Molecular Analysis of Cyst Fluid Aspiration in the Diagnosis and Risk Assessment of Cystic Lesions of the Pancreas

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1 Molecular Analysis of Cyst Fluid Aspiration in the Diagnosis and Risk Assessment of Cystic Lesions of the Pancreas Sagar S. Garud, M.D., M.S. and Field F. Willingham, M.D., M.P.H. Abstract Pancreatic cyst detection is increasing largely due to increasing use of cross-sectional imaging. The management of pancreatic cysts differs for true cysts, pseudocysts, mucinous cysts, nonmucinous cysts, and malignant lesions. Depending on the setting, diagnostic tests, such as cross-sectional imaging, endoscopic ultrasound, cyst fl uid chemistry, and cytology, have moderate accuracy in characterizing the cyst subtype. Molecular analysis of cyst fl uid aspirates has shown promise in preliminary studies and may require smaller fl uid volumes than is needed for carcinoembryonic antigen level and cytology. This article reviews published studies in which molecular analysis was performed in the evaluation of pancreatic cysts. The molecular studies are compared with the conventional tests. Most studies have had moderate sample sizes (16 124) and have characterized a high proportion of patients with malignant cysts. Evaluation of molecular analysis as a diagnostic tool merits larger prospective trials with long-term follow-up of patients who are not sent to surgery. Larger cysts may meet size criteria for resection, and it is the smaller cysts for which molecular analysis may be of benefi t if additional molecular testing results in a change in management. Clin Trans Sci 2012; Volume 5: Keywords: pancreas, endoscopy, molecular genetics Introduction The detection of pancreatic cysts has increased largely due to the increasing use of cross-sectional imaging. Pancreatic cysts often represent an incidental finding on an imaging study performed for unrelated reasons. A review of 24,000 abdominal computed tomography (CT) and magnetic resonance imaging (MRI) studies over an 8-year period revealed that 1.2% of the patients had pancreatic cysts. 1 Another study of patients undergoing CT scan for unrelated reasons demonstrated a prevalence of 2.6 per 100 patients. 2 A retrospective review of 1,944 pancreaticoduodenectomies showed 118 (6.1%) incidental pancreatic lesions. Eighty-six (4.4%) were identified on imaging and the rest by lab studies or endoscopy. 3 Pancreatic cysts were identified in 19.6% of 1,444 patients who underwent an MRI in another study. 4 A series of 475 pancreatic resections showed that 13.5% of these operations were performed for incidental pancreatic lesions. 5 The incidental finding of a cystic pancreatic lesion can cause concern for patients and for providers, and cross-sectional imaging is not able to characterize many cysts as benign or malignant. Cyst fluid aspirates for carcinoembryonic antigen (CEA) and amylase may suggest a mucinous or nonmucinous subtype but are not diagnostic. Cyst fluid cytology can be specific but has a very low sensitivity in the setting of cystic lesions of the pancreas. Cysts with malignant potential include mucinous cystic neoplasms, intraductal papillary mucinous neoplasms (IPMNs), and solid pseudopapillary neoplasms. There is minimal malignant potential with serous cystadenomas ( Table 1 ). 6 Improving the preoperative diagnosis and characterization of the malignant potential of pancreatic cysts would be of great benefit to the large number of patients with these lesions. This article describes the methods of molecular analysis, the interpretation of the results, and the various studies evaluating the use of molecular analysis for the diagnosis of pancreatic cysts. Methods of Molecular Analysis Pancreatic cyst fluid is obtained, often by endoscopic ultrasound (EUS) guided fine needle aspiration (FNA). The cyst fluid is inoculated into a specimen container and sent for molecular characterization. Approximately 0.4 ml of fluid is sufficient for molecular analysis. 