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Molecular Analysis to Detect Pancreatic Ductal Adenocarcinoma in High-Risk Groups

2005; Elsevier BV; Volume: 128; Issue: 7 Linguagem: Inglês

10.1053/j.gastro.2005.03.006

1528-0012

Li Yan, Christopher McFaul, Nathan Howes, Jane Leslie, Gillian Lancaster, Theresa Wong, Jane Threadgold, Jonathan Evans, Ian Gilmore, Howard Smart, Martin Lombard, John P. Neoptolemos, William Greenhalf,

RNA modifications and cancer

Background & Aims: Screening of high-risk groups for pancreatic cancer has not been adopted because of concerns regarding specificity and sensitivity. Suitability of a combination of 3 novel molecular screening techniques was investigated. Methods: Pancreatic juice was extracted from 146 patients with pancreatic ductal adenocarcinoma, chronic pancreatitis, or biliary tract stones. p53 mutations were analyzed by using a modified yeast functional assay, K-ras status was analyzed using mutation-specific real-time PCR and the proportion of p16INK4a promoter methylation was estimated using comparative methylation-specific real-time PCR. Results: p53 mutations were detected in 20 of 48 (42%) cancer cases, none of 49 controls, and 2 of 49 (4%) patients with pancreatitis. K-ras mutations were detected in 31 of 57 (54%) cancer patients, 13 of 61 (21%) controls, and 23 of 67 (34%) patients with pancreatitis. Twenty-six of 42 (62%) cancer patients had promoter methylation levels > 12%, compared with 3 of 24 (13%) controls, and 2 of 26 (8%) with pancreatitis. Mutations in p53 or high-level p16INK4a promoter methylation occurred in 29 of 36 (80%) patients with cancer, 3 of 24 (13%) controls, and 3 of 22 (13%) with pancreatitis. Three patients (8%) of 36 with cancer; 14 of 24 (58%) controls, and 13 of 22 (59%) patients with pancreatitis had no marker. The gallstone disease patients had a high rate of positive K-ras mutations, possibly reflecting the fact that they were not disease free. Conclusions: Combination molecular analysis increased the discrimination between patients with malignant and benign disease. This level of discrimination would allow patients in high-risk groups to be stratified from negligible risk to over 50% probability of an early cancer. Background & Aims: Screening of high-risk groups for pancreatic cancer has not been adopted because of concerns regarding specificity and sensitivity. Suitability of a combination of 3 novel molecular screening techniques was investigated. Methods: Pancreatic juice was extracted from 146 patients with pancreatic ductal adenocarcinoma, chronic pancreatitis, or biliary tract stones. p53 mutations were analyzed by using a modified yeast functional assay, K-ras status was analyzed using mutation-specific real-time PCR and the proportion of p16INK4a promoter methylation was estimated using comparative methylation-specific real-time PCR. Results: p53 mutations were detected in 20 of 48 (42%) cancer cases, none of 49 controls, and 2 of 49 (4%) patients with pancreatitis. K-ras mutations were detected in 31 of 57 (54%) cancer patients, 13 of 61 (21%) controls, and 23 of 67 (34%) patients with pancreatitis. Twenty-six of 42 (62%) cancer patients had promoter methylation levels > 12%, compared with 3 of 24 (13%) controls, and 2 of 26 (8%) with pancreatitis. Mutations in p53 or high-level p16INK4a promoter methylation occurred in 29 of 36 (80%) patients with cancer, 3 of 24 (13%) controls, and 3 of 22 (13%) with pancreatitis. Three patients (8%) of 36 with cancer; 14 of 24 (58%) controls, and 13 of 22 (59%) patients with pancreatitis had no marker. The gallstone disease patients had a high rate of positive K-ras mutations, possibly reflecting the fact that they were not disease free. Conclusions: Combination molecular analysis increased the discrimination between patients with malignant and benign disease. This level of discrimination would allow patients in high-risk groups to be stratified from negligible risk to over 50% probability of an early cancer. Only detection of very early pancreatic ductal adenocarcinoma (PDAC) gives any chance of curative treatment.1Ariyama J. Suyama M. Satoh K. Sai J. Imaging of small pancreatic ductal adenocarcinoma.Pancreas. 