SCREENING FOR EARLY PANCREATIC DUCTAL ADENOCARCINOMA IN HEREDITARY PANCREATITIS

INHERITED DISEASES OF THE PANCREAS

0025-7125/00 $15.00

+ .OO

SCREENING FOR EARLY PANCREATIC DUCTAL ADENOCARCINOMA IN HEREDITARY PANCREATITIS Nathan Howes, MB, ChB, FRCS, William Greenhalf, BSc, PhD, and John Neoptolemos, MA, MB, ChB, FRCS, MD

The risk of ductal adenocarcinoma is greatly increased in patients with hereditary pancreatitis (HP). Analysis of the biggest series of HP patients studied to date resulted in an estimated lifetime risk for the development of ductal adenocarcinoma of 40%. Only patients in whom the disease was transmitted paternally contracted cancer, giving a lifetime risk for this subset of individuals of 70%." Lerch et a142estab-. I that the risk of pancreatic cancer is also increased with maternal trans in of HP. Patients with sporadic clLIvILIc. pancreatitis also have an increased risk of developing pancreatic cancer, whch has been shown to be six times greater than that of unaffected i n d i v i d ~ a l s Lowenfels .~~ et al" showed that patients had chronic pancreatitis for at least 20 years before the development of pancreatic cancer. Patients who developed pancreatic cancer had more severe disease, manifested by increased complications of pancreatitis and increased calcification of the pancreas gland, compared with age-matched controls. This finding suggests that the relationship between the development of pancreatic cancer in patients with HP may be due to the duration and severity of the disease as a precursor to malignant pathogenesis, rather than the genetic predisposition of developing pancreatitis. Secondary screening for pancreatic ductal adenocarcinoma in patients with HP is desirable and feasible, given the high risk of cancer and the long lead time between the development of pancreatitis and pancreatic cancer. Conventional methods of diagnosis of pancreatic ductal adenocarcinoma are limited. Tumor markers are falsely elevated in 10% of patients with chronic pan~reatitis:~in

From the Department of Surgery, University of Liverpool, Liverpool, United Kingdom ~~

MEDICAL CLINICS OF NORTH AMERICA VOLUME 84 * NUMBER 3 * MAY 2000

719

cigarette smokers, and in the presence of jaundice.@They are of little use in the diagnosis of lesions suitable for curative surgery because the sensitivity for detection in early tumors is less than 50%.29, 48 The sensitivity of CA19-9 for the detection of early pancreatic cancer (< 20 mm and confined to the pancreas) is only 43% in pure pancreatic juice and 0% in the serumF Other tumor markers, such as CEA, SPAN-1, and CA50, used alone or in combination, have been assessed and have a lower sensitivity than CA19-9.59 The accuracy of imaging early pancreatic lesions is disappointing. The sensitivity of detecting pancreatic ductal adenocarcinoma using computed tomography (CT) scan alone is approximately 33% for tumors less than 2 cm and 50% for tumors less than 3 ~ m . Muller 2~ et a156found similar rates of detection in early pancreatic lesions, with 4 of 10 (40%) lesions less than 2 cm and 8 of 15 (67%)lesions less than 3 cm being visible on dynamic helical CT scan. Midwinter et a15' performed spiral CT scan on 24 patients with histologically proven ductal adenocarcinoma of the pancreas, who were being assessed for surgical resection. Mass lesions were detected in 23 of 26 pancreatic tumors of all stages; the remaining 3 tumors, which were missed, had a mean size of 22 mm. In another study, Phoa et a161used spiral CT scan for the preoperative staging of potentially resectable pancreatic cancer. CT scan showed a mass lesion in 54 of 56 cases, with a mean diameter of 28 mm. Only half of these patients, however, were suitable for resection because of undiagnosed metastatic disease or understaged local invasion. In five of the patients with pancreatic cancer, distinction from chronic pancreatitis by CT scan criteria alone was not possible, suggesting that the sensitivity for the detection of pancreatic cancer in a background of chronic pancreatitis is likely to be even less. Johnson and O ~ t w a t e rused ~ ~ dynamic magnetic resonance (MR) imaging with gadolinium to differentiate chronic pancreatitis from ductal adenocarcinoma of the pancreas. Both groups showed abnormal pancreatic enhancement indicative of disease; however, there was considerable overlap between the two groups, precluding distinction of the two entities. The similar appearance of chronic pancreatitis and pancreatic cancer on imaging may be explained, in part, by the abundant fibrosis that occurs in both conditions. Endoscopic techniques for the diagnosis of pancreatic cancer are well established. Endoluminal ultrasound for the detection of malignancy on a background of chronic pancreatitis may be more sensitive than CT scan and MR irnaging5I but because of poor specificity gives a positive predictive value of only 6O%.l, 5, 65 Endoscopic retrograde cholangiopancreatography (ERCP) used in isolation is not sufficiently sensitive to detect subtle pancreatic ductal changes associated with early neoplastic lesions. Bakkevold et a14 prospectively examined stage I ductal adenocarcinomas and ampullary lesions and found that ERCP was 78% sensitive for diagnosing these lesions. Diagnosis of pancreatic cancer by using cytology alone on pancreatic juice obtained at ERCP has a sensitivity of 30% to 70% for the diagnosis of pancreatic cancer at all stages.21,74 Studies that have correlated the stage of the pancreatic cancer have found that the sensitivity for detecting stage I and I1 pancreatic cancers using this method is only 5 6 7 0 . ~ ~ The sensitivity of conventional methods for diagnosing early pancreatic cancer, particularly in the presence of chronic pancreatitis, is disappointing. Advances in understanding of the molecular biology of chronic pancreatitis and pancreatic cancer have enabled the development of new strategies for the early diagnosis of pancreatic cancer, which are potentially more sensitive and specific than existing conventional modalities.

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SCREENING FOR EARLY PANCREATIC DUCTAL ADENOCARCINOMA IN HP

MOLECULAR MARKERS ASSOCIATED WITH PANCREATIC CANCER The mutations associated with pancreatic ductal adenocarcinoma have a distinct pattern, which is believed to reflect the stepwise pathogenesis of the disease (Fig. 1). K- ras

K-ras is a G protein normally involved in mitogen signal transduction, mutations of which cause guanosine triphosphate (GTP) independence of the molecule, resulting in constitutive activation. Such K-ras mutations occurring at codons 12 and 13 are observed in 70% to 100% of all pancreatic ductal 46, 52, which is the highest rate of K-ras mutation reported adenocarcinomas,3z~ in any human malignancy.1° K-ras mutations are not limited only to frankly neoplastic cells but are also present in preneoplastic cells. A study of microdissected areas of carcinoma in situ and atypical hyperplasia, adjacent to frankly invasive ductal adenocarcinomas, showed K-ras mutations in 17 of 17 (100Y0) in situ carcinoma lesions and in 19 (90%)of 21 atypical hyperplastic Matsubayashi et a149analyzed 317 carcinoma-associated ductal lesions from normal pancreatic tissue located around pancreatic tumor (mucinous cell hypertrophy type) and found 39% of these were positive for a K-ras mutation. Luttges et a146 in an analysis of ductal lesions found that 105 (29%) of 364 harbored K-ras mutations. These findings, together with the equal incidence of K-ras mutations in small compared with large pancreatic cancers,71suggest that these mutations occur early in the pathogenesis of pancreatic cancer and are likely to be precursor lesions of carcinoma. Although K-ras mutations are characteristic of pancreatic cancer, several groups have found that these mutations are not specific to cancer because they have also been found in patients with chronic pancreatitis. Furuya et alZ5studied pancreatic juice from 54 patients with chronic pancreatitis and found that 20 patients (37%) had a K-ras mutation detectable in pancreatic juice, none of whom, after a mean follow-up of 78 months, had any evidence of pancreatic

Ductal cell

1

K-Ras

lnvasive neodasia

~

Ductal cell hyperplasia

~

t

A

~

4

lntraductal neoplasia

Ductal cell pre-neoplasia Figure 1. Proposed stepwise progression of the development of pancreatic ductal adenocarcinoma starting with a normal pancreatic ductal cell. The key molecular events in tumorigenesis are shown and the order in which they may occur.

