Supplementary Figure 1. Study design and MRD sampling scheme of the NADIM II trial. Patient numbers shown for each treatment arm indicate the number of patients for whom MRD analysis was performed.

Single-cell DEG and GSEA by cell type and extraction B&Tcells

Supplementary figure 4. Kaplan–Meier curves for (A) event-free survival (EFS) and (B) overall survival (OS) in non-pCR patients according to MRD status. MRD positivity was defined as detection at any time point. Hazard ratios (HRs) and 95% confidence intervals were estimated with Cox proportional hazards models, and P values were derived from log-rank tests, as indicated in each plot. Abbreviations: pCR, pathological complete response; MRD, minimal residual disease.

BACKGROUND:Despite the incorporation of atezolizumab, an anti-programmed death-ligand 1 (PD-L1) antibody, into first-line chemotherapy regimen, survival in patients with extensive-stage small cell lung cancer (ES-SCLC) remains poor. Given the role of dendritic cells (DCs) in modulating responses to anti-programmed cell death protein 1 (PD-1) antibodies, a promising avenue is the development of vaccines based on DCs, together with the evaluation of how DC phenotypic modifications shape treatment efficacy. MATERIALS AND METHODS:This is a single-arm, phase Ib/II, open-label clinical trial investigating the safety and activity of combining autologous tumor lysate pulsed DCs administered intradermally with intravenous atezolizumab as maintenance therapy for patients with ES-SCLC. Following leukapheresis, monocytes were differentiated ex vivo into mature DCs and loaded with irradiated autologous tumor lysate. After standard induction therapy (four cycles of carboplatin, etoposide, and atezolizumab), patients received up to six doses of mature DCs intradermally, along with intravenous atezolizumab 1,200 mg every 3 weeks, until disease progression. RESULTS:20 patients were enrolled, including six patients with brain metastases. 18 patients received DC vaccination, with a median of three doses (range 1-6). Most grade 3-4 treatment-related adverse events were hematological and related to chemotherapy. DC vaccination was well tolerated, with only grade 1-2 transient skin irritation at the injection site. Among 18 evaluable patients, 17 (94.4%) achieved an objective response. After a median follow-up of 31.4 months, progression-free survival at 2 years (PFS2y) was 20% (95% CI 6.4% to 39.1%), median overall survival (OS) 11.2 months (95% CI 6.3 to 26.0) and OS3y 22.5% (95% CI 6.8% to 43.6%). Six patients achieved long survival (median 30.5 months, range 23.4-36.2). Expansion of conventional type DC1 (cDC1s) XCR1+ and stem-like progenitor-exhausted (CXCR5+PD-1+) CD8+ T cells following chemotherapy plus atezolizumab was associated with improved PFS (HR 0.94, 95% CI 0.90 to 0.99 and HR 0.59, 95% CI 0.39 to 0.89). CONCLUSION:DC immunization combined with atezolizumab maintenance following standard chemotherapy plus atezolizumab treatment is well tolerated. Its clinical activity cannot be definitively determined in this study, as it was administered as maintenance therapy; however, the observed rate of long-term survivors suggests a potential benefit. Our findings suggest a pivotal role for cDC1s in mediating the antitumor activity in long-term survivors. TRIAL REGISTRATION NUMBER:NCT04487756.

Supplementary Figure 2. BCR repertoire dynamics.

