Phase 1/2 trial of ATR inhibitor berzosertib plus immune checkpoint inhibitor avelumab in patients with advanced cancers with DNA damage response (DDR) gene alterations: Tumor microenvironment–mediated pathways of resistance from correlative data.

3141 Background: Herein we present novel correlative data from baseline and on-treatment tumor and blood sampling with associated therapeutic outcomes from a phase 1/2 trial of ATRi (berzosertib) plus ICI (avelumab) in patients (pts) with advanced cancers with pathogenic mutations in select DDR genes. Methods: 17 pts enrolled on trial, and they received berzosertib (day 1, 8, 15, 22) plus avelumab (day 1 and 15, q28 days). Paired tumor biopsies were collected at baseline and on Day 12 of treatment, with spatial gene expression profiling (Visium) performed on the tumor tissues. Spot-level bioinformatics and manual annotations were performed to identify regions enriched for tumor cells and key TME components. Blood was collected at baseline and longitudinally for high-dimensional CyTOF profiling for immune cell populations and Luminex for circulating cytokines. Results: The ATRi + ICI combination was safe and well tolerated as previously reported, with ORR of 12.5% in this heavily pre-treated study population, with 2 durable RECISTv1.1 partial responses observed in advanced HPV+ vaginal cancer pt with pathogenic somatic RAD51 mutation lasting 23 months and an advanced colorectal cancer with MSH2 mutation lasting >24 months. In liquid sampling (N=16 pts), CyTOF analysis revealed a common baseline monocyte “stress-brake” state marked by elevated MRE11 and impaired antigen presentation. Treatment resulted in relief of innate DNA stress and induction of a myeloid-to-lymphoid immune shift, with subsequent expansion of adaptive T-cell effectors (EM1 CD4 T cells), with the durable responses characterized by an early proliferative CD8 T-cell burst (D12–D28) followed by sustained lymphoid dominance. Luminex assay revealed substantial cytokine remodeling at D12–D28 relative to baseline, with multiple analytes exceeding the predefined effect-size threshold (|log2FC| ≥ 0.58; ~1.5-fold), suggesting an acute inflammatory response and myeloid infiltration. Spatial profiling in non-responders showed high baseline levels of cancer associated fibroblasts (CAFs) and intra-tumoral CNV heterogeneity, with treatment resulting in upregulation of immunosuppressive, pro-survival pathways (SPP1, MIF, MYC, and Annexin). In contrast, the available on-treatment pt tissue from the vaginal cancer responding tumor displayed higher lymphoid infiltrate and lower immune suppression genes. Conclusions: Despite universal acute immune activation in circulation following ATRi + ICI, resistant tumors displayed high CNV heterogeneity, CAFs, and increased immunosuppressive pathways, in contrast to responder tissue. Co-clinical studies will further elucidate targetable mechanisms of resistance and guide further DDRi and ICI combinations. Clinical trial information: NCT04266912 .