PFS and OS by detection of BRAF V600 alterations in ctDNA at (A and B) baseline and (C and D) on treatment. Only patients with BRAF V600 alteration detections at baseline are shown in C and D. Bini, binimetinib; D, detectable; Enco, encorafenib; ND, nondetectable; Vemu, vemurafenib.

PURPOSE:Treatment with encorafenib ± binimetinib is associated with improved survival versus vemurafenib in patients with BRAF V600E/K-mutant advanced melanoma. We retrospectively analyzed genomic and transcriptomic data from the phase III COLUMBUS trial to identify molecular correlates of benefit with encorafenib ± binimetinib. EXPERIMENTAL DESIGN:In COLUMBUS, patients with BRAF V600E/K-mutant locally advanced, unresectable, or metastatic melanoma (n = 921) were randomized to receive encorafenib plus binimetinib, encorafenib, or vemurafenib. We used whole-exome sequencing (n = 666), whole-transcriptome sequencing (RNA sequencing; n = 514), and assessment of circulating tumor DNA (ctDNA) at baseline (n = 336) and on treatment (cycle 2 day 1, n = 184) to evaluate biomarker associations with progression-free and overall survival. RESULTS:Survival benefits with encorafenib plus binimetinib versus vemurafenib were greatest in patients with higher tumor mutational burden (TMB) and those with evidence of tumor immune infiltration (i.e., higher cytolytic score, PD-L1 expression, or IFNγ gene signature scores). Clustering of gene expression profiles identified three tumor subgroups, including an "immune" subgroup associated with improved survival. Detection of BRAF V600 alterations in baseline ctDNA was associated with shorter survival; clearance of BRAF V600 alterations at cycle 2 day 1 was associated with improved survival across arms. CONCLUSIONS:The greatest benefits of encorafenib plus binimetinib were observed in patients with evidence of high TMB and/or tumor-immune infiltration, suggesting potential immune contributions to efficacy, which were not observed with vemurafenib. BRAF V600 detectability in ctDNA seems to have utility as a marker of prognosis and response in this population.

Supplemental Figure S9 | Supporting data for single cell RNA sequencing of CAR-T in 3D co-cultures with PDOTS.

PFS and OS for encorafenib plus binimetinib or encorafenib vs. vemurafenib by (A and B) TMB and (C and D) cytolytic score. E, Volcano plots of univariable gene expression associations with OS in each arm based on z-scored gene expression values. F, Gene-set enrichment analysis of gene expression associations against hallmark gene signatures; signatures with greatest average NES across arms shown. Bini, binimetinib; BM, biomarker; CYT, cytotoxic score; Enco, encorafenib; NES, normalized enrichment scores; Vemu, vemurafenib.

Supplemental Figure S10 | Supporting data for single cell RNA sequencing of native tumor-infiltrating lymphocytes following CAR-T challenge in 3D co-cultures with PDOTS.

Supplemental Figure S3 | Supporting data for ex vivo profiling of B7-H3.CAR-T cells using PDOTS.

Characterization of tumor samples to identify candidate molecular subtypes by K-means clustering and distribution of subtypes by biopsy site. A, Heatmap showing K-means cluster and the expression of key marker genes (AXL and MITF) and cell type scores generated by xCell. B, Distribution of K-means subgroups across biopsy sites. C, OS by K-means subgroup and treatment arm. Bini, binimetinib; Vemu, vemurafenib.

Supplemental Figure S5 | Supporting data that targeting TBK1 prevents CAR-T cell dysfunction.

Supplementary Figure S1 | Supporting data demonstrating B7-H3 expression in melanoma and other cancers.

PFS and OS for encorafenib plus binimetinib or encorafenib vs. vemurafenib by (A and B) ERBB2 expression level and (C and D) PI3K pathway mutation status. Bini, binimetinib; BM, biomarker; Enco, encorafenib; Vemu, vemurafenib.

Supplemental Figure S6 | Supporting data that targeting TBK1 prevents CAR-T cell dysfunction.

PDOTS patient data (accompanying Fig. 1)

Supplemental Figure S11 | Supporting data that targeting TBK1 sensitizes cancer cells to CAR-T cell-derived TNFα/IFNγ.

Supplemental Figure S7 | Supporting data that targeting TBK1 prevents CAR-T cell dysfunction.

Baseline patient characteristics by biomarker status.

Supplemental Figure S4 | Supporting data for ex vivo profiling of PD-1 blockade and TBK1 inhibition in combination with B7-H3.CAR-T cells using PDOTS.

