Masaya Suenaga

Digital next-generation sequencing identifies low-abundance mutations in pancreatic juice samples collected from the duodenum of patients with pancreatic cancer and intraductal papillary mucinous neoplasms

Objective Secretin-stimulated pancreatic juice contains DNA shed from cells lining the pancreatic ducts. Genetic analysis of this fluid may form a test to detect pancreatic ductal neoplasia. Design We employed digital next-generation sequencing (‘digital NGS’) to detect low-abundance mutations in secretin-stimulated juice samples collected from the duodenum of subjects enrolled in Cancer of the Pancreas Screening studies at Johns Hopkins Hospital. For each juice sample, digital NGS necessitated 96 NGS reactions sequencing nine genes. The study population included 115 subjects (53 discovery, 62 validation) (1) with pancreatic ductal adenocarcinoma (PDAC), (2) intraductal papillary mucinous neoplasm (IPMN), (3) controls with non-suspicious pancreata. Results Cases with PDAC and IPMN were more likely to have mutant DNA detected in pancreatic juice than controls (both p<0.0001); mutant DNA concentrations were higher in patients with PDAC than IPMN (p=0.003) or controls (p<0.001). TP53 and/or SMAD4 mutations were commonly detected in juice samples from patients with PDAC and were not detected in controls (p<0.0001); mutant TP53/SMAD4 concentrations could distinguish PDAC from IPMN cases with 32.4% sensitivity, 100% specificity (area under the curve, AUC 0.73, p=0.0002) and controls (AUC 0.82, p<0.0001). Two of four patients who developed pancreatic cancer despite close surveillance had SMAD4/TP53 mutations from their cancer detected in juice samples collected over 1 year prior to their pancreatic cancer diagnosis when no suspicious pancreatic lesions were detected by imaging. Conclusions The detection in pancreatic juice of mutations important for the progression of low-grade dysplasia to high-grade dysplasia and invasive pancreatic cancer may improve the management of patients undergoing pancreatic screening and surveillance.

Downregulation of DENND2D by promoter hypermethylation is associated with early recurrence of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide and its prognosis is poor. Novel targets for treating recurrence and progression along with associated biomarkers are urgently required. In this study, the expression and regulatory mechanism of DENN/MADD domain containing 2D (DENND2D) were investigated in an attempt to identify a tumor suppressor gene for HCC regulated by silencing through promoter hypermethylation. The levels of DENND2D expression in HCC cell lines and surgical specimens were determined using a quantitative polymerase chain reaction assay and the relationship between the expression levels of DENND2D mRNA and clinicopathological factors was evaluated. The expression and distribution of DENND2D were determined using immunohistochemistry. DNA methylation analysis was performed to determine the regulatory mechanisms of DENND2D expression in HCC. Most HCC cell lines (89%) and surgical specimens (78%) expressed lower levels of DENND2D mRNA compared with normal liver tissue. In contrast, there was no significant difference in the expression levels of DENND2D mRNA between normal tissues of HCC patients with and without cirrhosis. The expression patterns of DENND2D protein and mRNA were consistent. Patients with significantly lower levels of DENND2D mRNA in HCC tissues had remarkably earlier recurrences after hepatectomy and their prognosis worsened. The DENND2D promoter was methylated in eight out of nine HCC cell lines and DNA demethylation reactivated DENND2D mRNA expression. Hypermethylation of DENND2D was frequently detected in HCC tissues (75%) and was significantly associated with downregulation of DENND2D mRNA expression. DENND2D is a candidate tumor suppressor gene that is inactivated by promoter hypermethylation in patients with HCC and may serve as a novel biomarker of early recurrence of HCC.

