Genomic Anomalies in Pancreatic Tumors Other Than Common Adenocarcinoma

179

Genomic Anomalies in Pancreatic Tumors Other Than Common Adenocarcinoma

ALDO SCARPAa AND GIUSEPPE ZAMBONI

Istituto di Anatomia Patologica, Università di Verona, Verona, Italy

ABSTRACT: In recent years enormous advances have been made in the un-derstanding of the molecular mechanisms governing pancreatic ductal adeno-carcinoma. However, little is known about other pancreatic neoplasms, whichinclude intraductal papillary mucinous (IPMT), serous cystic (SCT), mucinouscystic (MCT), solid pseudopapillary (SPT), acinar and islet cell tumors. In ad-dition, the study of tumors grouped under the unfortunate term of periampul-lary cancers will help distinguish the pathogenetic features of these neoplasms,often confused with pancreatic head neoplasms. The available data suggestthat the less common pancreatic tumor types do not generally follow the samemolecular pathway as the more common ductal carcinoma. IPMT seems tocontain chromosomal anomalies similar to those found in ductal cancers,whereas papilla of Vater and duodenal cancers show genetic anomalies resem-bling those of gastrointestinal malignancies. The application of genome-widescreening techniques to these less common pancreatic tumors will undoubtedlyplay a central role in unraveling the complexity behind their pathology.

INTRODUCTION

Despite the conspicuous advances in our understanding of the molecular basis ofpancreatic ductal adenocarcinoma,1 very little is known about molecular abnormal-ities in pancreatic neoplasms other than common ductal adenocarcinoma. These aretumor entities with distinct clinicopathologic features and include neoplasms origi-nating from either pancreatic ducts or acinar or islet cell precursors.2 The tumorsconsidered as originating from ductal cell precursors include the common ductalcancer, intraductal papillary-mucinous tumor (IPMT), serous cystic tumor (SCT),mucinous cystic tumor (MCT) and solid pseudopapillary tumor (SPT).

The pancreatic cancer research group of Verona University is focusing on thestudy of genetic anomalies of carcinomas arising from the epithelium of pancreaticducts and their terminal excretory structure (papilla of Vater), as well as of tumorsoriginating from islet cells. The molecular characterization of a representative seriesof different pancreatic tumor types will lead to an understanding of their specific mo-lecular pathogenesis and clinical behavior. As a result, the identification of novel anduseful diagnostic/prognostic markers can be expected, as has been the case for can-cers of the papilla of Vater.3 In addition, IPMT, MCT and papilla of Vater tumorsrepresent useful models to search for transformation-related and progression-related

aAddress for correspondence: Aldo Scarpa, M.D., Istituto di Anatomia Patologica, Universitàdi Verona, Strada Le Grazie, 8, I-37134 Verona, Italy. Phone, +39 45-8098-617; fax, +39 45-8098-136; e-mail, [email protected]

180 ANNALS NEW YORK ACADEMY OF SCIENCES

genetic anomalies of pancreatic epithelia. In fact, and at variance with ductal adeno-carcinoma, the different stages of progression of malignancy are morphologicallyrecognized in these peculiar tumor types.

This presentation is divided into three parts. The first describes the ongoing ge-nomic screenings at Verona University, the second deals with genetic anomalies inthe different pancreatic tumors, and the last treats periampullary malignancies.

GENOME-WIDE SCREENING FOR DETECTION OF NONRANDOMCHROMOSOMAL ANOMALIES

The search for specific genes involved in the pathogenesis and progression of ma-lignancy of cancers may be driven by the discovery of nonrandom genomicanomalies.

The available genome-wide screenings for chromosomal anomalies in pancreatictumors and those that we are applying with the aim of obtaining genomic character-ization of different pancreatic tumor types are summarized in TABLE 1. We are usingtwo complementary genome-screening approaches to obtain a genomic fingerprintand a map of chromosomal losses of the different pancreatic tumors, namely, DNAfingerprinting by arbitrarily primed polymerase chain reaction (AP-PCR) andgenome-wide allelotyping.

AP-PCR amplifies DNA fragments that are chromosome specific,4–7 and it hasbeen demonstrated that decreased intensity of AP-PCR bands in tumor DNA reflectsallelic losses, whereas increased band intensity indicates the presence of extra copiesof these sequences.6–8 Being based on a PCR technique, this approach requires onlya limited amount of DNA (20 ng/test). This provides a rapid and sensitive tool forthe analysis of genetic material extracted from microdissected cancer samples.