7 Exfoliated epithelial cells in the small volume of cyst fluid are the source for the DNA used for the detection of underlying molecular changes. There is one commercially available test for pancreatic cyst fluid molecular analysis (PathFinderTG, RedPath Integrated Pathology, Pittsburgh, PA, USA). All the studies reviewed in this article have used this test for molecular analysis. See Appendix for details about the methods. The criteria for molecular diagnosis are outlined in Table 2. The presence of any one of the following is used to designate a cyst mucinous by molecular analysis criteria: DNA quantity 40 ng/ml, the presence of a K- ras point mutation, or the presence of 2 specified allelic imbalances. In addition to the criteria for a mucinous molecular diagnosis, if the K- ras or loss of heterozygosity (LOH) mutation is present at high amplitude (>75% of total DNA), suggesting a significant clonal expansion, then a malignant diagnosis is rendered. 8 Results of Molecular Analysis K-ras mutation in the diagnosis of pancreatic cysts The K- ras gene encodes the KRAS protein, which is a GTPase. It is involved early in many signal transduction pathways. When mutated, K- ras is an oncogene. The protein product of the normal K- ras gene performs an important function in normal tissue signaling, and the mutation of a K- ras gene is an essential step in the development of many cancers. Determination of a K- ras point mutation is accomplished by fluorescent-based direct sequencing of the amplified first exon of the gene. 9 Studies have shown that there is a sequential accumulation of genetic changes in the carcinogenesis of pancreatic mucinous cysts. These changes include K- ras mutation, p53 mutation, and loss of p16 and SMAD Distinct adenoma-to-carcinoma sequences exist in the pancreas in which invasive adenocarcinomas may arise from cystic neoplasia. 14 K- ras mutation has shown a high specificity (93 100%) but a low sensitivity (11 57%) for the diagnosis of mucinous cysts ( Table 3 ). Both specificity (71 93%) and sensitivity (20 53%) Division of Digestive Diseases, Emory University School of Medicine, Atlanta Georgia, USA. Correspondence: FF Willingham ( fwillin@emory.edu ) DOI: /j x 102 VOLUME 5 ISSUE 1

2 Malignant and nonmalignant cystic lesions of the pancreas Nonneoplastic cysts Pseudocyst Retention cyst Congenital cyst Foregut cyst Endometriotic cyst Cystic nonepithelial neoplasms Lymphangioma Hemangioma Primarily cystic epithelial neoplasms Serous cystadenoma Microcystic Macrocystic Mucinous cystic neoplasm Intraductal papillary mucinous neoplasm Miscellaneous cysts Lymphoepithelial cyst Epidermoid cyst in intrapancreatic heterotopic spleen Secondarily cystic solid neoplasms Solid pseudopapillary neoplasm Ductal adenocarcinoma Endocrine neoplasms Acinar cell neoplasms Table 1. Cysts of the pancreas. 6 decrease further when K- ras mutation is used for the diagnosis of malignant cysts ( Table 3 ). Only exception is one study that revealed a sensitivity of 83% for malignant cysts. 8 O n l y t w o studies have reported data on use of K- ras to differentiate between benign mucinous and malignant mucinous cysts. Both showed a high specificity (96 100%) and a low sensitivity (42 47%). 7,8 Overall, the studies show that K- ras has a low sensitivity and a high specificity in the differentiation of benign and malignant pancreatic cysts. Molecular diagnosis Benign nonmucinous Benign mucinous Malignant (in situ or invasive carcinoma) LOH = loss of heterozygosity. Criteria Table 2. Criteria for molecular diagnosis. 8 (1) DNA quantity/quality low to moderate; AND (2) K-ras gene point mutation not present; AND (3) LOH, <2 genomic loci present (1) DNA quantity/quality: high; OR (2) K-ras gene point mutation present; OR (3) LOH, 2 genomic loci present. (1) K-ras gene point mutation, high amplitude (>75%); OR (2) 2 more LOH, high amplitude (>75%) Allelic imbalance Humans have two copies of each gene. Normally, these two copies are expressed equally; thus, the mrna transcript will have roughly the same number of copies. When the ratio of the expression is not 1 to 1, this is referred to as allelic imbalance. LOH is a common form of allelic imbalance. LOH represents the loss of normal function of one allele of a gene. After an inactivating mutation in one allele of a tumor suppressor gene occurs in the parent s germline cell, it is passed on to the zygote resulting in an offspring that is heterozygous for that allele. LOH occurs when the remaining functional allele in a somatic cell of the offspring becomes inactivated by mutation. This could cause a normal tumor suppressor to no longer be produced that could result in tumorigenesis. The detection of LOH has been used to identify genomic regions that harbor tumor suppressor genes. 