1998; 16: 396-401Crossref PubMed Scopus (102) Google Scholar Although screening of the general population is not practical given the prevalence of PDAC and the accuracy of current diagnostic techniques, secondary screening in high-risk populations may be feasible.2Wong T. Howes N. Threadgold J. Smart H.L. Lombard M.G. Sutton R. Greenhalf W. Ellis I. Neoptolemos J.P. Molecular diagnosis of early pancreatic ductal adenocarcinoma in high risk patients.Pancreatology. 2001; 1: 486-509Abstract Full Text PDF PubMed Scopus (59) Google Scholar Combined modality screening of 35 patients from a familial PDAC kindred using endoluminal ultrasound and endoscopic retrograde cholangiopancreatography identified 12 patients for pancreatic resection. In all 12 patients there was evidence of pancreatic dysplasia,3Rulyak S.J. Brentnall T.A. Inherited pancreatic cancer surveillance and treatment strategies for affected families.Pancreatology. 2001; 1: 477-485Abstract Full Text PDF PubMed Scopus (87) Google Scholar, 4Brentnall T.A. Bronner M.P. Byrd D.R. Haggitt R.C. Kimmey M.B. Early diagnosis and treatment of pancreatic dysplasia in patients with a family history of pancreatic cancer.Ann Intern Med. 1999; 131: 247-255Crossref PubMed Scopus (329) Google Scholar but this approach is not reliable in the presence of chronic pancreatitis.5Rosch T. Schusdziarra V. Born P. Bautz W. Baumgartner M. Ulm K. Lorenz R. Allescher H.D. Gerhardt P. Siewert J.R. Classen M. Modern imaging methods versus clinical assessment in the evaluation of hospital in-patients with suspected pancreatic disease.Am J Gastroenterol. 2000; 95: 2261-2270Crossref PubMed Google Scholar Molecular analysis of pancreatic juice to detect key mutations in pancreatic carcinogenesis could increase specificity and sensitivity further. Estimates for the detection of K-ras mutations in pancreatic juice from PDAC and control patients vary widely, giving predicted specificities and sensitivities from 30% to 100%.6Kondoh S. Kaino M. Okita S. Ryozawa S. Akiyama T. Okita K. Detection of Ki-ras and p53 gene mutations in tissue and pancreatic juice from pancreatic adenocarcinomas.J Gastroenterol. 1998; 33: 390-396Crossref PubMed Scopus (21) Google Scholar, 7Yamashita K. Kida Y. Shinoda H. Kida M. Okayasu I. K-ras point mutations in the supernatants of pancreatic juice and bile are reliable for diagnosis of pancreas and biliary tract carcinomas complementary to cytologic examination.Jpn J Cancer Res. 1999; 90: 240-248Crossref PubMed Scopus (25) Google Scholar, 8Tada M. Omata M. Kawai S. Saisho H. Ohto M. Saiki R.K. Sninsky J.J. Detection of ras gene mutations in pancreatic juice and peripheral blood of patients with pancreatic adenocarcinoma.Cancer Res. 1993; 53: 2472-2474PubMed Google Scholar, 9Watanabe H. Yamaguchi Y. Ha A. Hu Y.X. Motoo Y. Okai T. Yoshimura T. Sawabu N. Quantitative determination of K-ras mutations in pancreatic juice for diagnosis of pancreatic cancer using hybridization protection assay.Pancreas. 1998; 17: 341-347Crossref PubMed Scopus (51) Google Scholar, 10Kondo H. Sugano K. Fukayama N. Kyogoku A. Nose H. Shimada K. Ohkura H. Ohtsu A. Yoshida S. Shimosato Y. Detection of point mutations in the K-ras oncogene at codon 12 in pure pancreatic juice for diagnosis of pancreatic carcinoma.Cancer. 1994; 73: 1589-1594Crossref PubMed Scopus (165) Google Scholar, 11Trumper L. Menges M. Daus H. Kohler D. Reinhard J.O. Sackmann M. Moser C. Sek A. Jacobs G. Zeitz M. Pfreundschuh M. Low sensitivity of the ki-ras polymerase chain reaction for diagnosing pancreatic cancer from pancreatic juice and bile a multicenter prospective trial.J Clin Oncol. 2002; 20: 4331-4337Crossref PubMed Scopus (40) Google Scholar, 12Furuya N. Kawa S. Akamatsu T. Furihata K. Long-term follow-up of patients with chronic pancreatitis and K-ras gene mutation detected in pancreatic juice.Gastroenterology. 1997; 113: 593-598Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 13Van Laethem J.L. Bourgeois V. Parma J. Delhaye M. Cochaux P. Velu T. Deviere J. Cremer M. Relative contribution of Ki-ras gene analysis and brush cytology during ERCP for the diagnosis of biliary and pancreatic diseases.Gastrointest Endosc. 