Asif Khalid

Pancreatic cyst fluid DNA analysis in evaluating pancreatic cysts: a report of the PANDA study

BACKGROUND The role of pancreatic cyst fluid DNA analysis in evaluating pancreatic cysts remains unclear. OBJECTIVE Our purpose was to evaluate the utility of a detailed DNA analysis of pancreatic cyst fluid to diagnose mucinous and malignant cysts. DESIGN Prospective, multicenter study. PATIENTS Patients with pancreatic cysts presenting for EUS evaluation. INTERVENTION EUS-guided pancreatic cyst aspirates cytology evaluation, carcinoembryonic antigen (CEA) level determination, and a detailed DNA analysis; incorporating DNA quantification, k-ras mutation and multiple allelic loss analysis, mutational amplitude, and sequence determination. MAIN OUTCOME MEASUREMENTS Cyst fluid analysis compared with surgical pathologic or malignant cytologic examination. RESULTS The study cohort consisted of 113 patients with 40 malignant, 48 premalignant, and 25 benign cysts. Cyst fluid k-ras mutation was helpful in the diagnosis of mucinous cysts (odds ratio 20.9, specificity 96%), whereas receiver-operator characteristic curve analysis indicated optimal cutoff points for allelic loss amplitude (area under the curve [AUC] 0.79; optimal value > 65%) and CEA (AUC 0.74; optimal value >148 ng/mL). Components of DNA analysis detecting malignant cysts included allelic loss amplitude over 82% (AUC 0.9) and high DNA amount (optical density ratio >10, AUC 0.79). The criteria of a high amplitude k-ras mutation followed by allelic loss showed maximum specificity (96%) for malignancy. All malignant cysts with negative cytologic evaluation (10/40) could be diagnosed as malignant by using DNA analysis. LIMITATIONS Limited follow-up, selection bias. CONCLUSIONS Elevated amounts of pancreatic cyst fluid DNA, high-amplitude mutations, and specific mutation acquisition sequences are indicators of malignancy. The presence of a k-ras mutation is also indicative of a mucinous cyst. DNA analysis should be considered when cyst cytologic examination is negative for malignancy.

ACG Practice Guidelines for the Diagnosis and Management of Neoplastic Pancreatic Cysts

The diagnosis and management of pancreatic cystic lesions is a common problem. At least 1% of hospitalized patients at major medical centers will have a pancreatic cystic lesion on cross sectional imaging. Up to a quarter of all pancreata examined in an autopsy series contained a pancreatic cyst, 16% of which were lined by an “atypical” epithelium and 3% of which had progressed to carcinoma-in-situ (high grade dysplasia). in the past, it was thought these cystic lesions were benign, but increasing evidence points to the cystic lesions as being the origin of some pancreatic malignancies.The most important clinical tools in the diagnosis and management of pancreatic cystic lesions are cross sectional imaging, endoscopic ultrasound, and cyst fluid analysis. The most important differential diagnosis is distinguishing mucinous (pre-malignant) and non-mucinous cystic lesions. The findings of a macrocystic lesion containing viscous fluid rich in CEA are supportive of a diagnosis of a mucinous lesion. Serous lesion are the most common non-mucinous cyst and are characterized by a microcystic morphology, non-viscous fluid and a low concentration of CEA in the cyst fluid.The following document includes a description of neoplastic pancreatic cysts, a critical review of relevant diagnostic tests, and a discussion of treatment options. We have proposed a set of guidelines for the diagnonis and management of patients with neoplastic pancreatic cysts. The guidelines are based on published data backed by an analysis of the quality of the data and are designed to address the most frequent and important clinical scenarios. In addition to providing a summary of the diagnostic data, we offer diagnostic and management suggestions based on 13 common clinical problems. Although the field is rapidly evolving, a set of core principles is provided based on a balance between the risk of malignancy and the benefit of pancreatic resection.