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cancer. Van Laethem et a P examined pancreatic brushings obtained from 49 patients with ductal adenocarcinoma of the pancreas and 76 patients with chronic pancreatitis. K-ras mutations were found in 38 (78%)of 49 patients with cancer and in 19 (25%) of 76 patients with chronic pancreatitis. In comparison, cytologic analysis of the pancreatic brushings from the same patients gave a sensitivity of only 66% for the detection of malignant cells in the pancreatic cancer group. There were no false-positive results in cytology of patients with chronic pancreatitis, however, yielding specificity of 100% for cytology in chronic pancreatitis compared with 72% for K-ras analysis.84K-ras mutations, when they occur in patients with chronic pancreatitis, seem to arise in hyperplastic cells of the pancreatic Rivera et aP4 studied patients with chronic pancreatitis and found that 2 (18%) of 11 patients with chronic pancreatitis and ductal hyperplasia harbored K-ras mutations, compared with 0 of 4 patients with chronic pancreatitis and no ductal hyperplasia. Tada et alsoexamined postmortem specimens of pancreas tissue and found K-ras mutations in 12 (32%) of 38 patients with benign inflammatory hyperplastic foci of the pancreatic duct, which are often seen in chronic pancreatitis. The lack of specificity of K-rus mutations in the presence of chronic pancreatitis limits the use of K-ras as a single marker for cancer detection. Given that K-ras mutations have been detected in 100% of pancreatic cancers and are an early event in the initiation of pancreatic carcinogenesis, however, K-ras mutation detection is still useful when allied with additional markers. Possible strategies for the detection of K-ras mutations in blood, stool, pancreatic juice, and bile are discussed in the following sections. Detection of Mutant K-ras DNA in Plasma

Soluble, extracellular DNA arises in the blood of healthy people, usually as a result of the breakdown of white cells. Levels of free DNA are low, ranging from 10 to 30 r ~ g / m L , ' but ~ , ~can ~ be greatly increased in malignancy. Shapiro et al" showed that more than 90% of individuals with pancreatic cancer had levels of soluble extracellular DNA greater than 100 ng/mL, which was significantly higher than in patients with chronic pancreatitis. Sensitive assays have been developed to detect the presence of K-ras mutations in soluble DNA as an adjunct to diagnosis in colorectal,2 lung,16 and head and neck malignan~ies.~~ The sensitivity for detection of mutant K-ras in the plasma of patients with pancreatic cancer of all stages is 33% to 100%. In all cases, mutations in soluble DNA were verified by sequencing K-ras from the primary tumor (Table 1).All of the assays were specific, with only one study showing detectable K-ras mutations in 2 of 35 patients with chronic pancreatitis. Studies have correlated the clinical stage of pancreatic cancer with the detection of K-ras mutations in serum. Yamada et a191 showed that only two patients of six with resectable pancreatic cancer had detectable K-rus mutations in soluble DNA compared with seven of nine nonresectable patients, with a mean tumor size of 2.6 cm and 4.2 cm. Mutations were detected by a polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) or a mutant-specific primer technique. In the former, a first round of PCR introduces a specific restriction enzyme site into normal DNA sequences but not on mutant sequences. After digestion with the restriction enzyme, part of the 5' end of amplified wild-type is removed; a second round of PCR then selectively amplifies mutant product but not wild-type. The size differential can be visualized by running the products out onto a gel. The principal disadvantage of this technique is that it does not type the mutation but only identifies that one is present. In

6 10 15 21 44

No. in Series

RFLP RFLP MASA RFLP RFLP

Method

10* 9 17 12

2

Samples With Mutant K-ras 33 100 60 81 27

("/I

Sensitivity

012 2/37

014

012

Mutant K-ras in Chronic Pancreatitis

*In this series, blood was obtained from patients postoperatively. No K-ras mutations were detectable in blood obtained preoperatively. RFLP = Polymerase chain reaction restriction fragment length polymorphism; MASA = mutation specific primers.

Tada et al,*I 1993 Nomoto et al?* 1996 Yamada et a1Y1 1998 Mulcahy et al,551998 Castells et aI,I5 1999

Series

Table 1. K-ras MUTATIONS IN FREE DNA IN PERIPHERAL BLOOD

100 100 100 95

("/I

Specificity

the latter technique, mutant-specific primers are used, which only anneal to and amplify a corresponding mutant sequence. By running multiple separate assays and visualizing the results on a gel, the mutant sequence can be inferred. In a separate study, Castells et all5 showed that 10 of 12 patients with detectable K-ras mutations in serum had stage IV pancreatic ductal adenocarcinoma of the pancreas. These studies show that the detection of mutations in soluble DNA lacks sensitivity for the detection of early pancreatic ductal adenocarcinoma. K-ras Mutations in Stool The detection of K-ras mutations in stool for the early diagnosis of malignancy has been successfully applied to the diagnosis of colorectal neoplasms. Sidransky et a176detected K-ras mutations in DNA from the stool of eight of nine patients with curable neoplasms of the proximal and distal bowel. Another study used highly sensitive molecular technology to detect K-ras mutations in the stools of patients with colorectal cancer. The Amplification Refractory Mutation System (ARMS) (Astra Zeneca Diagnostics, Northwich, U.K.) uses primers that are specific for K-ras mutations at their 3' end. If there is complementary binding to a mutant sequence, DNA polymerization can be initiated. K-ras sequence can be amplified by cycling polymerization using a mutation-specific primer and a complementary primer to a downstream region of the gene. Wildtype sequences do not anneal to the 3' end of the mutant-specific primer, and there should be no amplification. In practice, however, a low rate of amplification is still observed. The rate of amplification by the mutant-specific primer is compared with a control primer, which amplifies mutant and wild-type sequences equally. K-ras mutations were identified in a single stool sample in 14 (66%)of 21 patients with K-ras mutant tumors with 100% specificity (JP Neoptolemos et al: personal communication, 1999). The sensitivity of this technique was directly related to the quality of the human DNA in the stool, with highyield DNA stool samples allowing detection of K-ras mutations in seven (100%) of seven of the cases. This result compared with only 7 (50%) of 14 of the cases with lower-quality and lesser DNA yields. This difference can probably be explained by the presence of PCR inhibitors in the stool, which is supported by the lack of correlation of stage, grade, or site of the disease with mutant Kras status. Caldas et all3 used a plaque hybridization assay to overcome the vast background of wild-type K-ras sequences in stool. DNA was extracted from stool and amplified by PCR. The PCR products were then cloned into phage and transfected into Escherichia coli. The resultant clones were then hybridized with phosphorus 32-labeled probes, one for each K-ras mutation. A minimum of 10,000 clones were examined per stool sample; a positive result was at least 10 positive clones (0.1%). This technique demonstrated the presence of K-ras mutations in the stools of 6 (55%) of 11 patients with K-ras mutation-positive pancreatic adenocarcinomas. K-ras mutation was detected in one of three patients with chronic pancreatitis, the same mutation as in areas of ductal mucinous hyperplasia of the microdissected chronic pancreatitis tissue. Using mutantenriched PCR primers (as described previously) and reverse dot blot hybridization using mutant-specific oligonucleotides, Berndt et a18 showed K-ras mutations in the stool of 10 (40%) of 25 patients with mutation-positive ductal adenocarcinomas and in 2 (33%) of 6 patients with chronic pancreatitis. In all positive cases, the K-ras mutation was confirmed in tissue. Wenger et a P used PCR and hybridization to detect K-ras mutations in DNA extracted from the stool. Of 36 patients with ductal adenocarcinoma, K-ras mutations were detected

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in only 7 patients (20%) compared with a detection rate of 28 (78%)of 35 using matched pancreatic tissue. Without improvements in the quantity and quality of DNA extracted from stool, these results are not encouraging in terms of specificity and sensitivity for the early diagnosis of pancreatic ductal adenocarcinoma.