15 One-microliter aliquots are used for polymerase chain reaction (PCR) amplification of individual microsatellite markers. Fluorescent-labeled oligonucleotide primers are used for quantitative determination of allelic imbalance. Allelic imbalance has shown a moderate sensitivity (43 70%) and a wide range of specificity (66 100%) for the diagnosis of mucinous cysts ( Table 3 ). The sensitivity (75 100% except one study showing 50%) improves, while the specificity (36 83%) decreases when it is used for the diagnosis of malignant cysts ( Table 3 ). Both sensitivity (27 67%) and specificity (17 93%) are low for differentiating benign mucinous versus benign nonmucinous cysts. 7,8,16 DNA quantity/quality High DNA quantity/quality is used as an indicator of a mucinous cyst. DNA quantity can be determined by spectrophotometric analysis. Nucleic acids absorb ultraviolet light in a specific pattern. In a spectrophotometer, a DNA sample is exposed to ultraviolet light at 260 nm, and a photo-detector measures the light that passes through the sample. The more light absorbed by the sample, the higher the nucleic acid concentration in the sample. DNA is extracted from the fluid and then resuspended in a tris(hydroxymethyl)aminomethane (TRIS) buffer. The concentration of DNA is obtained according to the optical density (OD) ratio at 260 of 280 wavelength. About 200 μl of fluid is sufficient for DNA extraction. 7 DNA quantity/quality had a low sensitivity (29 46%) and a high specificity (68 100%) for the diagnosis of mucinous cysts (Table 3 ). The sensitivity (40 83%) and specificity (75 93%) are better for diagnosing malignant cysts ( Table 3 ). Only two studies have reported data on using DNA quantity/ quality for differentiating benign mucinous cysts from benign nonmucinous cysts and both show poor sensitivity (13 21%) and a moderate to high (68 100%) specificity. 7,8 Combinations of Markers K-ras mutation and CEA level One of the studies showed that a CEA level of 148 ng/ml yielded a sensitivity 67% (39/58) and specificity of 67% (12/18) for detection of mucinous cysts. Checking for a K- ras mutation in cysts with CEA <148 ng/ml, lead to detection of 10 additional mucinous cysts and increased the sensitivity to 84% (49/58) VOLUME 5 ISSUE 1 103

3 First author Khalid Sawhney Shen Study design Prospective/ multicenter N No. and percentage of malignant cysts /113 (35%) Mucinous versus nonmucinous K-ras mutation Alleilic imbalance DNA quantity/ quality Malignant vs. nonmalignant K-ras mutation Alleilic imbalance DNA quantity/ quality Sen Spe Sen Spe Sen Spe Sen Spe Sen Spe Sen Spe /19 (26%) /35 (17%) Sreenarasimhaiah Schoedel Khalid Prospective/ Sen = sensitivity; Spe = specifi city; N = number of study subjects; NR = not reported Table 3. Characteristics of studies of molecular analysis of pancreatic cyst fl uid. 20 9/20 (45%) NR NR 33 NR 50 NR NR NR 16 4/16 (25%) 25 NR 44 NR NR NR NR NR 36 11/36 (31%) NR NR NR NR NR NR NR NR NR NR without decreasing the specificity. 7 In the same study, in K- ras negative cysts, a CEA level >148 ng/ml was present in a higher number of mucinous cysts (69%) than nonmucinous cysts (31.5%, p =.01). 7 Cytology and DNA analysis A study reported that all malignant cysts with false-negative cytologic findings manifested at least one DNA analysis variable associated with malignancy. There were 10 of 40 (25%) malignant cysts in which the cytology evaluation did not reveal malignant cells. DNA analysis revealed allelic loss amplitude >80% in seven of 10 and OD >10 in six of 10 cases. 