Molecular Genetic Basis of PancreaticAdenocarcinoma

Werner Hilgers and Scott E. Kern*

Departments of Oncology and Pathology, Johns Hopkins Medical Institutions, Baltimore, Maryland

Pancreatic ductal adenocarcinoma is a complex genetic disease. As might be expected for a malignancy that is ratherhomogeneous in clinical presentation and behavior, a distinct subset of genes are found to be genetically inactivated in a majorityof the tumors. A yet larger subset of genes experiences genetic inactivation at much lower frequencies. The latter subset couldsolely reflect a somewhat trivial genetic heterogeneity of the tumor, but more likely will represent the initial insights intopathways whose more widespread importance will be shown in future work. Familial pancreatic cancer susceptibility underlies asignificant fraction of the overall incidence. Genetic testing is feasible for many of the causative genes, although the clinical utilityremains unsettled. The precursor lesion for pancreatic cancer shares some of the genetic lesions of the more advanced invasivestage, and follows a stepwise progression model both histologically and genetically. Genes Chromosomes Cancer 26:1–12,1999. r 1999 Wiley-Liss, Inc.

INTRODUCTION

Pancreatic cancer is among the best-describedgenetic diseases. The accumulation of past andrecent discoveries now offers an opportune time forconsideration of some broader issues, such as thelikely direction and difficulties of future research.The study of pancreatic cancer has also had generalutility in the understanding of the biology ofeukaryotic regulatory pathways and of other humanneoplasms, many of which are less completelydescribed in genetic terms. Clinically, pancreaticcancer is the fifth leading cause of cancer death inthe United States and is remarkably homogeneous,presenting late in the course of the disease andusually fatal within a year or so. Pathologically, mostcases are moderately differentiated with abortiveduct formation and an intense desmoplastic reac-tion. While unfortunately still of lagging interest inclinical circles, pancreatic cancer is of mainstreaminterest as a scientific model of a common cancertype in adults.

PREINVASIVE NEOPLASIA

Pancreatic ductal epithelium constitutes 5% orless of the total cellular population of the pancreas,yet is the origin of the common form of pancreaticcarcinoma (Cubilla and Fitzgerald 1976; Kozuka etal., 1979; Pour et al., 1982; Furukawa et al., 1994;Brat et al., 1998; Hruban et al., 1998). It is a lowcuboidal epithelium with basal nuclei. In the earli-est form of neoplasia, which is unfortunately buthistorically termed a ‘‘flat hyperplasia,’’ the epithe-lium becomes columnar. Half of these lesions,when microdissected, prove to contain K-ras muta-

tions. A more advanced stage, ‘‘papillary hyperpla-sia,’’ is distinguished by a crowded mucosa thatassumes a folded architecture. (This is reasonablyreminiscent of the colorectal adenoma, which be-comes so folded that it acquires its own inappropri-ate name, ‘‘villous adenoma.’’ In most cases thisappearance is an artifact of a cerebriform, infoldedsurface pattern that is viewed in cross-section.)Papillary hyperplasia can exhibit varying degrees ofcellular and nuclear atypia, or even an architectur-ally disordered amassing of cells sometimes termed(again, unfortunately) as ‘‘carcinoma in situ.’’ Thevast majority of papillary hyperplasias contain amutation of the K-ras (KRAS2) gene (Caldas et al.,1994b). These lesions become a bona fide carci-noma with the development of invasion throughthe wall of the duct. The carcinoma stage almostalways engenders a dense collagenous and inflam-matory reaction termed ‘‘desmoplasia’’ in the in-vaded tissue. Because of the desmoplastic re-sponse, the cancer cells, on average, representabout 25% of the cells of the tumor, and often manyfewer (Seymour et al., 1994). The genetic analysisof the primary tumor is therefore an analysis largelyof normal cells. Most genetic analyses of pancreaticcancer therefore must first enrich for the neoplasticcomponent, by microdissection or by establishment

Supported by: NIH SPORE (Specialized Program of ResearchExcellence) in Gastrointestinal Cancer; Grant number: CA62924;Deutsche Krebshilfe (W.H.).

*Correspondence to: Scott E. Kern, 632 Ross Building, JohnsHopkins University School of Medicine, Baltimore, MD 21205.E-mail: sk@welchlink.welch.jhu.edu

Received 30 December 1998; Accepted 17 March 1999

GENES, CHROMOSOMES & CANCER 26:1–12 (1999)

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r 1999 Wiley-Liss, Inc.

of cell lines and xenografts. Analysis of the earlier-stage intraductal neoplasms, of course, requiresmicrodissection or in situ labeling techniques.