Detection of K-ras Mutations in Pancreatic Juice and Bile K-rus mutations have been successfully identified in pancreatic and biliary samples obtained at ERCP. The sensitivity for the detection of pancreatic ductal adenocarcinoma ranges from 67% to 100% using pancreatic juice or brushings and 25% to 50% using bile (Table 2). Early studies indicated a high specificity for K-rus mutations in pancreatic ductal adenocarcinoma in the presence of chronic pancreatitis, but more recent studies have shown a lower specificity. Furura et alZ5used PCR-RFLP to show that 20 (37%) of 54 patients with chronic pancreatitis harboring a K-rus mutation did not go on to develop pancreatic ductal adenocarcinoma over a mean followup period of 78 months. In an ongoing study, Van Laethem et als4enrolled 76 patients with confirmed chronic pancreatitis and performed K-rus analysis on pancreatic juice taken at ERCP. K-rus mutations were found in 19 (25%) of 76 patients. A cohort of these patients, 12 with K-rus mutations and 43 with wildtype K-rus, was observed over a 2-year period. No pancreatic cancer occurred during the first 6 months, but two cancers were diagnosed in patients harboring K-rus mutations at 18 and 24 months, compared with none in patients with wild-type K-rus. The specificity of K-rus as a diagnostic test in differentiating malignant and benign conditions of the pancreas may be improved by estimating the amount of mutant K-rus DNA in pancreatic juice. New PCR-based technology that semiquantifies mutant K-rus sequences in pancreatic juice to differentiate pancreatic neoplasms from benign pancreatic disease has been applied with promising results. Tada et alS2showed that the K-rus gene was mutant in more than 1% of total DNA in 8 (53%)of 15 patients with pancreatic adenocarcinoma, in 3 of 3 patients with intraductal neoplasm of the pancreas, but in only 2 (11%) of 19 patients without neoplasm. Of the negative control group, four patients had chronic pancreatitis, none of whom were K-rus mutant positive. A comparative study using pancreatic juice showed that 1 (4%) of 26 patients with chronic pancreatitis had a K-YUSmutation by a PCR hybridization protection assay compared with 5 (19%) of 26 patients using a PCR-RFLP assay. Twenty-one (79%) of 28 patients with pancreatic cancer had K-rus mutations detected by the PCR-RFLP technique and 19 (66%) of 29 patients using the hybridization assay.86 p53 Mutations The normal function of p53 is to control cell cycle arrest and apoptosis in response to signals suggesting inappropriate cell division, such as DNA damage. Mutated p53 circumvents control mechanisms that would prevent tumor cell division and result in further accumulation of mutations in the tumor by impairing DNA repair mechanisms. p53 Mutations are the commonest mutations in human cancers, and in pancreatic cancer they are located largely in exons 5 a 3 *with a relative hot spot located at codon 273.60In pancreatic ductal adenocarcinoma, accumulation of p53 protein (indicative of mutant p53 protein) has been found in 23% to 80% of primary tumors (Table 3). This finding correlates with the finding of 27% to 76% incidence of p53 mutations in tissue, detected using single-stranded confirmational polymorphism (SSCP) and microdissection.

6 9 22 8 19 26 49 9 36 15 51 13

Series

Tada et al,al 1993 Kondo et al:a 1993 Berhelemy et al,7 1995 Lee et a1,4I 1995 Iguchi et al,= 1996 Watanabe et al:6 1996 Van Laethem et a1,841998 Ito et al:5 1998 Sturm et al,= 1998 Kondoh et al,”9 1998 Wilentz et al,a91998 Yamashita et al:3 1999

RFLP, P RFLP, P RFLP, P RFLP, B RFLP, P RFLP, P RFLP, PB RFLP, B MS-PCR, PB RFLP, P PCR hybrid, B SSCP, P

Method

6 6 17 4 12* 21 38 3 30 10 13 13

No. With K-ras Mutations 100 67 77 50 63 81 78 33 83 67 25 100

(%I . .

Sensitivity

0/1 0117 0124 013 2/32 016 015 0110 -

No. of Mutant K-ras in Controls

*Two patients in this series had a histologic diagnosis of intraductal papillary adenocardnoma. l7FLP = restriction fragment length polymorphism; P = pancreatic juice; B = bile; PB = pancreatic brushings; MS-PCR PCR hybrid = PCR hybridization; SSCP = single-stranded confirmatory polymorphism.

No. in Series

=

100 100 100 100 98 96 75 100 100 100 100 66

(“w

Specificity

mutation-specific polymerase chain reaction;

0/9 319

0/2 0110 0129 1/41 19/76 -

No. of Mutant K-ras in Chronic Pancreatitis

Table 2. K-ras MUTATIONS IN PANCREATIC DUCTAL ADENOCARCINOMA AND CHRONIC PANCREATITIS: PURE PANCREATIC JUICE, PANCREATIC BRUSHINGS, AND BILE

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SCREENING FOR EARLY PANCREATIC DUCTAL ADENOCARCINOMA IN HP

Table 3. p53 MUTATIONS IN PANCREATIC DUCTAL ADENOCARCINOMA Series

No. in Series

Method

No. With Mutant p53

IHC IHC SSCP IHC SSCP IHC MID IHC SSCPS IHC IHC IHC IHC

13/ 22 281124 14/34 21/34 8/30 621133 31/41 711136 51/74 33/ 76 23/57 20 142 13/15

Percent With Mutations ~

Barton et a1,6 1991

22

Scarpa et al,’O 1993

34

Berrozpe et a1,9 1994 Lundin et al,” 1996 Rozenblum et a1,661997 Ruggeri et al,671997 Dergham et a1,2O 1997 Harada et al,’8 1997 Coppola et al,I8 1998 Apple et al,3 1999

30 133

42 136 (35,* 91t) 76 57 42 15

60 23 41 62 27 47 76 56 70 43 40 48 80

*Tissue used for the analysis was either snap frozen in liquid nitrogen or used fresh for the analysis. tTissue for this series was obtained from archival tissue embedded in paraffin blocks. $Twenty-five of these analyses were suitable for direct sequencing. Mutations were detected in 23 of 25 cases. This gave an 80% concordance with the IHC findings. IHC = Immunohistochemistry; SSCP = single-stranded confirmatory polymorphism; M / D = microdissection and sequencing.