7 CEA level and molecular analysis A study reported that if CEA and molecular analysis were combined, in that cysts with either CEA level 192 ng/dl or meeting molecular analysis criteria were classified as mucinous, then all mucinous cysts were correctly identified. However, these results were based on a small number ( n = 19) of patients. 17 K-ras mutation and LOH For the presence of malignancy in a mucinous cyst, the sensitivity and specificity of a K- ras mutation were 91% and 86%, respectively, whereas the presence of allelic loss after K- ras mutation increased sensitivity and specificity to 91% and 93%, respectively. These findings were based on data analysis from 26 cysts. 17,18 Correlation Between Genetic Analysis, Histology, and Cyst Fluid CEA Level A retrospective study of 100 patients showed that there was a poor agreement between CEA and molecular analysis for the classification of mucinous cysts (kappa = 0.2, agreement 59.5%). Poor agreement existed between CEA and DNA quantity (Spearman correlation = 0.2; p = 0.1), K- ras mutation (kappa = 0.3), and 2 allelic imbalance mutations (kappa = 0.1). 17 Another study also reported a poor concordance between cyst fluid CEA level, DNA analysis, and histology. Consistency among all parameters was seen in only seven of 20 (35.0%) patients, and this was only noted in patients who had a benign cyst. 19 A better concordance was found between clinical consensus diagnosis and molecular diagnosis. The clinical consensus diagnosis was made using histologic diagnosis or a combination of two of three concordant characteristics: (1) EUS features, (2) cyst fluid CEA level, or (3) cytology. The concordance between clinical consensus diagnosis and molecular diagnosis was five of six (83%), 13 of 15 (87%), and 13 of 14 (93%), respectively, for malignant, benign mucinous, and benign nonmucinous cysts, with an overall Cohen kappa statistic of Discussion Patients with benign cysts who are resected have undergone major pancreatic surgery without a malignant diagnosis or symptoms, and patients with malignant cysts who are not resected may progress to pancreatic carcinoma. Gastroenterologists, primary care providers, and surgeons are seeing increasing numbers of patients with pancreatic cysts; many are asymptomatic and are referred for the finding of an incidental cyst on cross-sectional imaging. Establishing the diagnosis has been difficult in the preoperative setting. Clinical and demographic features are inadequate in differentiating mucinous and serous cysts A small number of serous cysts show a central scar on radiologic studies; however, most cysts do not have diagnostic radiographic features. 23,24 Analysis of cyst fluid has improved the characterization of the cysts but is criticized for moderate sensitivity and specificity. CEA level has been reported to have an 80% accuracy in differentiating mucinous versus nonmucinous cysts. 25,26 Adding to the challenge, not all small cysts in asymptomatic patients are benign and not all cysts >3 cm are malignant. 27 Molecular analysis of cyst fluid has been developed to provide additional information in the characterization of pancreatic cysts. Cysts contain DNA from degenerated cells. There have been six 104 VOLUME 5 ISSUE 1

4 Study Year N CEA sensitivity K-ras sensitivity CEA specificity K-ras specificity Khalid et al Sawhney et al Sreenarasimhaiah et al Shen et al Schoedel et al NR 25 NR NR Khalid et al NR NR NR NR NR = not reported. Table 4. Comparing sensitivity of CEA and K- ras for diagnosis of mucinous cysts. studies looking at the role of genetic analysis in the diagnosis of pancreatic cysts. 7,8, Five of the six studies were and all were performed at academic medical centers. Four studies were retrospective that did not allow them to fully measure the impact of DNA analysis on clinical decision making ( Table 3 ). Only one study had more than 100 patients; the remainders were small trials (16 36 patients). Small sample sizes can lead to imprecise estimates of test accuracy and limited ability to analyze test characteristics of different variations of the molecular analysis criteria. One commercial laboratory, RedPath Integrated Pathology performs this analysis. It is currently unknown whether these results can be reliably reproduced in other laboratories. There is a significant cost for molecular analysis. The analysis is often covered by the patient s insurance provider. RedPath Integrated Pathology was involved in three of the six studies. 