Because K-ras mutations are not required for theearly flat stage but have a high prevalence in thepapillary stage, it appears that K-ras mutations mayprovide a growth advantage for the middle stages ofthis neoplastic progression, but do not serve a truegatekeeper role. Similarly, alterations of p16 geneticstructure or a loss of expression tend to representonly a subset of lesions, probably those that mighthave higher biologic potential for progression tocancer (Moskaluk et al., 1997; Wilentz et al., 1998).BRCA2 may serve such a late role as well; inpatients with a germline BRCA2 mutation, thepancreas does not harbor an obviously increasednumber of intraductal neoplasms, and the loss ofthe remaining wild-type BRCA2 allele appears toarise well after the development of the hyperplasia(Goggins et al., 1997b). This is perhaps an insight toexplain the observation that in pancreas cancerfamilies, cancer does not arrive at an acceleratedage. If the wild-type allele is lost at later stages ofneoplasia, it represents not a key change but analternate change, i.e., just another in a series ofrate-limiting steps. These genetic lesions them-selves might be in part age-dependent. Theseparticulars would mask the tie that would otherwiselink the germline mutations with a younger age ofdisease onset, a connection seen so reproduciblywith both neoplastic and nonneoplastic diseases ofother organs.

Estimates have been made that as many as half ofthe normal elderly population develop flat hyperpla-sias of pancreatic ducts, whereas less than 10% ofpersons have lesions with significant cellular atypia(Cubilla and Fitzgerald 1976; Kozuka et al., 1979).Considering the low chance that each lesion has todevelop into a cancer (probably near 1 in 500lesions will so progress) and the potentially highrisk of cancer from episodic activation of the pancre-atic enzymes carried by these ducts (as seen inhereditary pancreatitis), humans have truly evolveda remarkable ability to thwart the malignant careerpotential of this neoplastic farm club. It is with thisas a backdrop that one should regard the geneticpatterns seen in pancreatic cancer.

MULTIPLE GENETIC LESIONS

In terms of known mutations per tumor, pancre-atic cancers harbor more than any other tumor type.More than 40% of the chromosomal arms suffer lossof heterozygosity (Hahn et al., 1995). Some tumorshave mutations in five or more genes (Rozenblum

et al., 1997). The changes in pancreatic cancer areoften mirrored in the related biliary carcinomas,although generally at somewhat lower frequenciesin the latter. The genes involved can be divided intwo groups on the basis of genetic alterations intheir signaling/regulatory pathways: those of highfrequency and those of low (Table 1).

High-Frequency Changes

Oncogenes.

Mutations of the K-ras gene are present in over90% of pancreatic cancers (Almoguerra et al., 1988).This is the highest fraction of K-ras alteration foundin any human tumor type.

It is interesting that the K-ras genes of pancreaticcancer do not share the same mutational spectrumseen for this gene in colorectal cancer, the codon 12serine allele being vanishingly rare in the pancreasbut common in the colorectum (Forrester et al.,1987; Vogelstein et al., 1988). This perhaps reflectsan undetermined but surprisingly specific muta-genic influence. On occasion, two differing K-rasmutations are identified in a pancreatic cancer.Most often, this probably represents a focal artifactproduced by a cancer with one type of mutation,invading and obliterating a precursor duct lesionhaving a different mutation. Rarely, however, itdoes appear that two K-ras mutations can coexist ina single cancer clone.

TABLE 1. Genetic Profile of Pancreatic Carcinomaa

GeneGene

locationsFrequency

(%)Mutation

origin

OncogenesK-ras 12p12 95 Som.AKT2 19q13.1 10–20 Som.MYB 6q24 10 Som.

Tumor Suppressorsp16/RB1 9p21/13q14 .90 Som. . germ.p53 17p31 75 Som.DPC4 18q21 55 Som.BRCA2 13q13 5–10 Germ. . som.MKK4 17p13 4 Som.LKB1/STK11 19p13 5 Som. . germ.ALK5 9q21 1 Som.b

RER2/TGFBR2 /3p22 1 Som.b

RER1/TGFBR2 /3p22 3 Som. . germ.c

aReferences are given in the text. Som., (rate of) somatic mutation ormethylation; germ., (rate of) germline mutation.bSingle examples of homozygous deletion of the ALK5 gene and of theTGFBR2 gene have been identified in pancreatic cancer. In colorectalcancer, a single family having a germline mutation of the TGFBR2 gene isreported (Lu et al., 1998).cIn RER1 tumors, the mismatch repair defect is usually somatic in origin;the TGFBR2 alterations are somatic.

2 HILGERS AND KERN

Unlike the seldom-mutated H-ras protein, theactivity of the K-ras protein is not solely dependenton farnesylation (James et al., 1995; Lerner et al.,1995). Consequently, the development of farnesyltransferase inhibitor drugs is of uncertain promisein the treatment K-ras mutant tumors. Many compo-nents of signal transduction that lie downstream ofthe ras genes have been discerned in artificialsystems, but no measurable downstream biologicalactivity is yet known that is reliably increasedamong K-ras mutant cells from human tumors.Finally, K-ras mutations are not specific for malig-nancy, being present in a significant number ofbenign human neoplasms and early clonal lesions(Vogelstein et al., 1988; Pretlow et al., 1993; Tada etal., 1993; Yanagisawa et al., 1993; Caldas et al.,1994b; Tada et al., 1996). This unfortunate factdampens (although does not eliminate) enthusiasmfor the application of K-ras mutation detection inscreening programs that will attempt to achieveearly cancer diagnosis. It has been fashionable towonder whether, in the K-ras wild-type cancers,another gene of the same pathway might have acomplementary oncogenic mutation. Yet, the oftendistinctive clinicopathological features of the K-raswild-type cancers suggests instead that they mightshare little in common with the K-ras-mutant tu-mors to hold such an expectation (Goggins et al.,1998a).

