j,, ,a, o,,lJo, ,,Jof ,cr ,Jolo VSl l l n0 [,I , UlUlilIt5 �9 Copyright 1995 by Humana Press Inc, All rights of any nature whatsoever reserved, 0169-4197/95/18:1--6/$5.20

State-of-the-Art

K-ras Mutation and Pancreatic Adenocarcinoma

Carlos Caldas 1'3 and Scott E. Kern*, ,2

Departments oflOncology and :Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD; 3Current address: The Institute of Cancer Research, Chester Beatty Laboratories, London

Key Words: Pancreatic adenocarcinoma; K-ras; mutation; ducts.

I n t r o d u c t i o n

Pancreatic cancer is one of the more common epi- thelial cancers. It is difficult to diagnose and to treat, and its poor prognosis is reflected in a very low 5-yr survival (1). A minority of patients are diagnosed while the disease is still resectable, and these patients can have a survival as high as 40% at 5 yr if the lymph nodes are not involved (2). However, even in this very select group of patients, the survival is consid- erably worse than that of patients of similar stage who have other common epithelial malignancies (breast and colon, for example), suggesting that pan- creatic cancer is more aggressive, This increased aggressiveness cannot be easily accounted for by any significant differences in classical "indicators" of worse prognosis, as for example, histologic differen- tiation or nuclear ploidy, thus the importance of defining other events that might help explain this clinical behavior.

The characterization of genetic changes in cancer cells has significantly helped our understanding of cancer pathogenesis. The knowledge of these changes in human malignancies has already had some applications in diagnosis and prognostica- tion &cancer (3). The profile of molecular genetic changes in pancreatic cancer was poorly charac- terized until recently.

*Author to whom all correspondence and reprint requests should be addressed: The Department of Ontology, The Johns Hopkins University School of Medicine, 628 Ross Research Bldg., Baltimore, MD 21205.

The high incidence of K-ras mutations (4) has been widely confirmed by several independent groups as the most common genetic change in human pancreatic cancer (5-8).

Over the past 3 yr, an explosion of new data has helped identify changes in other genes. A Southern blot-based allelotype of pancreatic cancer, using probes of the variable number of tandem repeats (VNTR) type, shoved acetic loss in chromosomal arms 17p and 18q in over 70% of the cases, as well as additional sites of less frequent loss of heterozy- gosity (LOH) (9). Mutations of the p53 gene are common, as predicted after the finding of frequent allelic loss at 17p (10,11). One of these studies, although numbers were small, suggested a particular type of mutation involving small deletions at homo- copolymer tracts (10). Mutations in the APC gene were reported by one group in Japan (12), but were absent in cases from the Johns Hopkins Hospital (9). Allelic loss at 18q and a report of loss of expression of the DCC gene (13) suggest involvement of this putative tumor-suppressor gene. The recently described "mutator" genes are probably altered in only a minority of pancreatic cancers (9,14). A high incidence of somatic mutations and homozygous deletions of the MTS 1 gene has just been reported, confirming that this cyclin-dependent kinase (Cdk) inhibitor is a potential tumor-suppressor gene (15). These observations support a genetic model of pan- creatic tumorigenesis: ductal cells driven by almost universal mutation of a dominant oncogene, K-ras, and deregulation of cell-cycle control, by frequent

mutation of two genes involved in Cdk inhibition, p53 and MTS 1.

