Oncological evaluation in the perioperative period using cfDNA with BRAF V600E mutation in patients with colorectal cancer

The detection of circulating cell-free DNA (cfDNA) by liquid biopsy is reported to provide prognostic information in colorectal cancer (CRC). Although the frequency of BRAF V600E mutation in CRC is less than 10%, it is associated with poor responses to conventional chemotherapy. We conducted a prospective study to investigate the relationship between the perioperative mutant allele frequency (MAF) of BRAF V600E and tumor recurrence, and to evaluate the possibility of early detection of recurrence. Among 362 patients who underwent radical resection, cfDNA was extracted from the perioperative blood of 11 CRC patients with BRAF V600E mutation and analyzed using the digital polymerase chain reaction (dPCR) system. The median follow-up time was 22 months, and there were four cases of recurrence. Although there was no correlation between recurrence and the perioperative MAF of BRAF V600E, tumor diameter was correlated with the MAF (p = 0.024), and the MAF increased with time in two patients from whom additional samples were obtained prior to recurrence. In this study, we identified a correlation between the pathological tumor diameter and the MAF, but it was difficult to predict recurrence by measuring cfDNA with BRAF V600E mutation in the perioperative period of radical resection of CRC.

www.nature.com/scientificreports/ of a cancer patient 16 . Analysis of these liquid biopsies can provide comprehensive real-time information of the tumor-associated changes in an individual cancer patient. These data can be used for the early detection of cancer, prognostic information (the risk for metastatic recurrence or progression), predictive information (sensitivity to anti-cancer agents), monitoring of treatment response, and identification of minimal residual disease (MRD) 17 .
In terms of CTCs, they are more sensitive than tumor markers, and they can be used to predict prognosis when combined with tumor markers 18 . The presence of postoperative CTCs predicts decreased disease-free survival 19 , and the presence of preoperative CTCs predict early recurrence and decreased disease-free survival 20 . A review by Peach et al. summarized that the presence of CTCs in peripheral blood at least 24 h after tumor resection was an independent prognostic marker of recurrence 21 . However, there are no reports of the correlation between perioperative changes in cfDNA with BRAF V600E and prognosis.
In this study, we evaluated the dynamics of BRAF V600E mutation during the perioperative period by liquid biopsy to test the hypothesis that measurement of BRAF V600E in cfDNA in the early postoperative period is useful for predicting recurrence.

Results
Patient characteristics. A total of 362 patients underwent surgery for CRC at our hospital between April 2018 and March 2020. Sixty-one patients who did not undergo genetic testing for KRAS/NRAS/BRAF were excluded, and 301 patients were included in the study. There were 139 patients with KRAS mutations and 12 with NRAS mutations, and 139 were wild-type. Among the 151 patients with KRAS or NRAS mutations, none carried the BRAF V600E mutation. BRAF V600E mutation was identified in 11 (3.7%) cases using resected specimens. All patients had complete blood samples and underwent molecular analysis. We were able to collect samples from two of the four patients with recurrence after 30 days postoperatively, and molecular analysis was performed (Fig. 1).
The characteristics of these 11 cases are shown in Table 1. The median age was 69 years (range, 46-83), and seven cases were in women. The median body mass index was 21.6 kg/m 2 . The Eastern Cooperative Oncology Group performance score (ECOG-PS) was 0 in six patients, 1 in two patients, and ≥ 2 in three patients. In one case, duodenal NET was recognized in synchrony, and simultaneous resection was performed by laparotomy. All other cases underwent laparoscopic surgery. The median diameter of the pathological primary tumor at the baseline was 45 mm (range: 30-80 mm). Pathologically, four patients had lymph node metastasis, and seven patients did not.
The progress of each patient is shown in Table 2. The mean observation period was 22 months. Five patients received postoperative adjuvant chemotherapy. Recurrence was observed in four cases. The recurrent organs were liver in one case and peritoneum in three cases. A patient with liver metastases died 17.2 months after treatment with liver resection and subsequent chemotherapy. Of the three patients with peritoneal disseminated metastases, one died at 9.4 months after surgery due to poor performance score (PS) and the inability to undergo chemotherapy. The remaining two patients were treated with chemotherapy, one of whom was resected for peritoneal dissemination and is still undergoing chemotherapy, and one of whom died at 11.6 months after surgery due to tumor progression.
Relationship between mutant allele frequency of BRAF V600E in the perioperative period and recurrence. We examined BRAF V600E in the perioperative cfDNA of all patients. The MAFs of BRAF V600E in all cases are shown in Fig. 2. Some patients had a high MAF even if they had not relapsed, while others had a low MAF but had relapsed. Changes in MAF did not fluctuate over time. The relationship between detection of BRAF V600E and recurrence was simplified using a cutoff value of 0.15% for MAF 22 (Table 3). In www.nature.com/scientificreports/ patients who experienced relapse, the BRAF V600E mutation was detected in all postoperative measurements. Conversely, the mutation was inconsistently detected in patients who did not experience relapse. There was no correlation between cfDNA in the perioperative period and recurrence. A comparison of the perioperative MAF of BRAF V600E with recurrence is shown in Fig. 3. There was no significant difference in recurrence and MAF preoperatively or on postoperative days 1 or 30. The preoperative BRAF V600E MAF and each factor were compared (Fig. 4). Tumor diameter, lymph node metastasis, preoperative CEA, and preoperative CA19-9 were examined; the MAF was significantly higher in patients with tumor diameters of 60 mm or more.

