Abstract INTRODUCTION: Liver disease occurs on a continuum from steatosis to fibrosis, cirrhosis and ultimately hepatocellular carcinoma (HCC), with a 30% lifetime risk of HCC among those with cirrhosis (LCr). If identified early, steatosis and fibrosis are potentially reversible, and in LCr, surveillance can reduce cancer morbidity and mortality. Despite these benefits, conventional LCr detection modalities are invasive or have limited performance. We previously demonstrated that cost-effective liquid biopsies of genome-wide cell-free DNA (cfDNA) fragmentomes enable early detection of HCC. Here, we use these technologies to detect liver steatosis, fibrosis, and cirrhosis towards improved pre-cancer intervention and HCC surveillance. METHODS: We performed low-coverage, whole genome sequencing of plasma cfDNA from separate Discovery (n=423) and Validation (n=221) cohorts including individuals with no known liver disease (n=397), chronic liver disease and early fibrosis (n=91) including viral hepatitis and metabolic associated steatotic liver disease, or advanced fibrosis/cirrhosis (n=156). We computed genome-wide fragment length, coverage, and repeat element features (DELFI and ARTEMIS), cross-validated a machine learning classifier for fibrosis and LCr detection in the Discovery Cohort and evaluated the locked model in the Validation Cohort. We then performed whole methylome sequencing (n=28) and cell-type deconvolution to reveal mechanisms of change to cfDNA fragmentomes in LCr. RESULTS: Individuals with early liver disease/fibrosis and advanced fibrosis/cirrhosis were detected with high performance (AUC=0.90, 95% CI=0.86-0.95 and AUC=0.95, 95% CI=0.93-0.98, respectively) in the Discovery Cohort. At an 80% specificity locked cutpoint, Validation Cohort sensitivity was 70.8% (90% CI=52.3%-87.5%) for early liver disease/fibrosis and 90.1% (90% CI=84.4%-94.4%) for advanced fibrosis/cirrhosis. The model displayed low cross-reactivity for other fibrotic origin conditions including benign lung nodules or chronic pancreatitis (median scores 0.087 and 0.068 respectively vs. 0.55 for LCr, p<0.0002). The approach outperformed the existing fibrosis index FIB-4, detecting 5.07x (95% CI=3.03-17.35) and 1.2x (95% CI=1.18-1.32) more cases of early liver disease/fibrosis and advanced fibrosis/cirrhosis in simulations. cfDNA methylome deconvolution revealed increased contributions of liver endothelium (p=0.00016) and blood monocytes (p=5.2x10-5) and decreased contribution of hepatocytes (p=0.00035) with shorter fragment lengths in LCr. CONCLUSIONS: A cfDNA fragmentome biomarker enabled early detection of liver disease including LCr and reflected both liver-derived and immune-cell related changes. These analyses may enable accessible early detection of pre-cancer conditions with potential to improve liver disease management and early detection of HCC. Citation Format: Akshaya Vijaya Annapragada, Zachariah Foda, Hope Orjuela, Carter Norton, Shashi Koul, Noushin Niknafs, Sarah Short, Keerti Boyapati, Adrianna Bartolomucci, Dimitrios Mathios, Michael Noe, Chris Cherry, Jacob Carey, Alessandro Leal, Bryan Chesnick, Nic Dracopoli, Jamie Medina, Nicholas Vulpescu, Daniel Bruhm, Sarah Bacus, Vilmos Adleff, Amy Kim, Steve Baylin, Greg Kirk, Andrei Sorop, Razvan Iacob, Speranta Iacob, Liana Gheorghe, Simona Dima, Katherine McGlynn, Manuel Ramirez-Zea, Claus Feltoft, Julia Johansen, John Groopman, Jillian Phallen, Rob Scharpf, Victor Velculescu. Non-invasive early detection of cancer-predisposing liver diseases using genome-wide cfDNA fragmentomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4074.

Abstract Background: Timely assessment of response to immune checkpoint inhibition (ICI) is critical but often limited by the heterogeneity of radiographic responses. Mutation-based analyses of cell-free DNA (cfDNA) circumvent these challenges, but are, in turn, prone to artifacts arising from clonal hematopoiesis. Plasma cfDNA fragmentome analyses using low-pass whole genome sequencing (WGS) may enable a scalable approach to evaluate systemic tumor burden in a tumor- and mutation-naïve manner. Methods: cfDNA fragmentome analyses were performed following low-pass WGS of 244 plasma samples from 62 patients treated with pembrolizumab +/- radiotherapy (NCT02492568). A locked instance of the DELFI-TF, a random forest regression model based on genome-wide fragmentation patterns and aneuploidy, was applied to determine cfDNA fragmentome-based estimates of tumor fraction. The 95th percentile of tumor fraction in a non-cancer reference set established the limit of blank (LOB). Fragmentome-based landmark molecular response was defined as ctDNA below LOB at 6 weeks. Baseline tumor samples (n=24) patients were analyzed by RNA sequencing to characterize transcriptomic profiles stratified by cfDNA fragmentome profiles. Clinical outcomes were evaluated by RECIST 1.1 (at 6 and 12 weeks), progression-free survival (PFS), and overall survival (OS). Results: At baseline, DELFI-TF values were correlated with radiographic tumor burden (R=0.32, P=0.017). Notably, tumors from patients with high DELFI-TF showed an enrichment in gene sets related to cell cycle, DNA replication and repair (adjusted P<0.05), suggesting that fragmentome TF accurately captured cellular turnover. At 6 weeks, 73% (8 out of 11) of patients with radiographic response attained fragmentome molecular response, while the subset of patients with radiographically stable or progressive disease was more heterogeneous in their fragmentome molecular response (24 out of 50, 48%). Fragmentome molecular response was more concordant with best overall response (BOR) at 12 weeks (Fisher’s exact P=0.018). Analysis of baseline tumors from patients achieving fragmentome molecular response revealed an inflamed tumor microenvironment (adjusted P-value <0.001). Among patients with stable or progressive disease at the first radiographic evaluation, fragmentome response predicted longer PFS (logrank P=0.0096) and OS (logrank P=0.012). Similarly, fragmentome molecular response predicted PFS (logrank P=7.4e-5) and OS (logrank P=0.00028) across the entire cohort. Conclusions: Plasma cfDNA fragmentome-derived tumor fraction reflects cellular turnover and lung cancer biology within the context of immunotherapy, while also enabling reliable, cost-effective, and scalable molecular response evaluations. Citation Format: Noushin Niknafs, Lavanya Sivapalan, Bahar Alipanahi, Gavin Pereira, Amna Jamali, Jaime Wehr, Daniel Rabizadeh, Christopher Cherry, Bryan Chesnick, Nicholas C. Dracopoli, Jamie Medina, Stephen Cristiano, Willemijn S. Theelen, Robert Scharpf, Lorenzo Rinaldi, Victor E. Velculescu, Valsamo (Elsa) Anagnostou. Cell-free DNA fragmentomes capture response to immuno-radiotherapy in metastatic non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1134.