K-ras Mutations in Pancreatic Cancer

Pancreatic cancer is the human tumor with the highest incidence ofras mutations identified to date (16,17). All the mutations reported are in the K-ras gene. Both findings remain unexplained, although there are sequence characteristics that might explain why mutations in N- and H-ras are uncommon in human cancer and so prevalent in rodent tumors (I 8). In the largest series reported to date, the incidence of K-ras mutations approaches 90% (8). Mutations are almost exclusively at codon 12, with only two cases of human pancreatic cancer reported with codon 13 mutations, and in both cases, the tumor also had a codon 12 mutation (19,20). There is one isolated report of a codon 61 mutation in a pancreatic carci- noma cell line (21). Even with the more sensitive techniques, there appear to be a small number of pancreas cancers that have wild-type K-ras alleles. The numbers are too small to know if these have a different biology and clinical behavior. There is some suggestion from one study that smoking is associated with a higher rate of K-ras mutation, but this finding was of borderline statistical significance (p = 0.046) (8). The study of other gene products of the ras path- way (hSOS, hGRB2, ERK, and so forth) in tumors with wild-type K-ras will have important implica- tions in our understanding of the biology of pancreas cancer in general.

ras gene mutations were originally identified using biological assays (22-24). Their inconvenience and low sensitivity have led to the use of direct sequence-specific assays. These assays were first performed using genomic DNA, and relied on the detection of mismatched base pairs by RNase I in nucleic acid hybrids (25) or by differential hybrid- ization to specific oligonucleotide probes (26). The advent of PCR permitted amplification of ras sequences prior to the detection of mutations, with use of much smaller amounts of genomic DNA. The ras "amplicon" is then analyzed for the presence of mutant sequences using one of several methods. The sensitivity of these methods is determined by the intrinsic characteristics of the test and by the percent-

age of mutant ras sequences in the amplicon mix (containing also amplified ras from normal cells present in the sample). Direct sequencing of the PCR product (27) appears to be the least sensitive method, requiring at least 20% of mutant sequences, but ulti- mately is the "gold" standard. The method more commonly used consists of the hybridization of allele-specific oligonucleotides to either Southern or dot blots (28), and has a better sensitivity (10% of mutant sequences). RNase I detection of mismatched base pairs in the ras amplicon has been used with success (4), but has the disadvantage of not identify- ing the type of mutation. Attempts to improve sensi- tivity have relied mainly on selective amplification of mutant sequences and better detection of these sequences in the amplicon (29-32).

We use a modified allele-specific ligation assay (33) that is very easy to perform, produces reliable results, has a sensitivity to mutant sequences of only 0.1--0.2%, and is convenient for the analysis of large numbers of samples (Caldas C, Kem SE, unpublished results).

An issue that has remained controversial is whether multiple K-ras mutations can be found in pancreatic cancer, indicating either multifocal or biclonal origin. The phenomenon is unusual, having been reported in 8 of 137 cancers where it was spe- cifically sought (7,19,20,34). A similar percentage was found at Johns Hopkins (2 of 53 cases studied). It has been noted that pancreatic ducts with epithelial lesions can have K-ras mutations different from the cancer (see next section). Therefore, a likely expla- nation for cases with two mutations is invasion of duct lesions with a certain K-ras mutation by a can- cer with a different mutation.

Pancreatic Ductal Lesions Precursors of Pancreatic Cancer?

Abnormal ducts are frequently seen in areas of the pancreas not directly involved by cancer. The duct cells have a mucinous morphology (fiat mucinous cell hyperplasia), and the mucosa can form papillary projections into the duct lumen (papillary mucinous cell hyperplasia). In extreme cases, the nuclei of the abnormal mucinous cells becomes frankly dysplas- tic (atypical mucinous cell hyperplasia), or in situ carcinoma is present. These duct lesions are believed to be the earlier precursors of pancreatic cancer