1998; 47: 479-485Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 14Tada M. Ohashi M. Shiratori Y. Okudaira T. Komatsu Y. Kawabe T. Yoshida H. Analysis of K-ras gene mutation in hyperplastic duct cells of the pancreas without pancreatic disease.Gastroenterology. 1996; 110: 227-231Abstract Full Text PDF PubMed Scopus (287) Google Scholar, 15Yanagisawa A. Ohtake K. Ohashi K. Hori M. Kitagawa T. Sugano H. Kato Y. Frequent c-Ki-ras oncogene activation in mucous cell hyperplasias of pancreas suffering from chronic inflammation.Cancer Res. 1993; 53: 953-956PubMed Google Scholar Detection of p16INK4a promoter CpG island methylation previously has been reported to give a low sensitivity for cancer (<20% to 40%).16Fukushima N. Walter K.M. Uek T. Sato N. Matsubayashi H. Cameron J.L. Hruban R.H. Canto M. Yeo C.J. Goggins M. Diagnosing pancreatic cancer using methylation specific PCR analysis of pancreatic juice.Cancer Biol Ther. 2003; 2: 78-83Crossref PubMed Google Scholar, 17Matsubayashi H. Sato N. Fukushima N. Yeo C.J. Walter K.M. Brune K. Sahin F. Hruban R.H. Goggins M. Methylation of cyclin D2 is observed frequently in pancreatic cancer but is also an age-related phenomenon in gastrointestinal tissues.Clin Cancer Res. 2003; 9: 1446-1452PubMed Google Scholar, 18Klump B. Hsieh C.J. Nehls O. Dette S. Holzmann K. Kiesslich R. Jung M. Sinn U. Ortner M. Porschen R. Gregor M. Methylation status of p14ARF and p16INK4a as detected in pancreatic secretions.Br J Cancer. 2003; 88: 217-222Crossref PubMed Scopus (51) Google Scholar Mutations in p53 have been detected in pancreatic juice by using single-stranded conformation polymorphism. In the largest of these studies, 11 of 26 (42%) patients with PDAC had a p53 mutation in comparison with none of 16 patients with chronic pancreatitis and 92% of patients with cancer had either a K-ras or p53 mutation.19Yamaguchi Y. Watanabe H. Yrdiran S. Ohtsubo K. Motoo Y. Okai T. Sawabu N. Detection of mutations of p53 tumor suppressor gene in pancreatic juice and its application to diagnosis of patients with pancreatic cancer comparison with K-ras mutation.Clin Cancer Res. 1999; 5: 1147-1153PubMed Google Scholar The same group subsequently reported no p53 mutations in the pancreatic juice of 25 chronic pancreatitis patients.20Wang Y. Yamaguchi Y. Watanabe H. Ohtsubo K. Motoo Y. Sawabu N. Detection of p53 gene mutations in the supernatant of pancreatic juice and plasma from patients with pancreatic carcinomas.Pancreas. 2004; 28: 13-19Crossref PubMed Scopus (35) Google Scholar We report the use of 3 techniques able to detect very low levels of the most common DNA abnormalities characteristic of PDAC (K-ras, p16INK4a, and p53) found in pancreatic juice. Patients with a suspicion of PDAC, chronic pancreatitis, or biliary tract stones were recruited prospectively between 1997 and 2004, after informed consent and before endoscopic retrograde cholangiopancreatography. The final classification of patients was based on tissue diagnosis or clinical diagnosis with imaging and a follow-up period of at least 12 months. Pancreatic juice was aspirated during endoscopic retrograde cholangiopancreatography, the first 1000 μL was discarded and the following 500 μL was spun (4000g, 10 min). DNA was purified from the supernatant by phenol-chloroform extraction and ethanol precipitation. The p53 assay (see Figure 1) is based on the system of Flaman et al21Flaman J.M. Frebourg T. Moreau V. Charbonnier F. Martin C. Chappuis P. Sappino A.P. Limacher I.M. Bron L. Benhattar J. et al.A simple p53 functional assay for screening cell lines, blood, and tumors.Proc Natl Acad Sci U S A. 1995; 92: 3963-3967Crossref PubMed Scopus (429) Google Scholar but uses DNA instead of RNA because of the instability of RNA in pancreatic juice. Exons 5–8 were chosen because these contain the mutation hotspots.22Olivier M. Eeles R. Hollstein M. Khan M.A. Harris C.C. Hainaut P. The IARC TP53 database new online mutation analysis and recommendations to users.Hum Mutat. 2002; 19: 607-614Crossref PubMed Scopus (1048) Google Scholar The assay gives 5%–10% red colonies with wild-type p53 using gap repair.21Flaman J.M. Frebourg T. Moreau V. Charbonnier F. Martin C. Chappuis P. Sappino A.P. Limacher I.M. Bron L. Benhattar J. et al.A simple p53 functional assay for screening cell lines, blood, and tumors.Proc Natl Acad Sci U S A. 1995; 92: 3963-3967Crossref PubMed Scopus (429) Google Scholar, 23Waridel F. Estreicher A. Bron L.R.I. Field cancerisation and polyclonal p53 mutations in the upper aero-digestive tract.Oncogene. 