Abstract Background: Circulating tumor DNA (ctDNA) has become a key biomarker for minimally invasive detection of residual disease during neoadjuvant immunotherapy. However, its integration into routine clinical decision-making remains limited by modest sensitivity and the practical constraints of current assays. Methods: We performed multi-modality matched tumor, white blood cell (WBC), and cell-free DNA (cfDNA) next-generation sequencing (NGS) of 520 biospecimens (56 tumor, 105 WBC, 359 plasma) from 32 patients with operable gastroesophageal (GE) cancer (NCT03044613) and 30 patients with resectable pleural mesothelioma (PM; NCT03918252). ctDNA residual disease analyses were performed at baseline, before each cycle of neoadjuvant immunotherapy, and preoperatively. For the tumor-informed approach, whole genome sequencing (WGS) data of matched tumor, WBC, and cfDNA (80x, 40x, and 30x coverage) were integrated through a random forest machine learning model and calibrated using a reference set of noncancerous cfDNA to determine cfDNA tumor fraction (TF). In parallel, we performed orthogonal tumor-naïve, fixed-gene-panel targeted error-correction NGS of cfDNA and WBC (30,000x), filtering germline and clonal hematopoiesis variants. Results: Overall, the tumor-informed assay showed significantly higher sensitivity, evidenced by a higher ctDNA detection rate at all evaluated timepoints compared to the tumor-naïve assay. In the GE cohort, the tumor-informed assay detected ctDNA for 22 of 25 (88%), 20 of 25 (80%), 18 of 26 (69%), and 5 of 21 (24%) patients at baseline, cycle 2, cycle 3, and preoperatively, respectively. By contrast, 13 of 30 (43%), 12 of 30 (40%), 11 of 30 (37%), and 5 of 25 (20%) had detectable ctDNA by the tumor-naïve assay at corresponding timepoints. In detectable cases, cfDNA TFs were highly concordant between approaches (R = 0.85, p < 0.001). In the PM cohort, 12 of 26 (46%), 11 of 25 (44%), 7 of 21 (33%), and 13 of 25 (52%) had ctDNA detected by the tumor-informed assay at baseline, cycle 2, cycle 3, and preoperatively, respectively. Having demonstrated higher analytical sensitivity with the tumor-informed approach in the GE cohort, we applied the tumor-naïve approach only in cases with detectable ctDNA by the tumor-informed assay. Of these, 6 of 14 (43%), 6 of 13 (46%), 5 of 8 (63%), and 7 of 15 (47%) had ctDNA detected at corresponding timepoints. cfDNA TFs were concordant at timepoints when ctDNA was detectable by both approaches (R = 0.63, p = 0.002). Tumor-informed ctDNA residual disease preoperatively was associated with shorter progression-free survival (log-rank, p = 0.0059). Conclusion: Tumor-informed WGS-based liquid biopsies reliably measure ctDNA residual disease during neoadjuvant immunotherapy, demonstrating greater sensitivity compared to a tumor-naïve approach, supporting their clinical value. Citation Format: Paul K. Lee, Blair V. Landon, Ezgi Oner, Jaime Wehr, Qiong Meng, Amna Jamali, Mimi Najjar, Gavin Pereira, Samira Hosseini-Nami, Rachel Keogh, Chen Hu, Ronan J. Kelly, Joshua E. Reuss, Patrick M. Forde, Mark Sausen, Vincent K. Lam, Robert B. Scharpf, Noushin Niknafs, Valsamo (Elsa) Anagnostou. Analytical and clinical sensitivity of tumor-informed and tumor-naïve ctDNA residual disease detection during neoadjuvant immune checkpoint inhibition in resectable cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1129.