International Journal of Pancreatology Volume 18, 1995

K-ras and Pancreatic Adenocarcinoma 3

(35,36). The problem is that duct lesions, in particu- lar, the first two described, can be seen frequently in the pancreas from patients with cancer in other parts of the gastrointestinal tract or in other organ systems, or in the pancreas with chronic pancreatitis (3 7,38). These lesions have also been described at autopsy in otherwise "normal" pancreas (3 7,38). The identifi- cation of mutations in some duct lesions has compli- cated the interpretation of their significance even further. Yanagisawa et al. first reported that duct lesions in chronic pancreatitis have K-ras mutations (39), characterizing them as clonal and, therefore, as neoplastic (40, 41). We have confirmed these results, and extended the observation by demonstrating K-ras mutations in duct lesions from patients with carcinoma of the binary tract and in duct lesions from patients with pancreas cancer (42). Moreover muta- tions have been seen in duct lesions in a patient with history of familial pancreas cancer (43). These observations provide strong arguments in favor of these lesions being precursors ofinvasive cancer, but some important questions remain, particularly the definition of events determining the progression to invasive cancer in some, but clearly not the majority, of cases. K-ras mutations have also been described in duct lesions associated with more uncommon forms of pancreatic neoplasia, including intraductal papillary neoplasms (44) and ductectatic-type muci- nous cystic adenomas and carcinomas (45).

The study of other genetic changes, namely the identification of mutations in p53 and MTS 1, will help define the molecular genetic profile of duct lesions that progress to invasive pancreatic cancer. Careful study of animal models might also provide some important insights--the Syrian golden hamster model in particular, since carcinogen-induced pan- creas cancer in this animal model is preceded by duct lesions identical to the ones described in humans, and these lesions have K-ras mutations (46).

Biology of r a s

Activating mutations ofras genes have been con- sidered dominant events, because they result in a gain of function (47). This has been confirmed by trans- fection experiments, or even more definitively by "knocking out" the mutant allele and showing a reversion of the malignant phenotype (48). Some

observations raise questions about the role of mutant ras. In at least one experimental setting, the genera- tion of a single mutant ras allele by two-step homolo- gous recombination produces transformation rarely, and all the transformed clones show amplification of the mutant allele (49). The rare amplification and/or overexpression of the mutant ras allele (25) and the loss of the wild-type allele (26) suggest that a similar finding might occur in some cancers. Loss of the wild-type allele in some pancreatic cancer cell lines with K-ras mutation (ASPC 1, Capan 1, MiaPaca) has been confirmed by sequencing and hybridization (Caldas C, Kern SE, unpublished results). A modifi- cation of the allele-specific ligation assay (50) was used to quantitate each K-ras allele in cell lines with mutant K-ras and intact wild-type allele. Using this assay in pancreatic carcinoma ATCC cell lines, no case with amplification of the mutant gene copy was seen. Quantification of RNA expression from both alleles showed no evidence ofoverexpression, and in two cell lines (Su8686 and Panc 1), there was under- expression of the mutant allele in comparison with the wild-type (Caldas C, Kern SE, unpublished results). Therefore, these observations, as well as the presence of mutations in nonmalignant duct lesions, are not conclusive in defining the precise role of activating K-ras mutations in pancreatic ductal carcinogenesis. It is conceivable that the K-ras mutation is important, but is best considered in the genetic (and epigenetic) context of the particular duct cell in which it occurs. When a genetic model for colorectal cancer was proposed and the multistep basis of tumorigenesis was highlighted, the accumu- lation of events, rather than order, was felt to be most important (51). The finding of frequent K-ras muta- tions in aberrant crypts (52), previously felt by many to be the earliest precursors of colon cancer, has called into question the model as originally proposed. It is now clear that those crypts that are most likely to progress into colon cancer likely have an APC muta- tion preceding the K-ras mutation (33). Lesions with K-ras mutation and normal APC appear to be unim- portant, in the sense that they do not show evidence of accumulation of other genetic changes or progres- sion to colon cancer. Similarly, in pancreatic cancer, the presence ofa K-ras mutation in a duct might be important only if a mutation in a pancreas "gate- keeper" gene has occurred first.

International Journal of Pancreatology Volume 18, 1995

4 Caldas and Kern

Animal models can also be used to study ras mutations in pancreatic ductal tumorigenesis. These models have inherent limitations, since they result from the overexpression ofras in transgenic animals (53), or from carcinogen exposure (46,54). Their advantage arises from the useful ability to manipu- late the context in which ras mutations exert their phenotypic effects.