Tumor suppressor genes

The p16 (INK4a, CDKN2, MTS1), p53 (TP53), andDPC4 (MADH4, SMAD4) genes are geneticallyinactivated in half or more of pancreatic cancers(Caldas et al., 1994a; Pellegata et al., 1994; Redstonet al., 1994; Hahn et al., 1996b; Schutte et al., 1996;Rozenblum et al., 1997). The tumors with andwithout these mutations are overwhelmingly simi-lar in clinical and pathological features (Rozenblumet al., 1997). This high frequency implies that formost pancreatic neoplasms, in their earlier benignstages, the presence of these genes essentiallyprevents the progression to an invasive stage. If thisholds for most precursors, might it not hold for all?That is, are there genes in these pathways that arealtered in the remaining cancers, so that essentiallyall pancreatic cancers embody inactivation of thesethree pathways? And might this genetic homogene-ity underlie the clinical and pathological homogene-ity of the disease? These are extremely influentialviewpoints and have served to guide the identifica-tion of candidate genes for mutational studies.

For the p16 gene, this expectation is almostentirely borne out. p16 is an inhibitor of the

cyclinD-Cdk4 and cyclinD-Cdk6 mitotic kinasecomplexes that downregulate the activity of the cellcycle inhibitor protein Rb (Serrano et al., 1993).Within this pathway, therefore, mutational activa-tion of any one of the cyclin D, CDK4, and CDK6oncogenes appears to produce a disregulation ofcell division cycle control that is similar to that seenwith mutational inactivation of the p16 or RB1tumor suppressor genes. Tumors such as lungcancer and melanoma almost uniformly select forclones having alterations of any one of these genes,implicitly to remove this regulatory system (Shapiroet al., 1995; Bartkova et al., 1996; Kinoshita et al.,1996; Kratzke et al., 1996; Zuo et al., 1996). Whilepancreatic cancer can sustain mutations of RB1 onoccasion (Ruggeri et al., 1992), it overwhelminglytargets the p16 gene for inactivation, found in over95% of the cancers (Caldas et al., 1994a; Schutte etal., 1997). Germline mutations of p16 predispose topancreatic cancer (Whelan et al., 1995; Ciotti et al.,1996). In sporadic tumors, homozygous deletionsand intragenic mutations (missense, nonsense, andframeshift) of p16 are nearly identical in frequency,accounting for nearly 85% of cases (Caldas et al.,1994a). In most of the remaining cases transcriptionof the p16 gene is turned off in association withmethylation of its promoter (Schutte et al., 1997).Mutations of the CDK4 gene have not been identi-fied (Schutte et al., 1997). Cyclin D amplificationwas observed at a high rate in one study, but not inanother (Schutte et al., 1997).

p53 gene mutations are seen in 50%–75% of cases(Pellegata et al., 1994; Redston et al., 1994; Rozen-blum et al., 1997). Missense mutations vastly out-number the nonsense and frameshift mutations,and homozygous deletions are not described inpancreatic and in most other tumor systems. Theupstream controls and downstream actions of p53appear to be highly complex. The expression of p53is stimulated by a wide variety of cellular stresses.Mdm2 is a major downregulator of p53 that consti-tutes a negative feedback loop for controlling p53activity (Momand et al., 1992; Oliner et al., 1993;Chen et al., 1994) and serves as an oncogene whenactivated by amplification (usually in p53 wild-typetumors of mesodermal origin; Oliner et al., 1992;Leach et al., 1993). There are a large number ofgenes whose transcription is controlled by p53,including an arm effecting cell cycle control (thep21WAF1/CIP1 gene; El-Deiry et al., 1993), a separateapoptotic arm (Yonish-Rouach et al., 1991; Polyaket al., 1997), and many other genes whose tumor-related roles remain yet undefined (Polyak et al.,1997). Perhaps because of this complexity, it has

3MOLECULAR GENETIC BASIS

remained unclear whether there is a definable p53pathway such that mutations upstream or down-stream of the MDM2/p53 axis would be observed inthose carcinomas that retain a wild-type p53 gene.

p53 mutations may represent a special case intumor genetics. The ability of p53 to bind DNA atspecific sequences is lost in essentially all mutantsthat are found in tumors, indicating that DNAbinding is central to its tumor suppressive role. Theability of mutant proteins to complex with andinhibit the DNA binding of wild-type p53 isthought to produce an effective impairment of theprotein that is expressed from the remaining wild-type allele (Kern et al., 1992). Consequently, thecompetitive survival of clones having sustained amutation of p53 is aided by the dominant negativeaction of the mutant p53 proteins. The specificityof this mechanism may account for the paucity ofnonsense, frameshift, and homozygous deletions inthe p53 gene, since the development of the firstnull allele would then not allow the formation ofinhibitory complexes and an immediate growthadvantage. The strength of the p53 selection sys-tem (i.e., that biallelic genetic inactivation is notrequired to exert a growth advantage) is not gener-ally considered to be common among tumor suppres-sor genes, and selective pressures for other genes ofan extended p53 suppressive pathway cannot beexpected to match those for p53. Thus, for manyreasons, it may prove difficult technically to demon-strate a universal involvement of the p53 pathwayin pancreatic cancer, although such an effort wouldbe of high impact if successful.