Abstract Introduction: Overcoming immunotherapy (IO) failure in advanced NSCLC requires distinguishing molecular features of acquired vs primary resistance. Here we used comprehensive multi-omic analyses to define resistance mechanisms. Methods: Analyses were performed on the largest-to-date IO resistance cohort: 1819 biospecimens from 892 patients with NSCLC (371 primary, 521 acquired) from the Phase 2 HUDSON study (NCT03334617) of combination regimens after progression on anti-PD-(L)1/chemotherapy. Targeted next-generation sequencing of unpaired tumor biopsies pre- (n=226) and post-IO (n=497) was used to profile mutation signatures, clonality, aneuploidy and genomic instability. Post-IO tumor burden was assessed by plasma ctDNA (n=445); peripheral T-cell repertoires were analyzed (n=335). Bulk (n=205) and single-cell (sc, n=75) RNA sequencing (RNAseq) of unpaired post-IO tumor biopsies enabled gene set enrichment analysis (GSEA) and high-resolution cell type annotation. Results: Comprehensive analyses revealed acquisition of sub-clonal genomic alterations at the time of acquired resistance. Despite lower systemic ctDNA burden at progression, activating FGF10 and RICTOR and inactivating RBM10 and MSH6 mutations were enriched in acquired vs primary resistance (false discovery rate [FDR] p<0.05). Post-IO tumors harbored more CDK4, CDK6, and CD22 activating mutations, and inactivating mutations in KDM6A, SMARCA4, and CDKN2A (FDR p<0.05). Genomic instability (increased homologous recombination deficiency signatures, elevated large-scale transitions, telomeric allelic imbalance) was noted in both primary and acquired resistant tumors. Bulk RNAseq GSEA detected upregulation of epithelial-to-mesenchymal transition (EMT), IFNγ response, and inflammatory pathways (FDR p<0.05) in acquired vs primary resistant tumors. Single-cell transcriptomics showed enrichment of tumor-reactive, tissue-resident memory CD8+ T-cell clusters in acquired resistant tumors. Notably, a naïve/stem-like CD8+ T-cell cluster was also enriched in acquired resistant tumors. GSEA in early, central, and tissue-resident memory CD4+ T-cell clusters revealed an upregulation of naïve/stem-like gene sets and a downregulation of antigen processing/presentation gene sets in acquired resistant tumors. scRNAseq and differential expression analysis of epithelial populations highlighted pronounced EMT activation, increased lineage plasticity, and neuroendocrine differentiation gene signatures, implicating cellular reprogramming and phenotypic plasticity as potential contributors to acquired IO resistance. Conclusion: Acquired IO resistance in NSCLC involves dynamic and unique genomic and transcriptomic remodeling, encompassing EMT, lineage plasticity, stem-like programs, and immune reprogramming—highlighting potential avenues for therapeutic intervention. Citation Format: Archana Balan, Sonia Iyer, James Conway, Christopher Cherry, Noushin Niknafs, Mohamed Reda Keddar, Avinash Reddy, Robert McEwen, James White, Grace Kim, Anissa Dallmann, Nima Boluriaan, Sreeharsha Gunda, Mark Awad, Glenwood Goss, Se-Hoon Lee, Keunchil Park, Martin Reck, Michael Thomas, Rachel Karchin, Jane Peters, John F. Kurland, Giuseppe Galletti, Simon T. Barry, Jan Cosaert, J. Carl Barrett, Benjamin Besse, John V. Heymach, Patrick M. Forde, Valsamo Anagnostou. Multi-modal multi-omic analyses reveal mechanisms of immunotherapy resistance in non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr CT233.

Abstract Introduction: Circulating tumor DNA (ctDNA) has been established as a promising biomarker for detecting residual disease and monitoring therapy response across the cancer care continuum, however its utility to assess response to neoadjuvant immune checkpoint inhibition (ICI) is still being investigated. We utilized a tumor-informed cell-free DNA (cfDNA) whole genome sequencing (WGS) approach to molecularly monitor tumor burden dynamics and link clinical outcomes with transcriptomic assessment of baseline tumors based on ctDNA status in gastroesophageal cancer treated with neoadjuvant ICI. Methods: We performed WGS on tumor (n=28), matched white blood cell (WBC, n=28), and serial plasma samples (n=97) from patients with resectable gastroesophageal cancer treated with neoadjuvant ICI and chemoradiation prior to surgical resection (NCT03044613). Tumor and WBC sequence data was used to identify tumor-specific single nucleotide variants (SNVs). High quality SNVs were utilized to determine the presence of ctDNA in plasma through a random forest machine learning model. ctDNA status and tumor fraction (TF) were assessed across four timepoints (baseline, post-ICI cycle 1, post-ICI cycle 2, and pre-operatively). Results were compared with tumor-naïve gene panel ctDNA targeted NGS (n=32 patients, n=152 samples) and correlated with clinical outcomes. Additionally, bulk RNA sequencing (RNAseq) was performed on baseline tumor samples (n=28) and utilized for gene set enrichment analyses (GSEA). Results: Patients with pre-operative ctDNA TF above the median had a significantly shorter overall survival (OS) compared to those with ctDNA TF below the median or undetectable ctDNA (logrank p < 0.0001). Among patients who were undetectable pre-operatively via the tumor-naïve targeted NGS panel, individuals with detectable ctDNA via the tumor-informed WGS approach attained a numerically shorter OS compared to individuals with undetectable ctDNA (logrank p=0.1). Similarly, patients with ≥95% reduction in cfDNA TF pre-operatively had longer recurrence-free (logrank p=0.002) and OS (logrank p= 0.0001). GSEA of RNAseq data from baseline tumors revealed enrichment of G2/M cell cycle checkpoint and E2F targets in tumors from individuals with detectable baseline ctDNA via the tumor-informed WGS approach (FDR-adjusted p=2.31e-12), suggesting that cfDNA TF detection captures cellular turnover in the blood stream. The upregulation of proliferation and cell cycle progression-associated gene sets was not observed in stratified analyses by ctDNA detection utilizing the tumor-naïve approach. Conclusions: Tumor-informed cfDNA WGS analyses accurately capture tumor burden dynamics and cellular turnover during neoadjuvant immunotherapy, opening a window of opportunity for further therapeutic intervention and optimization. Citation Format: Blair V. Landon, Jaime Wehr, Rachel Keogh, Noushin Niknafs, Christopher Cherry, Nisha Rao, Gavin Pereira, Mark Sausen, Richard J. Battafarano, Stephen C. Yang, Stephen Broderick, Jinny Ha, Russell K. Hales, K. Ranh Voong, Kristen A. Marrone, Chen Hu, Josephine L. Feliciano, Ali H. Zaidi, Ronan J. Kelly, Vincent K. Lam, Valsamo (Elsa) K. Anagnostou. Longitudinal tumor-informed cell-free DNA whole genome sequencing coupled with transcriptomic analysis captures tumor burden dynamics in resectable gastroesophageal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1140.