K-ras as a Diagnostic and Therapeutic Target The high prevalence of K-ras mutations and the

ease of their detection have made this gene the pre- ferred candidate for gene-based diagnostic and thera- peutic approaches. In pancreatic cancer, this is particularly appealing since, as discussed above, nearly 90% of pancreatic cancers have mutation at codon 12 of K-ras, and 75% of these mutations are of only two types (12val and 12asp).

Detection of K-ras mutations in cytology speci- mens from fine-needle aspirates has been reported to be very sensitive and specific to pancreatic cancer, with 43 positives in 60 cases studied to date (55-57). In these preliminary studies, all 23 "controls" (pan- creatic mass caused by chronic pancreatitis and benign disease) were negative.

Detection of K-ras mutations in pancreatic juice obtained at ERCP from patients with pancreatic can- cer and intraductal pancreatic neoplasm has been reported (58,59). The use of this technique as a screening test would be impractical and expensive. The demonstration that K-ras mutations can be detected in the stool of patients with pancreatic can- cer and pancreatic ductal hyperplasia (60) raises the possibility of early detection and screening of pan- creatic cancer and precursor duct lesions. K-ras mutations have also been detected in the blood of patients with pancreatic cancer (58, 61).

Unfortunately, there are problems with the use of K-ras as a target for the detection of pancreatic can- cer cells in clinical specimens, because as discussed, K-ras mutations are present in preinvasive neoplas- tic lesions of unknown significance and these muta- tions can be detected in clinical specimens (60, 62). Until the significance of K-ras mutations found in clinical specimens is understood fully, the wide- spread clinical application of these tests will be premature (63).

The inhibition of ras farnesylation had been pro- posed as a therapeutic strategy in cancers with acti- vating mutations of ras. Synthetic inhibitors of farnesyltransferase were shown to decrease the growth of cells with ras mutations (64,65). More recently, these inhibitors have been used success- fully to treat pancreatic carcinoma xenografts implanted into nude mice (66). Phase I studies of these compounds in human pancreatic cancer will soon be a reality.

Conclusion

Pancreatic adenocarcinoma, the human tumor with the highest incidence of K-ras mutations, is one of the leading causes of cancer death and has a very poor prognosis. Over the past few years, a molecular genetic profile of pancreatic cancer has started to emerge: K-ras mutations in at least 90% of cases, MTS 1 alterations (somatic mutations and homozy- gous deletions) in 80%, p53 mutations in about 70%, as well as multiple sites ofallelic loss in cancer cells. The detection of K-ras mutations is done by using one of several PCR-based methods. A modified allele-specific ligation assay recently described appears particularly simple and reproducible. The possibility of pancreatic ductal lesions being the early precursors of pancreatic cancer has gained support from the finding of frequent K-ras mutations in these lesions. Although K-ras mutations are a defining event in pancreatic ductal carcinogenesis, it is unclear in which genetic context they occur. K-ras mutations as markers of cancer cells have been detected in clini- cal specimens from patients, including stool and blood, raising the possibility of early diagnosis. In addition K-ras can be a molecular target for thera- peutic intervention, as illustrated with the use of farnesylation inhibitors.

References 1 Warshaw AL, Castillo CFD. Pancreatic carcinoma. NEngl

JMed 1991; 326: 455-465. 2 Cameron JL, Crist DW, Sitzmann JV, Hruban RH, Boitnott

JK, Seidler A J, Coleman J. Factors influencing survival after pancreatoduodenectomy for pancreatic cancer. Ann Surg 1992; 161: 120-125.

3 Sidransky D, Tokino T, Hamilton SR, Kinzler KW, Levin B, Frost P, Vogelstein B. Identification of ras oncogene mutations in the stool of patients with curable colorectal tumors. Science 1992; 256: 102-105.