The tumor suppressor DPC4 was cloned uponthe mapping of a hotspot of homozygous deletionsin pancreatic and biliary cancer (Hahn et al., 1996b).It was the fourth marker to be investigated forhomozygous deletions in pancreatic cancer (DPC;Hahn et al., 1996a). DPC4 is inactivated in about55% of pancreatic cancers, being homozygouslydeleted in nearly 35% and suffering intragenicmutations in another 20% (Schutte et al., 1996).Germline mutations of DPC4 cause not pancreaticcancer but a number of nonneoplastic erosive andinflammatory lesions of the intestinal mucosa thatbecome polypoid with time, termed juvenile polypo-sis (Howe et al., 1998). As with any chronic inflam-matory lesions of the bowel, these polyps canstimulate the development of dysplasia and cancer(Giardiello et al., 1991). Here, a direct role for DPC4(due to its mutation) in this progression is sug-gested in studies of accelerated progression of

colorectal neoplasia in transgenic mice mutant forboth the APC and DPC4 genes (Takaku et al., 1998).

DPC4 is a member of the Smad family of genesrelated to the Drosophila Mad and C. elegans Smagenes (Sekelsky et al., 1995; Hahn et al., 1996b;Savage et al., 1996). It is essential for mammaliandevelopment (Sirard et al., 1998). Smad proteinsmediate signals initiated by the binding of TGFb-like cytokines to their receptors (Sekelsky et al.,1995; Savage et al., 1996). Dpc4 protein is cytoplas-mic, redistributes to the nucleus upon receptoractivation (Liu et al., 1997; Zhang et al., 1997), andbinds to a short specific sequence of DNA termedthe Smad-binding element (SBE; Zawel et al.,1998). These functions probably involve the homo-oligomerization of Dpc4 as well as formation offunctional heteromeric complexes with other Smadproteins and with more general transcription factors(Liu et al., 1996; Zhang et al., 1996; Shi et al., 1997).The tumorigenic mutations fall in three majorcategories. The missense mutations of the N-terminal third of the protein abrogate its DNA-binding function, the truncating mutations of theC-terminus remove its ability to activate geneexpression while preserving its DNA-binding prop-erty, and the missense mutations of the C-terminalregion appear to interfere with its nuclear localiza-tion in response to ligand-initiated signaling (Dai etal., 1998). The ability of Dpc4 to act as a transcrip-tional activator at SBE sites is uniformly lost in allmutations found in tumors, strongly suggesting thatit is this property which provides its tumor suppres-sive role (Dai et al., 1998). When Dpc4 is artificiallydirected to the nucleus, it arrests cells in G1 phaseof the cell cycle and induces apoptosis (Dai et al.,1999).

Dpc4 is suspected to act in the TGFb suppres-sive pathway; in some cells Dpc4 has been conclu-sively proven to be required for important TGFb

effects (Zhou et al., 1998). Nonetheless, the geneticinactivation of both DPC4 and a TGFb receptorgene has been observed to coexist in the sametumors (Goggins et al., 1998b). This suggests thatDPC4 has functions that are sufficiently distinctfrom TGFb to require its separate inactivation—one should not simplistically consider them torepresent the same pathway. If the inactivation ofthe DPC4 pathway were indeed to be universallyrequired in pancreatic carcinogenesis, we do not yetknow enough of the upstream and downstreamcomponents to identify the most promising candi-date genes for mutational searches.

4 HILGERS AND KERN

Low-Frequency Changes

Oncogenes

Gene amplification is seen at multiple sites.Chromosomal band 19q13 is the most commontarget, being amplified in 10%–20% of the cancers.This hotspot of amplification was first found in ascreen for overexpressed genes (Batra et al., 1991a,1991b). Many years later it was confirmed by acandidate gene approach and separately also by anunbiased genome landmark scanning method(Cheng et al., 1996; Miwa et al., 1996).

When speaking of gene amplification, whichaffects large genomic regions, it is often difficult tospecify with certainty the target gene, and indeedmultiple genes may together provide the survivaladvantage that allows selection for clones that carrythe amplification. A number of genes are known tobe amplified at 19q13, but the signal transducerAKT2 is the leading candidate target gene in thisregion (Cheng et al., 1996).

Amplifications of the MYB gene has also beenobserved in as many as 10% of cases in systematic(positional mapping) studies (Wallrapp et al., 1997).Reports also implicate the amplification of othergenes and chromosomal regions (Yamada et al.,1986; Solinas-Toldo et al., 1996; Ghadimi et al.,1999).