The wide variability of the studies can be, in part, explained by the type of tissue used, fresh tissue giving higher rates of mutation detection than archived paraffin-embedded tissue, as well as the methodology used to detect mutations. Methods using microdissection and sequencing tend to be more sensitive in the detection of low copy numbers of mutant p53 sequence. Evidence suggests that p53 mutations occur at a relatively late stage of carcinogenesis. Apple et a13 examined 15 cases of ductal adenocarcinoma of the pancreas using immunohistochemistry. They found that p53 protein staining was detected in 13 (87%)of 15 areas of invasive carcinoma, rarely in dysplastic cells (3 [20%] of 15), and virtually never in hyperplastic or normal cells (1 [3%]of 15). The same study examined p21 protein staining (K-ras gene product) and found that 11 (73%)of 15 patients had p21 protein staining in areas of dy~plasia.~ The finding of p53 mutations seems to be specific to pancreatic cancer because most studies have not found p53 mutations in chronic pancreatitis in the absence of malignan~y.~, 14,62 Gansauge et alZ6used SSCP analysis to detect p53 mutations in 80 tissue samples obtained from patients with chronic pancreatitis who had undergone resection surgery. They identified eight cases with a p53 mutation; however, four of these mutations were neutral polymorphisms, and two mutations were located in the intron sequence. Only two mutations resulted in an alteration to the amino acid sequence, and no comment was made on whether these changes led to an altered function or a loss of function in the p53 gene product. Detection of p53 Mutations in Pancreatic Juice

Immunocytochemistryhas been applied to pancreatic brushings obtained at ERCP. Iwao et aP6 studied 44 cases of pancreatic cancer and found 36 (82%) of 44 were positive for p53 protein (indicative of a p53 mutation). Microdissection and direct sequencing of the DNA of the positively stained cells showed point mutations in 12 (86%) of 14 cases. There was no staining for p53 protein from

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HOWES et a1

pancreatic brushings in any of 30 patients with chronic pancreatitis or in any of . ~ ~ higher rate of p53 detection in the 9 patients with papillary a d e n ~ m a The pancreatic brushings compared with tissue cannot be explained entirely by patient selection because only 13 of 44 patients had locally advanced or metastatic disease. Iwao et a136showed that positive immunocytochemistry for mutant p53 protein was more accurate than cytology alone for differentiating benign mucin-producing adenomas from malignant mucin-producing carcinomas. In contrast to K-ras, there are.few published studies for the direct detection of p53 mutations in pancreatic juice. Identification of p53 mutations in pancreatic juice is made difficult by the existence of at least 200 different mutations combined with the low mutant copy number compared with wild-type. Yamaguchi et a192 used SSCP to detect p53 mutations in pancreatic juice obtained at ERCP. Of 26 patients with ductal adenocarcinoma of the pancreas, 11 (42%) of the samples harbored a mutation. Direct confirmatory sequencing was possible on only 3 of 11 of the samples in which sufficient DNA from the PCR reactions could be recovered and purified. Identical mutations were found in the corresponding pancreatic tissue of the three sequenced mutations, and in the remaining eight samples, an identical SSCP band pattern was seen in tissue and juice. Nine of 11 of the p53 mutations detected in pancreatic juice were stage IV tumors, with no mutations detected in stage I disease. The same group also analyzed 4 patients with adenoma of the pancreas and 16 patients with chronic pancreatitis and found no detectable p53 mutations in either the pancreatic juice or the corresponding pancreatic tissue?* This technology is promising for the detection of p53 mutations in pancreatic juice, but further studies are required to determine the sensitivity for detecting early cancers of the pancreas. A similar technique, that has the advantage of allowing the use of larger PCR fragments, is enzymatic mutation detection (EMD),19which is based on the specific cleavage of mismatched DNA fragments. The technique is sensitive enough to detect 2.5% mutant DNA in a wild-type background but is untested in pancreatic juice. SSCP and EMD are handicapped by their inability to distinguish polymorphisms, functionally silent mutations, and inactivating mutations. Flaman et alZ3have described a functional p53 assay based on yeast for screening low-concentration mutant p53 without prior knowledge of the mutant sequence. The technique (Fig. 2) uses a reverse transcriptase PCR (RT-PCR) amplification with a proofreading Taq-polymerase. Exons are amplified from RNA, then PCR products are transformed into yeast (Saccharomyces cerevisiae), which contains a plasmid with the complete coding sequence for wild-type p53 protein. The PCR product recombines with the coding sequence, such that mutant p53 protein is produced if the PCR product contained a gene mutation and, conversely, wild-type p53 if the PCR product contained no mutation. When wild-type p53 protein is produced, there is activation of the ADE2 gene via a preinserted p53 recognition sequence in an artificial promoter sequence inserted in front of the ADE2 gene. Activation of the gene allows production of adenine in the yeast, allowing efficient growth of white yeast colonies on medium deficient in adenine. Conversely, mutant p53 protein does not switch on the ADE2 gene; no additional adenine is produced; and in a medium with low adenine concentrations, the resultant colonies are small, and because of buildup of a metabolic intermediate, they are red. Extraction of the DNA from the red clones allows direct sequencing for detection of mutant sequences. A shortcoming with this technique is the difficulty of extracting good enough quality RNA from pancreatic juice.

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Figure 2. Adapted yeast functional assay initially described by Flaman et aLZ3In the original assay, RNA is recombined with a plasmid containing the coding sequence for p53 protein. If the RNA contains wild-type p53 sequence,wild-type p53 protein is produced, which binds to a p53 receptor site on the ADEZ promotor. Activation of this gene produces clonal growth of white yeast. Conversely, if the RNA contained mutant p53 sequence, mutant p53 protein is produced, which does not activate the ADEZ gene complex, resulting in clonal growth of red yeast. The figure shows the adapted assay, in which RNA is substituted for p53 DNA exon sequences of 5-8,which are combined using polymerase chain reaction.

p53 Mutations in the Serum

Measurement of antibodies to mutant p53 in serum has been applied to the diagnosis of neoplasms, but the sensitivity of detection is low. In head and neck squamous carcinoma, p53 antibodies were found in the serum of 15 (18%)of 80 of patients, whereas matched tissue stained for the presence of p53 protein in 43 (59%)of 75 cases." Similar studies in lung cancer have shown antibodies to p53 in 13 (21%)of 62 cases compared with mutant p53 in 24 (40%)of 62 matched tissue samples.- Laurent-Puig et a140 showed positive antibodies in 8 (28%) of 29 patients with pancreatic ductal adenocarcinoma, and none in 11 patients with chronic pancreatitis. Of the 29 patients with pancreatic ductal adenocarcinoma, only 3 (10%) cases had potentially resectable disease, 8 had metastatic disease, and 18 (62%) had locally advanced disease. None of the three patients with resectable disease had detectable levels of p53 protein antibodies in their serum. In another study, Suwa et a179showed that 23 (22%)of 104 patients with ductal adenocarcinoma of the pancreas had elevated levels of p53 protein antigen in the serum. A higher proportion of patients with metastatic disease, 16 (34%) of 47, had elevated antigens, compared with only 7 (12%) of 57 without metastasis. These studies suggest that although this technique has a high specificity, it lacks sensitivity, particularly in the diagnosis of early pancreatic ductal adenocarcinoma. The detection of p53 mutations in the serum has been reported in colorectal tumors. Hibi et a130 used an oligonucleotide-mediated mismatch ligation assay to detect mutations in matched samples of tumor and serum. This technique relies on ligating two oligonucleotides together. The two oligonucleotides hybridize to a template at abutting positions. The ligation is efficient only if the abutting bases match the template perfectly. In the assay, amplified DNA from patients is used as a template on which ligation can occur. By designing one oligonucleotide to end with a mutant base and labeling an oligonucleotide that begins at the adjacent base, label incorporation can be taken as a measure of ligation efficiency and, in turn, as a measure of mutant template concentration. To bias label incorporation further, an unlabeled blocking primer specific for the

730

HOWES et a1

wild-type sequence is also added to the ligation mix. Of 10 patients with sequence-confirmed mutations of p53 in tissue, 7 patients (70%) had identical mutations in the serum: 5 with Dukes’ B and 2 with Dukes’ C. Detection of mutations using this method requires prior knowledge of the target sequence, which may limit the usefulness of the technique. No studies have yet been published for the detection of p53 mutations in pancreatic ductal adenocarcinoma using this technique, but it may improve the sensitivity of existing methods.