International Journal of Pancreatology Volume 18, 1995

K-ras and Pancreatic Adenocarcinoma 5

4 Almoguera C, Shibata D, Forrester K, Martin J, Arnheim N, Perucho M. Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes. Cell 1988; 53: 54%554.

5 Smit VTHBM, Boot AJM, Smits AMM, Fleuren G J, Comelisse CJ, Bos JL. K-ras codon 12 mutations occur very frequently in pancreatic adenocarcinomas. Nucleic Acids Res 1988; 16: 7773-7782.

6 Motojima K, Tsunoda T, Kanematsu T, Nagata Y, Urano T, Shiku H. Distinguishing pancreatic carcinoma from other periampullary carcinomas by analysis of mutations in the Kirsten-ras oncogene. Ann Surg 1991; 214: 657-662.

7 Grunewald K, Lyons J, Frohlich A, Feichtinger H, Weger RA, Schwab G, Janssen JWG, Bartram CR. High frequency of Ki-ras codon 12 mutations in pancreatic adenocar- cinomas, lnt J Cancer 1989; 43:1037-1041.

8 Hruban RH, van Mansfeld ADM, Offerhaus GJA, van Weering DHJ, Allison DC, Goodman SN, Kensler TW, Bose KK, Cameron JL, Bos JL. K-ras oncogene activation in adenocarcinoma of the human pancreas. A study of 82 carcinomas using a combination of mutant-enriched poly- merase chain reaction analysis and allele-specific oligo- nucleotide hybridization. A m JPathol 1993; 143: 545-554.

9 Seymour AB, Hruban RH, Redston MS, Caldas C, Powell SM, Kinzler KW, Yeo C J, Kern SE. Allelotype of pancreatic adenocarcinoma. Cancer Res 1994; 54:2761-2764.

10 Redston MS, Caldas C, Seymour AB, Hruban RH, da Costa L, Yeo C J, Kern SE. p53 mutations in pancreatic adeno- carcinoma and evidence of common involvement of homocopolymer tracts in DNA microdeletions. Cancer Res 1994; 54: 3025-3033.

11 Pellegata NS, Sessa F, Renault B, Bonato B, Leone BE, Solcia E, Ranzani GN. K-ras and p53 gene mutations in pancreatic cancers: ductal and nonductal tumors progress through different lesions. CancerRes 1994; 54:1556-1560.

12 Horii A, Nakatsuru S, Miyoshi Y, Ichii S, Nagase H, Ando H, Yanagisawa A, Tsuchiya E, Kato Y, Nakamura Y. Frequent somatic mutations of the APC gene in human pancreatic cancer. Cancer Res 1993; 52: 669645698.

13 Hohne MW, Halatseh ME, Kahl GF, Weinel RJ. Frequent loss of expression of the potential tumor suppressor gene DCC in ductal pancreatic adenocarcinoma. Cancer Res 1993; 52: 2616-2619.

14 Han H-J, Yanagisawa A, Kato Y, Park J-G, Nakamura Y. Genetic instability in pancreatic cancer and poorly differ- entiated type of gastric cancer. Cancer Res 1993; 53: 5087-5089.

15 Caldas C, Hahn SA, da Costa LT, Redston MS, Schutte M, Seymour AB, Weinstein CL, Hruban RH, Yeo C J, Kern SE. Frequent somatic mutations and homozygous deletions of the p 16 (MTS 1) gene in pancreatic adenocarcinoma. Nature Gen 1994; 8: 27-32.

16 Bos JL. The ras gene family and human carcinogenesis. MutatRes 1988; 195: 255-271.

17 Bos JL. ras oncogenes in human cancer: a review. Cancer Res 1989; 49: 4682-4689.

18 Smith SS. Species-specific differences in tumorigenesis and senescence. Trends Gen 1994; 10: 305,306.

19 Nagata Y, Abe M, Motoshima K, Nakayama E, Shiku H. Frequent glycine-to-aspartic acid mutations at codon 12 of

c-Ki-ras gene in human pancreatic cancer in Japanese. Jpn JCancerRes 1989; 81: 135-140.

