Yasuyuki Yokoyama

Utility of KRAS mutation detection using circulating cell‐free DNA from patients with colorectal cancer

In this study, we evaluated the clinical utility of detecting KRAS mutations in circulating cell‐free (ccf)DNA of metastatic colorectal cancer patients. We prospectively recruited 94 metastatic colorectal cancer patients. Circulating cell‐free DNA was extracted from plasma samples and analyzed for the presence of seven KRAS point mutations. Using the Invader Plus assay with peptide nucleic acid clamping method and digital PCR, KRAS mutations were detected in the ccfDNA in 35 of 39 patients previously determined to have primary tumors containing KRAS mutations using the Luminex method, and in 5 of 55 patients with tumors containing wild‐type KRAS. Curative resection was undertaken in 7 of 34 patients with primary and ccfDNA KRAS mutations, resulting in the disappearance of the mutation from the cell‐free DNA in five of seven patients. Three of these patients had tumor recurrence and KRAS mutations in their ccfDNA reappeared. Epidermal growth factor receptor blockade was administered to 24 of the KRAS tumor wild‐type patients. Of the 24 patients with wild‐type KRAS in their primary tumors, three patients had KRAS mutations in their ccfDNA and did not respond to treatment with epidermal growth factor receptor (EGFR) blockade. We also detected a new KRAS mutation in five patients during chemotherapy with EGFR blockade, before disease progression was detectable with imaging. The detection of KRAS mutations in ccfDNA is an attractive approach for predicting both treatment response and acquired resistance to EGFR blockade, and for detecting disease recurrence.

Oxaliplatin-induced increase in splenic volume; irreversible change after adjuvant FOLFOX

Oxaliplatin can cause hepatic sinusoidal obstruction syndrome (SOS). SOS can cause chemotherapy‐related adverse effects or morbidity after liver resection. Conventionally, SOS is diagnosed using liver biopsy. Recently, it was reported that increased splenic volume (SV) can be used to detect SOS. In this study, we evaluated the changes in SV during adjuvant chemotherapy.

Feasibility of Neoadjuvant FOLFOX Therapy Without Radiotherapy for Baseline Resectable Rectal Cancer

Background/Aim: The combination of oxaliplatin, leucovorin and fluorouracil (FOLFOX) has been established as postoperative adjuvant chemotherapy for stage III colon cancer. However, the safety and efficacy of neoadjuvant FOLFOX in patients with rectal cancer are still controversial. This prospective pilot study aimed to evaluate the feasibility of neoadjuvant FOLFOX therapy without radiation for baseline resectable rectal cancer (RC). Patients and Methods: The study included 30 patients with clinical stage II/III RC between February 2012 and December 2015. The patients were treated with six cycles of FOLFOX followed by elective surgery. The primary endpoint was the R0 resection rate. The secondary endpoints were the scheduled treatment completion rate, adverse events, pathological response and the disease-free survival (DFS) rate. Results: All the patients underwent elective R0 resection after neoadjuvant FOLFOX therapy. The completion rate of the 6-cycle regimen was 93.3% (28/30 patients). Grade 3-4 adverse events occurred in seven patients (23.3%). Pathological complete response was noted in two patients (6.7%). The 3-year DFS rate was 77.5% (95% confidence interval, 61.4%-93.7%). Conclusion: Neoadjuvant FOLFOX therapy without radiation is a feasible therapeutic strategy for baseline resectable RC.