SMAD4

SMAD4 (or DPC4) is the common factor controlling pathway-specific transforming growth factor-p signaling. It forms a hetero-oligomeric complex with other phosphorylated SMAD members, which then regulates the transcription of target genes. The central role of the SMAD4 gene in all SMAD signaling pathways is consistent with the finding that most mutations found to date affect only the SMAD4 gene and not other SMAD members.63SMAD4 is homozygously deleted in approximately 30% of pancreatic adenocarcinomas and inactivated by an iatrogenic mutation in a further 2O%.” This is the highest rate of mutation yet reported in human cancers and reflects the importance of this gene in pancreatic carcinogenesis. SMAD4 mutations have been detected in pancreatic juice using fluorescence in-situ hybridization (FISH). This technique uses specific fluorescent DNA probes that hybridize to a specific region of interest on a chromosome, together with control fluorescent DNA probes that are specific for the centromere region. The percentage loss or gain of the region of interest can be implied by the fraction of nuclei having fewer or more signals for the specific probe than those for the centromeric probe. Fukushige et aIz4used FISH on cells from pancreatic juice obtained at ERCP from 32 patients with pancreatic diseases; 12 (63%) of 19 of the samples obtained from patients with pancreatic neoplasms showed chromosomal abnormalities. When the locus 18,, where the SMAD4 gene is located, was probed, 11 (92%)of 12 patients showed complete or partial loss at that locus. Similar results were observed in DNA extracted from paired pancreatic tissue in four of four cases, providing verification of the results seen in pancreatic juice. By contrast, a control group of 13 patients (1 normal, 12 with chronic pancreatitis) showed no chromosomal a b n o r m a l i e ~ FISH . ~ ~ relies on intact cells for successful analysis of mutations, but pancreatic juice often contains degraded cells. The technique also depends on the skill of the investigator and so may not easily be extended to general use, especially when only small numbers of cancer cells are present, as with early ductal adenocarcinoma. SSCP and EMD also could be used to detect SMAD4 mutations in pancreatic juice, but there are the same limitations in terms of sensitivity and poor selectivity for functional rather than polymorphic mutations as for mutant p53 detection. An alternative would be to use loss of heterozygosity (LOH) analysis as an indication of mutation. This LOH analysis could be achieved using comparative analysis of microsatellite markers in pancreatic juice and blood. To increase the sensitivity of this approach, multiple PCR reactions could be performed using multiple primer pairs that specifically amplify the chromosomal region of interest in blood and pancreatic juice DNA. Allelic loss of one or both copies at that locus in the pancreatic juice compared with blood could then be determined. As well as increasing sensitivity, multiple PCR primers for the area of interest can

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be used to exclude template degradation, inhibitors, and primer site polymorphisms, whch cause false-positive results. This technology has already been used to detect p16 mutations in pancreatic tissue,12 and the possibility remains that accurate determination of LOH may be possible, even with the high wildtype background expected in pancreatic juice.

The p16 gene (INK4p’6) inhibits activation of the cyclin-dependent kinase CDK4 by cyclin D. Loss of p16 function prevents normal inhibition of passage from Go to G,/S; p16 is deleted or mutated in 85% of pancreatic adenocarcinomas.l2S66 Moskaluk et a154showed p16 mutations in microdissected preneoplastic pancreatic intraductal lesions, suggesting that p16 might be an early event in carcinogenesis. The same study also showed that p16 mutations were never found in normal pancreatic tissue; when these mutations were present, they were always in association with a K-ras mutation, suggesting that K-rus mutations precede p16 mutations in tumorigenesis. In another study, Wilentz et a190 analyzed 126 microdissected intraductal lesions from 33 infiltrating, pl6-positive ductal adenocarcinoma specimens. Using p16 immunohistochemistry, 60 of 126 showed loss of p16 expression. More significantly, the loss of expression was seen in atypical lesions three times more often than in nonatypical lesions, suggesting that loss of p16 expression occurs more frequently in higher-grade duct lesions. There are few reports of direct detection of p16 mutations in pancreatic juice. Fukushige et alZ4used FISH to analyze pancreatic juice from 19 patients with pancreatic neoplasia and found that 6 of 19 had deletion at the 9, site, indicative of a complete or partial deletion of p16. Analysis of 13 patients with benign pancreatic disease showed no deletion of the 9, region. The sensitivity of this method for the detection of early pancreatic cancer in chronic pancreatitis is likely to be limited in the same way as in SMAD4 mutation detection, described previously. Mutations of p16 may be detectable in pancreatic juice using LOH technology in a similar manner to SMAD4. p 16 Mutations in Serum

Detection of hypermethylation of the promoter region in p16 has been successfully applied to the diagnosis of other tumors. Esteller et a122used PCR to amplify selectively methylated regions of the promoter region in patients with lung cancer.. Hypermethylated markers at the p16 promoter were detected in the serum of three (33%)of nine patients with lung cancer. Identical methylation was found in matched tumor specimens. Of the positive results, two patients had stage I disease, and one patient had stage I11 disease; p16 was hypermethylated in tissue in 9 (41%) of 22 cases, which is similar to previous studies. None of the 22 control patients had detectable methylation in serum or tissue. Methylation mutations in the promoter region have been reported in pancreatic cancer. Schutte et al” found methylation of the p16 promoter region in tissue from five of seven patients with pancreatic ductal adenocarcinoma. All seven samples had been shown to have LOH at the 9p allele and wild-type sequence of the remaining allele. Protein expression of p16 was shown to be absent, suggesting that methylation had silenced the expression of the remaining functioning copy of p16. In that series, the overall incidence of methylation of p16

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in tissue was 5 (10%) of 50. Bigger studies are needed to evaluate the usefulness of this technique in the diagnosis of pancreatic ductal adenocarcinoma. STRATEGY FOR SECONDARY SCREENING IN HEREDITARY PANCREATITIS The European Registj of Hereditary Pancreatitis and Familial Pancreatic Cancer (EUROPAC) was established to identify families with HP and develop strategies for the early detection of pancreatic ductal adenocarcinoma. EUROPAC has developed an ethical committee-approved protocol for the secondary screening of HP patients. After multidisciplinary counseling and informed consent, affected family members older than age 30 years are enrolled. All patients undergo an ERCP, at which time pancreatic juice is aspirated to undertake molecular tests. Conventional imaging techniques of endoluminal ultrasound scan and high-resolution spiral CT scan performed at the same time are used as a benchmark to compare the results of the molecular analysis. Mutation detection for K-ras, p53, p16, and SMAD4 is performed on the DNA extracted from the pancreatic juice obtained at ERCP. It is likely that individual markers lack the sensitivity and specificity to be diagnostically useful in isolation, but used together in tandem with conventional imaging modalities, they may greatly assist in the diagnosis of early pancreatic ductal adenocarcinoma.