Deleterious Germline Mutations in Patients With Apparently Sporadic Pancreatic Adenocarcinoma

Purpose Deleterious germline mutations contribute to pancreatic cancer susceptibility and are well documented in families in which multiple members have had pancreatic cancer. Methods To define the prevalence of these germline mutations in patients with apparently sporadic pancreatic cancer, we sequenced 32 genes, including known pancreatic cancer susceptibility genes, in DNA prepared from normal tissue obtained from 854 patients with pancreatic ductal adenocarcinoma, 288 patients with other pancreatic and periampullary neoplasms, and 51 patients with non-neoplastic diseases who underwent pancreatic resection at Johns Hopkins Hospital between 2000 and 2015. Results Thirty-three (3.9%; 95% CI, 3.0% to 5.8%) of 854 patients with pancreatic cancer had a deleterious germline mutation, 31 (3.5%) of which affected known familial pancreatic cancer susceptibility genes: BRCA2 (12 patients), ATM (10 patients), BRCA1 (3 patients), PALB2 (2 patients), MLH1 (2 patients), CDKN2A (1 patient), and TP53 (1 patient). Patients with these germline mutations were younger than those without (mean ± SD, 60.8 ± 10.6 v 65.1 ± 10.5 years; P = .03). Deleterious germline mutations were also found in BUB1B (1) and BUB3 (1). Only three of these 33 patients had reported a family history of pancreatic cancer, and most did not have a cancer family history to suggest an inherited cancer syndrome. Five (1.7%) of 288 patients with other periampullary neoplasms also had a deleterious germline mutation. Conclusion Germline mutations in pancreatic cancer susceptibility genes are commonly identified in patients with pancreatic cancer without a significant family history of cancer. These deleterious pancreatic cancer susceptibility gene mutations, some of which are therapeutically targetable, will be missed if current family history guidelines are the main criteria used to determine the appropriateness of gene testing.

Excess Weight Adversely Influences Treatment Length of Postoperative Pancreatic Fistula: A Retrospective Study of 900 Patients.

Objectives Pancreatectomy is still associated with a high morbidity rate, even in high-volume centers, and a leading cause of morbidity is represented by postoperative pancreatic fistula (POPF). Many previous studies have evaluated the risk factors for the occurrence of POPF, but protracted courses of POPF have not been fully discussed. Methods This study included 900 patients who underwent pancreatectomy between January 1991 and June 2013 after exclusion of patients who underwent total pancreatectomy. Subgroup analysis of the duration of drain placement was conducted among patients with POPF to identify predictive factors for a protracted course of POPF. Results Overall, 292 patients (32.4%) had clinically relevant POPF (grade B/C). The length of drain placement in patients with a body mass index (BMI) of 25 kg/m2 or greater was significantly longer than that in patients with a BMI of less than 25 kg/m2 (44.8 ± 25.2 vs 33.8 ± 21.2 days, respectively; P = 0.001). The operative procedure, duct diameter, and pancreatic texture, which were independent risk factors for clinically relevant POPF, did not delay removal of the drainage tubes. Conclusions A BMI of 25 kg/m2 or greater was the only factor associated with delayed POPF healing. Vigilant postoperative management after pancreatectomy should be considered in obese patients.

Identification of intragenic methylation in the TUSC1 gene as a novel prognostic marker of hepatocellular carcinoma

Patients with hepatocellular carcinoma (HCC) have a poor prognosis, and novel molecular targets for treating recurrence and progression of the disease along with associated biomarkers are urgently required. In the present study, expression and the regulatory mechanism of TUSC1 (tumor suppressor candidate 1) were investigated to determine if it is a candidate tumor suppressor gene for HCC, which shows repressed transcription that involves aberrant DNA methylation. TUSC1 mRNA expression levels in HCC cell lines and 94 pairs of surgical specimens were determined using quantitative real-time reverse transcription polymerase chain reaction assay. Methylation status of HCC cell lines and clinical samples were analyzed to investigate the regulatory mechanism of TUSC1 transcription and the relationship between the methylation status of the TUSC1 gene and clinicopathological factors. The expression and distribution of the TUSC1 protein in liver tissues were determined using immunohistochemistry. A majority of HCC cell lines (89%) and surgical specimens (84%) demonstrated reduced expression levels of TUSC1 mRNA compared with paired non-cancerous liver tissues. The mean mRNA expression level in HCC was significantly lower than in corresponding non-cancerous liver. In contrast, no significant difference was found in TUSC1 mRNA expression level between adjacent normal and cirrhotic liver tissue from HCC patients. The TUSC1 protein expression pattern in HCC and liver tissues was consistent with TUSC1 mRNA expression. Twenty-nine (31%) of 94 patients showed intragenic hypermethylation of the TUSC1 gene in HCC, and hypermethylation was significantly associated with advanced pathological stage. Subsequently, patients with hypermethylation of the TUSC1 gene had a significantly poorer prognosis than patients without hypermethylation. Our results suggest that TUSC1 is a candidate tumor suppressor gene and intragenic hypermethylation is one of the suppressive mechanisms that regulate TUSC1 transcription in HCC. Intragenic methylation of the TUSC1 gene may serve as a novel prognostic marker of HCC.