Accessible liquid biopsies, including analyses of genome-wide cell-free DNA (cfDNA) fragmentation, are emerging for early detection of cancer but remain largely unexplored in other diseases. Here, we used whole-genome sequencing to examine cfDNA fragmentomes in 1576 individuals, including those with liver disease or with other morbidities such as vascular, autoimmune, and neurodegenerative conditions. As a prototype for disease-specific cfDNA fragmentomic biomarkers, we developed a machine learning classifier that detected early liver disease, advanced fibrosis, and cirrhosis with high sensitivity in separate discovery (n = 423) and validation cohorts (n = 221) and had limited cross-reactivity for other diseases. Genome-wide fragmentome and methylome analyses revealed liver-derived and immune-mediated changes in cfDNA in the circulation of individuals affected with liver disease. Fragmentomic changes were also observed across a range of other human morbidities and reflected disease-specific changes in the circulation. A machine learning model using cfDNA fragmentomes predicted overall survival in separate morbidity discovery (n = 571) and validation cohorts (n = 231). These analyses demonstrate the connection between cfDNA fragmentomes and an individual's physiologic state and provide previously unrecognized possibilities for cfDNA liquid biopsies across human disease.

Abstract Introduction: Spatial and temporal heterogeneity and evolution of the T cell receptor (TCR) landscape may influence immunotherapy outcomes. Studying TCR repertoires and their antigen specificities, together with T cell dynamics, could provide important insights into the quality of anti-tumor immune responses and their impact on cancer immunotherapy effectiveness. Methods: We performed serial and multi-region sampling at critical timepoints (n=52 samples) during the clinical course of 7 patients with metastatic lung and head and neck cancer, who received immunotherapy-containing regimens. To study the TCR repertoire, we performed bulk TCR sequencing of TCR-β CDR3 regions and recovered 69,135 unique productive clones (ImmunoSeq assay, Adaptive Biotechnologies). To determine differential responses to immunotherapy across tumor sites and patients, we evaluated TCR repertoire similarity using Morisita’s overlap index (MI) based on clonotype frequency and identity. Next, we clustered TCR clones of similar antigen specificities using GIANA (15,476 clusters) and performed differential T cell abundance analyses at the clone- and cluster-level to assess T cell dynamics. Statistically significant TCR expansions and regressions were determined using Fisher’s exact test (p≤0.05). We used the VDJDB and CEDAR databases to de-orphanize 2,019 TCR clusters, mapping to TCRs with known antigen specificities. Results: We found significant TCR repertoire heterogeneity between patients (MIbetween patients ≈ 0), suggesting private TCR landscapes at a patient level. In individual patients, a higher heterogeneity was noted between primary and metastatic sites (MIbaseline-metastatic = 0.01 - 0.34) while TCR repertoires of metastatic sites from proximal anatomic locations shared similarities (MIproximal metastasis = 0.47 - 0.96). Despite the largely private expanded clusters and clones in each patient, similar dynamics were observed in regressing compared to progressing tumors in the context of immunotherapy, while at the autopsy sites, significantly expanded clusters dominated their respective TCR repertoires (14-28% abundance). TCR sequences recognizing viral antigen epitopes, such as GLCTLVAML (EBV), KLGGALQAK (CMV) and RAKFKQLL (EBV) were identified across patients and were part of the significantly expanded clusters in progressing metastatic sites. Several significantly expanding TCR clusters (FDR p≤0.05) were detected; while these had unmapped antigen specificity, they could recognize tumor antigens and mutation-associated neoantigens relevant for mounting anti-tumor immune responses. Conclusions: Spatial and temporal TCR repertoire analyses provided a better understanding of the patients’ immune landscape during therapy and offered insights that can be used to further dissect the heterogeneity of adaptive immune responses in the context of immunotherapy. Citation Format: Asimina Zoitou, Stefan Velculescu, Gavin Pereira, Archana Balan, Mimi Najjar, Amna Jamali, James R. White, Rachel Karchin, Noushin Niknafs, Hyunseok Kang, Patrick M. Forde, Christine L. Hann, Jody E. Hooper, Julie R. Brahmer, Valsamo (Elsa) Anagnostou. Longitudinal and spatial heterogeneity of intra-tumoral TCR repertoires during immunotherapy in metastatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7424.