Evaluation and Management of Autoimmune Pancreatitis: Experience at a Large US Center

OBJECTIVES:Autoimmune pancreatitis (AIP) is increasingly recognized as a form of chronic pancreatitis. Systematic evaluation and management of AIP in the United States is reported only from one center. Our aim was to review the evaluation and management of AIP at a large tertiary center.METHODS:We retrospectively reviewed information on demographics, clinical presentation, laboratory and imaging findings, extrapancreatic involvement, treatment response, and recurrence in 26 patients with AIP treated at the University of Pittsburgh Medical Center from 1998 to 2007.RESULTS:The median age at presentation was 62.5 years (range: 23–86), 65% were men, and 88% were Caucasians. The most common presentation included new-onset mild abdominal pain (65%), jaundice (62%), and weight loss (42%). Pancreatic mass, enlargement, or prominence on imaging was present in 85% of the patients. Serum IgG4 (immunoglobulin-4) was elevated (>140 mg/dl) in 44% (8/18) at presentation. The most common extrapancreatic finding was extrapancreatic/intrahepatic biliary strictures (35%). Peri-pancreatic vascular complications were noted in 23% of the patients. Six patients underwent partial or complete pancreatectomy. Partial or complete response was observed for initial steroid treatment in 19 patients and for methotrexate in 1 patient. Recurrences were common, especially in patients with extrapancreatic manifestations, and usually responded to a combination of steroids and azathioprine. Any one of the commonly used diagnostic criteria (Mayo Clinics HISORt criteria, the Japanese Pancreas Society criteria, Korean diagnostic criteria) was fulfilled in 85% of cases.CONCLUSIONS:In this second major US series, we confirm several findings previously reported in AIP. Our study highlights the presence of vascular complications in a subset of patients with AIP. The current diagnostic criteria may not identify all AIP patients.

The Role of Pancreatic Cyst Fluid Molecular Analysis in Predicting Cyst Pathology

BACKGROUND & AIMS Current methods to detect malignancy in mucinous cystic neoplasms of the pancreas remain inadequate. The role of detailed molecular analysis in this context was investigated. METHODS Endoscopic ultrasound-guided pancreatic cyst aspirates were prospectively collected during a period of 19 months and studied for cytology, carcinoembryonic antigen level, and molecular analysis. Molecular evaluation incorporated DNA quantification (amount and quality), k-ras point mutation, and broad panel tumor suppressor linked microsatellite marker allelic loss analysis by using fluorescent capillary electrophoresis. The sequence of mutation acquisition was also calculated on the basis of a clonal expansion model, and comparison was made to the final pathology. RESULTS Thirty-six cysts with confirmed histology were analyzed. There were 11 malignant, 15 premalignant, and 10 benign cysts. Malignant cysts could be differentiated from premalignant cysts on the basis of fluid carcinoembryonic antigen level (P=.034), DNA quality (P=.009), number of mutations (P=.002), and on the sequence of mutations acquired (P<.001). Early k-ras mutation followed by allelic loss was the most predictive of a malignant cyst (sensitivity, 91%; specificity, 93%). CONCLUSIONS Malignant cyst fluid contains adequate DNA to allow mutational analysis. A first hit k-ras mutation followed by allelic loss is most predictive of the presence of malignancy in a pancreatic cyst. This approach should serve as an ancillary tool to the conventional work-up of pancreatic cysts. Cumulative amount and timing of detectable mutational damage can assist in diagnosis and clinical management.

Elevated Serum Creatinine as a Marker of Pancreatic Necrosis in Acute Pancreatitis

OBJECTIVES:Pancreatic necrosis is a serious complication of acute pancreatitis. The identification of simple laboratory tests to detect subjects at risk of pancreatic necrosis may direct management and improve outcome. This study focuses on the association between routine laboratory tests and the development of pancreatic necrosis in patients with acute pancreatitis.METHODS:In a cohort of 185 patients with acute pancreatitis prospectively enrolled in the Severity of Acute Pancreatitis Study, patients with contrast-enhanced computerized tomography performed were selected (n=129). Serum hematocrit, creatinine, and urea nitrogen on admission and peak values within 48 h of admission were analyzed. The volume of intravenous fluid resuscitation was calculated for each patient.RESULTS:Of 129 patients, 35 (27%) had evidence of pancreatic necrosis. Receiver operating characteristic curves for pancreatic necrosis revealed an area under the curve of 0.79 for admission hematocrit, 0.77 for peak creatinine, and 0.72 for peak urea nitrogen. Binary logistic regression yielded that all three tests were significantly associated with pancreatic necrosis (P<0.0001), with the highest odds ratio, 34.5, for peak creatinine. The volume of intravenous fluid resuscitation was similar in patients with and without necrosis. Low admission hematocrit (≤44.8%) yielded a negative predictive value of 89%; elevated peak creatinine (>1.8 mg/dl) within 48 h yielded a positive predictive value of 93%.CONCLUSIONS:We confirm that a low admission hematocrit indicates a low risk of pancreatic necrosis (PNec) in patients with acute pancreatitis. In contrast, an increase in creatinine within the first 48 h is strongly associated with the development of PNec. This finding may have important clinical implications and warrants further investigation.