Cyst Fluid Telomerase Activity Predicts the Histologic Grade of Cystic Neoplasms of the Pancreas.

Purpose: Pancreatic cysts frequently pose clinical dilemmas. On one hand, cysts with high-grade dysplasia offer opportunities for cure, on the other hand, those with low-grade dysplasia are easily over treated. Cyst fluid markers have the potential to improve the evaluation of these cysts. Because telomerase activity is commonly activated in malignant cells, we evaluated the diagnostic performance of cyst fluid telomerase activity measurements for predicting histologic grade. Experimental Design: Telomerase activity was measured using telomerase repeat amplification with digital-droplet PCR in surgically aspirated cyst fluid samples from 219 patients who underwent pancreatic resection for a cystic lesion (184 discovery, 35 validation) and 36 patients who underwent endoscopic ultrasound fine-needle aspiration. Methodologic and clinical factors associated with telomerase activity were examined. Results: Telomerase activity was reduced in samples that had undergone prior thawing. Among 119 samples not previously thawed, surgical cyst fluids from cystic neoplasms with high-grade dysplasia ± associated invasive cancer had higher telomerase activity [median (interquartile range), 1,158 (295.9–13,033)] copies/μL of cyst fluid than those without [19.74 (2.58–233.6) copies/μL; P < 0.001)]. Elevated cyst fluid telomerase activity had a diagnostic accuracy for invasive cancer/high-grade dysplasia of 88.1% (discovery), 88.6% (validation), and 88.2% (merged). Among cysts classified preoperatively as having “worrisome features,” cyst fluid telomerase activity had high diagnostic performance (sensitivity 73.7%, specificity 90.6%, accuracy, 86.1%). In multivariate analysis, telomerase activity independently predicted the presence of invasive cancer/high-grade dysplasia. Conclusions: Cyst fluid telomerase activity can be a useful predictor of the neoplastic grade of pancreatic cysts. Clin Cancer Res; 22(20); 5141–51. ©2016 AACR. See related commentary by Allen et al., p. 4966

Vein resections >3 cm during pancreatectomy are associated with poor 1-year patency rates

BACKGROUND Resection of the superior mesenteric vein (SMV) and portal vein (PV) involved in pancreatic neoplasms improves the chances of complete tumor removal. No consensus exists, however, on the optimal reconstructive approach, and postoperative venous stenosis in the first 6 months to 1 year can cause patient morbidity. We investigated medium-term patency after direct, end-to-end venous anastomosis and evaluate predictive factors for stenosis or occlusion. METHODS We analyzed retrospectively the records of 810 patients who underwent pancreatectomy at our institution from January 2000 through April 2014, and 197 patients who underwent concurrent SMV/PV resection were selected. The venous anastomosis was assessed every 4 or 6 months postoperatively by the use of portography with computed tomography. Preoperative and intraoperative variables were evaluated for their possible association with the development of severe anastomotic stenosis (≥70% occlusion). RESULTS Among patients whose cancer did not recur during the 1-year follow-up period, 18 developed severe stenosis. Univariate analyses showed that operation time ≥520 minutes and length of SMV/PV resection ≥31 mm were associated with the development of severe anastomotic stenosis. Multivariate analysis showed that length of SMV/PV resection ≥31 mm was among independent predictors of medium-term, severe anastomotic stenosis (hazard ratio, 5.96; 95% confidence interval 1.79-22.69; P = .003). CONCLUSION Direct end-to-end anastomosis of the PV system is safe and offers patients with periampullary neoplasia improved chances of complete tumor excision. When tension-free anastomosis cannot be guaranteed, generally in cases requiring ≥31 mm of SMV/PV resection, venous autografting may decrease the likelihood of anastomotic stenosis.