Circulating tumor DNA (ctDNA) analyses are informative as an early indicator of immunotherapy response in advanced non-small cell lung cancer (NSCLC); however, the clinical value of ctDNA molecular response requires further validation. As part of a prospective clinical protocol ( NCT05995821 ), we conducted targeted error-correction sequencing of ctDNA (n=328) and matched WBC DNA (n=109) from 109 patients with metastatic NSCLC who received anti-PD-(L)1 either as monotherapy or in combination. Following cellular origin resolution of 2,818 variants, landmark molecular response (mR) was defined as undetectable ctDNA within 3-9 weeks of treatment initiation. Pre-treatment ctDNA burden, but not blood tumor mutation burden, predicted survival. Implementing a tumor-naïve WBC DNA-informed approach increased the number of evaluable cases without compromising the overall accuracy of landmark ctDNA molecular responses. A direct comparison of single-timepoint on-therapy ctDNA assessment with ctDNA dynamics from baseline to the 3-9-week interval, along with an analysis of heterogeneity in molecular response within the 3-9-week window, showed that undetectable ctDNA at the landmark timepoint can effectively predict survival outcomes. A significant enrichment in landmark ctDNA mR was noted among patients with progression-free survival (PFS) ≥6 months with immunotherapy (p=2.5e-05) and chemo-immunotherapy (p=0.02). Patients in the landmark mR group had longer progression-free (p=1.6e-06) and overall survival (p=2.5e-05) than those with molecular progression. Landmark ctDNA molecular response provides a real-time, accurate approach for monitoring immunotherapy clinical outcomes. Although not currently validated for regulatory use, these findings demonstrate the potential utility of ctDNA as an early endpoint in clinical trials. Employing circulating tumor DNA (ctDNA) dynamics as an early indicator of immunotherapy response requires a roadmap for the next-generation sequencing approach, definition of molecular response and establishment of its clinical sensitivity. In this study, we introduce the concept of a landmark ctDNA molecular response, determined 3-9 weeks after initiation of immunotherapy, that maximizes the number of evaluable patients without sacrificing the specificity of the approach. Notably, when evaluating heterogeneity in ctDNA detection within the landmark 3-9-week window and assessing the impact of landmark interval dynamics on survival, we found that a single ctDNA assessment performed similarly to multiple ctDNA measurements within the landmark window (most notably, regardless of whether the timepoints were concordant or discordant). Our findings demonstrate that a single assessment of early on-therapy landmark ctDNA molecular response, can identify patients at risk of disease progression and enable future intervention and therapy optimization.

Abstract Background: Immunotherapy-containing therapies have shown promise for patients with unresectable diffuse pleural mesothelioma (DPM), yet there are no reliable strategies to monitor therapy response. By expanding the compendium of cancer-associated alterations profiled, including genome-wide fragmentation patterns, cell-free DNA (cfDNA) whole genome sequencing (WGS) enables tracking of tumor dynamics. In tandem, while understudied, T cell clone dynamics may be informative in capturing early immunotherapy response. Methods: Using 314 tumor and peripheral blood biospecimes, we analyzed serial plasma cfDNA samples (n=135) from 55 patients with unresectable DPM, who received durvalumab with platinum-based chemotherapy (NCT02899195). Following cfDNA extraction and genomic library preparation, cfDNA at baseline (C1D1), Cycle 2 Day 1 (C2D1), and Cycle 5 Day 1 (C5D1) underwent low-coverage (1-2x) whole genome sequencing. Tumor- and mutation-naive estimates of tumor fraction were derived by applying the DELFI tumor score (DELFI-TS) model, which integrates genome-wide fragmentation patterns and chromosomal arm aneuploidy. In parallel, we performed TCR Vβ CDR3 next-generation sequencing on tumor (n=43) and peripheral serial blood (n=136) samples. The TCR repertoire was characterized using clonality, TCR clonotype/cluster dynamics, and the Morisita-Horn similarity index to assess repertoire similarity across samples. Clinical outcomes were assessed by radiographic response, progression-free (PFS), and overall survival (OS). Results: Patients with distant metastasis (M1) had higher DELFI-TS levels (p=0.038). At baseline, DELFI-TS levels were numerically higher for radiographic non-responders (SD/PD) vs responders (CR/PR). Using the 92th percentile of DELFI-TS in non-cancer cfDNA control samples to determine the limit of blank, patients with detectable baseline DELFI-TS (ctDNA+) had shorter PFS and OS (log-rank p<0.001). Patients that attained a radiographic response had more clonal peripheral TCR repertoires at both baseline and on-therapy timepoints compared to non-responders (p=0.037 at C1D1; p=0.007 at C2D1; p=0.042 at C5D1). Morisita-Horn similarity between C1D1 and on-therapy was lower in non-responders than responders (p=0.019 for C1D1 vs C2D1; p=0.036 for C1D1 vs C5D1). In contrast, a more diverse intra-tumoral TCR repertoire was noted for patients with an OS of 12 or more months (p=0.018). Conclusions: Our findings provide proof-of-concept that cfDNA fragmentomic analyses can quantify pre-treatment cfDNA tumor fraction that may capture clinical outcomes with chemo-immunotherapy response for patients with DPM. Longitudinal analyses of peripheral TCR repertoires can further differentiate responding from non-responding DPM, supporting the notion that joint analyses may more accurately capture immunotherapy response. Citation Format: Jinny Huang, Jennifer Li, James R. White, Shashikant Koul, Gavin Pereira, Nisha Rao, Jennie Yao, Julie R. Brahmer, Robert B. Scharpf, Rachel Karchin, Victor E. Velculescu, Zhouxin Sun, Suresh S. Ramalingam, Patrick M. Forde, Noushin Niknafs, Valsamo (Elsa) K. Anagnostou. Integrative analyses of the cfDNA fragmentome and TCR repertoires capture chemo-immunotherapy response in diffuse pleural mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 109.