MAF of BRAF V600E at diagnosis of recurrence.
We were able to collect a liquid biopsy from two cases of recurrence (case 4 and case 11) until the recurrence was confirmed on imaging. Case 4 had poor PS and could not receive postoperative adjuvant chemotherapy. At 7.2 months postoperatively, peritoneal dissemination  www.nature.com/scientificreports/ was diagnosed. The MAF was 0.22% at 30 days postoperatively, 0.23% at 5 months postoperatively, and 0.85% at diagnosis of recurrence. However, case 11 had idiopathic thrombocytopenic purpura and could not receive adjuvant chemotherapy. At 2.7 months after surgery, liver metastasis was diagnosed. The MAF on postoperative day 30 was 0.61%, while the MAF at the start of chemotherapy was 0.85%. In both cases, the cfDNA levels showed an increasing trend over time (Fig. 5).   23 . To the best of our knowledge, this is the first prospective study to evaluate the oncological changes of perioperative cfDNA levels patients with the BRAF V600E mutation over time. In the present study, we showed two main findings. First, it was difficult to predict recurrence by measuring cfDNA with BRAF V600E in the perioperative period. Second, the perioperative MAF of BRAF V600E was not correlated with tumor markers or lymph node metastasis but was correlated with tumor diameter.  www.nature.com/scientificreports/ Previous reports demonstrated that cfDNA is a prognostic marker in metastatic CRC patients [24][25][26] . However, these studies were conducted in patients already diagnosed with distant metastasis. There have been reports that preoperative and pre-chemotherapy cfDNA levels are useful prognostic markers, but no mutation-specific reports have been published [27][28][29] . Although Shimada et al. reported that BRAF V600E positivity in resection specimens is an important molecular marker for predicting prognosis and the feasibility conversion surgery (conversion surgery defined as a surgical treatment aiming at a curable intention after tumors initially deemed technically or oncologically unresectable or only marginally resectable respond to chemotherapy) in patients with stage IV CRC 30 , no report describe the early prediction of recurrence using cfDNA containing BRAF V600E in the perioperative period after radical surgery. In this study, we examined the MAF of BRAF V600E preoperatively and on postoperative days 1 and 30 and found no correlation between MAF and recurrence. Therefore, it was difficult to predict recurrence after radical resection of CRC by perioperative BRAF V600E.
This study showed that the MAF of BRAF V600E did not correlate with recurrence or tumor markers or lymph node metastasis, but with tumor diameter. Bando et al. compared the concordance rate of RAS mutations between cfDNA in plasma and tumor tissue DNA in patients with metastatic CRC 31 . They reported that patients with pulmonary metastases only, with a maximum lesion diameter of 20 mm and fewer than 10 lesions, had less tumor released into the plasma, which decreased the detection sensitivity of cfDNA. Because the detection rate varies depending on the site of metastasis, tumor diameter, and number of metastases, it may be difficult to predict recurrence using cfDNA in the early postoperative period.
Early studies established that many cancer cells are disseminated into the blood from an early stage, but metastasis is a highly inefficient process [32][33][34] . These studies showed that within 24 h after entry into the circulation, less than 0.1% of tumor cells are still viable, and less than 0.01% of these cells, when introduced into the circulation, survive to produce metastases. Therefore, only a few cells in a primary tumor can give rise to a metastasis.
We were able to collect samples from two recurrent cases. In these cases, the MAF of BRAF V600E mutation had increased over time. This may be due to the increased amount of tumor in the body caused by the recurrence. Although recurrence could not be predicted from cfDNA in the perioperative period, it may be possible to detect recurrence early by measuring it in succession. If the detection sensitivity of liquid biopsy is improved by further research and the accumulation of knowledge, BRAF mutated cases may be detected earlier, permitting earlier therapeutic intervention.
The present study has several limitations. First, it was conducted at a single institution. Second, only 3.7% of patients had BRAF V600E, which was a small number of eligible patients. Third, because the observation period of our study was 22 months, it is possible that other patients may relapse in the future. Finally, we analyzed only BRAF V600E by dPCR. Cancer is spatially and temporally heterogeneous, and recurrent tumors may not have the BRAF V600E mutation. In the future, other genetic mutations should be studied.
In conclusion, it is difficult to predict early recurrence by measuring cfDNA for BRAF V600E in the perioperative period. Metastasis is known to be a very inefficient process, and this may make an early prediction of recurrence difficult.