Tumor suppressor and DNA repair genes

There is a considerable and growing number ofgenes known to be inactivated at frequencies of4%–10% of tumors. Some of these clearly fall underthe high-frequency category considered above, onthe basis of pathway memberships. The remainderare in some ways even more exciting, because theyoften represent novel and unexplored regulatorysystems, and also simply because of the sheernumber of such mutant genes. They illustratevividly that pancreatic cancer is not a four-chordsong, but a symphony of large and small parts.

There is a technical point that needs to be made,one of extreme importance. These low-frequencygenetic targets are not random. Genes chosen atrandom have a somatic mutation rate expected atless than 1% of tumors examined (with appropriateexceptions made for tumors having defects in DNAsynthetic fidelity). There are also simple statisticalmeans to discriminate between random mutationsand those patterns that imply the action of selectivepressures during tumorigenesis. Statistical ap-proaches are feasible because random mutationsindeed do occur in dividing cell populations, andthese random mutations have a known pattern. For

example, one could imagine a gene whose muta-tional spectrum included many silent changes (suchas third-base nucleotide substitutions that do notchange the amino acid or other substitutions thatproduce a conservative amino acid change). Onewould be justified to assume that differing degreesof selective pressures had not acted on the sub-clones that sustained these initial random muta-tions. On the other hand, if a mutational spectrumnearly exclusively comprised a skewed spectrum ofnonsense mutations and other inactivating muta-tions, the conclusion becomes the opposite. Itwould then be clear that, of the initial randommutations that are expected to occur in any growingcell population, only those that removed the geneactivity had been able to provide the cells with aselective growth advantage. In other words, a spec-trum statistically enriched in inactivating mutationsclearly indicates a tumor suppressor gene. As in allstatistics, it is a matter of N, the number of samplesstudied. Lower mutational frequencies require ahigher N, but in theory present no special difficul-ties.

Intragenic mutations and homozygous deletionsof the MKK4 (MAP kinase kinase 4) gene are seenin about 4% of pancreatic cancers (Teng et al., 1997;Su et al., 1998). MKK4 is a component of a stress-activated protein kinase cascade and has roles inapoptosis and growth control (Derijard et al., 1995;Lin et al., 1995). MKK4 was localized to a homozy-gous deletion site that was first identified in apancreatic cancer, but is inactivated in other cancertypes as well (Teng et al., 1997). The geneticinactivation of MKK4 can be found to coexist withmutations of p16, p53, and DPC4, and so is thoughtto serve a distinct pathway of tumor suppression(Su et al., 1998).

A somatic homozygous deletion was identified ina pancreatic cancer by an unbiased genomic dele-tion cloning method, the representational differ-ence analysis (Schutte et al., 1995a, 1995b). Thisencompassed the BRCA2 gene, and the map of thisdeletion (markers that included coding sequencesof the BRCA2 gene) was instrumental in the posi-tional cloning of the gene (Wooster et al., 1994).Somatic mutations, however, appear rare. About 5%of unselected pancreatic cancers, and about 10% ofthose from Ashkenazi Jewish patients, have germ-line mutations (Goggins et al., 1996; Ozcelik et al.,1997). This is remarkable in that few of thesepatients had a family history of breast cancer andnone had a relative with pancreatic cancer. Thus,among pancreatic cancers that appear clinically to

5MOLECULAR GENETIC BASIS

be sporadic, one can observe an inherited basis. Ifinherited mutations can indeed give rise to ‘‘spo-radic’’ cancer, one must wonder to what extent thecontribution of heritable cancer susceptibility tendsto be overlooked. This brings up the speculativebut undeniable possibility that inherited mutationsof low penetrance might account for much or evenmost of the cancers in the general population.Whatever the wider epidemiologic and theoreticimplications, the precise role of BRCA2 in pancre-atic and other cancers remains largely unknown.The gene is not yet firmly established in either agrowth regulatory or a DNA repair function inhuman tumors, although both are suggested fromexperimental systems.

The Peutz-Jeghers syndrome (PJS) is associatedwith an increased risk of cancer, including pancre-atic (Giardiello et al., 1987). Inherited inactivatingmutations of the LKB1/STK11 gene cause thesyndrome (Hemminki et al., 1998; Jenne et al.,1998). In a pancreatic cancer of a patient havingPJS, loss of the remaining wild-type allele wasobserved, suggestive that the LKB1/STK11 geneacts as a typical tumor suppressor gene in neo-plasms (remember that Peutz-Jeghers polyps arenot neoplastic, but classified as hamartomatous; Suet al., 1999). In pancreatic cancers that are notassociated with the syndrome, somatic mutationsaccompanied by of the LKB1/STK11 gene are seenin about 5% (Su et al., 1999). The gene encodes aserine/threonine kinase, for which the componentsof the signaling pathway and its cellular role arecurrently undefined (Hemminki et al., 1998; Jenneet al., 1998).