1997; 14: 163-169Crossref PubMed Scopus (166) Google Scholar The use of uncut vector decreases the ratio of red to white colonies but reduces false positives because of self-ligation.21Flaman J.M. Frebourg T. Moreau V. Charbonnier F. Martin C. Chappuis P. Sappino A.P. Limacher I.M. Bron L. Benhattar J. et al.A simple p53 functional assay for screening cell lines, blood, and tumors.Proc Natl Acad Sci U S A. 1995; 92: 3963-3967Crossref PubMed Scopus (429) Google Scholar DNA was prepared using the QIAamp DNA mini kit (Qiagen Hilden, Germany). Amplification and direct sequencing of p53 exons confirmed that CAPAN-2 was wild-type for p53 and MiaPaCa2 had the R248W mutation. Real-time polymerase chain reaction (PCR) was performed using the LightCycler with SYBR Green detection (Roche Diagnostics, Pinzberg, Germany). Because there is amplification of the wild-type sequence with mutation-specific primers, the threshold cycles with mutant-specific primers (Supplementary Table 2, see supplemental material online at www.gastrojournal.org) were plotted against threshold cycles using control primers that amplified both wild-type and mutant sequences. PCR of 100 blood samples allowed 98% confidence intervals to be produced on a linear regression curve for each mutation-specific primer. Samples were analyzed in triplicate; if all 3 points were less than the 98% confidence limit then the sample was classified as mutant. Pancreatic-juice DNA was sodium-bisulfite modified.24Herman J.G. Merlo A. Mao L. Lapidus R.G. Issa J.P. Davidson N.E. Sidransky D. Baylin S.B. Inactivation of the CDKN2/p16/MTS1 gene is frequently associated with aberrant DNA methylation in all common human cancers.Cancer Res. 1995; 55: 4525-4530PubMed Google Scholar PCR amplification was performed using the LightCycler (Roche Diagnostics) as shown in Figure 2. The methylation index was [M/(M + U)] × 100%, where M is the quantity of methylated p16INK4a promoter and U is the quantity of unmethylated p16INK4a promoter measured by real-time PCR.25Lo Y.M. Wong I.H. Zhang J. Tein M.S. Ng M.H. Hjelm N.M. Quantitative analysis of aberrant p16 methylation using real-time quantitative methylation-specific polymerase chain reaction.Cancer Res. 1999; 59: 3899-3903PubMed Google Scholar The sensitivity was the percentage of PDAC cases correctly identified by the test and the specificity percentage controls correctly identified. Cut-off values for the p16INK4a test were determined by a receiver operating characteristic curve analysis. Bayes' theorem was used to determine the posttest probability of cancer, ie, positive likelihood ratio = sensitivity/(1 − specificity); negative likelihood ratio = (1 − sensitivity)/specificity. Posttest odds = pretest odds multiplied by the likelihood ratio. Posttest probability = odds/1 + odds.26Greenberg R. Daniels S. Flanders W. Eley J. Boring J. Medical epidemiology. McGraw-Hill, London, UK2001Google Scholar The Confidence Interval Analysis (CIA) Software (BMJ publishing, UK) program was used to calculate 95% confidence intervals for sensitivity, specificity, and the likelihood ratios.27Altman D. Machin D. Bryant T. Gardner M. Statistics with confidence. 2nd ed. BMJ Books, London, UK2000Google Scholar The Bayesian approach used all samples but assumed test independence. A logistic regression analysis was performed for comparison on a subset of patients for whom all 3 test results were known, giving conditional probability of PDAC after a p53 test in isolation or with subsequent p16INK4a and/or K-ras tests, assuming test dependency. The screening techniques were introduced as they were developed and were standardized. For this reason and because there was insufficient DNA extracted from some samples, not all samples were tested with all 3 modalities. Three hundred transformations were performed using CAPAN-2 (p53 wild-type) DNA; the number of transformants ranged from 225 to 2500 (median, 400 white colonies). Red colonies were seen in 101 transformations (presumably as a result of PCR error). The minimum number of white colonies that gave a red colony was 423, so a value greater than .25% red colonies was taken as indicating the presence of p53 mutations. p53 was amplified from mixtures of CAPAN-2 and MiaPaCa2 (p53 mutant) cell line DNA. By using any concentration between 10% and 100% MiaPaCa2 DNA, approximately 40% of the colonies were red, consistent with known cotransformation frequencies for yeast with lithium acetate.28Gietz R.D. Schiestl R.H. Applications of high efficiency lithium acetate transformation of intact yeast cells using single-stranded nucleic acids as carrier.Yeast. 