K-ras Mutation Analysis Although K-ras mutations occur in patients with pancreatitis, the high prevalence of K-ras mutations in pancreatic ductal adenocarcinoma (approaching ~OO'XO) means that this analysis remains a sensitive initial screening test. All patients are screened for the presence of K-rus mutations in codons 12 and 13 using ARMS technology, which employs primers that are specific for the various mutations at their 3' end. If there is complementary binding to a mutant sequence, DNA polymerization can be initiated. By cycling polymerization using a mutation-specific primer and a complementary primer to a downstream region of the gene, a K-ras sequence can be amplified. A molecular beacon is attached to an oligonucleotide in such a way that it fluoresces only if bound to the amplified K-ras sequence. The quantity of amplified product can be assessed continuously by increase in fluorescence. Wild-type sequences do not anneal to the 3' end of the mutant-specific primer, not resulting in amplification. In practice, however, a low rate of amplification is still observed. Threshold times (the time at which fluorescence becomes detectable) are plotted for wild-type against mutant primers and the plots compared with control curves prepared with varying concentrations of wild-type and mutant templates. Deviation from the wild-type curve beyond the 98% confidence level indicates the presence of mutant K-ras, and the degree of deviation gives the relative mutant concentration. Control studies of K-rus analysis in healthy individuals and patients with pancreatic cancer are already in progress. The system is sensitive enough to detect one mutant copy of DNA in 5000 copies of wild-type and has the additional advantage of being quantitative for mutant DNA in the sample, without reducing the overall sensitivity of the technique.

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p53 Mutation Detection

To overcome the sensitivity problem of p53 mutation detection in pancreatic juice, EUROPAC is working in close collaboration with Astra Zeneca Diagnostics to develop a p53 version of the ARMS technique described earlier. The system will use a bank of more than 80 p53 mutation specific primers, based on the commonest point mutations found in pancreatic ductal adenocarcinoma. It will not, however, allow detection of mutations outside of this spectrum or deletions or insertions. The authors are also using a development of the yeast functional assay for p53 described previously (see Fig. 2). This technique is specific for the detection of functional p53 mutations. The technique as applied by EUROPAC involves an initial screen of clones for the lack of functional p53 as described in Figure 2, followed by a PCR screen of colonies to eliminate those that contain p53 with rearrangements or large deletions, neither of which are typically found in pancreatic cancer. Clones are sequenced to eliminate mixed populations that are found with PCR error. The sensitivity of this technique for the detection of mutant p53 in a background of wild-type is 1 in 3000.53

INK4Pl6 and SMAD4

Identification of p16 and SMAD4 abnormalities in pancreatic juice is not a simple task. EUROPAC is in the process of establishing the sensitivity of LOH analysis using banks of microsatellite markers. The threshold at which absence of LOH can be confidently assigned needs to be determined in comparison to appropriate controls. For the technique to be effective, the levels of LOH in known pancreatic cancer patients must be significantly higher than in the control groups. The long-term strategy would be to combine a number of techniques including LOH and methylation-specific PCR of the p16 promoter region in pancreatic juice and peripheral blood to optimize detection.

CONCLUSION Technology is advancing at a fast rate, and clinicians will soon be able to detect molecular ,markers for the early diagnosis of pancreatic ductal adenocarcinoma. The question arises as what to do with the information obtained. It is already evident that the identification of K-uas mutation, which is the simplest and most sensitive of the available molecular approaches, is unsuitable for diagnosis in isolation because it lacks specificity for cancer. The molecular diagnosis of pancreatic ductal adenocarcinoma must involve the analysis of more than one molecular marker. As yet, insufficient data have been obtained as to the sensitivity of molecular analysis in the diagnosis of early pancreatic cancer. EUROPAC has made a decision to adopt and concentrate on a specific set of molecular examinations. The implications of these assays need to be established by longitudinal clinical correlation and require the integrated collaboration of all groups caring for patients with HP.

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References 1. Akahoshi K, Chijiiwa Y, Nakano I, et al: Diagnosis and staging of pancreatic cancer by endoscopic ultrasound. Br J Radio1 71:492496, 1998 2. Anker P, Lefort F, Vasioukhin V, et al: K-Ras mutations are found in DNA extracted from the plasma of patients with colorectal cancer. Gastroenterology 112:1114-1120, 1997 3. Apple S, Hecht R, Lewin D, et al: Immunohistochemical evaluation of K-ras, p53, and HER-2 / neu expression in hyperplastic, dysplastic, and carcinomatous lesions of the pancreas: Evidence for multistep carcinogenesis. Hum Pathol 30:123-129, 1999 4. Bakkevold KE, Arnesjo B, Kambestad B: Carcinoma of the pancreas and papilla of Vater-assessment of resectability and factors influencing resectability in stage I carcinomas. A prospective multicentre trial in 472 patients. Eur J Surg Oncol 18:494507, 1992 5. Barthet M, Portal L, Boujaoude J, et al: Endoscopic ultrasonographic diagnosis of pancreatic cancer complicating pancreatic cancer. Endoscopy 28:487491, 1996 6. Barton CM, Staddon SL, Hughes CM, et al: Abnormalities of the p53 tumour suppressor gene in human pancreatic cancer. Br J Cancer 9410761082, 1991 7. Berhelemy P, Bouisson M, Escourrou J, et al: Identification of K-Ras mutations in pancreatic juice in the early diagnosis of pancreatic cancer. Ann Intern Med 123:188191, 1995 8. Berndt C, Haubold K, Wenger F, et al: K-Ras mutations in stools and tissue samples from patients with malignant and non-malignant pancreatic diseases. Clin Chem 44~2103-2107,1998 9. Berrozpe G, Schaeffer J, Peinado MA, et al: Comparative analysis of mutations in the p53 and K-Ras genes in pancreatic cancer. Int J Cancer 58:185-191, 1994 10. Bos JL: Ras oncogenes in human cancer: A review. Cancer Res 49:46824689, 1989 11. Bourhis J, Lubin R, Roche 8, et al: Analysis of p53 serum antibodies in patients with head and neck squamous cell carcinoma. J Natl Cancer Inst 88:1228-1233, 1996 12. Caldas C, Hahn SA, da Costa L, et al: Frequent somatic mutations and homozygous deletions of the p16 (MTS1) gene in pancreatic adenocarcinoma. Nat Genet 8:27-32, 1994 13. Caldas C, Hahn SA, Hruban RH, et al: Detection of K-Ras mutations in the stool of patients with pancreatic adenocarcinoma and pancreatic ductal hyperplasia. Cancer Res 543568-3573, 1994 14. Casey G, Yamanaka Y, Fries H, et al: P53 mutations are common in pancreatic cancer and are absent in chronic pancreatitis. Cancer Lett 69:151-160, 1993 15. Castells A, Puig P, Mora J: K-Ras mutations in DNA extracted from the plasma of patients with pancreatic carcinoma: Diagnostic utility and prognostic significance. J Clin Oncol 17:578-584, 1999 16. Chen XQ, Stroun M, Magenat JL, et al: Microsatellite alterations in the plasma of DNA of small cell lung cancer patients. Nat Med 2:1033-1035, 1996 17. Cohen JJ, Duke RC, Fadok VA, et al: Apoptosis and programmed cell death in immunity. AMU Rev Immunol 10:267-293, 1992 18. Coppola D, Lu L, Fruehauf J, et al: Analysis of p53, p21WAF1, and TGF-beta 1 in human ductal adenocarcinoma of the pancreas: TGF-beta 1 protein expression predicts longer survival. Am J Clin Pathol 110:16-23, 1998 19. Del Tito BJ, Poff HE, Novotny MA, et al: Automated fluorescence analysis procedure for enzymatic mutation detection. Clin Chem 44731-739, 1998 20. Dergham S, Dugan M, Kucway R, et al: Prevalence and clinical significance of combined K-Ras mutation and p53 aberration in pancreatic adenocarcinoma. Int J Pancreato1 21:127-143, 1997 21. Endo Y, Morii T, Tamura H, et al: Cytodiagnosis of pancreatic malignant tumours by aspiration, under direct vision, using a duodenal fiberscope. Gastroenterology 67944-951, 1974 22. Esteller M, Sanchez-Cespedes M, Rose11 R, et al: Detection of aberrant promoter hypermethylation of tumour suppressor genes in serum DNA from non-small cell lung cancer patients. Cancer Res 59:67-70, 1999