Combined use of preoperative lymphocyte counts and the post/preoperative lymphocyte count ratio as a prognostic marker of recurrence after curative resection of stage II colon cancer

Purpose Diagnostic markers for recurrence of colorectal cancer have not been established. The aim of the present study was to identify new diagnostic markers for recurrence after curative surgery of stage II colon cancer. Materials and Methods In this study, the prognostic values of the preoperative lymphocyte count and the post/preoperative lymphocyte count ratio (PPLR) were evaluated in 142 patients with localized colon cancer treated with surgery at a single medical center. The associations of patient demographics, blood chemistry, and serum biochemical indices with recurrence-free survival (RFS) and cancer-specific survival (CSS) were examined by univariate and multivariate analyses. Results Receiver operating characteristic (ROC) curve analysis showed that the optimal cut-off values of the lymphocyte count and PPLR were, respectively, 1555.2/μl and 1.151 for RFS. On univariate analysis, tumor depth of invasion, carbohydrate antigen 19-9 (CA19-9), and preoperative low lymphocyte count (≤1555.2/μl) were all correlated with poorer RFS (p < 0.05). On multivariate analysis, T4, low lymphocyte count, and low PPLR were independent predictors of poor RFS. Furthermore, the patients were categorized into four categories based on preoperative lymphocyte count high/low and PPLR high/low. Patients with a low preoperative lymphocyte count and low PPLR had the poorest RFS and CSS compared to the other patients. Conclusion The combination of the preoperative lymphocyte count and the PPLR appears to be a potential marker for predicting recurrence of stage II colon cancer.

Negative Effects of Mechanical Bowel Preparation on the Postoperative Intestinal Motility of Patients with Colorectal Cancer

Several processes can occur as a reaction to surgery, including postoperative intestinal hypoperistalsis, which normally recovers over several hours to days. Postoperative ileus (POI), a transient impairment of bowel motility after abdominal surgery, is characterized by nausea, vomiting, inability to tolerate oral diet, abdominal distension, and delayed passage of flatus and stool. The pathophysiology of POI is multifactorial, but the detailed underlying mechanisms are unknown. This complication should be prevented; however, no single technique or agent has been found to prevent POI effectively. A multidirectional approach is therefore needed to prevent POI. Operative management following the enhanced recovery after surgery (ERAS) approach, including minimally invasive methods, optimal pain control, aggressive postoperative rehabilitation, and early oral nutrition, reportedly exerts a positive effect on the recovery speed of gastrointestinal motility after colon surgery. Although, traditionally, mechanical bowel preparation (MBP) has been thought to decrease the prevalence of surgical site infection and anastomotic leakage, no benefit of MBP has been reported in clinical trials. Some studies have associated MBP with polyethylene glycol (PEG) with poor anastomosis healing and decreased intestinal motility. We showed that MBP with PEG negatively affects motility of the small intestines after both open and laparoscopic colon surgeries. As nutrient absorption occurs in the small intestines, it is important to promote its prompt recovery. In the ERAS approach, omission of MBP is recommended and may be the most important element of ERAS, allowing for early small intestinal motility recovery.

Preoperative Bowel Preparation in ERAS Program: Would-Be Merits or Demerits

For over a century, surgeons have used preoperative mechanical bowel preparation (MBP) to decrease fecal mass within the large bowel. However, over the past 2 decades, several randomized trials and a large meta-analysis have failed to demonstrate reduced rates of surgical site infection (SSI) after elective colorectal surgery in patients who received MBP alone. It was reported in 1971 that MBP removed gross feces but did not alter the number of microorganisms in the colonic lumen. MBP with oral antibiotics, but not alone, reduces the prevalence of SSI. MBP does not affect the prevalence of anastomotic leakage in colon surgery. However, we should not equate rectal surgery with colon surgery because the rate of anastomotic leakage is higher in the former. Also, omitting MBP may be a risk factor for anastomotic leakage in elderly patients. MBP does not reduce morbidity, including SSI, in patients undergoing digestive tract surgery (not for colorectal cancer), such as esophagocoloplasty, hepatectomy or pancreaticoduodenectomy. MBP can negatively affect intestinal motility after surgery. Of note, omission of MBP may negatively affect long-term survival; however, this hypothesis is controversial.