7,16,18 All three components of molecular analysis (K- ras mutation, LOH, and DNA quantity/quality) have shown a high specificity (66 100%) but a low sensitivity (25 70%) for diagnosing mucinous cysts. For diagnosis of malignant cysts, K- ras mutation showed moderate sensitivity (20 91%); LOH and DNA quantity showed a higher sensitivity (40 100%). The specificity was high for all of the molecular markers. Use in diagnosis of mucinous cysts CEA level has been the most sensitive test for the diagnosis of mucinous cysts. A CEA level of 192 ng/ml had a sensitivity of 73%, specificity of 84%, and accuracy of 79%. 25 How does molecular analysis do in comparison to this? Presence of K- ras mutation, by itself is not as sensitive as CEA level for the diagnosis of mucinous cysts ( Table 4 ). In one study where CEA level was available for 76 patients, combining these two tests increased the sensitivity to 84%. 7 Another study indicated that combining CEA level and results of molecular analysis (as opposed to just the K- ras component of it), increased the sensitivity to 100%, in a series with 19 patients. 17 Another study of 35 patients suggested that molecular analysis was 87% sensitive for the diagnosis of mucinous cysts. 8 The above data may be hypothesis forming, with small studies and retrospective designs suggesting a role for powered prospective trials. Use in diagnosis of malignant cysts After diagnosing a cyst as mucinous, is it important to determine if malignancy is present. If all mucinous cysts are considered for surgical resection then the distinction may be less relevant clinically. Cytology is the most specific test to diagnose malignant cysts. However, it has a low sensitivity ranging between 34% and 50% for the diagnosis of mucinous cysts and 22% for the diagnosis of malignant mucinous cysts. 25,28,29 Many times this is due to the small amount of cyst fluid aspirated. CEA levels do not reliably differentiate benign from malignant mucinous cysts. Of the three components of molecular analysis, allelic imbalance is the most sensitive ( Table 3 ). Combining K-ras mutation with allelic imbalance increased the sensitivity and specificity in one study. 7,18 However, K-ras mutation by itself is not very sensitive in the diagnosis of malignant cysts. If cyst cytologic examination is negative for malignancy, a detailed DNA analysis can be performed in this situation. Future directions Molecular analysis of cyst fluid has shown promise in previous studies, though studies to date have primarily involved larger lesions. Patients with smaller lesions who do not meet established criteria for surgical resection would stand to benefit most if genetic markers could be used for risk stratification. The greatest clinical need is for longitudinal studies that could provide data on the large population of patients with incidentally detected cysts who do not undergo resection. Which patients progress over follow-up and which patients may be safely observed? Several additional technologies are being examined. One study evaluated two proteins, olfactomedin-4 and mucin-18, using proteomics to differentiate between benign and malignant cystic lesions. 30 Another study of 20 patients demonstrated that two homologs of amylase, solubilized molecules of four mucins, four solubilized CEA-related cell adhesion molecules, and four S100 homologs may be candidate biomarkers to facilitate pancreatic cyst diagnosis and risk stratification. The analysis required less than 40 μl of cyst fluid. 31 A small study demonstrated IL1β as a potential biomarker to differentiate low- from high-risk IPMN. 32 Identification of new biomarkers and novel biomarker combinations may lead to improved risk stratification. Summary Frequent abdominal imaging is contributing to the increasing identification of asymptomatic pancreatic cysts. Characterization of the cysts with imaging, chemistry, and cytology has shown moderate success. If a cyst has a solid component, this should be specifically targeted for cytologic evaluation. CT scan and MRI may reveal characteristic findings that help in determining the cyst subtype; however, cross-sectional imaging has not been shown to be diagnostic. In patients for whom the management is in question after cross-sectional imaging, analysis of cyst fluid may provide additional information. EUS is the procedure of choice to obtain cyst fluid by FNA. The cyst fluid should be analyzed VOLUME 5 ISSUE 1 105