Methods
Study design and patients. This was a prospective study that included patients who underwent colorectal surgery without distant metastasis at Fukuoka University Hospital between April 2018 and March 2020. A total of 362 patients diagnosed with primary colorectal adenocarcinoma who underwent primary tumor resection were included. For tissue KRAS, NRAS, and BRAF tests, the MEBGEN RASKET-B kit (MBL, Tokyo, Japan), which applies the polymerase chain reaction-reverse sequence-specific oligonucleotide method, was used in accordance with the manufacturer's protocol 35 . Cases with wild type and KRAS/NRAS mutations were excluded. The institutional review board of Fukuoka University Faculty of Medicine approved this study (2017M35, U19-09-001). Written informed consent was obtained from all patients. All procedures were performed in accordance with the Declaration of Helsinki.
Blood collection procedures. Peripheral blood was collected within 1 week before surgery and on postoperative days 1 and 30. For each patient, 10 ml blood was collected in a BD Vacutainer® PPT plasma preparation tube (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). The sample was centrifuged at 1100×g for 10 min at 4 °C within 2 h after blood collection. The plasma was transferred to a microtube and stored at − 80 °C until use. cfDNA extraction from frozen plasma samples. Plasma samples kept at − 80 °C were re-centrifuged at 16,000×g for 10 min at 4 °C to eliminate debris. cfDNA was extracted from 1 ml plasma using the Maxwell® RSC cfDNA plasma kit (Promega Corporation, Madison, USA) and Maxwell® RSC Instrument (Promega Corporation, Madison, USA) in accordance with the manufacturer's protocol, as described previously 36 . Mutation detection by dPCR and sequencing data analysis. The quantity of cfDNA was calculated using the QuantStudio™ 3D Digital PCR System (Applied Biosystems, South San Francisco, CA) 37 . Each polymerase chain reaction (PCR) mixture was prepared with 9 μL QuantStudio 3D Digital PCR Master Mix, 0.45 μL TaqMan™ assays, and 8.1 μL cfDNA. We loaded 15 μL of the 17.1 μL reaction mixture onto a QuantStudio™ 3D Digital PCR 20 K Chip using the automatic chip loader. The DNA amplification reaction using the ProFlex™ PCR System (Applied Biosystems, South San Francisco, CA) was 96 °C for 10 min followed by 39 cycles of 56 °C for 2 min and 98 °C for 30 min, and 39 cycles at 56 °C for 2 min and 98 °C for 30 s, with a final extension step at 60 °C for 2 min. For dPCR, predesigned dual-probe TaqMan assays were purchased from ThermoScientific for BRAF V600E (c.1799T>A; Assay Hs000000004_rm). Results were analyzed using QuantStudio 3D Analysis