Supplementary Figure S21 shows Venn diagrams of CNV-confirmed cancer cells and their coexpression of DLL3, SEZ6, B7H3 for tumors in Cohort C.

Supplementary Figure S24 shows the distribution of DLL3 positive cells based on cell type for Cohort B and patient 37.

Supplementary Figure 4. Predicted HLA alleles (A) and their associated immunogenic epitopes (B) in patients treated with mRNA-4157 monotherapy.

Supplementary Figure 8. Study design for Part A and Part D.

Supplementary Figure S2 shows the CTC-iCHIP workflow and images of staining optimization using control cancer cell lines.

Supplementary Figure S5 shows single-cell transcriptional landscape of SCLC tumor biopsies and CNV for Cohort B.

Supplementary Figure 7 shows CNV of SCLC tumor biopsies for patient-2.

Supplementary Figure S17 shows graphs of CD4+ T cells phenotypic profiles for memory, activation and exhaustion.

Supplementary Data S1 contains characteristic descriptions for Cohort B.

9564 Background: Intismeran autogene (intismeran) is an individualized mRNA-based neoantigen (neoAg) therapy designed to enhance endogenous antitumor T-cell responses by targeting patient-specific tumor mutations. In the phase 2 KEYNOTE-942 trial of resected high-risk melanoma, intismeran combined with pembrolizumab (pembro) significantly improved recurrence-free survival (RFS) vs pembro alone. Previously, it was shown that the combination induced greater expansion of de novo T-cell clonotypes, which correlated with RFS in the intismeran arm. Here, we show that de novo T-cell clonotypes are reactive to intismeran-encoded neoAgs. Methods: Peripheral leukapheresis samples longitudinally collected from a subset of intismeran plus pembro treated participants (pts) with melanoma from phase 1 and 2 studies (NCT3313778 and NCT03897881) were assessed at single-cell level to characterize neoAg-specific T cells. In 7 pts, functional assessment of neoAg-reactive CD8⁺ T cells was performed by intracellular cytokine staining and activation-induced marker assays. To define antigen specificity and phenotype of intismeran-induced T cells, we combined longitudinal bulk and single-cell T-cell receptor (TCR) sequencing with functional validation assays from 3 pts. De novo expanded TCRs were tested in Jurkat NFAT-luciferase reporter cells for reactivity against pt-specific intismeran mRNA cassettes and individual neoAgs. For reactive TCRs, minimal epitopes were mapped and mutant vs wild-type peptide reactivity was quantified. Results: Using the combination of bulk and single-cell TCR sequencing and Jurkat NFAT-luciferase reporter assays in 3 pts, we identified 20 TCRs expanded following intismeran therapy across all pts mapping to 11 neoAgs, providing direct evidence of intismeran-driven polyclonal T-cell expansion. These responses targeted multiple neoAgs with persistence >100 days after last intismeran dose. Reactive TCRs showed stronger recognition of mutant vs wild-type peptides, confirming antigen specificity. Functional assessment of T cells from 7 pts showed that neoAg-reactive CD8⁺ T cells secreted IFNγ and TNFα and coexpressed markers for degranulation/activation upon peptide restimulation, demonstrating functional activity. Flow cytometry–based characterization of neoAg-reactive T cells ex vivo revealed that these cells predominantly exhibited an effector-memory phenotype. Upon stimulation, they were polyfunctional (IFNγ⁺ TNFα⁺), mobilized CD107a, and were granzyme B⁺, consistent with cytotoxic effector function relevant to tumor control. Conclusions: Complementary sequencing and functional analyses demonstrated that adjuvant therapy with intismeran plus pembro induced durable, polyclonal, neoAg-specific T-cell responses with effector-memory characteristics, providing mechanistic data consistent with the clinical benefit observed in pts with melanoma. Clinical trial information: NCT3313778 & NCT03897881 .

Supplementary Figure S6 shows the CNV of SCLC tumor biopsies for Cohort C.

