Patients and study design
We prospectively collected data from 22 patients who were diagnosed with unresectable PDAC. They underwent chemotherapy between June 2015 and December 2017 at Saitama Medical Center, Jichi Medical University, Japan. One hundred and four blood samples collected from the 22 patients were available for assessments including 22 samples before drug treatments and 82 samples during drug treatments. In addition, we retrospectively collected data from 104 patients with PDAC who underwent surgery between March 2005 and March 2013 at our hospital. The characteristics of the 22 patients who underwent chemotherapy and 104 patients who underwent surgery are shown in Supplementary Tables S1 and S3, respectively. Of the 104 patients with PDAC who underwent surgery, three were not diagnosed with PDAC, 10 were suspected of being positive for Lewis antibody, and 31 patients with hyperbilirubinemia of 3 mg/dL or more were excluded from the evaluation of CA19-9 regardless of the presence or absence of bile drainage. Consequently, the remaining 60 patients were enrolled in the evaluation. In this study, patients with 2 U/mL of CA19-9 or less were defined as Lewis antibody-positive. After surgery, evidence of recurrence was confirmed based on imaging findings. The median follow-up time for patients in the surgery group was 22.7 months. All patients provided written informed consent for the examination of their tissue and plasma and the use of their clinical data. The study protocol was approved by the research ethics committee of Jichi Medical University and conformed to the ethical guidelines of the World Medical Association Declaration of Helsinki.
Analysis of KRAS status in PDAC tissues
KRAS status in PDAC tissues of the chemotherapy group was evaluated by RASKET with a sensitivity of 1 – 5% and droplet digital polymerase chain reaction (ddPCR) with a sensitivity of 0.01 – 0.1%, using endoscopic ultrasound-guided fine-needle aspiration samples. KRAS status was analyzed in 22 tumor tissues by a clinical testing company (Special Reference Laboratories, Tokyo, Japan) using RASKET. Tissue DNA was extracted from 22 formalin-fixed paraffin-embedded (FFPE) tissues using the QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Early reports have shown that point mutations at codon 12 of the KRAS oncogene mostly include G12V, G12D, and G12R, while other types of KRAS point mutations are rarely detected in patients with PDAC [33,52,53]. Therefore, these three types of KRAS mutations were predominantly identified by ddPCR. In addition, Q61H, another type of KRAS mutation that emerged prior to drug resistance, was verified in four patients by ddPCR after initial determination by RASKET. KRAS status in two patients could not be assessed because of insufficient DNA samples.
Plasma sample collection and processing
A total of 106 blood samples were collected from patients with locally advanced and metastatic PDAC in the chemotherapy group at Saitama Medical Center, Jichi Medical University. From each patient, 7 mL of whole blood was drawn into EDTA-containing tubes, and plasma was collected by centrifugation at 3000 × g for 20 min at 4°C, followed by centrifugation at 16000 × g for 10 min at 4°C in a fresh tube. Plasma samples were separated from peripheral blood cells and stored at -80°C until DNA extraction.
Extraction of circulating cell-free DNA
Circulating cell-free DNA was extracted from 2 mL of plasma using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.
Droplet digital polymerase chain reaction analyses
KRAS status in tumor tissues and plasma was analyzed using the Bio-Rad QX200 ddPCR system (Bio-Rad Laboratories, Hercules, CA, USA). We used a commercially available PrimePCR KRAS kit for ddPCR. KRAS mutations in each blood sample were verified according to the corresponding mutation (C12V, G12D, G12R, and Q61H) in matched tumor tissues determined by ddPCR. The reaction mixture comprised 10 µL of 2× ddPCR Supermix, 1 µL of each reference and variant 20× Bio-Rad PrimePCR KRAS for ddPCR, and 10 µL of sample eluted from plasma in a final volume of 22 µL. The mixture was loaded onto a DG8 cartridge (Bio-Rad Laboratories, Hercules, CA, USA) with 70 µL of droplet generation oil, and the cartridge was placed into a droplet generator. The generated droplets (approximately 15000 generated droplets per well) were transferred to a 96-well reaction plate, heat-sealed with a foil lid, and subjected to thermocycling in a Veriti thermal cycler (Thermo Fisher Scientific, Waltham, MA, USA) under the following cycling conditions: 95°C for 10 min and 40 cycles at 95°C for 30 s and 55°C for 90 s. Amplified droplets were analyzed using a QX200 droplet reader (Bio-Rad Laboratories, Hercules, CA, USA) for the fluorescence measurement of FAM and HEX probes for wild-type and mutant genes, respectively. QuantaSoft software (Bio-Rad Laboratories, Hercules, CA, USA) was used to measure the number of positive and negative droplets. Samples with two or more positive droplets were considered positive according to the threshold values, as previously reported . For data reproducibility, analysis of KRAS status in tumor tissues and plasma was performed in duplicate or triplicate.
ROC curve analysis was plotted to determine the cutoff value of CA19-9 corresponding to the presence of KRAS-mutated ctDNA. To assess prognosis in the surgery group, we measured RFS and OS as endpoints. RFS was defined as the time from surgery to confirmation of recurrence based on radiological findings. OS was defined as the time from surgery to event occurrence. A Cox proportional hazards regression model was used to evaluate the association between overall mortality and other factors in univariate and multivariate analyses. The following variables were analyzed in patients: sex; age at surgery (≤67 years versus >67 years); adjuvant chemotherapy (yes versus no); tumor location (head versus body and tail); tumor size (≤2 cm versus >2 cm); pathological differentiation (well and moderate versus others); Union for International Cancer Control (UICC) T factor (T1+T2 versus T3), lymph node metastasis (negative versus positive), preoperative CA19-9 level (≤median CA19-9 value, 221.1 U/mL versus >median CA19-9 value, 221.1 U/mL); preoperative CA19-9 level (≤new cutoff of CA19-9 value; 949.7 U/mL versus > new cutoff of CA19-9 value; 949.7 U/mL). RFS and OS curves were constructed using the Kaplan-Meier method. Several factors with a P-value of <0.1 in univariate analysis were subjected to multivariate analysis. Statistical significance was set at P < 0.05. Fisher’s exact test was used for categorical variables, such as the presence of KRAS-mutated ctDNA, CA19-9 level (≥37 U/mL versus <37 U/mL), and outcome (dead or alive). All statistical analyses were performed using EZR version 1.31 (Saitama Medical Center, Jichi Medical University, Saitama, Japan). R version 3.1.1 (The R Foundation for Statistical Computing, Vienna, Austria) was used for the graphical interface.