5 Symptoms attributable to the cyst (e.g., abdominal pain, pancreatitis) Dilatation of the main pancreatic duct Cyst size 30 mm Presence of intramural nodules Cyst fluid cytology suspicious or positive for malignancy Table 5. Clinicopathologic criteria considered for pancreatic resection. 33 for CEA, amylase, and cytology if of sufficient quantity. Several parameters have been determined in which setting resection is often considered ( Table 5 ). The current data for molecular markers have included a larger proportion of patients who have undergone surgical resection, and malignant cysts may be overrepresented in the studies to date ( Table 3 ) 7,18,19 An average proportion of malignant cysts of 30% is not in keeping with the indolent nature of the majority of incidental pancreatic cysts. So, we have less data on molecular analysis in the population of patients with smaller lesions not meeting current criteria for resection. This may be the population for whom additional data could be most helpful. Prospective trials with long-term follow-up of patients who are not sent to surgery would be very helpful. Molecular analysis is a promising approach that may add information in selected patients and should be studied in patients with lesions who do not meet current clinocopathologic criteria for resection ( Table 5 ). Conflicts of interest None. Funding source None. References 1. Spinelli KS, Fromwiller TE, Daniel RA, Kiely JM, Nakeeb A, Komorowski RA, Wilson SD, Pitt HA. Cystic pancreatic neoplasms: observe or operate. Ann Surg. May 2004 ; 239 ( 5 ): ; discussion Laffan TA, Horton KM, Klein AP, Berlanstein B, Siegelman SS, Kawamoto S, Johnson PT, Fishman EK, Hruban RH. Prevalence of unsuspected pancreatic cysts on MDCT. AJR Am J Roentgenol. Sep 2008 ; 191 ( 3 ): Winter JM, Cameron JL, Lillemoe KD, Campbell KA, Chang D, Riall TS, Coleman J, Sauter PK, Canto M, Hruban RH, et al. Periampullary and pancreatic incidentaloma: a single institution s experience with an increasingly common diagnosis. Ann Surg. May 2006 ; 243 ( 5 ): ; discussion Zhang XM, Mitchell DG, Dohke M, Holland GA, Parker L. Pancreatic cysts: depiction on singleshot fast spin-echo MR images. Radiology. May 2002 ; 223 ( 2 ): Lahat G, Ben Haim M, Nachmany I, Sever R, Blachar A, Nakache R, Klausner JM. Pancreatic incidentalomas: high rate of potentially malignant tumors. J Am Coll Surg. Sep 2009 ; 209 ( 3 ): Pitman MB, Lewandrowski K, Shen J, Sahani D, Brugge W, Fernandez-del Castillo C. Pancreatic cysts: preoperative diagnosis and clinical management. Cancer Cytopathol. Feb ; 118 ( 1 ): Khalid A, Zahid M, Finkelstein SD, LeBlanc JK, Kaushik N, Ahmad N, Brugge WR, Edmundowicz SA, Hawes RH, McGrath KM. Pancreatic cyst fl uid DNA analysis in evaluating pancreatic cysts: a report of the PANDA study. Gastrointest Endosc. May 2009 ; 69 ( 6 ): Shen J, Brugge WR, Dimaio CJ, Pitman MB. Molecular analysis of pancreatic cyst fl uid: a comparative analysis with current practice of diagnosis. Cancer Cytopathol. Jun ; 117 ( 3 ): Yoshizawa K, Nagai H, Sakurai S, Hironaka M, Morinaga S, Saitoh K, Fukayama M. Clonality and K-ras mutation analyses of epithelia in intraductal papillary mucinous tumor and mucinous cystic tumor of the pancreas. Virchows Arch. Nov 2002 ; 441 ( 5 ): Izeradjene K, Combs C, Best M, Gopinathan A, Wagner A, Grady WM, Deng CX, Hruban RH, Adsay NV, Tuveson DA, et al. Kras(G12D) and Smad4/Dpc4 haploinsuffi ciency cooperate to induce mucinous cystic neoplasms and invasive adenocarcinoma of the pancreas. Cancer Cell. Mar 2007 ; 11 ( 3 ): Jimenez RE, Warshaw AL, Z Graggen K, Hartwig W, Taylor DZ, Compton CC, Fernandez-del Castillo C. Sequential