Endpoints
tumor, death, Tumor, TILs, T-cell infiltration, Safety/tolerability
DISEASE PROGRESSION, TUMOR, ADVERSE EVENT, TUMOR, PAD, SECONDARY
In Phase I, the primary endpoints were safety/tolerability and identification of the MTD or PAD. The MTD was defined as the highest dose at which less than one-third of patients (out of a minimum of six patients) experienced a DLT (see Additional file 2: Supplementary Table SIn Phase II, investigator-assessed ORR per RECIST v1.1 was the primary endpoint, and safety/tolerability was a secondary endpoint. Tumor assessments were performed every 9 weeks or more frequently if clinically indicated. Either computed tomography or magnetic resonance imaging could have been used, but investigators were instructed to use the same imaging technique throughout the study. After 12 months of study treatment, tumor assessments could have been performed every 12 weeks. Imaging was performed until documented disease progression, the start of new anticancer treatment, withdrawal of consent, death, or the end of the study. Safety/tolerability was evaluated throughout the study. TEAEs were coded per Medical Dictionary for Regulatory Activities v19.1 and graded per Common Terminology Criteria for Adverse Events (CTCAE) v4.03.Changes in T-cell infiltration in the tumor microenvironment was a secondary endpoint in both Phase I and Phase II. To determine expression of PD-L1 in the tumor microenvironment, chromogenic immunohistochemistry was performed at Indivumed (Hamburg, Germany) using the 22C3 pharmDx assay (Agilent/Dako; Santa Clara, California, USA). Membranous anti-PD-L1 staining was semi-quantitatively evaluated using the H-score and tumor proportion score (TPS) per the manufacturer’s instructions. Changes in T-cell infiltration were assessed using 5-color, multiplex immunohistochemistry and analyzed by Indivumed. To quantify T cells, tissue sections were stained with antibodies specific to CD3 (2GV6; Roche/Ventana, Cat # 05278422001), CD8 (SP16; DCS, Cat # CI008C002), and FoxP3 (SP97; Spring Biotech, Cat # M3970; LS Bio, Cat # LS-C210349; Thermo Fisher, Cat # MA5-16365). To identify tumor regions, sections were stained with antibodies specific to pan-cytokeratin (polyclonal; Agilent/Dako, Cat # Z0622) or PMEL (HMB45; Leica, Cat # NCL-L-HMB45). Nuclei were stained with 4′,6-diamidino-2-phenylindole. TILs positive for CD3, CD8, and FoxP3 were quantified by counting the number of cells present within the tumor, as assessed by co-localization with pan-cytokeratin. Cell densities (cells/mm