1991; 7: 253-263Crossref PubMed Scopus (376) Google Scholar At a concentration less than 10% there was a decrease in red colonies, with 2 red colonies observed per 600 white colonies at a relative concentration of 1:1000 MiaPaCa2:CAPAN-2 DNA. Mutant p53 was detected in 20 of 48 (42%) patients with PDAC, in 2 of 49 (4%) patients with chronic pancreatitis, and 0 of 49 patients with benign biliary disease (Supplementary Table 3, see supplemental material online at www.gastrojournal.org). One of the positive pancreatitis patients was 43 years old and died shortly after surgery (no cancer), the other was 21 years old and was well 2 years after surgery. Point mutations were predominant (90%), with similar numbers of transition (n = 9; 41%) and transversion (n = 10; 45%) mutations. There also were 3 different frameshift mutations (13%) resulting from single base-pair deletions (Supplementary Table 4, see supplemental material online at www.gastrojournal.org). All of the mutations recorded have been described previously and are inactivating mutations.29Hollstein M. Rice K. Greenblatt M.S. Soussi T. Fuchs R. Sorlie T. Hovig E. Smith-Sorensen B. Montesano R. Harris C.C. Database of p53 gene somatic mutations in human tumors and cell lines.Nucleic Acids Res. 1994; 22: 3551-3555PubMed Google Scholar All but 1 mutation (R213L) were dominant (cotransformation of wild-type and mutant sequence gave red colonies). Tissue samples from 22 patients were tested by microdissection, PCR amplification, and sequencing. Twelve samples were mutant in tissue and 8 of these (66%) also had a mutation in pancreatic juice. Ten samples were wild-type for p53 in tissue, 8 of which (80%) also were wild-type in pancreatic juice. The remaining 2 samples might reflect heterogeneity in the tumor because other tumor cells than those dissected may have had the mutation. Mutant K-ras was detected in 31 of 57 (54%) PDAC, 23 of 67 (34%) chronic pancreatitis, and 13 of 61 (21%) biliary stone control patients (χ2, P < .001) (Supplemental Table 3, see supplemental material online at www.gastrojournal.org). Restricting the mutation analysis to the most common mutations (G12D and G12V) gave a higher specificity at the expense of reduced sensitivity (Supplemental Table 3, see supplemental material online at www.gastrojournal.org). Exon 1 of K-ras was amplified and sequenced from 26 microdissected tumor samples. Only 10 (38%) had the same K-ras result in juice and tissue. In 5 cases (19%) a K-ras mutation in tissue was not found in the juice whereas in 11 cases (43%) a K-ras mutation was detected in pancreatic juice that apparently was not present in the tumor. Two of 5 tissues were wild-type on the basis of microdissection and sequencing but had a K-ras mutation in gross tissue specimens (detected by the amplification refractory mutation system), indicating heterogeneity within the tumor. The median (range) level of p16INK4a promoter methylation was 20% (.07%–57%) for 42 PDAC patients, .3% (.06%–51%) for 24 chronic pancreatitis patients, and .2% (.03%–55%) in 26 control patients (Kruskal-Wallis, P < .001). A receiver operating characteristic analysis identified 12% as the level of methylation that maximally differentiated PDAC from the other diagnosis (see supplemental Figure 4 and Table 3, see supplemental material online at www.gastrojournal.org). Twenty-six (62%) of 42 cancer patients had p16INK4a promoter methylation levels greater than 12% compared with 3 of 24 (13%) controls and 2 of 26 (8%) with chronic pancreatitis (χ2, P < .001). Despite the association of p16INK4a promoter methylation with a diagnosis of cancer, most DNA does not come from cancer cells and methylation levels in pancreatic juice do not