The replication error (RER1) phenotype, signify-ing a defect in DNA mismatch repair function, isfound in about 3% of pancreatic cancers (Goggins etal., 1998a). This is an extremely distinctive subsetof pancreatic cancer. Very few tumors have beendescribed, but they are characterized by a sheet-like growth pattern with pushing borders, cells thatare almost syncytial without differentiation, and awild-type K-ras gene. Long-term survivors havebeen observed in this subgroup. In the one tumorcharacterized in detail, p16, p53, and DPC4 genealterations were not found. However, as with theRER1 cancers of the colorectum, alterations ofsimple repeated sequences, including those of thepolyA tract (a mononucleotide run) within thecoding sequence of the TGFBR2 (TGFb type IIreceptor) gene, are widespread (Goggins et al.,1998b). The precise genetic lesions that cause themismatch repair defect in pancreatic cancer are notyet described. Pancreatic cancers are reported in

families with inherited mutations in the knowngenes of this repair system, and some patients havepancreatic cancer combined with multiple colorec-tal cancers, thus showing a connection in somecases with hereditary nonpolyposis colorectal can-cer (Lynch et al., 1985). Some of the repair defectstherefore are undoubtedly on an inherited basis,while others are of somatic origin.

Except for in the RER1 cancers where biallelicinactivation of the TGFBR2 gene is seen, alter-ations in the TGFb receptors are quite rare (Gog-gins et al., 1998b). A single homozygous deletion ofthe TGFBR2 gene is known in a pancreatic cancer.A homozygous deletion of the ALK5 (TGFb type Ireceptor) gene in one pancreatic and one biliarycancer constitutes the only known somatic geneticinactivation of this gene in human cancer. Intra-genic mutations of ALK5 are not identified, and onecould indeed raise questions that challenge theevidence to support ALK5 as a bona-fide tumorsuppressor gene. Homozygous deletions and muta-tions of other related (TGFb superfamily-binding,serine/threonine kinase) receptors, however, havenot been found in pancreatic cancer. Also, defectsin TGFb receptor expression and function arecommon in many human cancers including pancre-atic (Baldwin et al., 1996), and the TGFb receptorsare widely considered to be tumor suppressors.

Abnormalities of one of the chromosome fragilesites (FRA3B) are seen in pancreatic cancers(Shridhar et al., 1996; Simon et al., 1998). Thisregion of chromosomal band 3p14.2 is also the siteof the diadenosine hydrolase gene FHIT, which inmultiple tumors types is affected by occasion homo-zygous deletions (Kastury et al., 1996). Such dele-tions of the FHIT gene were found in about 6% ofpancreatic cancers. In tumors that are competent inmismatch DNA repair (i.e., not RER1), the rate isabout 4%. Intriguingly, two of three RER1 tumorshad such deletions (P , 0.01), suggesting a selec-tive pressure that favors the evolution of clones thathave lost the FHIT gene or that contain a tendencyto sustain breakage in the FRA3B region. Intra-genic mutations of FHIT in pancreatic cancer are sorare as to be considered of no consequence. Thereis one report that found a high rate of FHIThomozygous deletion in a series of pancreatic can-cer (Simon et al., 1998), but it is unconfirmed inother series.

It is important to note that most candidate genesthat are studied are found not to have mutations.Genes known to lack mutations include the tumorsuppressors and related genes: PTEN1 (Kong et al.,1997; Okami et al., 1998), SCH (Hahn et al., 1995),

6 HILGERS AND KERN

SMAD genes 1, 2, 3, 5, and 6 (Riggins et al., 1997a,1997b), APC (McKie et al., 1993; Smith et al., 1993;Seymour et al., 1994), b-catenin (Goggins et al.,1997a), ALK1 (Goggins et al., 1998b), p18INK4c

(Rozenblum et al., 1996), p19INK4d (Rozenblum etal., 1996), (in tumors lacking homozygous deletionof the neighboring p16 gene) p15INK4b (Rozenblumet al., 1996), and (in tumors having an intact copy ofthe overlapping p16 gene but without mutationsthat inactivate the p16 reading frame) p14ARF (Cal-das et al., 1994a; Rozenblum et al., 1997).

FAMILIAL PANCREATIC CANCER

It is estimated that as many as 10% of pancreaticcancer may occur in a familial form. Unlike in otherfamilial cancer syndromes, pancreatic cancer doesnot often occur in association with a recognizableclinical syndrome. Kindreds of affected individualsare small. The age of onset in such families isunchanged from that of sporadic pancreatic cancer,about 63 years (Lynch et al., 1990). The recognitionthat familial pancreatic cancer indeed had an inher-ited basis was therefore first proved not by the useof LOD score analysis or other positional ap-proaches, but by the mutational analysis of candi-date tumor suppressor genes. Three such genes(the p16, BRCA2, and LKB1/STK11 genes), as wellas inherited syndromes associated with deficientDNA mismatch repair, are known to be directlyassociated with susceptibility to pancreatic cancer.Inherited mutations of the p16 gene are seen inpatients with pancreatic cancer in the setting of theFAMMM (familial atypical multiple mole and mela-noma) syndrome (Lynch and Fusaro 1991; Gruis etal., 1995; Whelan et al., 1995); but p16-mutantpedigrees having pancreatic cancer alone have notbeen reported. In breast cancer families and in apopulation study of Iceland, breast cancers due toinherited mutations of BRCA2 accompany an in-creased risk of pancreatic cancer (Thorlacius et al.,1996). Unlike with the p16 gene, inherited muta-tions of BRCA2 have such a low penetrance that afamily history may not be apparent. Inheritedoncogenes are not known to predispose to pancre-atic ductal cancer.