Primary small intestinal volvulus after laparoscopic rectopexy for rectal prolapse: Volvulus after laparoscopic rectopexy

Primary small intestinal volvulus is defined as torsion in the absence of congenital malrotation, band, or postoperative adhesions. Its occurrence as an early postoperative complication is rare. A 40‐year‐old woman presented with rectal prolapse, and laparoscopic rectopexy was uneventfully performed. She could not have food on the day after surgery. She started oral intake on postoperative day 3 but developed abdominal pain after the meal. Contrast‐enhanced CT revealed torsion of the small intestinal mesentery. An emergent laparotomy showed small intestinal volvulus, without congenital malformation or intestinal adhesions. We diagnosed it as primary small intestinal volvulus. The strangulated intestine was resected, and reconstruction was performed. The patient recovered uneventfully after the second surgery. To the best of our knowledge, this is the first report of primary small intestinal volvulus occurring after rectopexy for rectal prolapse. Primary small intestinal volvulus could be a postoperative complication after laparoscopy.

A case of ulcerative colitis with squamous cell carcinomas and multiple foci of squamous dysplasia

Squamous cell carcinoma (SqCC) in ulcerative colitis (UC) is rare. A 38 year‐old Japanese woman, who suffered from left‐sided UC for 18 years, underwent total colectomy due to SqCCs in the rectum and the sigmoid colon. They were well differentiated SqCC, and metastasis was found in the paracolic lymph nodes. Multiple small foci of squamous dysplasia (SD) were noted in the rectal mucosa. Glandular dysplasia was not found. TP53 was not detected in SD. Approximately 40% of cells were immunostained with TP53 in SqCC, however no mutation was found in TP53 gene. Human papilloma virus and Epstein Barr virus were negative in SD and SqCCs. The patient is free of the disease at one and half years after surgery and chemotherapy. SD may be a precursor of SqCC. It appeared that TP53 does not play a vital role in the development of SqCCs in the current case. Careful attention should be paid to SD in UC patients. Viral infection may need to be examined. The pathogenesis of SqCC in patients of UC needs to be elucidated.

Abstract 3134: Prediction of early recurrence after resection of metastatic liver tumors from colorectal cancer using circulating cell-free DNA

Background: We have reported that the amount of total circulating cell-free DNA (ccfDNA) increases with tumor growth and decreases upon tumor shrinkage. However, adverse effects of drugs and surgical stress can increase total ccfDNA because ccfDNA is derived from both normal and cancer cells. Thus, the ability to determine how much ccfDNA is derived from cancer cells is a critical issue. It has been reported that the length of ccfDNA derived from cancer cells is greater than 200 bp while that from normal cells undergoing apoptosis is less than 200 bp. Furthermore, the ratio of ccfDNA to β-globin reflects the amount of mitochondrial DNA derived from normal cells undergoing stress-induced apoptosis. We developed a new biomarker readout, the LINE-1 long fragment (longer than 200 bp) to β-globin ratio (LBR), based on this principle. In this study, we evaluated the clinical utility of the LBR to detect early recurrence of liver metastasis from colorectal cancer after liver resection. Methods: We enrolled 20 patients who underwent curative liver resection of metastatic liver tumors from colorectal cancer. Total ccfDNA and LBR were measured pre-surgery, and at 1 week and 1 month post-surgery. ccfDNA was purified from 1 mL serum using the QIAamp Circulating Nucleic Acid Kit. Total ccfDNA was measured using Qubit Fluorometer. LINE-1 long fragment and β-globin in ccfDNA were measured using real time PCR. The Ethics Review Committee of our institution approved the study protocol. Written informed consent was obtained from each patient. Results: We completed 1-year follow-up in 13 of 20 patients. Recurrence was detected in 7 patients and no signs of recurrence were detected in the other 6. Total ccfDNA increased 1 week after surgery in all 13 patients, which could have been caused by surgical stress. Total ccfDNA 1 month after surgery increased in 5 of 7 patients with recurrence. Total ccfDNA 1 month after surgery increased in 3 of 6 patients without recurrence. The 3 patients with increased ccfDNA had post-operative complications or drug-induced liver dysfunction. Notably, LBR increased in the 7 patients with recurrence and decreased in the 6 patients without recurrence 1 month post-surgery. Conclusion: LBR has potential as a novel biomarker readout for early detection of recurrence after liver resection of metastatic liver tumors from colorectal cancer. Citation Format: Takuma Iwai, Takeshi Yamada, Hayato Kan, Michihiro Koizumi, Seiichi Shinji, Yasuyuki Yokoyama, Goro Takahashi, Shiro Kitano, Masato Nakayama, Zenya Naito, Keiichiro Ohta, Eiji Uchida. Prediction of early recurrence after resection of metastatic liver tumors from colorectal cancer using circulating cell-free DNA. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3134.