Abstract Background: Nanomechanical properties of tumors are emerging biomarkers reflecting extracellular matrix (ECM) remodeling, immune exclusion, and aggressiveness. The AFM-based ARTIDIS platform measures nanomechanical signatures (NS) from fresh biopsies within hours, providing rapid diagnostic and prognostic information while preserving tissue for conventional analyses. Prior work showed that ARTIDIS NS distinguishes malignant from uninvolved lung tissue and predicts early postoperative recurrence. However, the relationship between NS and oncogenic drivers such as KRAS remains unclear. As KRAS influences ECM composition, matrix remodeling, and stromal activation, we evaluated the preliminary nanomechanical and microenvironmental features of KRAS-mutant NSCLC in our prospective cohort. Methods: Fresh tumor and paired uninvolved lung tissue were collected from 70 early-stage NSCLC patients undergoing curative resection. NS were measured on the ARTIDIS platform. Samples were formalin-fixed for quantitative immunohistochemistry (IHC) of α-SMA, picrosirius red, Ki-67, CD31, CD4, CD8, FOXP3, CD68, and PD-1. Clinical characteristics, driver mutations, and outcomes were recorded with a 36-month follow-up. A focused analysis was performed in KRAS-mutant cases. Results: Preliminary analyses showed that ARTIDIS NS distinguished malignant from uninvolved tissue with 88% sensitivity, 86% specificity, and 87% accuracy (n = 52 paired samples). Eleven patients carried KRAS mutations (G12A/D/F/S, G13D, Q16H). Median age was 68.5 years; 63.4% were former smokers. Most tumors were early-stage (IA-IIB), and 45.5% were poorly differentiated. Across the full cohort, 17 patients recurred, including 3 patients (27.3%) with KRAS mutations. ARTIDIS NS predicted progression within 10 months after surgery with 100% sensitivity and 93% specificity, including predictions derived from uninvolved tissue. KRAS-mutant tumors showed higher stiffness than KRAS-wild-type or driver-negative tumors. Quantitative IHC demonstrated a higher percentage of proliferating cancer cells together with increased Treg infiltration and PD-1 expression in KRAS-mutant samples compared with uninvolved lung tissue, suggesting immune exhaustion. Conclusions: This study provides the first evidence that KRAS-mutant NSCLC displays a distinct, stiffer nanomechanical phenotype. These findings support tumor stiffness as a functional characteristic of KRAS-driven biology and highlight ARTIDIS NS as a rapid biomarker complementing histopathology and molecular profiling. A 200-patient prospective validation study is underway to confirm the clinical utility of NS for risk stratification and personalized treatment planning. Citation Format: Jordi Alcaraz, Elba Marin, Héctor Sanz-Fraile, Marina Querol, Paula Gausa, Keat Neal, Marselina Arshakyan, Marc Rico-Pastó, Marc Boada, David Sanchez, Carolina Ortiz Velez, Reinier Oropesa Nunez, Gitika Srivastava, Ahmed Jizawi, Sara Nizzero, Tobias Appenzeller, Philipp Oertle, Marko Loparic, Marija Plodinec, Noemi Reguart. Tumor stiffness as a functional biomarker of KRAS-mutant NSCLC: Insights from the ARTIDIS prospective clinical cohort [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 767.

Abstract More than 20% of individuals ≥70 harbor clonal hematopoiesis (CH) mutations without overt malignancy. CH has been linked to elevated risk of non-hematologic cancers, particularly lung cancer, although causality remains unresolved. The TRACERx study showed that CH in blood and tumor predicts higher recurrence and mortality in NSCLC, and recent reports suggest that TET2-mutant CH may enhance immunotherapy responsiveness. However, how CH shapes lung tumorigenesis and modulates neoadjuvant immunotherapy outcomes in early-stage lung cancer remains unclear. We performed integrated analyses in murine models and NSCLC patients, identifying a CH-driven immunosuppressive program. To test whether age-associated Tet2-mutant hematopoiesis accelerates lung cancer, KP (KrasG12D;Trp53-/-) lung cancer cells were orthotopically implanted into young (8-week) and aged (30-week) Tet2-null (Tet2fl/fl;VavCre), Tet2-heterozygous (Tet2fl/+;VavCre), and WT mice. Aged Tet2-deficient mice developed tumors earlier and with greater burden compared to young counterparts. Spleen and tumor profiling showed expansion of CD11b+ myeloid cells, Tregs, and PD-1+ T cells, with profound NK-cell depletion in older Tet2-deficient mice, indicating enhanced immune suppression in aged Tet2-deficient hosts. We next profiled PBMCs from 59 NSCLC patients using scRNA-seq. Individuals with TET2-mutant CH (n=5) exhibited enrichment of MDSCs, Tregs, effector and exhausted T cells, along with significant reductions in naïve T cells and activated NK cells compared with CH-negative patients (n=45). Long-read variant calling from scRNA-seq further revealed that within CH-positive individuals, both wild-type and CH-mutant immune cells adopted similarly aberrant immune states, unlike those from CH-negative individuals. Ligand-receptor modeling indicated that myeloid cells predominantly drove CD4 T-cell alterations, whereas CD8 T-cell remodeling arose from B- and T-cell interactions—highlighting global immune rewiring triggered by limited CH-derived clones through repeated immune synaptic interactions. Finally, in the real-NADIM cohort treated with neoadjuvant immunotherapy (n=161), patients with CH mutations showed comparable response rates but improved cancer-specific and overall survival compared with CH-negative patients, with this preliminary evidence supporting the idea that CH-induced immune alterations can be therapeutically reprogrammed. Conclusion: age-associated TET2-mutant CH remodels systemic immunity to create tumor-promoting microenvironments. These effects appear reversible with immunotherapy. Ongoing work is evaluating combination strategies targeting both myeloid and T-cell pathways to intercept CH-mediated immune escape in age-associated Tet2-deficient CH and includes spatial transcriptomic analysis of early-stage NSCLCs. Citation Format: Kernyu Park, Eunyoung Kim, Maria Belen Sierra-Rodero, Yohan Byun, Tarrant McPherson, Shivani Kapadia, Derrick Bayer, Julia Yoo, Suresh S. Ramalingam, Woong-Yang Park, Bosch Bosch, Noemi Reguart, Bartomeu massuti_, Alberto Cruz-Vermudez, Mariano Provencio, Janghee Woo. Age-related clonal hematopoiesis drives immune remodeling that promotes lung cancer and alters neoadjuvant chemo-immunotherapy outcomes [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 6820.