20 Motojima K, Urano T, Nagata Y, Shiku H, Tsurifune T, Kanematsu T. Detection of point mutations in the Kirsten- ras oncogene provides evidence for the multicentricity of pancreatic carcinoma. Ann Surg 1993; 217:138-143.

21 Hirai H, Okabe T, Anraku Y, Fusiwava M, Urabe A, Takaku F. Activation of the c-K-ras oncogene in a human pancreas carcinoma. Bioch Biophys Res Commun 1985; 127:168-174.

22 Parada LF, Tabin C J, Shih C, Weinberg RA. Human EJ bladder carcinoma oncogene is homologue of Harvey sarcoma virus ras gene. Nature 1982; 297: 474-478.

23 Santos E, Tronick SR, Aaronson SA, Pulciani S, Barbacid M. T24 human bladder carcinoma oncogene is an activated form of the normal human homologue of BALB- and Harvey-MSV transforming genes. Nature 1982; 298: 337-341.

24 Der C J, Krontiris TG, Cooper GM. Transforming activity of human tumor DNAs. Proc Natl Acad Sci USA 1982; 79: 3637-3640.

25 Winter E, Yamamoto F, Almoguera C, Perucho M. A method to detect and characterize point mutations in transcribed genes: amplification and overexpression of the mutant c-Ki-ras allele in human tumor cells. Proc NatlAcad Sci USA 1985; 82: 7575-7579.

26 Bos JL, Verlaan-de Vries M, Jansen AM, Veeneman GH, van Boom JH, Van der Erb AJ. Three different mutations in codon 61 of the human N-ras gene detected by synthetic oligonucleotide hybridization. Nucleic Acids Res 1984; 12: 9155-9163.

27 Gonzalez-Cadavid NF, Zhou D, Battifora H, Bar-Eli M, Cline MJ. Direct sequencing analysis of exon 1 of the c-K-ras gene shows a low frequency of mutations in human pancreatic adenocarcinomas. Oncogene 1989; 4: 1137-1140.

28 Verlaan-de-Vries M, Bogaard ME, van den Elst H, van Boom JH, van der Eb A J, Bos JL. A dot-blot screening procedure for mutated ras oncogenes using synthetic oligodesoxynucleotides. Gene 1986; 50:313-320.

29 Stork P, Loda M, Bosari S, Wiley B, Poppenhusen K, Wolfe H. Detection of K-ras mutations in pancreatic and hepatic neoplasms by non-isotopic mismatched polymerase chain reaction. Oncogene 1991; 6: 857-862.

30 Kahn SM, Jiang W, Culbertson TA, Weinstein IB, Williams GM, Tomita N, Ronai Z. Rapid and sensitive non-radio- active detection of mutant K-ras genes via "enriched" PCR amplification. Oncogene 1991; 6:107%1083.

31 Levi S, Urbano-Ispizua A, Gill R, Thomas DM, Gilbertson J, Foster C, Marshall CJ. Multiple K-ras codon 12 mutations in cholangiocarcinomas demonstrated with a sensitive polymerase chain reaction technique. CancerRes 1991 ; 51 : 3497-3502.

32 van Mansfeld ADM, Bos JL. PCR-based approaches for detection of mutated ras genes. PCR Methods Applications 1992; 1: 211-216.

33 Jen J, Powell SM, Papadopoulos N, Smith KJ, Hamilton SR, Vogelstein B, Kinzler KW. Molecular determinants of dysplasia in colorectal lesions. Cancer Res 1994; 54: 5523-5526.

International Journal o f Pancreatology Volume 18, 1995

6 Caldas and Kern

34 Mariyama M, Kishi K, Nakamura K, Obata H, Nishimura S. Jap frequency and types of point mutation at the 12th codon of the c-Ki-ras gene found in pancreatic cancers from Japanese patients. J Cancer Res 1989; 80: 622-626.