SCREENING FOR EARLY PANCREATIC DUCTAL ADENOCARCINOMA IN HP

735

23. Flaman JM, Frebourg T, Moreau V, et al: A simple p53 functional assay for screening cell lines, blood and tumours. Genetics 92:3963-3967, 1995 24. Fukushige S, Furukawa T, Satoh K, et al: Loss of chromosome 18, is an early event in pancreatic ductal tumorigenesis. Cancer Res 58:42224226, 1999 25. Furuya N, Kawa S, Akamatsu T, et al: Long-term follow up of patients with chronic pancreatitis and K-Ras gene mutation detected in pancreatic juice. Gastroenterology 113:593-598, 1997 26. Gansauge S, Schmid RM, Muller G, et al: Genetic alterations in chronic pancreatitis: Evidence for early occurrence of p53 but not K-Ras mutations. Br J Surg 85:337-340, 1998 27. Graham RA, Bankoff M, Hediger R, et al: Fine needle aspiration biopsy of pancreatic ductal adenocarcinoma: Loss of diagnostic accuracy with small tumours. J Surg Oncol 55:92-94, 1994 28. Harada N, Gansauge F, Gause H, et al: Nuclear accumulation of p53 correlates significantly with clinical features and inversely with the expression of the cyclindependent p21 in pancreatic cancer. Br J Cancer 76:299-305, 1997 29. Hayakawa T, Kondo T, Shibata T, et al: Sensitive serum markers for detecting pancreatic cancer. Cancer 61:1827, 1988 30. Hibi K, Robinson R, Booker S, et al: Molecular detection of genetic alterations in the serum of colorectal cancer patients. Cancer Res 58:1405-1407, 1998 31. Hollstein M, Sidransky D, Vogelstein B, et al: p53 mutations in human cancers. Science 253:49-53, 1991 32. Hruban RH, van Mansfield ADM, Offerhaus GJA, et al: K-Ras mutations in adenocarcinoma of the pancreas. Am J Pathol 143:545-554, 1993 33. Iguchi H, Sugano K, Fukayama N, et al: Analysis of Ki-ras codon 12 mutations in the duodenal juice of patients with pancreatic cancer. Gastroenterology 110:221-226, 1996 34. Iizasa T, Fujisawa T, Saitoh Y, et al: Serum anti-p53 autoantibodies in primary resected non-small-cell lung carcinoma. Cancer Immunol Immunother 46:345-349, 1998 35. Ito R, Tamura K, Ashida H, et al: Usefulness of K-Ras gene mutation at codon 12 in bile for diagnosing biliary strictures. Int J Oncol 121019-1023, 1998 36. Iwao T, Tsuchida A, Hanada K, et al: Immunocytochemical detection of p53 protein as an adjunct in cytologic diagnosis from pancreatic duct brushings in mucin-producing tumors of the pancreas. Cancer 81:163-171, 1997 37. Johnson P, Outwater E: Pancreatic carcinoma versus chronic pancreatitis: Dynamic MR imaging. Radiology 212:213-218, 1999 38. Kondo H, Sugano K, Fukayama N, et al: Detection of point mutations in the k-Ras oncogene at codon 12 in pure pancreatic juice for the diagnosis of pancreatic carcinoma. Cancer 73:1589-1594, 1993 39. Kondoh S, Kaino M, Okita S, et al: Detection of K-ras and p53 mutations in tissue and pancreatic juice from pancreatic adenocarcinomas. J Gastroenterol33:390-396, 1998 40. Laurent-Puig P, Lubin R, et al: Antibodies against p53 protein in serum of patients with benign or malignant pancreatic and biliary diseases. Gut 36:455458, 1995 41. Lee JG, Leung JW, Cotton PB, et al: Diagnostic utility of K-ras mutational analysis on bile obtained by endoscopic retrograde cholangiopancreatography.Gastrointest Endosc 42:317-320, 1995 42. Lerch MM, Ellis I, Whitcomb DC, et al: Maternal inheritance pattern of hereditary pancreatitis in patients with pancreatic carcinoma. J Natl Cancer Inst 91:723-724, 1999 43. Lowenfels A, Maisonneuve P, Cavallini G, et al: Pancreatitis and the risk of pancreatic cancer. N Engl J Med 328:1433-1437, 1993 44. Lowenfels A, Maisonneuve P, DiMagno E, et al: Hereditary pancreatitis and the risk of pancreatic cancer. J Natl Cancer Inst 89:442-446, 1997 45. Lundin J, Nordling S, von Boguslawsky K, et al: Prognostic value of immunohistochemical expression of p53 in patients with pancreatic cancer. Oncology 53:1OPlll, 1996 46. Liittges J, Schlehe B, Menke M, et al: The K-Ras mutation pattern in pancreatic ductal adenocarcinoma usually is identical to that in associated normal, hyperplastic and metaplastic epithelium. Cancer 85:1703-1710, 1999 47. Malescia A, Tommasini MA, Bonato C, et al: Determination of CA19-9 antigen in