Abstract Background: Circulating tumor DNA (ctDNA) analyses are informative as an early indicator of immunotherapy response in advanced non-small cell lung cancer (NSCLC), however the clinical role of ctDNA molecular response requires further validation. Methods: As part of a prospective clinical protocol (NCT05995821), we performed ctDNA (n=328) and matched white blood cell DNA (WBC; n=109) targeted 521 fixed gene panel next-generation sequencing (Elio Plasma Complete) from 109 patients with advanced/metastatic NSCLC who received anti-PD-(L)1 as monotherapy or in combination with chemotherapy. Following variant cellular origin resolution, landmark molecular response (mR) was defined as undetectable ctDNA within 3-9 weeks of treatment initiation. For a subset of patients (n=34), whole-exome sequencing of baseline tumors was performed and used in benchmarking ctDNA detection and therapy response assessment. Results: Among 2,818 plasma variants, 23% were clonal haematopoiesis-related, which confounded the interpretation of driver gene associations with clinical outcomes and assessment of molecular responses. Implementing a tumor-naïve WBC DNA-informed approach increased the number of evaluable cases while maintaining the overall accuracy of landmark ctDNA molecular responses. Pre-treatment ctDNA burden but not blood tumor mutation burden, predicted survival. Overall, 77 patients were evaluable for landmark molecular response assessment; of these, 29 patients (38%) attained a mR. Landmark evaluation of molecular response enabled evaluation of all cases, was highly specific (92%), and achieved a higher sensitivity (66%) compared to clearance or ctDNA reduction from baseline (sensitivity 59%). In predicting landmark progression-free survival (PFS) at 6 months (durable clinical benefit, DCB), the tumor-agnostic WBC-informed approach strikes a balance between sensitivity (71.4%) and specificity (100%) compared to plasma-only (sensitivity=14.3%, specificity=100%) or tumor-informed (sensitivity=78.6%, specificity=71.4%) approaches. A significant enrichment in landmark ctDNA mR was noted among patients with DCB with immunotherapy (p=2.5e-05) and chemo-immunotherapy (p=0.02). Patients in the landmark mR group attained longer PFS (p=1.6e-06) and overall survival (p=2.5e-05) compared to those with molecular progression. The association between landmark molecular response and survival remained significant (PFS and OS, P < 0.001) after accounting for clinical covariates, line of therapy, and baseline ctDNA levels. Conclusions: Our findings indicate that landmark ctDNA molecular response at 3-9 weeks on treatment provides a real-time and accurate approach for monitoring immunotherapy clinical outcomes. Although not currently validated for regulatory use, these findings demonstrate the potential utility of ctDNA as an early endpoint in clinical trials. Citation Format: Jaime Wehr, Noushin Niknafs, Lavanya Sivapalan, Archana Balan, Gavin Pereira, Samira Hosseini-Nami, Iiasha Beadles, Aliyah Pabani, Kristen Marrone, Qing K. Li, Joseph Christopher Murray, Mark Sausen, Bryan Chesnick, Lorenzo Rinaldi, Christine L. Hann, Susan Combs Scott, Josephine Feliciano, Vincent K. Lam, Benjamin Levy, Patrick M. Forde, Julie R. Brahmer, Valsamo (Elsa) Anagnostou. Clinical utility of landmark ctDNA molecular response as an early indicator of immunotherapy outcomes in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 110.

Abstract Background: Microbiome-derived metabolites have demonstrated significant impact on immunotherapy response, particularly in the context of immune checkpoint blockade (ICB). Trimethylamine-N-oxide (TMAO), an amine oxide generated when gut bacteria metabolize dietary choline, has been identified in preclinical studies to enhance anti-tumor immune responses. Here, we evaluated circulating choline metabolites with respect to clinical outcomes and circulating tumor DNA (ctDNA) residual disease in patients with resectable cancers receiving neoadjuvant ICB. Methods: Pre-treatment choline, trimethylamine (TMA) and TMAO plasma concentrations from 51 patients with stage I-III diffuse pleural mesothelioma (DPM; n = 25 patients; NCT03918252) or stage II/III esophageal/gastro-esophageal junction cancer (E/GEJ; n = 26 patients; NCT03044613) on neoadjuvant ICB, were quantified via liquid chromatography-mass spectrometry (SCIEX Triple Quad 6500+). Clinical outcomes were compared across metabolite thresholds, as were ctDNA residual disease through tumor-informed cell-free DNA (cfDNA) whole-genome sequencing (WGS). ctDNA residual disease in the DPM cohort was measured by intersecting tumor (n = 28), white blood cell (n = 28), and plasma (n = 97) WGS. Results: In the DPM cohort, median baseline choline, TMA and TMAO concentrations were 3970, 2840 and 256 ng/mL, respectively. Higher baseline TMAO levels were associated with lower ctDNA residual disease at C2D1 (Fisher’s exact, p = 0.028), C3D1 (Fisher’s exact, p = 0.057) and pre-surgery (Fisher’s exact, p = 0.01). Further, patients with higher TMAO had either undetectable ctDNA throughout the neoadjuvant window or ≥95% reduction in cfDNA tumor fraction from baseline to pre-surgery (Fisher’s exact, p = 0.028). In this cohort, ctDNA residual disease strongly correlated with progression-free and overall survival (log-rank, p < 0.05). In the E/GEJ cohort, median baseline choline, TMA and TMAO concentrations were 279, 2945 and 4175 ng/mL, respectively. Patients who attained a major pathologic response had numerically higher TMAO levels (Fisher’s exact, p = 0.078). In line with the findings in the DPM cohort, patients in the E/GEJ cohort with lower TMAO (lowest quintile; <197 ng/mL) had shorter overall survival (log-rank, p = 0.034) compared to those with higher TMAO concentration ( ≥197 ng/mL), an association that remained significant after adjusting for clinicopathological variables (Cox multivariable, p = 0.04). Conclusion: Our findings indicate that higher plasma TMAO levels may be linked to molecular ctDNA response and better clinical outcomes with neoadjuvant ICB, further supporting a potential immunomodulatory role of choline metabolites in enhancing immunotherapy response. Citation Format: Rachel J. Keogh, Paul K. Lee, N.V. Rajeshkumar, Blair V. Landon, Joshua E. Reuss, Jaime Wehr, Gavin Pereira, Amna Jamali, Noushin Niknafs, Ronan J. Kelly, Ali H. Zaidi, Josephine L. Feliciano, Julie R. Brahmer, Vincent K. Lam, Patrick M. Forde, Chi V. Dang, Valsamo Anagnostou. Elevated plasma trimethylamine-N-oxide (TMAO) levels correlate with better clinical and molecular responses in immunotherapy-treated resectable gastroesophageal cancer and pleural mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4724.

