Andrew H. Ko

Appendix 1. Full clinical trial eligibility from UCSF CC#154524 protocol.

Purpose:Immune checkpoint inhibitors (ICI) have limited activity as monotherapy in biliary cancers. Granulocyte–macrophage colony-stimulating factor (GM-CSF) is a pleiotropic immune cell growth factor that achieved prolonged survival when combined with ipilimumab in melanoma. We conducted a single-center, phase II trial to evaluate the efficacy and safety of combining GM-CSF with pembrolizumab in patients with advanced biliary cancers after prior chemotherapy but no prior ICI. Patients and Methods:Pembrolizumab 200 mg was administered intravenously in 21-day cycles, along with two cycles of GM-CSF 250 μg subcutaneously on days 1 through 14. The primary endpoint was the objective response rate (ORR). Results:Among 42 patients enrolled, the median age was 61 years; 67% had intrahepatic cholangiocarcinoma, 90% had stage IV disease, and 24% had underlying viral hepatitis. The confirmed ORR was 12% (95% confidence interval, 4–26), including two patients with a complete response, and 26% of patients had progression-free survival (PFS) ongoing at 6 months. Treatment was well tolerated, with treatment-related grade 3 to 4 events in 7% and treatment-related serious adverse events in 10%. Tumor programmed cell death ligand 1 expression was present in 46% and was associated with a higher rate of 6-month PFS. Paired tumor biopsies showed upregulation of CD8+ T-cell populations and antigen processing pathways after the addition of GM-CSF. Conclusions:The addition of GM-CSF to pembrolizumab was well tolerated but did not meet the prespecified response rate for efficacy. A subset of patients experienced deep responses and prolonged stable disease. GM-CSF elicited changes in the tumor immune microenvironment that could guide future combination approaches.

Circulating myeloid cell populations.

Correlation scores indicate co-localized immune cell types in pre and post biopsies (n=6 pairs). Pre=post-pembrolizumab and pre-GM-CSF; post=post-GM-CSF and pembrolizumab.

Representative PD-L1 staining in tissue samples.

Proportion of each cell type in pre versus post biopsies (n=6 each) as determined with spatial transcriptomics. Pre=post-pembrolizumab and pre-GM-CSF; post=post-GM-CSF and pembrolizumab.

Supplementary Table 1. Peripheral blood samples used in single cell RNA sequencing analysis.

Supplementary Table 2. Biopsy samples used in spatial transcriptomics analysis.

Supplementary Table 4. Representativeness of study participants.