INTRODUCTION:Thyroid transcription factor-1 (TTF-1) expression, routinely assessed through immunohistochemistry in the diagnostic evaluation of lung adenocarcinomas (LUADs), is negative (TTF-1Neg) in approximately 15% to 20% of cases. Although worse outcomes have been reported for these tumors compared with TTF-1-positive (TTF-1Pos) LUAD, a comprehensive characterization of TTF-1 negativity is currently lacking. METHODS:Patients with LUAD and available TTF-1 immunohistochemistry from five institutions, The Cancer Genome Atlas, the Stand Up To Cancer-Mark Foundation, and the POPLAR/OAK data sets, were included. Features and outcomes were analyzed according to TTF-1 expression. RESULTS:Among 3297 patients, TTF-1Neg (15%, n = 496), compared with TTF-1Pos (85%, n = 2801), was associated with a more frequent tobacco use history and lower PD-L1 expression. TTF-1Neg LUAD was enriched for STK11, KEAP1, SMARCA4, NKX2-1, CDKN2A, and KRAS mutations (q < 0.05). Patients with metastatic TTF-1Neg LUAD treated with immune checkpoint inhibitors (n = 233), compared with TTF-1Pos cases (n = 1179), had worse objective response rates (ORR, 17% versus 28%, p = 0.001), median progression-free survival (mPFS, 2.5 versus 4.4 mo, p < 0.0001), and median overall survival (mOS, 9.6 versus 20.2 mo, p < 0.0001). Similarly, TTF-1Neg cases had worse outcomes to chemoimmunotherapy (ORR, 26% versus 41%, p < 0.0001; mPFS, 4.6 versus 8.2 mo, p < 0.0001; mOS, 11.2 versus 23.4 mo, p < 0.0001), durvalumab after chemoradiation for unresectable stage III disease (mPFS, 8.0 versus 24.8 mo, p = 0.016; mOS, 20.0 mo versus not reached, p = 0.004), and KRASG12C inhibitors in KRASG12C-mutant LUAD (ORR, 13% versus 36%, p = 0.03; mPFS, 2.7 versus 5.9 mo, p < 0.0001; mOS, 4.4 versus 12.1 mo, p < 0.0001). CONCLUSIONS:TTF-1 negativity identifies a subset of LUAD with worse outcomes to immunotherapy, chemoimmunotherapy, and KRASG12C inhibitors.

Supplementary Figure S9 shows that the fraction of DLL3-positive tumor cells does not significantly correlate with the distribution of SCLC molecular subtypes in primary tumors across cohorts.

Supplementary Figure S8 shows fraction of DLL3 negative tumor cells based on single-cell RNA-seq “drop out” of rare transcripts.

Supplementary Figure S12 shows isoform characterization of DLL3 including protein structure, expression and isoform profiles for specific patients.

Supplementary Figure S4 shows the size distribution of the CTCs based on their marker expression profiles.

Supplementary Table S1 shows patient demographics and clinical characteristics for Cohort A.

Supplementary Figure S16 shows CD4+ T cells phenotypic profiling for individual markers in control sample HD2.

Markers of T-cell dysfunction in patients with acquired tarlatamab resistance despite persistent DLL3 expression. A, Box plot representing flow cytometric analysis of PBMCs showing the percentage of CD8+ (left) or CD4+ (right) live T cells. Blood specimens analyzed from healthy donors (HD: gray, N = 3), tarlatamab-untreated advanced SCLC (Pre-Tx: orange, N = 5), SCLC cases at the time of tarlatamab response (Resp.: blue, N = 4), and patients with SCLC with acquired resistance to tarlatamab despite persistent DLL3 expression in CTCs (Prog.: red, N = 2, open circles denote two distinct measurements from one case). For A–E and G–I, multiple measurements from a single patient (where available) were averaged for statistical comparison using the Student unpaired t test between Resp. vs. Prog. groups. B, Percentage of central memory (CM) CD8+ T cells (phenotype: CD45RA−CCR7+CD28+) versus effector memory subtype 1 (EM1) CD8+ T cells (phenotype: CD45RA−CCR7−CD28+). C, Percentage of KLRG1+ cells within activated (PD-1+) CD8+ T cells. D, Percentage of GZMB+ cells within activated (PD-1+) CD8+ T cells. E, Percentage of CD39+ cells within activated (PD-1+) CD8+ T cells. F, Percentage of GZMB+ cells within PD-1+CD8+ T cells, with and without CD39 expression, across all SCLC samples. Statistical comparison between CD39+ (dark red) and CD39− (gray) cells was done using the Student paired t test (N = 9 SCLC cases, including Pre-Tx, Resp., and Prog.). Samples with fewer than 50 recorded T cells in either the CD39+ or the CD39− group were excluded. G, Percentage of PD-1+ cells within total CD8+ T cells. H, Percentage of TIM3+ cells within activated (PD-1+) CD8+ T cells. I, Percentage of TOX+ cells within activated (PD-1+) CD8+ T cells.

Supplementary Figure S19 shows Venn diagrams of CNV-confirmed cancer cells and their coexpression of DLL3, SEZ6, B7H3 for tumors in Cohort B.