Endoscopic Ultrasound Fine Needle Aspirate DNA Analysis to Differentiate Malignant and Benign Pancreatic Masses

OBJECTIVES:Accurate diagnosis of malignant and benign pancreatic masses can be challenging, potentially delaying treatment for cancer and subjecting patients with benign disease to unnecessary surgery. Endoscopic ultrasound fine needle aspirate (EUS-FNA) of pancreatic masses remains inconclusive in a subset of patients. The role of EUS-FNA molecular analysis in this context is studied.METHODS:Patients with benign pancreatic masses (6 cases, 4 autoimmune pancreatitis, 2 focal chronic pancreatitis) and malignant pancreatic masses (15) with inconclusive cytology (5 cases) and positive cytology (10 controls) were selected. All cases had definitive pathology. Representative cells were microdissected from each EUS-FNA sample and subjected to PCR for analysis of 16 microsatellite allele loss markers situated at 1p, 3p, 5q, 9p, 9q, 10q, 17p, 17q, 21q, and 22q. Loss of heterozygosity analysis used fluorescent capillary electrophoresis for quantitative determination of allelic imbalance. k-ras-2 point mutation analysis was also performed. Mean fractional mutation rate (FMR) was calculated and compared for each group.RESULTS:All malignant cases carried multiple mutations (FMR 0.50), regardless of positive cytology (FMR 0.52) or suspicious cytology (FMR 0.47) (p = NS). Five of the 6 benign cases carried no mutations whereas 1 case of autoimmune pancreatitis and coexisting PanIN lesions exhibited a k-ras mutation (FMR 0.01). The mean FMR for the malignant and benign samples was significantly different (p < 0.0001).CONCLUSIONS:Broad panel microsatellite loss and k-ras point mutation analysis can be reliably performed on EUS-FNA samples from pancreatic masses and improves the diagnostic accuracy. Furthermore, it accurately differentiates between malignant and benign pancreatic masses.

EUS-guided fiducial placement for stereotactic body radiotherapy in locally advanced and recurrent pancreatic cancer

BACKGROUND Stereotactic body radiotherapy (SBRT) has been approved for the treatment of locally advanced pancreatic cancer. Placement of gold fiducials is required for real-time tracking and delivery of a high-dose therapeutic beam of radiation to the tumor. Traditionally, fiducials have been placed either intraoperatively or percutaneously. Recently, EUS-guided fiducial placement has been reported, but the safety and feasibility of this approach is not well defined. OBJECTIVE The aim of this study was to determine the safety, feasibility, and limitations of EUS-guided placement of 0.8 x 5.0 mm fiducials via a 19-gauge needle for locally advanced and recurrent pancreatic cancer. DESIGN Prospective study of patients with either locally advanced or recurrent pancreatic cancer referred for EUS-guided fiducial placement for SBRT at our institution over a 3-year period. SETTING Tertiary referral center conducting >1800 EUS procedures annually. MAIN OUTCOME MEASUREMENTS Primary outcome measurements included success, complications, and technical limitations of EUS-guided fiducial placement in pancreatic cancer. In addition, the percentage of patients successfully completing SBRT after EUS-guided fiducial placement was determined. RESULTS A total of 51 patients (mean age 73 years; 57% male) with locally advanced (n = 36) and recurrent (n = 15) pancreatic cancer were referred for EUS-guided fiducial placement. Fiducials were successfully placed in 46 patients (90%), with technical failures occurring in 4 patients (8%) with recurrent cancer after pancreaticoduodenectomy. In 3 patients (7%), the fiducials spontaneously migrated from the original site of injection, thereby requiring a second EUS procedure for placement of additional fiducials. Of the 46 patients with fiducials placed under EUS guidance, 42 patients (91%) successfully completed SBRT. Two patients experienced disease progression before SBRT, 1 patient was lost to follow-up, and 1 patient experienced a complication at ERCP that precluded further therapy. Only 1 complication (2%), of mild pancreatitis, occurred in a patient undergoing simultaneous placement of fiducials and celiac plexus neurolysis for intractable abdominal pain. LIMITATIONS Single-center experience and lack of a formal follow-up protocol to assess for complications. CONCLUSION EUS-guided fiducial placement for SBRT in locally advanced and recurrent pancreatic cancer is safe and feasible. Successful placement was achieved in 90% of patients, with a low complication rate (2%). Furthermore, 91% of patients successfully completed SBRT after EUS-guided fiducial delivery. Although fiducials can spontaneously migrate from the initial injection site, the rate of migration is relatively low (7%), and no migration-related complications occurred over the course of this study. Limitations to EUS-guided fiducial placement may include surgically altered anatomy (pancreaticoduodenectomy) in patients with recurrent pancreatic cancer.

