br Treatment br Six patients were enrolled
Six patients were enrolled at DL1 and received SBRT 8 Gy and cyclophosphamide 200 mg/m2 of body surface area (BSA) intrave-nously (I.V.) on day 0, followed by AMP-224 10 mg/kg I.V. on day 1 and then every 14 days for a total of 6 doses. The next 9 patients received SBRT 8 Gy on days 2 to and cyclophosphamide 200 mg/ m2 of BSA I.V. on day (DL2), followed by AMP-224 10 mg/kg I.V. on day 1 and then every 14 days for a total of 6 doses. For both dose levels, an option to continue treatment with AMP-224 was consid-ered in responding patients until disease progression. The lesion subjected to irradiation was chosen by the consulting radiation oncologist. Imaging studies were done using contrast-enhanced
computed tomography (CT) or magnetic resonance imaging (when CT was contraindicated) at baseline, 2 weeks after completion of treatment; and every 8 weeks thereafter. Tumor biopsies from the liver lesions were performed optionally before the beginning of the study treatment (baseline) and at day 29 (post-treatment). Patients were assessed for toxicity before each treatment cycle.
End Points and Assessments
The primary objective was to determine the feasibility and safety of AMP-224 in combination with SBRT and chemotherapy in patients with advanced, unresectable CRC who had disease pro-gression during or after oxaliplatin and irinotecan-containing chemotherapy. Secondary objectives included evaluation of the response rate (assessed using RECIST guidelines31 by the investi-gator, in lesions not subjected to SBRT), progression-free survival (PFS; defined as time from first day of treatment to first docu-mented disease progression or death), and OS (defined as time from the first day of treatment to death from any cause) after the trial treatment. The Bradykinin (acetate) evaluable for response was defined as all patients who had received at least 1 dose of therapy and had at least 1 postbaseline tumor response assessment.
Safety and toxicity were monitored and managed accordingly. The safety population was defined as all patients who received at least 1 dose of AMP-224. The assessment period for dose-limiting toxicities (DLTs) was the first 4 weeks of the study. All adverse events (AEs) that occurred within 30 days of the last dose of treatment were reported according to the NCI Common Terminology Criteria for Adverse Events version 4.0.32
Fresh-frozen paraffin-embedded tumor samples from the core biopsies were processed for RNA isolation. Libraries for RNA sequencing were prepared using Illumina TruSeq Stranded Total RNA Library Prep and were pooled and sequenced on Illumina HiSeq3000/4000 using 150-base pair paired-end protocol following the manufacturer’s protocol. The obtained short sequence reads were aligned to the human hg38 genome using STAR33 and pro-cessed using RSEM34 to compute raw and normalized counts per gene and in all samples.
RNA sequencing data were analyzed for differential gene expres-sion with DESeq2.35 Differentially expressed genes with unadjusted P value < .05 were used for Gene Set Enrichment Analysis (GSEA; available at: http://software.broadinstitute.org/gsea/msigdb/index. jsp), compared against the canonical pathways gene sets, the Kyoto Encyclopedia of Genes and Genomes gene sets, the Reactome Pathway Knowledgebase gene sets, the oncogenic signatures gene sets, and the immune signatures gene sets.36 To study the changes in the CD8þ tumor infiltrating lymphocytes after treatment, we used the RNA expression data of the tumor samples from pre- and post-treatment biopsies, and used the CIBERSORT algorithm (available at: https://cibersort.stanford.edu) to enumerate the immune cells.37