Abstract Introduction: The phase 3 randomized DREAM3R clinical trial evaluated durvalumab + chemotherapy (D+Ch) compared to chemotherapy alone (Ch) for patients with pleural mesothelioma (PM) and showed an improvement in overall response rate (ORR) with D+Ch (Clinical trial identifier: NCT04334759). The study enrolment was terminated early due to slow accrual, and the primary endpoint of overall survival (OS) was not met. Here, we report translational analyses of genomic landscapes and PD-L1 expression and their association with clinical endpoints in the DREAM3R trial. Methods: Between February 2021 and November 2023, 173 participants with unresectable PM were randomized to D+Ch (n=113) vs Ch (n=60); most (94%) had epithelioid PM. Overall survival (OS) was the primary endpoint; secondary endpoints included progression-free survival (PFS) and objective response rate (ORR; by modified RECIST for PM). Pre-treatment tumor PD-L1 expression was evaluated by immunohistochemistry (pharmDx 22C3) in participants with available tissue. Tumor-positive score (TPS) was recorded, and PD-L1+ vs PD-L1- cohorts (≥1% vs <1%) were compared for OS, PFS, and ORR. Tumor mutational burden (TMB) estimates were derived by counting sequence alterations in coding regions using whole exome sequencing (n=91 in the D+Ch and n=35 in the Ch arm). Mutations were characterized by recurrence and functional consequence, and enrichment analyses were performed for individual mutations and for mutations that converged within gene families and cancer hallmarks. Genome-wide copy number profiles were used to derive tumor aneuploidy metrics and were intersected with sequence alterations to compute mutation clonality and the number of mutations in aneuploid regions. Results: Across the entire cohort, ORR among patients with PM expressing PD-L1 ≥1% vs <1% was 65% (31/48) vs 49% (45/92; Pearson’s chi-squared p=0.077). PD-L1 expression or overall TMB was not associated with PFS or OS in the whole cohort or in the D+Ch and Ch subsets. Patients with tumors harboring a higher number of clonal multicopy mutations had longer OS (median survival 24.44 vs 17.61 months, p=0.0082). In the D+Ch arm, patients with PM who had a higher fraction of genome-wide loss of heterozygosity had a shorter OS (median survival 15.77 vs 24.57 months, p=0.0042). The effect of D+Ch vs Ch on PFS was more apparent among patients with tumors harboring inactivating BAP1 mutations (median survival 9.89 vs 6.21 months, p=0.064) and homozygous deletions in DNA damage repair genes (median survival 6.14 vs 5.75 months, p=0.069). Conversely, patients with LATS2 wild-type tumors attained longer PFS with D+Ch than with Ch alone (median survival 7.79 vs 5.9 months, p=0.027). Conclusions: Our findings suggest that durvalumab and chemotherapy warrant further investigation in biomarker-defined subsets of patients with unresectable PM. Citation Format: Valsamo (Elsa) Anagnostou, Anna K. Nowak, Chris Brown, Brett G. Hughes, Noushin Niknafs, Tracy Hoang, Jaime Wehr, Connull Leslie, Qiong Meng, Asimina Zoitou, Kennet J. O’Byrne, Wee L. Chin, Thomas John, Peter Illei, Sonia Yip, Nick Pavlakis, Julie Brahmer, Martin R. Stockler, Patrick M. Forde, Alistair Cook. Translational analyses from the randomized phase 3 DREAM3R trial: DuRvalumab with chEmotherapy as first line treAtment in advanced pleural Mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr LB105.