35 Cubilla AL, Fitzgerald PJ. Morphological lesions associated with human primary invasive nonendocrine pancreas cancer. Cancer Res 1976; 36: 2690-2698.

36 Kozuka S, Sassa R, Taki T, Masamoto K, Nagasawa S, Saga S, Hasegawa K, Takeuchi M. Relation of pancreatic duct hyperplasia to carcinoma. Cancer 1979; 43: 1418-1428.

37 Sommers SC, Murphy SA, Warren S. Pancreatic duct hyperplasia and cancer. Gastroenterology 1954; 27: 629--640.

38 Kloppel G, Bommer G, Ruckert K, Seifert G. Intraductal proliferation in the pancreas and its relationship to human and experimental carcinogenesis. Virchows Arch A Pathol Anat Histol 1980; 387: 221-233.

39 Yanagisawa A, Ohtake K, Ohashi K, Hori M, Kitagawa T, Sugano H, Kato Y. Frequent Ki-ras oncogene activation in mucous cell hyperplasias of pancreas suffering from chronic inflammation. Cancer Res 1993; 53: 953-956.

40 Nowell PC. The clonal evolution of tumor cell populations. Science 1976; 194: 23-28.

41 Kern SE. Clonality: more than just a tumor-progression model. J Natl Cancer lnst 1993; 85:1020,1021.

42 CaldasC, Kern SE, Hruban RH. K-rasmutationsinpancreatic ductal cell hyperplasias. Ann Oncol 1994; 5(8): 75 (abst).

43 DiGiuseppe JA, Hruban RH, Offerhaus GJA, Clement M J, van der Berg FM, Cameron JL, van Mansfeld ADM. Detection of K-ras mutations in mucinous pancreatic duct hyperplasia from a patient with a family history of pan- creatic carcinoma. Am JPathol 1994; 144: 889-895.

44 Tada M, Omata M, Ohto M. ras gene mutations in intra- ductal papillary neoplasms of the pancreas. Cancer 1990; 67: 634-637.

45 Yanagisawa A, Kato Y, Ohtake K, Kitagawa T, Ohashi K, Hori M, Takagi K, Sugano H. c-Ki-ras point mutations in ductectatic-type mucinous cystic neoplasms of the pan- creas. Jpn J Cancer Res 1991; 82: 1057-1060.

46 Cerny WL, Mangold KA, Scarpelli DG. K-ras mutation is an early event in pancreatic duct carcinogenesis in the Syrian golden hamster. Cancer Res 1992; 52: 4507-4513.

47 Reddy EP, Reynolds RK, Santos E, Barbacid M. A point mutation is responsible for the acquisition of transforming properties by the T24 bladder carcinoma oncogene. Nature 1982; 300: 149-152.

48 Shirasawa S, Furuse M, Yokoyama N, Sasazuki T. Altered growth of human colon cancer cell lines disrupted at activated K-ras. Science 1993; 260: 85--87.

49 Finney RE, Bishop JM. Predisposition for neoplastic transformation caused by gene replacement of H-ras 1. Science 1993; 260: 1524-1527.

50 Levi DB, Smith KJ, Beazer-Barclay Y, Hamilton SR, Vogelstein B, Kinzler KW. Inactivation of both APC alleles in human and mouse tumors. Cancer Res 1994; 54: 5953-5958.

51 Fearon E, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell 1990; 61: 759-767.

52 Pretlow TP, Brasitus TA, Fulton C, Cheyer C, Kaplan EL. K-ras mutations in putative preneoplastic lesions in human colon. J Natl Cancer lnst 1993; 85: 2004-2007.

53 Quaife C J, Pinkert CA, Omitz DM, Palmiter RD, Brinster RL. Pancreatic neoplasia induced by ras expression in acinar cells oftransgenic mice. Cell 1989; 48: 1023-1034.

