Study design and patient recruitment
PA.7 received institutional Ethics Review Committee approval and was conducted in accordance with the Declaration of Helsinki and International Ethical Guidelines for Biomedical Research Involving Humans (NCT02879318). The study protocol was approved by the participating centers’ REB, the Ontario Cancer Research Ethics Board and the UBC BC Cancer Research Ethics Board. The patients gave a written informed consent. PA.7 was a randomized phase II clinical trial conducted at 28 centers across Canada. After a safety run involving 11 patients, 180 patients diagnosed with metastatic pancreatic ductal adenocarcinoma (mPDAC) were enrolled in the study. The study inclusion criteria were: Patients must have histologically or cytologically confirmed PDAC that is metastatic, have measurable or evaluable disease as defined in the Response Evaluation Criteria in Solid Tumors (RECIST 1.1), have as considered suitable candidates and able to receive treatment primarily chemotherapy for metastatic disease with gemcitabine and nab-paclitaxel, must consent to the provision of a formalin-fixed paraffin block of tumor tissue and blood, serum and plasma samples, must be at least 18 years of age be old and have an ECOG performance status of zero, or one with a life expectancy of at least 12 weeks, have not received prior anti-metastatic treatment, have reasonable normal organ and marrow function, have had an imaging study including CT/MRI of chest/ abdomen/pelvis to identify the disease foci in the Documented within 28 days prior to randomization, they must be able to complete quality of life questionnaires and be accessible for treatment and follow-up. Exclusion criteria included: history of other malignancies, prior treatment with PD1 or PD-L1 inhibitors, history of primary immunodeficiency, active or previously documented autoimmune or inflammatory disorders, and active or uncontrolled concomitant disorders. Patients were randomized to receive gemcitabine (1000 mg/m2 D1, 8, 15), nab-paclitaxel (125 mg/m2 D1, 8, 15), durvalumab (1500 mg D1 every 28 days), and tremelimumab (75 mg D1 for the first 4 cycles) versus gemcitabine and nab-paclitaxel alone in a 2:1 ratio (each). Randomization was dynamically balanced according to ECOG performance status (0 vs. 1) and receipt of prior adjuvant therapy (yes vs. no) using the minimization method. Randomization was performed centrally by the Canadian Cancer Trials Group (CCTG) headquarters. Overall survival (OS) was the primary endpoint. Secondary endpoints included progression-free survival (PFS), safety, overall response rate (ORR) and quality of life as assessed by EORTC QLQ-C30. Patient enrollment began on August 22, 2016. The randomized phase II component of this study opened on April 10, 2017 after reviewing the results of 11 run-in safety patients and closed on July 28, 2018 after the last patient was randomized had been . The CCTG Data and Safety Monitoring Committee regularly assessed the conduct and safety of the study.
cfDNA extraction, library preparation, probe acquisition and sequencing
Circulating cell-free DNA was extracted from plasma samples using the QIAamp Circulating Nucleic Acid Kit. The quantity and quality of the purified cfDNA were checked using Qubit Fluorimeter and Bioanalyzer 2100. For samples with severe peripheral blood cell genomic contamination, bead-based sizing was performed to remove large genomic fragments. Five to 30 ng of the extracted cfDNA were subjected to library construction, including end repair, dA tailing and adapter ligation. Ligated library fragments with appropriate adapters were amplified via PCR. The amplified DNA libraries were then further checked with the Bioanalyzer 2100 and samples with sufficient yield are sent to hybrid capture.
The 600 gene predicateATLASTM Panel with biotin labeled DNA probes was used for target enrichment. Briefly, the library was hybridized overnight with Predicine NGS panel and paramagnetic beads. The unbound fragments were washed away and the enriched fragments were amplified via PCR amplifications. Similar to library construction, the purified product was checked on the Bioanalyzer 2100 and then loaded into Illumina NovaSeq 6000 for NGS sequencing using paired-end 2×150 bp sequencing kits.