Novel diagnostics for aggravating pancreatic fistulas at the acute phase after pancreatectomy

AIM To identify sensitive predictors of clinically relevant postoperative pancreatic fistula (POPF) at the acute phase after pancreatectomy. METHODS This study included 153 patients diagnosed as having POPFs at postoperative day (POD) 3 after either open pancreatoduodenectomy or distal pancreatectomy between January 2008 and March 2013. The POPFs were categorized into three grades based on the International Study Group on Pancreatic Fistula Definition, and POPFs of grades B or C were considered to be clinically relevant. The predictive performance for the clinically relevant POPF formation of values at PODs 1, 3 and 5 as well as time-dependent changes in levels of inflammatory markers, including white blood cell count, neutrophil count, total lymphocyte count, C-reactive protein (CRP), procalcitonin level, platelet-to-lymphocyte ratio and neutrophil-to-lymphocyte ratio, and amylase content in the drain fluid were compared using the receiver operating characteristic (ROC) curve and multivariable analyses. A scoring system for the prediction of clinically relevant POPFs was created using five risk factors identified in this study, and its diagnostic performance was also evaluated. RESULTS Over time, 77 (50%) of 153 enrolled patients followed a protracted course and were categorized as having clinically relevant POPFs. ROC curve analyses revealed that changes in CRP levels from POD 1 to POD 3 had the greatest area under the curve value (0.767) and that an elevated CRP level of 28.4 mg/L yielded the most optimal predictive value for clinically relevant POPFs. Multivariable analyses for the risk factors of clinically relevant POPFs identified invasive carcinomas of the pancreas and elevation of the CRP level (≥ 28.4 mg/L, from POD 1 to POD 3) as independent diagnostic factors for clinically relevant POPFs (OR 2.94, 95%CI: 1.08-8.55, P = 0.035 and OR 4.82, 95%CI: 1.25-20.2, P = 0.022, respectively). A gradual increase in the prevalence of clinically relevant POPFs in proportion to the risk classification score was confirmed. A highly elevated CRP level and a risk score ≥ 8 were significantly associated with a prolonged duration of drain placement and postoperative hospitalization. CONCLUSION A steep rise in the serum CRP level from POD 1 to POD 3 was a highly predictive factor for subsequent clinically relevant POPFs.

Preoperative internal biliary drainage increases the risk of bile juice infection and pancreatic fistula after pancreatoduodenectomy: a prospective observational study.

Objectives The objective of this study was to identify the most appropriate endoscopic biliary drainage method in patients with pancreatic head cancer. Methods A prospectively collected database comprising 122 consecutive patients who underwent pancreatoduodenectomy, including 72 patients treated by endoscopic retrograde biliary drainage (ERBD) and 50 patients treated by endoscopic nasobiliary drainage (ENBD) procedures, was analyzed. Results All bile cultures collected intraoperatively were positive in the ERBD group, and the positive rates of drainage fluid cultures on postoperative days 1, 3, and 5 and the incidence of postoperative abdominal abscess formation were significantly higher than those in the ENBD group. Moreover, ERBD was identified as an independent predictive factor for postoperative pancreatic fistula (POPF) formation (hazards ratio, 11.81; P < 0.001). The receiver operating characteristic curve analysis for the preoperative drainage period in the ERBD group revealed that the determined cutoff level for the onset of POPF was 29 days. Conclusions Endoscopic retrograde biliary drainage resulted in more frequent postoperative complications, including POPF, compared with ENBD. Postoperative pancreatic fistula is more likely to occur if the ERBD period exceeds 1 month in patients scheduled to undergo pancreatoduodenectomy.

Evaluation of MAGE-D4 expression in hepatocellular carcinoma in Japanese patients.

Though Melanoma‐associated antigen (MAGE) family genes have received lots of attention as cancer‐related genes and targets for immunotherapy, MAGE‐D4 expression in hepatocellular carcinoma (HCC) has not yet been evaluated.