54 Fujii H, Egami H, Chancy W, Pour P, Pelling J. Pancreatic adenocarcinomas induced in Syrian hamsters by N-nitro- sobis(2-oxopropyl)amine contain a c-Ki-ras oncogene with a point-mutated codon 12. Mol Carcinog 1990; 3:296-301.

55 Shibata D, Almoguera C, Forrester K, Dunitz J, Martin SE, Cosgrove MM, Perucho M, Amheim N. Detection ofc-K-ras mutations in fine needle aspirates from human pancreatic adenocarcinomas. Cancer Res 1990; 50: 1279-1283.

56 Tada M, Omata M, Ohto M. Clinical applications of ras gene mutation for diagnosis of pancreatic adenocarcinoma. Gastroenterology 1991; 100: 233-238.

57 Urban T, Ricci S, Grange J-D, Lacave R, Boudghene F, Breittmayer F, Languille O, Roland J, Bernaudin J-F. Detection ofc-Ki-ras mutation by PCR/RFLP analysis and diagnosis of pancreatic adenocarcinomas. J Natl Cancer Inst 1993; 85: 2008-2012.

58 Tada M, Omata M, Kawai S, Saisho H, Ohto M, Saiki RK, Sninsky JJ. Detection of ras gene mutations in pancreatic juice and peripheral blood of patients with pancreatic adenocarcinoma. Cancer Res 1993; 53: 2472-2474.

59 Watanabe H, Sawabu N, Ohta H, Satomura Y, Yamakawa O, Motoo Y, Okai T, Takahashi H, Wakabayashi T. Identification of K-ras oncogene mutations in the pure pancreatic juice of patients with ductal pancreatic cancers. Jpn J Cancer Res 1993; 84: 961-965.

60 Caldas C, Hahn SA, Hruban RH, Redston MS, Yeo C J, Kern SE. Detection of K-ras mutations in the stool of patients with pancreatic adenocarcinoma and pancreatic ductal hyperplasia. Cancer Res 1994; 54: 3568--3573.

61 Sorenson GD, Pribish DM, Valone FH, Memoli VA, Bzik D J, Yao S-L. Soluble normal and mutated DNA sequences from single-copy genes in human blood. Cancer Epidemiol Biomarkers Prev 1994; 3: 67-71.

62 Suzuki H, Yoshida S, Ichikawa Y, Yokota H, Mutoh H, Koyama A, Fukazawa M, Todokori T, Fukao K, Uchida K, Miwa M. Ki-ras mutations in pancreatic secretions and aspirates from two patients without pancreatic cancer. J Natl Cancer Inst 1994; 86: 1547-1549.

63 Fearon ER. K-ras gene mutation as a pathogenetic and diagnostic marker in human cancer. JNatl Cancer Inst 1993; 85: 1978-1980.

64 Kohl NE, Mosser SD, deSolms S J, Giuliani EA, Pompliano DL, Graham SL, Smith RL, Scolnick EM, OliffA, Gibbs JB. Selective inhibition of ras-dependent transformation by a famesyltransferase inhibitor. Science 1993; 260:1934-1937.

65 James GL, Goldstein JL, Brown MS, Rawson TE, Somers TC, McDowell RS, Crowley CW, Lucas BK, Levinson AD, Marsters JC. Benzodiazepine peptidomimetics: potent inhibitors ofras famesylation in animal cells. Science 1993; 260: 1937-1942.

66 Kohl NE, Wilson FR, Mosser SD, Giuliani E, deSolms S J, Conner MW, Anthony N J, Holtz W J, Gomez RP, Lee T-J, Smith RL, Graham SL, Hartman GD, Gibbs JB, Oliff A. Protein famesylation inhibitors block the growth of ras- dependent tumors in nude mice. Proe Natl Acad Sei USA 1994; 91: 9141-9145.

International Journal o f Pancreatology Volume 18, 1995