correspond to levels in cancer tissue (Supplemental Table 5, see supplemental material online at www.gastrojournal.org). We conclude that methylation is indicative of a pancreas diseased with pancreatic cancer. In 36 patients with PDAC, 22 with chronic pancreatitis and 24 biliary stone controls, data were available for all 3 tests; logistic regression analysis was performed on this subset of patients (see supplementary Tables 6 and 7, see supplemental material online at www.gastrojournal.org). Taking the 3 tests independently (Bayesian approach) gave similar estimates of the proportion of cancer cases as the regression analysis (see supplemental Figure 5, see supplemental material online at www.gastrojournal.org). The regression is based on a subset of individuals that has an unrealistic proportion of cancer cases in a screening context (65%). The Bayesian approach uses specificity and sensitivity values, which were obtained using all individuals and considering cancer and control groups independently. Therefore, the effect of combination screening for hereditary pancreatitis was shown using Bayes' theorem (Figure 3A). The pretest probability of cancer was taken as 1% based on the 3-year risk for a 42-year-old patient with hereditary pancreatitis.30Howes N. Lerch M.M. Greenhalf W. Stocken D.D. Ellis I. Simon P. Truninger K. Ammann R. Cavallini G. Charnley R.M. Uomo G. Delhaye M. Spicak J. Drumm B. Jansen J. Mountford R. Whitcomb D.C. Neoptolemos J.P. Clinical and genetic characteristics of hereditary pancreatitis in Europe.Clin Gastroenterol Hepatol. 2004; 2: 252-261Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar Combination screening also was evaluated with biliary stone controls (Figure 3B). A number of high-risk groups for PDAC have been identified (described in the definition of high-risk groups). Although there is consensus that screening is appropriate in high-risk patients, knowledge is lacking as to the optimum method.31Ulrich C.D. Pancreatic cancer in hereditary pancreatitis consensus guidelines for prevention, screening and treatment.Pancreatology. 2001; 1: 416-422Abstract Full Text PDF PubMed Scopus (80) Google Scholar To argue that molecular analysis could be applied to high-risk groups, 3 modalities were selected but alternatives exist that also could be applied. The amplification-refractory mutation-system technique was chosen to assay K-ras after comparison with restriction fragment length polymorphism, and in our hands the amplification-refractory mutation system allows the detection of K-ras mutations missed with other techniques as also reported by other investigators.32Clayton S.J. Scott F.M. Walker J. Callaghan K. Haque K. Liloglou T. Xinarianos G. Shawcross S. Ceuppens P. Field J.K. Fox J.C. K-ras point mutation detection in lung cancer comparison of two approaches to somatic mutation detection using ARMS allele-specific amplification.Clin Chem. 2000; 46: 1929-1938PubMed Google Scholar Single-strand conformational polymorphism to detect p53 mutations lacks sensitivity (detecting approximately 1 mutant copy per 100 wild-type copies) and cannot distinguish between polymorphisms, functionally silent mutations, and inactivating mutations.33Howes N. Greenhalf W. Neoptolemos J. Screening for early pancreatic ductal adenocarcinoma in hereditary pancreatitis.Med Clin North Am. 2000; 84: 719-738Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar Therefore, a functional p53 assay was adopted. The assay for p16INK4a CpG island methylation was based on the same PCR amplification of sequences as previously described,16Fukushima N. Walter K.M. Uek T. Sato N. Matsubayashi H. Cameron J.L. Hruban R.H. Canto M. Yeo C.J. Goggins M. Diagnosing pancreatic cancer using methylation specific PCR analysis of pancreatic juice.Cancer Biol Ther. 2003; 2: 78-83Crossref PubMed Google Scholar, 17Matsubayashi H. Sato N. Fukushima N. Yeo C.J. Walter K.M. Brune K. Sahin F. Hruban R.H. Goggins M. Methylation of cyclin D2 is observed frequently in pancreatic cancer but is also an age-related phenomenon in gastrointestinal tissues.Clin Cancer Res. 2003; 9: 1446-1452PubMed Google Scholar, 18Klump B. Hsieh C.J. Nehls O. Dette S. Holzmann K. Kiesslich R. Jung M. Sinn U. Ortner M. Porschen R. Gregor M. Methylation status of p14ARF and p16INK4a as detected in pancreatic secretions.Br J Cancer. 