Abstract 3135: Prediction of acquired resistance in colorectal cancer patients treated with EGFR blockade by detection of a new KRAS mutation in ccfDNA

Background: Oncogenic KRAS mutations can be used to predict a lack of response to epidermal growth factor receptor (EGFR) blockade. KRAS status is usually determined by biopsy from the primary site of colorectal cancer (CRC). However, the genomic profiles of primary tumors and metastases are not always concordant because of intrinsic molecular heterogeneity. Furthermore, chemotherapeutic agents and targeted drugs can alter the tumor molecular landscape. We exploited circulating tumor DNA (ctDNA) to genotype colorectal tumors and track clonal evolution during treatment involving EGFR blockade. To account for these spatial and temporal changes, the genomic profiles of patients with CRC can be repeatedly evaluated during the course of therapy. In this study, we evaluated the utility of KRAS mutation detection using ctDNA before and during chemotherapy. Method: Experiment 1: We enrolled 46 metastatic colorectal cancer patients. Before starting chemotherapy, ctDNA was purified from 1 mL serum using the QIAamp circulating nucleic acid kit. We detected nine KRAS (G12A, G12R, G12D, G12C, G12S, G12V, G13D, Q61H, and Q61R) mutations using the Invader method and digital PCR. Experiment 2: Fifteen patients were treated with systemic chemotherapy including EGFR blockade. ctDNA was extracted from these patients every 2 months until disease progression, and the KRAS mutation was detected in nine. Results: Experiment 1: KRAS mutations in circulating tumor DNA were detected in 88% (14/16) of patients with KRAS mutations in their primary tumor, but in 10% (10/30) of patients without KRAS mutations in their primary tumors. Experiment 2: The response rate was 87% (13/15). In two non-responders, KRAS mutations in ctDNA were detected before chemotherapy. Disease progression was identified in five patients and KRAS mutations in ctDNA were detected in all patients; the five patients with disease progression included the two non-responders in whom KRAS mutations in ctDNA were detected before chemotherapy. However, the genotypes detected after disease progression were different from those detected before chemotherapy (G13D to G12C and Q61R to Q61H). New KRAS mutations occurred in codon 12 in the other three patients in whom no KRAS mutations in ctDNA were detected before chemotherapy. New KRAS mutations in codon 61 disappeared during chemotherapy and disease progression was not detected at that time in two patients. Discussion: It has been reported in a retrospective study that new KRAS mutations were detected in patients who acquired resistance to EFGR blockade, and many of them were in codon 61. However, new KRAS mutations in codon61 can disappear during treatment involving EGFR blockade. This phenomenon may indicate that new KRAS mutations in ctDNA do not always indicate acquired resistance to chemotherapy involving EGFR blockade. Citation Format: Takeshi Yamada, Hayato Kan, Takuma Iwai, Goro Takahashi, Michihiro Koizumi, Akihisa Matsuda, Seiichi Shinji, Yasuyuki Yokoyama, Atsushi Tatsguchi, Tetsuro Kawagoe, Shiro Kitano, Masato Nakayama, Satoshi Matsumoto, Keiichiro Ohta, Eiji Uchida. Prediction of acquired resistance in colorectal cancer patients treated with EGFR blockade by detection of a new KRAS mutation in ccfDNA. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3135.