Abstract Non-small cell lung cancer (NSCLC) exhibits strikingly different responses to antiangiogenic therapies between its two major histologic subtypes, lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC), suggesting an histotype-specific regulation of angiogenesis. Cancer-associated fibroblasts (CAFs) commonly exhibit an activated/myofibroblast-like phenotype in NSCLC, and are emerging as key modulators of tumor progression; however, their contribution to angiogenic control remains undefined. Here, we investigated angiogenesis and hypoxia signatures in NSCLC and integrated bulk RNA-seq, scRNA-seq, CAF secretome profiling, genetic perturbations, and functional in vitro and in vivo assays to dissect the histotype-dependent production of pro-angiogenic factors by CAFs. We observed greater angiogenesis and reduced necrosis/hypoxia in LUAD compared to LUSC across multiple patient cohorts. The LUAD-CAF secretome was primed for angiogenesis through SMAD3-dependent overproduction of key regulators, most notably TIMP-1 and VEGF-A. We also uncovered a previously unrecognized role for TIMP-1 in promoting endothelial hyper-branching. In contrast, LUSC-CAFs displayed attenuated angiogenic activity despite robust HIF-1α upregulation and an hypoxia-associated transcriptional program, due to their epigenetic repression of SMAD3 and compensatory increase in SMAD2. Collectively, these results reveal that CAFs critically shape the distinct angiogenic landscapes of LUAD and LUSC through opposing SMAD2/3 regulation of TIMP-1, VEGF-A, and hypoxia signaling. These results further highlight the therapeutic potential of targeting stromal SMAD3/TIMP-1 in LUAD or microenvironmental stressors such as hypoxia and acidosis in LUSC. In addition, these findings provide a biological framework for understanding histotype-specific patterns of dissemination, immune evasion, and response to antiangiogenic therapies in NSCLC. Citation Format: Jordi Alcaraz, Natalia Isabel Diaz Valdivia, Paula Duch, Marselina Arshakyan, Amelia Parker, Alejandro Bernardo Suarez, Danielle Park, Erik Sahai, Noemi Reguart, Derek C. Radisky, Oriol Casanovas. Antagonistic SMAD2/3 control of TIMP-1, VEGF-A, and hypoxia signaling in myofibroblasts shapes histotype-specific angiogenesis 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 4797.

Abstract The tumor stroma in non-small cell lung cancer (NSCLC) is highly desmoplastic/fibrotic and is emerging as a key regulator of tumor progression, immune evasion and therapy resistance. Tumor fibrosis is characterized by the aberrant accumulation of collagen. However, our understanding of collagen turnover and its potential dependence on the histologic subtype is very limited. Here, we combined transcriptional profiling and ELISA-based assays for neoepitope collagen fragments to analyze collagen production (fibrogenesis) and degradation (fibrolysis) in tumor-associated fibroblasts (TAFs) and in tumor samples from the major NSCLC histologic subtypes: lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). In culture, LUSC-TAFs exhibited enhanced collagen turnover with increased markers of both production (exPRO-C1, PRO-C3 and PRO-C6) and degradation (C1M, C3M, C6M). Consistently, LUSC-TAFs exhibited higher RNA levels of both fibrillar collagens and collagenolytic MMPs, whereas TIMP-1 (a natural inhibitor of major collagenolytic MMPs) showed opposite patterns at the RNA and secreted protein levels. Consistent hystotype-dependent collagen-turnover signatures were observed in bulk RNA-seq data (TCGA) and in histologic analyses of TMAs from surgical specimens. Our results indicate that fibrogenesis is favored in LUAD through increased production of TIMP-1 and lower levels of collagenolytic MMPs, whereas fibrolysis is dominant in LUSC through increased production of collagenolytic MMPs (notably MMP1) and reduced production of TIMP-1. Mechanistically, TGF-β1 is a major driver of tumor fibrosis, and knocking-down SMAD2 or SMAD3 in normal fibroblasts —to mimic previously reported histotype-specific SMAD2/3 expression patterns in lung TAFs (Ikemori et al, Cancer Res 2020) — was sufficient to recapitulate key differential features of collagen turnover between LUAD and LUSC. Together, these data reveal opposite collagen-turnover programs in NSCLC, in which LUAD are predominantly fibrogenic, whereas LUSC are largely fibrolytic through histotype-dependent regulatory processes. Targeting these divergent programs may help revert pathologic fibrosis and improve responses to chemotherapy and/or immunotherapy in NSCLC. Citation Format: Victoria Batto, Marselina Arshakyan, Rasmus Sund Pedersen, Neel Ingemann Nissen, Enrico Almici, Noemi Reguart, Morten Karsdal, Nicholas Willumsen, Jordi Alcaraz. Collagen turnover is dominated by fibrogenesis in lung adenocarcinoma and fibrolysis in lung squamous cell carcinomas [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 761.