Supplementary Figure S2 | Supporting data for the In vitro characterization and efficacy of B7-H3.CAR-T cells.

Supplemental Figure S8 | Supporting data that targeting TBK1 prevents CAR-T cell dysfunction.

3045 Background: In the NCI-MATCH trial (NCT02465060), tumor tissue from 5,954 patients with advanced cancers underwent next-generation sequencing using the Oncomine Comprehensive Assay v2 (OCAv2) to determine eligibility. Most tumors lacked a qualifying mutation of interest (MOI) for assignment. Plasma from 1,301 patients with common cancers – [colorectal (COADREAD), breast (BREAST), non–small cell lung (NSCLC), and prostate (PRAD) (OncoTree codes are shown in parentheses)] was analyzed to characterize circulating tumor DNA (ctDNA) and assess its utility for detecting clinically relevant MOIs. Methods: Cell-free DNA was extracted from plasma collected in Streck tubes at enrollment. ctDNA profiling was performed using the NCI ctDNA Research v2 assay (523 genes) on the Illumina NovaSeq 6000. Matched tumor tissue was analyzed using OCAv2 (143 genes). Positive percent agreement (PPA) was calculated using tissue as the reference. Blood-based microsatellite instability (bMSI) was assessed across ~2,400 loci, with bMSI-high (bMSI-H) defined as sum Jensen-Shannon Distance (sumJSD) ≥0.2. Blood-based tumor mutation burden (bTMB) was defined as total SNVs and indels per Mb. Results: Of 1,301 patients, 1,148 (88%) yielded evaluable ctDNA results (COADREAD, n=487; BREAST, n=367; NSCLC, n=220; PRAD, n=74). Overall PPA with matched tissue was 90.3%. Discordant samples had significantly lower median tumor fraction by maximum somatic allele frequency (MSAF; 0.39%) than concordant samples (12.04%). Median MSAF by histology was 14% (COADREAD), 7% (BREAST), 8% (NSCLC), and 5% (PRAD). Clinically relevant fusions detected exclusively in ctDNA included RET (1% NSCLC), EML4::ALK (2% NSCLC), FGFR2 (1% COADREAD; 2% BREAST), and NTRK1 (<1% COADREAD and BREAST), corresponding to actionable NCI-MATCH arms [ FGFR2/3 - arm K (erdafitinib), ALK - arm F (crizotinib), NTRK - arm Z1E (larotrectinib)]. Actionable ctDNA-only mutations in PRAD included ATM and MLH1. Twenty-eight cases were bMSI-H (MSAF ≥0.02; sumJSD ≥0.2), of which 13 were mismatch repair-deficient by tissue testing (MLH1/MSH2 nuclear stain-negative). bMSI-H was most frequent in COADREAD (7%). Ten cases (seven COADREAD, two NSCLC, one PRAD) were MMR-proficient by tissue but bMSI-H by ctDNA. Twenty-four cases had high bTMB (≥20 mut/Mb; MSAF ≥0.02), all of which were also bMSI-H. Conclusions: ctDNA - tissue concordance NCI-MATCH in these four cancer histologies was high (90.3%), supporting liquid biopsy as a practical alternative when tissue is unavailable. Detection of ctDNA-only alterations highlights tumor heterogeneity in advanced cancers and identifies additional therapeutic opportunities.

The COMBI-I trial (ClinicalTrials.gov identifier: NCT02967692) evaluating spartalizumab plus dabrafenib and trametinib (sparta-DabTram, n = 267) versus placebo plus dabrafenib and trametinib (placebo-DabTram, n = 265) for BRAF V600-mutant unresectable or metastatic melanoma failed to reach its primary end point of progression-free survival at 24 months. This final analysis reports overall survival (OS) during at least 5 years of extended follow-up. At the end of the trial (August 21, 2024), the median duration of follow-up was 76.9 months (range, 73.7-83.3 months). The median OS was 61.5 months (95% CI, 41.6 to not evaluable) for the sparta-DabTram arm and 41.6 months (95% CI, 30.6 to 56.9) for the placebo-DabTram arm (hazard ratio, 0.760 [95% CI, 0.598 to 0.966]). The safety findings were consistent with the known safety profile for sparta-DabTram. The most common treatment-related adverse event (TRAE) was pyrexia (65.9% v 46.2%, respectively, in the two study arms). Grade ≥3 TRAEs were reported in 57.3% and 36.7% of patients in the two arms, respectively. The combination of sparta-DabTram appears to improve OS compared with dabrafenib and trametinib alone in patients with BRAF V600-mutant metastatic melanoma.