There needs to be made special mention of aspecial type of cancer susceptibility gene. Onecould observe that the above list of inheritedmutations is restricted to genes that cause selectiveadvantages for individual cell clones; that is, theyare cell-autonomous. But cells do not live byinternal signals alone. Environmental influences oncancer rates are well known, such as when there isexposure to exogenous carcinogens or mitogenic

agents. This view can be extended to influencesarising within the body, acting on not only indi-vidual cells but entire tissues. In imprecise terms,this can be called a form of field effect. But for moreprecision, we would need an example. The cationictrypsinogen gene, PRSS1, provides it. Inheritedmutations in this gene underlie familial pancreatitis(Whitcomb et al., 1996), wherein there is an inappro-priate activation of trypsinogen to produce theprototype hydrolytic digestive enzyme, trypsin.

Inherited PRSS1 mutations underlie the form ofinherited pancreatic cancer susceptibility havingthe greatest penetrance. The relative risk for thedevelopment of pancreatic cancer in a carrier of aPRSS1 mutation is about 50-fold, the overall life-time risk is nearly 40%, and the average age of onsetis dramatically reduced to 39 years (Lowenfels etal., 1997). This tumor susceptibility is presumablydue to mitogenic stimulation and clonal outgrowthof pancreatic ductal cells as part of the normalhealing responses that occur subsequent to re-peated rounds of tissue destruction. If we were todesignate tumor suppressors as genes whose pres-ence in the germline prevents the formation oftumors (a definition sometimes referred to as thegold standard), then trypsinogen would be in nu-merical terms one of the most convincing examplesof a tumor suppressor gene yet identified.

Obviously, a number of interesting and conten-tious points are hereby raised. The first is thatinherited cancer susceptibility indeed does involvemore than the classic trio of tumor suppressorgenes, repair genes, and oncogenes. The second isthat the study of this particular class of tumorprevention is underdeveloped as a scientific field,but could have major implications for our under-standing of how human populations develop neopla-sia. The arguments that could be offered are quiteopen-ended, and anticipate a fascinating future ingenetic epidemiology. For example, what if a statis-tically higher mean body temperature were to becorrelated to the incidence of cancers of internalorgans? Would the gene(s) that control body tem-perature now be considered oncogenes and suppres-sor genes? The cationic trypsinogen gene is merelythe first clear example of this other form of tumorsuppression. If some genes can be classified asgatekeepers (Kern, 1993; Kinzler and Vogelstein,1997), DNA repair genes become caretaker genes(Kinzler and Vogelstein, 1997), and tumor suppres-sors that act late in tumorigenesis could be termedguardsmen genes (indicating a role after breachingof the neoplasm’s initial regulatory gate), then thecategory of genes represented by PRSSS1 needs a

7MOLECULAR GENETIC BASIS

designation as well. Since the activity is specificallyto reduce the destructive processes and inflamma-tion in neighboring or distant tissues, they could betermed diplomat genes.

In the absence of a known gatekeeper function inpancreatic ductal epithelium that controls the pro-pensity to initiate neoplasia, the inherited tumorsuppressor mutations in pancreatic cancer (p16,BRCA2, LKB1/STK11) fit the guardsmen designa-tion. Indeed, a role in later stages is suggested bystudies of these genes in pancreatic tumor progres-sion (Goggins et al., 1997b; Moskaluk et al., 1997;Wilentz et al., 1998). The penetrance of thesegenes is low, producing pancreatic cancer in per-haps 5%–10% of persons carrying the mutant genes.This corresponds to a relative risk of 10- to 20-foldover the incidence rate (less than 0.5% lifetimerisk) of the general population. To date, the knownelevations in incident rates of pancreatic carcinomain African Americans has no association with theknown somatic or germline genetic alterations ofthe cancers (Rozenblum et al., 1997).

It had been estimated that as many as 10% ofpancreatic cancer might occur in a familial form(Lynch, 1994). This estimate was based on theobservation of familial pancreatic cancer, definedwhen at least two first-degree relatives exhibit thedisease. But such an estimate would exclude inci-dents of susceptibility such as demonstrated by theBRCA2 gene mutations, wherein the patient almostinvariably has no familial history of pancreaticcancer. Consequently, the proportion of pancreaticcancer that is inherited is presumably even higherthan that of the above estimate.

Thus, even when we restrict consideration to thepatients that have known germline mutations, someremarkable facts exist for pancreatic cancer. For noother common adult cancer are there as manyknown pathways whose defects give rise to inher-ited susceptibility, nor is there any other organ forwhich the fraction of cancers having an inheritedbasis is higher than for the pancreas.