736

HOWES et al

serum and pancreatic juice and serum for the differential diagnosis of pancreatic adenocarcinoma and chronic pancreatitis. Gastroenterology 92:60, 1987 48. Manabe T, Miyashita T, Ohshio G, et al: Small carcinomas of the pancreas. Cancer 62:135, 1988 49. Matsubayashi H, Watanabe H, Nishikura K, et al: Determination of pancreatic ductal carcinoma histogenesis by analysis of mucous quality and K-Ras mutation. Cancer 82:651460, 1998 50. Matsumoto S, Harada H, Tanaka J, et al: Evaluation of cytology and tumour markers of pure pancreatic juice for the diagnosis of pancreatic cancer at early stages. Pancreas 6:741-747, 1994 51. Midwinter M, Beveridge C, Wilswdon J, et al: Correlation between spiral computed tomography, endoscopic ultrasonography and findings at operation in pancreatic and ampullary tumours. Br J Surg 86:189-193, 1999 52. Montijima K, Tsunoda T, Kanematsu T Distinguishing pancreatic carcinoma from other periampullary carcinomas by analysis of mutations in the Kirsten ras oncogene. Ann Surg 214:657-662, 1991 53. Moshinsky DJ, Wogan GN: UV-induced mutagenesis of human p53 in a vector replicated in saccharomyces cerevisiae. Biochemistry 94:2266-2271, 1997 54. Moskaluk CA, Hrubran RH, Kern SE: P16 and K-Ras gene mutations in the intraductal precursors of human pancreatic adenocarcinoma. Cancer Res 572140-2143, 1997 55. Mulcahy HE, Lyautey J, Lederrey C, et al: A prospective study of K-Ras mutations in the plasma of pancreatic cancer patients. Clin Cancer Res 4:271-275, 1998 56. Muller MF, Meyenberger C, Bertschinger P, et al: Pancreatic tumors: Evaluation with endoscopic US, CT, and MR imaging. Radiology 190:745-751, 1994 57. Nawroz H, Koch W, Anker P, et al: Microsatellite alterations in serum DNA of head and neck patients. Nat Med 2:1035-1037, 1996 58. Nomoto S, Nakao A, Kasai Y, et al: Detection of ras gene mutations in perioperative blood with pancreatic adenocarcinoma. Jpn J Cancer Res 87:793-797, 1996 59. Palson 8, Masson P, Andren-Sandberg A: Tumour marker CA50 levels compared to signs and symptoms in the diagnosis of pancreatic cancer. Eur J Surg Oncol 23:151-161, 1997 60. Pellagata NS, Sessa F, Renault B, et al: K-Ras and p53 gene mutations in pancreatic cancer: Ductal and nonductal tumours progress through different genetic lesions. Cancer Res 54:1556-1560, 1994 61. Phoa S, Reeders W, Rauws A, et al: Spiral computed tomography for preoperative staging of potentially resectable carcinoma of the pancreatic head. Br J Surg 86:789794, 1999 62. Rall C, Yan Y, Graeme Cook F, et al: K-Ras and p53 mutations in pancreatic ductal adenocarcinoma. Pancreas 12:7-10, 1996 63. Riggins GJ, Kinzler KW, Vogelstein B, et al: Frequency of smad4 gene mutations in human cancer. Cancer Res 57:2578-2580, 1997 64. Rivera JA, Rall CJN, Graeme-Cook F, et al: Analysis of K-Ras oncogene mutations in chronic pancreatitis and ductal hyperplasia. Surgery 121:4249, 1997 65. Rosch T, Lorenz R, Braig C, et al: Endoscopic ultrasound in pancreatic tumour diagnosis. Gastrointest Endosc 37347-352, 1991 66. Rozenblum E, Schutte M, Goggins M, et al: Tumour suppressive pathways in pancreatic cancer. Cancer Res 571731-1734, 1997 67. Ruggeri BA, Huang L, Berger D, et al: Molecular pathology of primary and metastatic ductal pancreatic lesions. Cancer 79:700-716, 1997 68. Safi F, Schlosser W, Falkenreck S, et al: CA19-9 serum course and prognosis in pancreatic cancer. Int J Pancreatol 20:155-161, 1996 69. Savill JS, Wyllie AH, Henson JE, et al: Macrophage phagocytosis of aging neutrophils in inflammation: Programmed cell death in the neutrophil leads to its recognition by macrophages J Clin Invest 83:865-875, 1989 70. Scarpa A, Capelli P, Mukai K, et al: Pancreatic adenocarcinoma frequently show p53 gene mutations. Am J Pathol 142:1534-1541, 1993 71. Schaeffer BK, Glasner S, Kuhlmann E, et al: Mutated c-K-Ras in small pancreatic adenocarcinomas. Pancreas 9:161-165, 1994

SCREENING FOR EARLY PANCREATIC DUCTAL ADENOCARCINOMA IN HP

737

72. Schutte M, Hruban RH, Geradts J, et al: Abrogation of the Rb/pl6 tumour-suppressive pathway in virtually all pancreatic carcinomas. Cancer Res 573126-3130, 1997 73. Schutte M, Ralph H, Hedrick L, et al: DPC4 in various tumour types. Cancer Res 56~2527-2530,1996 74. Seifert E, Urakami Y, Elster K Duodenoscopic guided biopsy of the biliary and pancreatic duct. Endoscopy 9:154-161, 1977 75. Shapiro B, Chakrabarty M, Cohn EM, et al: Determination of circulating DNA levels in patients with benign or malignant disease. Cancer 51:2116-2120, 1983 76. Sidransky D, Tokino T, Hamilton SR, et al: Identification of ras oncogene mutations in the stool of patients with curable colorectal tumours. Science 256:102-105, 1992 77. Sturm PD, Hruban RH, Ramsoekh TB, et al: The potential diagnostic use of K-Ras codon 12 and p53 alterations in brush cytology from the pancreatic head region. J Pathol 186:247-253, 1998 78. Sugio K, Molberg K, Albores-Saavedra J, et al: K-Ras mutations and allelic loss at 5q and 18q in the development of human pancreatic cancers. Int J Pancreatol21:205-217, 1997 79. Suwa H, Ohshio N, Okada N, et al: Clinical significance of serum p53 antigen in patients with pancreatic carcinomas. Gut 40:647-653, 1997 80. Tada M, Ohashi M, Shiratori Y, et al: Analysis of K-Ras mutation in hyperplastic duct cells of the pancreas without pancreatic disease. Gastroenterology 110:227-231, 1996 81. Tada M, Omata M, Kawai S, et al: Detection of ras gene mutations in pancreatic juice and peripheral blood of patients with pancreatic adenocarcinoma. Cancer Res 53:2472-2474, 1993 82. Tada M, Teratani T, Komatsu Y, et al: Quantitative analysis of ras gene mutation in pancreatic juice for diagnosis of pancreatic adenocarcinoma. Dig Dis Sci 43:15-20, 1998 83. Van Laethem J L K-Ras mutations in chronic pancreatitis: Which discriminating ability for malignant potential? In: Cell and Molecular Biology of Pancreatic Carcinoma. New York, New York Academy of Sciences, 1999, pp 210-218 84. Van Laethem JL, Bourgeois V, Parma J, et al: Relative contribution of K-Ras gene analysis and brush cytology during ERCP for the diagnosis of biliary and pancreatic diseases. Gastrointest Endosc 47479485, 1998 85. Wao T, Hanada K, Tsuchida A, et al: The establishment of a preoperative diagnosis of pancreatic carcinoma using cell specimens from pancreatic duct brushings with special attention to p53 mutations. Cancer 82:1487-1494, 1998 86. Watanabe H, Sawabu N, Songur Y, et al: Detection of K-ras point mutations at codon 12 in pure pancreatic juice by PCR-RFLP analysis. Pancreas 1:18-24, 1996 87. Watanabe H, Yamaguchi Y, Ha A, et al: Quantitative determination of K-Ras mutations in pancreatic juice for the diagnosis of pancreatic cancer using hybridisation protection assay. Pancreas 17341-347, 1998 88. Wenger F, Zieren J, Peter F, et al: K-Ras mutations in tissue and stool samples from patients with cancer and chronic pancreatitis. Langenbecks Arch Surg 384:181-186, 1998 89. Wilentz R, Chung C, Sturm P, et al: K-ras mutations in the duodenal fluid of patients with pancreatic carcinoma. Cancer 82:96-103, 1998 90. Wilentz RE, Geradts J, Maynard R, et al: Inactivation of the p16 (INK4A) tumour suppressor gene in pancreatic duct lesions: Loss of intranuclear expression. Cancer Res 58:4710-4714, 1998 91. Yamada T, Nakamori S, Ohzato H, et al: Detection of K-Ras gene mutations in plasma DNA of patients with pancreatic adenocarcinoma: Correlation with clinicopathological features. Clin Cancer Res 4:1527-1532, 1998 92. Yamaguchi Y, Watanabe H, Yrdiran S, et al: Detection of mutations of p53 tumour suppressor gene in pancreatic juice and its application to diagnosis of patients with pancreatic cancer: Comparison with K-ras mutation. Clin Cancer Res 5:1147-1153, 1999 93. Yamashita K, Kida Y, Shinoda H, et al: K-Ras point mutations in the supernatants of pancreatic juice and bile are reliable for diagnosis of pancreas and biliary tract carcinomas complementary to cytological examination. Jpn J Cancer Res 90:240-248, 1998

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HOWES et a1

94. Yanagisawa A, Ohtake K, Ohnishi K, et al: Frequent c Ki-ras oncogene activation in mucous cell hyperplasia of pancreas suffering from chronic inflammation. Cancer Res 53:953-956, 1993

Address reprint requests to John Neoptolemos, MA, MBChB, FRCS, MD Department of Surgery Royal Liverpool University Hospital Daulby Street Liverpool L69 3GA UK e-mail: J.P.NeoptolemosQliv.ac.uk