accumulation of K-ras mutations and p53 overexpression in the progression of pancreatic mucinous cystic neoplasms to malignancy. Ann Surg. Oct 1999 ; 230 ( 4 ): ; discussion Gerdes B, Wild A, Wittenberg J, Barth P, Ramaswamy A, Kersting M, Luttges J, Kloppel G, Bartsch DK. Tumor-suppressing pathways in cystic pancreatic tumors. Pancreas. Jan 2003 ; 26 ( 1 ): Biankin AV, Biankin SA, Kench JG, Morey AL, Lee CS, Head DR, Eckstein RP, Hugh TB, Henshall SM, Sutherland RL. Aberrant p16(ink4a) and DPC4/Smad4 expression in intraductal papillary mucinous tumours of the pancreas is associated with invasive ductal adenocarcinoma. Gut. Jun 2002 ; 50 ( 6 ): Hruban RH, Adsay NV, Albores-Saavedra J, Anver MR, Biankin AV, Boivin GP, Furth EE, Furukawa T, Klein A, Klimstra DS, et al. Pathology of genetically engineered mouse models of pancreatic exocrine cancer: consensus report and recommendations. Cancer Res. Jan ; 66 ( 1 ): Mei R, Galipeau PC, Prass C, Berno A, Ghandour G, Patil N, Wolff RK, Chee MS, Reid BJ, Lockhart DJ. Genome-wide detection of allelic imbalance using human SNPs and high-density DNA arrays. Genome Res. Aug 2000 ; 10 ( 8 ): Schoedel KE, Finkelstein SD, Ohori NP. K-ras and microsatellite marker analysis of fi ne-needle aspirates from intraductal papillary mucinous neoplasms of the pancreas. Diagn Cytopathol. Sep 2006 ; 34 ( 9 ): Sawhney MS, Devarajan S, O Farrel P, Cury MS, Kundu R, Vollmer CM, Brown A, Chuttani R, Pleskow DK. Comparison of carcinoembryonic antigen and molecular analysis in pancreatic cyst fl uid. Gastrointest Endosc. May 2009 ; 69 ( 6 ): Khalid A, McGrath KM, Zahid M, Wilson M, Brody D, Swalsky P, Moser AJ, Lee KK, Slivka A, Whitcomb DC, et al. The role of pancreatic cyst fl uid molecular analysis in predicting cyst pathology. Clin Gastroenterol Hepatol. Oct 2005 ; 3 ( 10 ): Sreenarasimhaiah J, Lara LF, Jazrawi SF, Barnett CC, Tang SJ. A comparative analysis of pancreas cyst fl uid CEA and histology with DNA mutational analysis in the detection of mucin producing or malignant cysts. JOP ; 10 ( 2 ): Brugge WR, Lauwers GY, Sahani D, Fernandez-del Castillo C, Warshaw AL. Cystic neoplasms of the pancreas. N Engl J Med. Sep ; 351 ( 12 ): Scheiman JM. Cystic lesion of the pancreas. Gastroenterology. Feb 2005 ; 128 ( 2 ): Warshaw AL, Compton CC, Lewandrowski K, Cardenosa G, Mueller PR. Cystic tumors of the pancreas. New clinical, radiologic, and pathologic observations in 67 patients. Ann Surg. Oct 1990 ; 212 ( 4 ): ; discussion Curry CA, Eng J, Horton KM, Urban B, Siegelman S, Kuszyk BS, Fishman EK. CT of primary cystic pancreatic neoplasms: can CT be used for patient triage and treatment? AJR Am J Roentgenol. Jul 2000 ; 175 ( 1 ): Koito K, Namieno T, Ichimura T, Yama N, Hareyama M, Morita K, Nishi M. Mucin-producing pancreatic tumors: comparison of MR cholangiopancreatography with endoscopic retrograde cholangiopancreatography. Radiology. Jul 1998 ; 208 ( 1 ): Brugge WR, Lewandrowski K, Lee-Lewandrowski E, Centeno BA, Szydlo T, Regan S, del Castillo CF, Warshaw AL. Diagnosis of pancreatic cystic neoplasms: a report of the cooperative pancreatic cyst study. Gastroenterology. May 2004 ; 126 ( 5 ): O Toole D, Palazzo L, Hammel P, Ben Yaghlene L, Couvelard A, Felce-Dachez M, Fabre M, Dancour A, Aubert A, Sauvanet A, et al. Macrocystic pancreatic cystadenoma: the role of EUS and cyst fl uid analysis in distinguishing mucinous and serous lesions. Gastrointest Endosc. Jun 2004 ; 59 ( 7 ): Pitman MB, Michaels PJ, Deshpande V, Brugge WR, Bounds BC. Cytological and cyst fl uid analysis of small (< or = 3 cm) branch duct intraductal papillary mucinous neoplasms adds value to patient management decisions. Pancreatology ; 8 ( 3 ): Sperti C, Pasquali C, Guolo P, Polverosi R, Liessi G, Pedrazzoli S. Serum tumor markers and cyst fl uid analysis are useful for the diagnosis of pancreatic cystic tumors. Cancer. Jul ; 78 ( 2 ): Sperti C, Pasquali C, Pedrazzoli S, Guolo P, Liessi G. Expression of mucin-like carcinomaassociated antigen in the cyst fl uid differentiates mucinous from nonmucinous pancreatic cysts. Am J Gastroenterol. Apr 1997 ; 92 ( 4 ): Cuoghi A, Farina A, Z Graggen K, Dumonceau