Use of microsatellite marker loss of heterozygosity in accurate diagnosis of pancreaticobiliary malignancy from brush cytology samples

Background: Brush cytology of biliary strictures to diagnose pancreaticobiliary malignancy suffers from poor sensitivity. Aim: To improve the diagnostic yield of pancreaticobiliary brush cytology through analysis of tumour suppressor gene linked microsatellite marker loss of heterozygosity (LOH) and k-ras codon 12 mutation detection. Methods: Twenty six patients with biliary strictures underwent endoscopic retrograde cholangiography with brush cytology. A panel of 12 polymorphic microsatellite markers linked to six tumour suppressor genes was developed. Genomic DNA from cell clusters acquired from brush cytology specimens and microdissected surgical malignant and normal tissue underwent polymerase chain amplification reaction (PCR). PCR products were compared for LOH and k-ras codon 12 mutations. Results: Seventeen patients were confirmed to have pancreaticobiliary adenocarcinoma. Nine patients had benign strictures (eight proven surgically, one by follow up). Cytomorphological interpretation was positive for malignancy (n = 8), indeterminate (n = 10), and negative for malignancy (n = 8). Selected malignant appearing cytological cell clusters and microdissected histological samples from cancer showed abundant LOH characteristic of malignancy while brushings from nine cases without cancer carried no LOH (p<0.001). LOH and k-ras mutations profile of the cytological specimens was almost always concordant with the tissue samples. Presence of k-ras mutation predicted malignancy of pancreatic origin (p<0.001). Conclusion: LOH and k-ras codon 12 mutation analysis of PCR amplified DNA from biliary brush cytology discriminates reactive from malignant cells, with 100% sensitivity, specificity, and accuracy. Minor variations in LOH in brushings and in different sites within the same tumour likely reflect intratumoral mutational heterogeneity during clonal expansion of pre- and neoplastic lineages.

American Gastroenterological Association guidelines are inaccurate in detecting pancreatic cysts with advanced neoplasia: a clinicopathologic study of 225 patients with supporting molecular data

BACKGROUND AND AIMS The American Gastroenterological Association (AGA) recently reported evidence-based guidelines for the management of asymptomatic neoplastic pancreatic cysts. These guidelines advocate a higher threshold for surgical resection than prior guidelines and imaging surveillance for a considerable number of patients with pancreatic cysts. The aims of this study were to assess the accuracy of the AGA guidelines in detecting advanced neoplasia and present an alternative approach to pancreatic cysts. METHODS The study population consisted of 225 patients who underwent EUS-guided FNA for pancreatic cysts between January 2014 and May 2015. For each patient, clinical findings, EUS features, cytopathology results, carcinoembryonic antigen analysis, and molecular testing of pancreatic cyst fluid were reviewed. Molecular testing included the assessment of hotspot mutations and deletions for KRAS, GNAS, VHL, TP53, PIK3CA, and PTEN. RESULTS Diagnostic pathology results were available for 41 patients (18%), with 13 (6%) harboring advanced neoplasia. Among these cases, the AGA guidelines identified advanced neoplasia with 62% sensitivity, 79% specificity, 57% positive predictive value, and 82% negative predictive value. Moreover, the AGA guidelines missed 45% of intraductal papillary mucinous neoplasms with adenocarcinoma or high-grade dysplasia. For cases without confirmatory pathology, 27 of 184 patients (15%) with serous cystadenomas (SCAs) based on EUS findings and/or VHL alterations would continue magnetic resonance imaging (MRI) surveillance. In comparison, a novel algorithmic pathway using molecular testing of pancreatic cyst fluid detected advanced neoplasias with 100% sensitivity, 90% specificity, 79% positive predictive value, and 100% negative predictive value. CONCLUSIONS The AGA guidelines were inaccurate in detecting pancreatic cysts with advanced neoplasia. Furthermore, because the AGA guidelines manage all neoplastic cysts similarly, patients with SCAs will continue to undergo unnecessary MRI surveillance. The results of an alternative approach with integrative molecular testing are encouraging but require further validation.

Preoperative GNAS and KRAS Testing in the Diagnosis of Pancreatic Mucinous Cysts

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. Clin Cancer Res; 20(16); 4381–9. ©2014 AACR.