2003; 88: 217-222Crossref PubMed Scopus (51) Google Scholar but because the LightCycler is very sensitive at identifying low (late) amplification of product our method of assay was more sensitive; this also meant that amplifiable sequences could be identified in control patients. Although in previous studies specificity was reported to be high in pancreatic juice, this was because of the low detection rate of the tests because the investigators confirmed the presence of methylated sequences in normal tissue.16Fukushima N. Walter K.M. Uek T. Sato N. Matsubayashi H. Cameron J.L. Hruban R.H. Canto M. Yeo C.J. Goggins M. Diagnosing pancreatic cancer using methylation specific PCR analysis of pancreatic juice.Cancer Biol Ther. 2003; 2: 78-83Crossref PubMed Google Scholar, 17Matsubayashi H. Sato N. Fukushima N. Yeo C.J. Walter K.M. Brune K. Sahin F. Hruban R.H. Goggins M. Methylation of cyclin D2 is observed frequently in pancreatic cancer but is also an age-related phenomenon in gastrointestinal tissues.Clin Cancer Res. 2003; 9: 1446-1452PubMed Google Scholar, 18Klump B. Hsieh C.J. Nehls O. Dette S. Holzmann K. Kiesslich R. Jung M. Sinn U. Ortner M. Porschen R. Gregor M. Methylation status of p14ARF and p16INK4a as detected in pancreatic secretions.Br J Cancer. 2003; 88: 217-222Crossref PubMed Scopus (51) Google Scholar The decrease in specificity was compensated for in this study by the use of comparative quantification. In hereditary pancreatitis the risk for developing PDAC within a 3-year period increases linearly with age from a negligible value at 40 years to 9% at age 70.30Howes N. Lerch M.M. Greenhalf W. Stocken D.D. Ellis I. Simon P. Truninger K. Ammann R. Cavallini G. Charnley R.M. Uomo G. Delhaye M. Spicak J. Drumm B. Jansen J. Mountford R. Whitcomb D.C. Neoptolemos J.P. Clinical and genetic characteristics of hereditary pancreatitis in Europe.Clin Gastroenterol Hepatol. 2004; 2: 252-261Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar With a pretest prevalence of 1% undiagnosed PDAC, the use of combination molecular testing could allow stratification of risk from less than .1% to in greater than 50%, assuming specificity and sensitivity from the symptomatic patients (Figure 3). The frequency of mutations in resectable patients was not lower than in patients with more advanced cancer (Supplemental Table 3, see supplemental material online at www.gastrojournal.org), but detection of the markers may be more difficult in earlier disease. None of our participants were subjected to repeated prospective evaluation, which may lead to overestimation of diagnostic performance.34Lijmer J.G. Mol B.W. Heisterkamp S. Bonsel G.J. Prins M.H. van der Meulen J.H. Bossuyt P.M. Empirical evidence of design-related bias in studies of diagnostic tests.JAMA. 1999; 282: 1061-1066Crossref PubMed Scopus (1514) Google Scholar In compensation, in practice, screening would include imaging. The nature of the tests recommended would be determined by the cancer risk that would trigger intervention and will depend on clinical evaluation of the patient. For example, if a greater than 50% risk for cancer is required to change the clinical management of a 45-year-old hereditary pancreatitis patient, then on the basis of Figure 3A there would be no value performing additional molecular tests if a p53 result were negative. There is the need for a prospective study that will establish if the sensitivity of the tests is applicable to asymptomatic patients. This also would allow a comparison of these techniques with conventional imaging approaches such as computed tomography and endoluminal ultrasound.Tables 5–7 and figures 4–5 The authors thank Richard Iggo of ISREC for the supply of vectors and strains for use in the yeast functional assay and all those members of the Royal Liverpool University Hospital who have supplied samples to this study. In particular, we would like to acknowledge the contribution of Drs B. Azadeh and F. Campbell from the department of Pathology and W. Prime from the Candis tissue bank for their support and Dr L. Vitone who has provided ongoing clinical information. Download .pdf (.45 MB) Help with pdf files