Abstract Introduction: Immune checkpoint inhibition (ICI) is gaining momentum as peri-operative therapy for resectable cancers, however early changes in the tumor microenvironment (TME) during and after neoadjuvant ICI have yet to be characterized. Methods: Bulk RNA sequencing (RNAseq) was performed on 71 serial tumors sampled at baseline, post 2 cycles of neoadjuvant ICI (post-ICI), and at the time of resection, in a cohort of 32 patients with resectable gastroesophageal cancer treated with neoadjuvant nivolumab or nivolumab + relatlimab, followed by concurrent ICI and chemoradiation (NCT03044613). We performed gene set enrichment analyses (GSEA) of RNAseq data, stratified by pathological response and clinical outcomes. Complete (pCR) and major pathological response (MPR) were defined as 0% and ≤10% residual tumor at resection. Results: GSEA revealed an upregulation of interferon alpha, interferon gamma, antigen processing and presentation, and pro-inflammatory M1 macrophage gene sets post-ICI compared to baseline (FDR-adjusted p < 0.006). After chemoradiation, resected tumors showed depletion of DNA repair, chromosome maintenance, cell proliferation and cell cycle progression gene sets, reflective of the cytotoxic effect of therapy (FDR-adjusted p < 10e-06). Notably, in the nivolumab + relatlimab arm, induction of adaptive immune response gene sets and TNFa signaling through NF-kB was noted in resected specimens (FDR-adjusted p < 10e-05). Among patients who attained a pCR, there was an upregulation of interferon alpha, interferon gamma, and antigen processing and presentation gene sets post-ICI (FDR adjusted p < 10e-06). Similarly, in those who attained an MPR there was an upregulation of inflammatory response, interferon gamma, and B cell receptor gene sets (FDR adjusted p < 10e-09), while cellular metabolism and oxidative phosphorylation gene sets were down regulated post-ICI (FDR adjusted p < 0.05). Linking transcriptomic profiles with disease recurrence, we found a downregulation of inflammatory related gene sets post-ICI in recurrent cases (FDR adjusted p < 0.0006). Depletion of oxidative phosphorylation, metabolism, and chromatin regulation gene sets was noted in baseline tumors of patients that attained long overall survival (FDR adjusted p < 10e-05). In post-ICI tumors, there was an upregulation of inflammatory and adaptive response gene sets in patients with longer overall survival (FDR adjusted p < 10e-08), while the oxidative phosphorylation and metabolism gene sets remained suppressed (FDR adjusted p < 0.003). Conclusions: Neoadjuvant ICI induces differential inflammatory and metabolism expression programs that are reflective of pathological responses, recurrence and survival, broadening our understanding of actionable mechanisms of ICI efficacy. Citation Format: Blair V. Landon, Christopher Cherry, Rachel Keogh, Jaime Wehr, Gavin Pereira, Noushin Niknafs, Richard J. Battafarano, Stephen C. Yang, Stephen Broderick, Jinny Ha, Russell K. Hales, K. Ranh Voong, Kristen A. Marrone, Chen Hu, Josephine L. Feliciano, Ali H. Zaidi, Ronan J. Kelly, Vincent K. Lam, Valsamo (Elsa) K. Anagnostou. Serial transcriptomic analyses capture the effects of neoadjuvant immune checkpoint inhibition in resectable gastroesophageal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2822.

Supplementary Table S1. Summary of clinical information. Supplementary Table S2. Summary of sample characteristics. Supplementary Table S3. Summary of whole-genome sequencing and fragment analyses. Supplementary Table S4. Summary of protein analyses. Supplementary Table S5. Summary of machine learning models and scores. Supplementary Table S6. Summary of performance for ovarian cancer detection.

Sequence alterations that were classified as germline (median 44.9%, range 27.3% - 73.1%) had the highest overall MAFs, compared to both tumor-derived (median 10.2%, range 0.1% - 75.7%) and clonal-hematopoiesis-derived (CH-derived) variants (median 0.8%, range 0.1 – 86.7%). Tumor-derived variants had significantly higher MAFs compared to CH-derived variants. P values were calculated using the Mann-Whitney U test. **** p < 0.0001

(A) PA scores were assessed across sequenced plasma (n=139) and matched white blood cell (n=32) samples from patients analyzed in the study cohort to determine a threshold for determination of aneuploidy in plasma. A PA score of 4.90, which was the maximum score observed in WBC DNA samples, was selected as the threshold for determination of aneuploidy in plasma. (B) Distribution of PA scores in the panel of 32 matched normal WBC samples analyzed for this study and an independent held out set of 56 WBC normal samples that were processed and analyzed using the same targeted panel and sequencing approach (Methods). The maximum PA score observed for both normal panels was 4.90, supporting the use of this value as the threshold for determination of aneuploidy in plasma. No significant differences were observed in the median PA score (indicated by dotted lines) for each normal panel. (C) Comparison between plasma aneuploidy scores and tumor fraction scores determined using an orthogonal method (ichorCNA) in matched normal DNA (blue) and plasma samples from all timepoints (yellow), plasma samples with detectable tumor-derived sequence mutations (orange) and plasma timepoints with undetectable sequence alterations (grey). Non-zero ichorCNA tumor fraction values were assigned to most samples resulting in significant overlap between the distributions of tumor fractions in plasma and matched normal DNA samples. In contrast, comparisons performed using plasma aneuploidy scores revealed significant differences in the distribution of assigned values across matched normal DNA samples (reflecting normal ploidy) and plasma samples (reflecting tumor aneuploidy). (D-I) Statistical modelling of MAF dynamics was performed using a set of known tumor-specific mutations previously characterized in Phallen et al, Science Transl Med, 2017 from a mixture of DNA from tumor cell lines spiked into unrelated wild-type DNA at dilutions ranging from 0.1-100%. Mutations in this set with replicate data were evaluated for (D, G) variability in MAF estimate followed by (E, H) calculation of the coefficient of variation (CV), and (F, I) finally projection of the relative percentage uncertainty in the MAF estimate. Analyses of mutation data from Phallen et al (2017) are shown in (D-F) and results from a simulation study of 30 mutations ranging in MAF from 0.1% to 80% using a simple statistical model of variability are shown in (G-I). Both analyses of mutation data from Phallen et al (2017) and simulation data showed a significant increase in CV as MAF decreases. For example, given a maximal MAF of 1%, the real 95% uncertainty in MAF estimate was shown to range from -50% to 50% (0.5% MAF to 1.5% MAF). Thus, a greater relative reduction is required to determine elimination of ctDNA for lower maximal MAF values. Based on these results, complete elimination of cell-free tumor load (to either 0% max MAF or undetectable PA) was deemed to be the most appropriate threshold for precise determination of molecular response.

Rearrangements of ovarian endometrioid and ovarian mucinous carcinomas identified by TRELLIS. Rearrangements present in ovarian endometrioid carcinomas CGOV161T and CGOV172T (a, b) and an ovarian mucinous tumor CGOV173T (c). Split reads that span the fusion junction are shown in black, while read pairs that reside on either side of the junction are shown in green and blue.