JM, Tomasi A, Hochstrasser DF, Genevay M, Lescuyer P, Frossard JL. Role of proteomics to differentiate between benign and potentially malignant pancreatic cysts. J Proteome Res. May ; 10 ( 5 ): Ke E, Patel BB, Liu T, Li XM, Haluszka O, Hoffman JP, Ehya H, Young NA, Watson JC, Weinberg DS, et al. Proteomic analyses of pancreatic cyst fl uids. Pancreas. Mar 2009 ; 38 ( 2 ): e33 e Maker AV, Katabi N, Qin LX, Klimstra DS, Schattner M, Brennan MF, Jarnagin WR, Allen PJ. Cyst fl uid interleukin-1beta (IL1beta) levels predict the risk of carcinoma in intraductal papillary mucinous neoplasms of the pancreas. Clin Cancer Res. Mar ; 17 ( 6 ): Tanaka M, Chari S, Adsay V, Fernandez-del Castillo C, Falconi M, Shimizu M, Yamaguchi K, Yamao K, Matsuno S. International consensus guidelines for management of intraductalpapillary mucinous neoplasms and mucinous cystic neoplasms of the pancreas. Pancreatology ; 6 ( 1 2 ): VOLUME 5 ISSUE 1

6 Appendix: Methods of Molecular Analysis 18 DNA was extracted from 200 μl of cyst fluid by column separation according to manufacturer s directions (Qiagen kit; Qiagen, Valencia, CA, USA). The extracted DNA was resuspended in 50 μl of dilute Tris buffer (ph 7.0). The concentration of DNA was obtained according to OD at 260 of 280 wavelength to document quantity and purity of extraction. One-microliter aliquots were removed for PCR amplification of individual microsatellite markers and direct sequencing of the first exon of the K-ras-2 gene. Nucleic acid amplification was carried out according to manufacturer s instructions (GeneAmp kit; Applied Biosystems, Foster City, CA, USA). Fluorescent-labeled oligonucleotide primers were used for quantitative determination of allelic imbalance on the basis of the peak height ratio of polymorphic microsatellite alleles. The microsatellite marker D17S1289 was used in quantitative PCR reactions to assess the amount of amplifiable DNA from each specimen. On the basis of the OD measurement, each sample was normalized to 5 ng/ μl and then amplified on a quantitative thermocycler system (Icycler; Bio-Rad Laboratories, Hercules, CA, USA), and the cycle threshold value was noted. This provided a measure of the amount of amplifiable DNA in each sample. Because the samples had been normalized to a set level, qpcr cycle threshold values provided a measure of DNA integrity reflected by the degree of amplifiability. All remaining amplifications were performed in standard thermocyclers (Promega USA, Madison, WI, USA) by using standard cycle profiles optimized for individual markers. Postamplification products were electrophoresed, and relative fluorescence was determined for individual alleles (GeneScan ABI3100; Applied Biosystems). The ratio of peaks was calculated by dividing the value for the shorter sized allele by that of the longer sized allele. Thresholds for significant allelic imbalance were determined by using normal (nonneoplastic) specimens for every marker used in the panel. Peak height ratios falling outside of two standard deviations (SDs) beyond the mean for each polymorphic allele pairing were assessed as showing significant allelic imbalance. In each case, a buccal brush or alternative source of nonneoplastic DNA was available to establish informativeness status and then to determine the exact pattern of polymorphic marker alleles. Having established significant allelic imbalance, it was then possible to calculate the proportion of cellular DNA that was subject to hemizygous loss. For example, a polymorphic marker pairing whose peak height ratio was ideally 1.00 with an SD of 0.23 in normal tissue could be inferred to have 50% of its cellular content affected by hemizygous loss if the peak height ratio was 0.5 or 2.0, as previously described. This requires that a minimum of 50% of the DNA in a given sample is derived from cells possessing deletion of the specific microsatellite marker. The deviation from ideal normal ratio of 1.0 indicated that specific allele was affected. In a similar fashion, allele ratios below 0.5 or above 2.0 could be mathematically correlated with the proportion of cells affected by genomic loss. VOLUME 5 ISSUE 1 107