Supplementary Figure 8. Deconvolution scores reflect spatial tissue architecture.

Visium DEG per main phenotype by response

Supplementary Figure 2. Kaplan–Meier curves for event-free survival (EFS) and overall survival (OS) according to minimal residual disease (MRD) status. (A) EFS in the experimental arm. (B) OS in the experimental arm. (C) EFS in the control arm. (D) OS in the control arm. MRD positivity was defined as MRD detection at any time point. Hazard ratios (HR) with 95% confidence intervals were estimated using Cox proportional hazards models, and P values were calculated with the log-rank test.

Supplementary Figure 4. Normalized expression of B cell related ChIO-induced genes.

Techniques performed for each patient and sample

Lung cancer remains the leading cause of cancer-related deaths worldwide. Despite advances to improve treatment in resectable early-stage and locally advanced non-small cell lung cancer (NSCLC), survival rates after 5 years remain low, with a high risk of disease progression. Patients who fail to achieve a pathological complete response (pCR) after neoadjuvant treatment have unfavorable outcomes. ARIAN is a phase III, multicenter, open-label, randomized trial evaluating adjuvant treatment in adults with stage IB-IIIB (N2) NSCLC who have undergone neoadjuvant chemo-immunotherapy but did not achieve pCR after completing surgical resection. Patients will be stratified based on programmed death receptor-ligand 1 (PD-L1) tumor expression and randomized (1:1:1) to observation for 10 months (Arm 1), zimberelimab for 13 cycles (Arm 2), or sacituzumab govitecan and zimberelimab for 8 cycles followed by zimberelimab monotherapy for 5 cycles (Arm 3). The primary endpoint is disease-free survival, and secondary endpoints include overall survival and safety.

TPS8675 Background: Despite advances in treatment of NSCLC with EGFR classical mutations, no universally accepted standard-of-care treatment exists for patients with EGFR uncommon mutations including PACC mutations. Treatment options include EGFR tyrosine kinase inhibitors (TKIs) (osimertinib, afatinib), chemotherapy, or other targeted therapies (amivantamab for exon 20 insertion mutations). EGFR PACC mutations comprise approximately 12.5% of all NSCLC EGFR mutations (Robichaux et al 2021, Nilsson et al 2024). Firmonertinib is a once daily oral, highly brain penetrant, broadly active mutant-selective EGFR inhibitor that targets classical and uncommon mutations (Musib et al., NACLC 2022). In the phase 1b FURMO-002 study (FURTHER), first-line locally advanced or metastatic EGFR PACC mutation NSCLC patients treated with firmonertinib 240 mg daily achieved a confirmed ORR of 68.2%, best ORR of 81.8%, disease control rate (DCR) of 100%, and median progression-free survival (mPFS) of 16.5 months by blinded independent central review (BICR) (Le et al., WCLC 2025). Firmonertinib was generally well-tolerated with manageable EGFR TKI-associated adverse events. Methods: ALPACCA (FURMO-006; NCT07185997) is a global, phase 3, randomized, open-label study investigating firmonertinib vs investigator’s choice of osimertinib or afatinib. Eligible patients have locally advanced or metastatic NSCLC with EGFR PACC mutations. Key inclusion criteria include documented presence of EGFR PACC mutation and measurable disease per RECIST v1.1. Patients with asymptomatic CNS metastases are allowed. Key exclusion criteria include prior systemic anticancer therapy in the locally advanced or metastatic setting or any prior EGFR TKI therapy. Approximately 480 patients will be randomized 1:1 to receive firmonertinib 240 mg daily or investigator’s choice of osimertinib 80 mg daily or afatinib 40 mg daily. Primary endpoints are PFS and ORR per RECIST v1.1 by BICR. Key secondary endpoints include OS, investigator assessed PFS and ORR, and safety and tolerability. Enrollment is ongoing. Clinical trial information: NCT07185997 .

Supplementary Figure 3. Blood BCR repertoire according to isotype distribution.

Supplementary figure 3. Incremental prognostic value of MRD status beyond pathological response in multivariable models. Likelihood ratio (LR) tests show the added predictive contribution of MRD (defined as detection in at least two samples) when incorporated into models including pathological response for (A) event-free survival (EFS) and (B) overall survival (OS). P values from likelihood ratio tests are indicated above each plot.