutations and glutaminase inhibition may allow personalized radiosensitization of KEAP1/NFE2L2-mutant tumors.This article is highlighted in the In This Issue feature, p. 1775.

Integrating genomic features for non-invasive early lung cancer detection.

Radiologic screening of high-risk adults reduces lung-cancer-related mortality1,2; however, a small minority of eligible individuals undergo such screening in the United States3,4. The availability of blood-based tests could increase screening uptake. Here we introduce improvements to cancer personalized profiling by deep sequencing (CAPP-Seq)5, a method for the analysis of circulating tumour DNA (ctDNA), to better facilitate screening applications. We show that, although levels are very low in early-stage lung cancers, ctDNA is present prior to treatment in most patients and its presence is strongly prognostic. We also find that the majority of somatic mutations in the cell-free DNA (cfDNA) of patients with lung cancer and of risk-matched controls reflect clonal haematopoiesis and are non-recurrent. Compared with tumour-derived mutations, clonal haematopoiesis mutations occur on longer cfDNA fragments and lack mutational signatures that are associated with tobacco smoking. Integrating these findings with other molecular features, we develop and prospectively validate a machine-learning method termed 'lung cancer likelihood in plasma' (Lung-CLiP), which can robustly discriminate early-stage lung cancer patients from risk-matched controls. This approach achieves performance similar to that of tumour-informed ctDNA detection and enables tuning of assay specificity in order to facilitate distinct clinical applications. Our findings establish the potential of cfDNA for lung cancer screening and highlight the importance of risk-matching cases and controls in cfDNA-based screening studies.