Analysis of NGS data from cfDNA
NGS data was analyzed using the Predicine DeepSea NGS analysis pipeline, which starts from the raw sequencing data (BCL files) and outputs the final mutation calls. Briefly, the pipeline first performs adapter trimming, barcode verification, and correction. Purified paired FASTQ files are aligned to the hg19 human reference genome build using the BWA alignment tool. Consensus BAM files are then derived by merging paired-end reads originating from the same molecules (based on association site and unique molecular identifiers) as single-stranded fragments. Single-stranded fragments from the same double-stranded DNA molecules were further annealed as double-stranded. By using the error suppression method described by Newman and colleagues33Both sequencing and PCR errors were mostly corrected during this process.
Candidate variants were retrieved by comparison to local variant background (defined based on health donor plasma samples and historical data). The variants were further filtered according to log-odds (LOD) thresholds34Base and mapping quality thresholds, repeat regions, and other quality metrics.
bTMB score estimation
Blood-based tumor mutation burden (bTMB) was defined as the number of somatic-coding single nucleotide variants (SNVs), including synonymous and non-synonymous variants, within the panel target regions. The bTMB score was then normalized by the total effective target panel size within the coding region35. Since no matching normal samples such as peripheral blood mononuclear cells (PBMCs) were available for germline variant filtering, germline variants were inferred based on variant annotation, variant allelic frequency, and other variant information such as variant copy number status. Variants in Common Clonal Hematopoietic Mutations of Genes with Undetermined Potential (CHIP) (DNMT3A, TET2, ASXL1 and JAK2) were excluded from the bTMB estimation.
Estimation of the circulating tumor fraction
Circulating tumor fractions were estimated based on the allelic fractions of autosomal somatic mutations as previously described36. In short, the mutant allele fraction (MAF) and the ctDNA fraction are related as MAF = (ctDNA*1)/[(1-ctDNA)*2 + ctDNA *1], and so ctDNA = 2/((1/MAF) + 1). Somatic mutations in genes with a detectable copy number change were not included in the estimation of the circulating tumor fraction.
For somatic and germline variants, frameshift and in-frame insertion/deletions (indels), missense and nonsense mutations were included in our analysis. For the secondary survival analysis of KRAS In wild-type samples, CTF was used as a metric to filter samples conservatively. To determine the CTF threshold above which samples are excluded, we iteratively tested increasing CTF thresholds. At each iteration, samples with CTF values below the threshold were excluded and the frequency of KRAS Wild type status was recalculated. A KRAS At CTF = 1.68%, a wild-type frequency of about 10% was achieved while retaining a large proportion of patients, and this value was therefore chosen as the threshold above which samples in the secondary group are excluded KRAS Wild-type survival analysis as a rate of KRAS Wild-type status was more similar to previous studies12,13,14.
The primary endpoint OS was defined as the time from the date of randomization to the date of death from any cause. The secondary endpoint PFS was defined as the time from the date of randomization to the first date that disease progression was objectively documented or the date of death from any cause, and the ORR as the proportion of randomized patients with a documented complete or partial response. This study was designed to detect a HR of 0.65 between two treatment arms with a power of 80% and a two-sided 10% level, resulting in an increase in median OS from 8.5 months for the chemo arm to 13, 1 month for the chemo arm corresponded to the chemo+ICI arm and required observation of 150 deaths prior to final analysis.
OS and PFS were summarized using the Kaplan-Meier method and compared using a log-rank test, stratified by ECOG performance status and prior adjuvant therapy. Stratified Cox models were used to calculate hazard ratios (HRs) and 90% confidence intervals (CIs). ORR between treatment groups was compared using a Cochran-Mantel-Haenszel test stratified by ECOG performance status and prior adjuvant therapy. Fisher’s exact test was used to compare adverse event rates between treatment arms as well as primary QOL endpoints, expressed as proportions of patients with worsening (defined as a change from baseline that is -10 points or less) Physical function and global health status were pre-specified at 8 weeks and 16 weeks after randomization. The two-tailed Wilcoxon mean-rank sum test was used to compare the CTF values between them KRAS wild-type and mutant groups. The primary analyzes included all randomized patients, while the ctDNA analysis included patients with successful ctDNA sample sequencing. The statistical software SAS (version 9.0; SAS Institute, Inc) and R v3.6.3 were used for the analyses.
Summary of Reporting
For more information on the research design, see the Nature Research Reporting Summary linked to this article.