NO NEWS, JUST SPECULATIVE VIEWS

It is entirely possible that all the major tumorsuppressor genes have already been found. In thisview, for pancreatic and other cancer types, nogenes that have high frequencies of mutation (above50% of cases) await discovery. The future of cancergenetics would then rely in large part in theuncovering of the low-frequency mutation targets.We will piece together entire mutant pathways, butwill do so piecemeal and perhaps at a snail’s pace,5% here and 3% there. Even so, the wonderful

variety and novelty of these new pathways willserve as the fountainhead for individualized riskassessment and for rational drug development.

As this science progresses, low-penetrance muta-tions carried in the germline will be realized toaccount for ever increasing proportions of the adult-onset cancers. This is likely to be an extremelyproductive search, in that evolution has had littleneed to eliminate such mutations from the genepool. Evolution having dropped the ball, the insur-ance companies will take up the offensive task ofgenetic exclusion, to the response of a compensa-tory legislative barrage. Because translational re-search involves real people, researchers will beaffected by these battles.

Positional cloning will become trivialized for twomajor reasons. First, the gene map soon will becompleted. Second, the low-frequency and low-penetrance genetic lesions, those left to be discov-ered, do not readily lend themselves to such posi-tional approaches. While it is true that homozygousdeletions can pinpoint a genetic target where LODscores will fail, not all suppressor and repair genesare affected by these deletions, and not all suchdeletions can reasonably implicate the existence ofan important gene. The use of homozygous dele-tions in a positional strategy is historically vindi-cated as productive, but will benefit greatly fromsome anticipated technical advances.

The consensus mapping of LOH (loss of hetero-zygosity) will prove to be largely disappointing. Ithas perhaps been underappreciated how little hasbeen accomplished through this technique to date.If we restrict ourselves to genes that are somaticallymutated (since germline mutations benefit fromLOD scores and other practical advantages), itmight be interesting to calculate the numerical ratioof LOH-mapping papers to convincing tumor sup-pressor genes cloned as the direct consequence ofthese maps. Our interest is frustrated by the impos-sibility of dividing by zero.

If so, then, how can one use LOH data? Itappears that it is the frequency of deletion at alocus, rather than the map of individual deletionsthat is most helpful. This distinction exists for atleast two major reasons. First, because only raretumor suppressor genes have a pattern of near-universal somatic inactivation when LOH is pres-ent. (A sidelight: The p53 gene is one such ex-ample, but was cloned long before LOH mappingbecame available; nevertheless, one cannot easilyexpect another such universal target. In anotherexample, the DPC4 gene is inactivated in onlyabout half of the pancreatic cancers having LOH of

8 HILGERS AND KERN

chromosome 18, but did not emerge from theattempted mapping of consensus LOH.) Second,the existence of a tumor suppressor gene does notimmunize the chromosome against additional ge-netic lesions, whose analysis would assemble astreet map of blind alleys.

For the low-frequency genetic targets, the muta-tion of the known gene constitutes only a minorityof the overall LOH frequency. Indeed, at the lowfrequencies often used in LOH consensus map-ping, the confounding occurrence of random (unse-lected) deletions will not simply overlap, but willcompletely obscure the real gene targets. The highpromise offered by LOH mapping perhaps remainsin special cases only, when a frequency of LOH isfar above background rates (as judged by studies ofother chromosomes) and when a consensus seemsto emerge from a vast majority of the deletionsmapped (in high-resolution studies of a particularchromosome). Chromosomal arm 1p may meetthese criteria in pancreatic cancer (Hilgers et al.,1999). Soberingly, to date the use of hundreds ofgenomic markers has not suggested another tempt-ing locale for such a mapping project in this disease.

Increasingly then, we will see the emergence ofnew low-frequency genetic targets through theapplication of brute-force technologies. Batch muta-tional screening of the hundreds of rational candi-date genes will follow, pursuing the putative candi-date members of signaling pathways, especiallythose pathways already known to have low-frequency mutations in a member gene. As thetranscript map of the genome becomes known, thelists of the gene content of the homozygousdeletions will become triaged as candidate genes.The requirement for technical advances will beacute in the study of inherited susceptibility, wheresmall family sizes and absence of a recognizableclinical syndrome renders the positional approachesineffective. Eventually, tumor studies will imple-ment the automated screening of tens of thousandsof AORGs (any old random genes). This mutationaldatabase will identify a busy grid of genes andnovel pathways, representing both the blind alleysand the true avenues of genetic research. Only theapplication of descriptive genetics, epidemiology,experimentation, and skeptical intellectual argu-ment will appropriately direct the research traffictoward the one as opposed to the other.

As ever, the initial glare and inelegance of thetechnology will fade into the background as thechallenges of progressive discovery remain thesame. Our implicit pact with the taxpayers thatfund this research provides an obligation not for

fascinating and engaging science, but for the actualidentification of clinical breakthroughs. Notwith-standing the gratification in reviewing the accumu-lated progress, by this measure, there is consider-ably more left to do.

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