ObjectiveDNA-based testing of pancreatic cyst fluid (PCF) is a useful adjunct to the evaluation of pancreatic cysts (PCs). Mutations in KRAS/GNAS are highly specific for intraductal papillary mucinous neoplasms (IPMNs) and mucinous cystic neoplasms (MCNs), while TP53/PIK3CA/PTEN alterations are associated with advanced neoplasia. A prospective study was performed to evaluate preoperative PCF DNA testing.DesignOver 43-months, 626 PCF specimens from 595 patients were obtained by endoscopic ultrasound (EUS)-fine needle aspiration and assessed by targeted next-generation sequencing (NGS). Molecular results were correlated with EUS findings, ancillary studies and follow-up. A separate cohort of 159 PCF specimens was also evaluated for KRAS/GNAS mutations by Sanger sequencing.ResultsKRAS/GNAS mutations were identified in 308 (49%) PCs, while alterations in TP53/PIK3CA/PTEN were present in 35 (6%) cases. Based on 102 (17%) patients with surgical follow-up, KRAS/GNAS mutations were detected in 56 (100%) IPMNs and 3 (30%) MCNs, and associated with 89% sensitivity and 100% specificity for a mucinous PC. In comparison, KRAS/GNAS mutations by Sanger sequencing had a 65% sensitivity and 100% specificity. By NGS, the combination of KRAS/GNAS mutations and alterations in TP53/PIK3CA/PTEN had an 89% sensitivity and 100% specificity for advanced neoplasia. Ductal dilatation, a mural nodule and malignant cytopathology had lower sensitivities (42%, 32% and 32%, respectively) and specificities (74%, 94% and 98%, respectively).ConclusionsIn contrast to Sanger sequencing, preoperative NGS of PCF for KRAS/GNAS mutations is highly sensitive for IPMNs and specific for mucinous PCs. In addition, the combination of TP53/PIK3CA/PTEN alterations is a useful preoperative marker for advanced neoplasia.

Purpose: Management guidelines for pancreatic intraductal papillary mucinous neoplasms (IPMN) and mucinous cystic neoplasms (MCN) are based on the assumption that mucinous cysts can be accurately distinguished from other pancreatic cystic lesions. Previous studies using surgical material have identified recurrent mutations in GNAS and KRAS in pancreatic mucinous neoplasms. Yet, the diagnostic utility of testing for both genes in pancreatic cyst fluid obtained by endoscopic ultrasound-fine-needle aspiration (EUS-FNA) remains unclear.Experimental Design: GNAS and KRAS testing was performed on EUS-FNA pancreatic cyst fluid from 91 pancreatic cysts: 41 IPMNs, 9 IPMNs with adenocarcinoma, 16 MCNs, 10 cystic pancreatic neuroendocrine tumors (PanNET), 9 serous cystadenomas (SCA), 3 retention cysts, 2 pseudocysts, and 1 lymphoepithelial cyst.Results: Mutations in GNAS were detected in 16 (39%) IPMNs and 2 (22%) IPMNs with adenocarcinoma. KRAS mutations were identified in 28 (68%) IPMNs, 7 (78%) IPMNs with adenocarcinoma, and 1 (6%) MCN. Mutations in either gene were present in 34 (83%) IPMNs, 8 (89%) IPMNs with adenocarcinoma, and 1 (6%) MCN. No mutations were found in cystic PanNETs, SCAs, retention cysts, pseudocysts, and a lymphoepithelial cyst. GNAS and KRAS mutations had 100% specificity [95% confidence interval (CI), 0.83-1.00] but 65% sensitivity (95% CI, 0.52-0.76) for mucinous differentiation. Among IPMNs, mutations in either gene had 98% specificity (95% CI, 0.86-1.00) and 84% sensitivity (95% CI, 0.70-0.92).Conclusions: The combination of GNAS and KRAS testing was highly specific and sensitive for IPMNs; however, the lack of sensitivity for MCNs highlights the need for additional markers to improve the detection of pancreatic mucinous neoplasms.

ObjectiveDespite improvements in imaging, serum CA19-9 and pathological evaluation, differentiating between benign and malignant bile duct strictures remains a diagnostic conundrum. Recent developments in next-generation sequencing (NGS) have opened new opportunities for early detection and management of cancers but, to date, have not been rigorously applied to biliary specimens.DesignWe prospectively evaluated a 28-gene NGS panel (BiliSeq) using endoscopic retrograde cholangiopancreatography-obtained biliary specimens from patients with bile duct strictures. The diagnostic performance of serum CA19-9, pathological evaluation and BiliSeq was assessed on 252 patients (57 trainings and 195 validations) with 346 biliary specimens.ResultsThe sensitivity and specificity of BiliSeq for malignant strictures was 73% and 100%, respectively. In comparison, an elevated serum CA19-9 and pathological evaluation had sensitivities of 76% and 48%, and specificities of 69% and 99%, respectively. The combination of BiliSeq and pathological evaluation increased the sensitivity to 83% and maintained a specificity of 99%. BiliSeq improved the sensitivity of pathological evaluation for malignancy from 35% to 77% for biliary brushings and from 52% to 83% for biliary biopsies. Among patients with primary sclerosing cholangitis (PSC), BiliSeq had an 83% sensitivity as compared with pathological evaluation with an 8% sensitivity. Therapeutically relevant genomic alterations were identified in 20 (8%) patients. Two patients with ERBB2-amplified cholangiocarcinoma received a trastuzumab-based regimen and had measurable clinicoradiographic response.ConclusionsThe combination of BiliSeq and pathological evaluation of biliary specimens increased the detection of malignant strictures, particularly in patients with PSC. Additionally, BiliSeq identified alterations that may stratify patients for specific anticancer therapies.

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With improvements in abdominal imaging, detection of incidental pancreatic cysts are becoming increasingly common. Analysis of pancreatic cyst fluid from fine-needle aspiration is particularly important in identifying intraductal papillary mucinous neoplasms (IPMNs) and mucinous cystic neoplasms (MCNs), which have significant implications in clinical intervention and follow-up. Previous controlled studies have shown that KRAS mutations in cyst fluid are highly specific for mucinous differentiation in pancreatic cysts; however, this has not been examined in the clinical setting. Over a 6-year study period, 618 pancreatic cyst fluids obtained by fine-needle aspiration at the time of endoscopic ultrasound were tested for KRAS mutations as part of routine evaluation for a cystic neoplasm. Of the 618 specimens, 603 (98%) from 546 patients were satisfactory for molecular analysis. Patients ranged in age from 17 to 90 years (mean, 63.9 years) and were predominantly female (68%). Pancreatic cysts were relatively evenly distributed throughout the pancreas and ranged in size from 0.6 to 11.0 cm (mean, 2.3 cm). Mutations in KRAS were detected in 232 of 603 (38%) aspirates. Although sufficient for molecular analysis, 320 of 603 (53%) specimens were either less than optimal (38%) or unsatisfactory (15%) for cytopathologic diagnosis. Surgical follow-up information was available for 142 (26%) patients and consisted of 53 KRAS-mutated and 89 KRAS-wild-type cysts. Overall, KRAS mutations had a specificity of 100%, but a sensitivity of 54% for mucinous differentiation. When stratified by cyst type, KRAS had a sensitivity of 67% and 14% for IPMNs and MCNs, respectively. In summary, KRAS mutations were highly specific for mucinous differentiation, but were inadequate in identifying MCNs. Future molecular studies and the combination of other fluid markers are required to improve the detection and classification of pancreatic mucinous neoplasms by endoscopic ultrasound fine-needle aspiration.