Allelotyping studies are based on the detection of loss of heterozygosity (LOH)at chromosomal-specific polymorphic sites in DNA extracted from tumor, whencompared to DNA from matched normal tissues. Such “allelotyping” is feasible byPCR amplification of microsatellite repeats, provided the heterozygosity for thestudied loci and a neoplastic cellularity higher than 60% in the cancer sample. Weare using a set of oligonucleotide primers to amplify 350 chromosomal locus-specif-ic microsatellite repeats. This approach allows the analysis of all chromosomes at anapproximate 10-cM resolution. The set of primers was chosen among those fur-

TABLE 1. Complete or partial studies on chromosomal anomalies of pancreatic tumors

Cystic Ductal Intraductal Papilla Mucinous

Karyotype Yes No No No

Allelotype Yesc Noc Noc Noc

Amplotype (ap-pcr)a Yesc Noc Yesc Noc

CGHb Yes No No No

aap-pcr, arbitrarily primed-polymerase chain reaction.bcgh, comparative genomic hybridization.cOngoing studies at Verona University.

181SCARPA & ZAMBONI: GENOMIC ANOMALIES

nished by the “MapPairs collection” (Research Genetics, Huntsville, AL, USA) andfrom “ABI PRISM Linkage mapping set panels” (Perkin-Elmer). The preliminarydata obtained from complete and/or partial allelotyping studies of different pancre-atic tumors is summarized in FIGURES 1 and 2.6, 7, 9–14

Tumor Cell Enrichment for Genetic Analysis

In the last few years, we have established a frozen tissue bank and a DNA bankfrom different cryostat-enriched pancreatic tumor types, and we have generated invivo and in vitro models of different pancreatic tumors (ductal, papilla of Vater andmucinous cystic tumors), through nude mice-xenografting and in vitro culture ofprimary tumors.

In fact, the main limitation for molecular studies in pancreatic neoplasms is thatcancer cellularity is low compared to other tumor types. To overcome this problem,neoplastic cell-enrichment techniques have been developed, such as cryostat dissec-tion of primary cancers6,15 or xenografting in nude mice.10,16,17 Each of these meth-ods introduces some bias in the selection of cases. Cryostat enrichment is successfulin only about 20% of cases, which represent those with a neoplastic cellularity high-er than the average common pancreatic cancer. Xenografting, which allows the neo-

FIGURE 1. Frequency of allelic losses (LOH) on different chromosomal arms in pan-creatic cancer cells enriched with different methods. The panel from xenograft-enrichedcells is from Hahn et al.10 (publicly available: http://www.path.jhu.edu/pancreas). The pan-el below is drawn from preliminary data (only for the chromosomes shown) obtained froma panel of cryostat-enriched cancers at Verona University and partially published.6,43 Notethe concordance of the results for chromosomes 17, 18, 21 and 22, whereas chromosomes3, 7, 9, and 13 show discordant results in the two series.


plastic enrichment of at least 2–5 times, may also introduce some bias in the panelsobtained. For instance, all xenografts show Ki-ras mutations and p53 mutation ratesare higher than those found in primary cancer samples,18 suggesting either a selec-tion for that particular 60% of pancreatic cancers containing a p53 mutation,19–21 orperhaps a selection for subclones within a cancer population.10, 22

Based on this consideration, the results obtained with either type of material maydiffer (FIG. 2). In addition, the DNA anomalies found in cryostat-enriched neoplastictissues represent an average of the anomalies in the different tumor areas, and thusthe heterogeneity of the cancer cell population will be hidden. However, consistentanomalies occurring in the large majority of cancer cells are nonetheless observed.The use of microdissection on additional frozen or paraffin-embedded samples then

FIGURE 2. Available data on the frequency of chromosome allelic losses (LOH) indifferent pancreatic tumors. Information is available only for the chromosomal arms shownand is recovered from published data12,14 and from partially published preliminary resultsobtained at Verona University.7,13,54 Note the low frequency of LOH in endocrine tumorswith respect to those of exocrine tumors shown in this figure and in FIGURE 2.


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GENETIC ANOMALIES

Known Defects

The known genetic anomalies described in ductal, intraductal and papilla of Vaterneoplasia, also with the contribution of the group from Verona University, are sum-marized in FIGURE 3 and discussed below.

In common ductal cancer, these mainly include mutation of the Ki-ras, p53,p16INK4a and DPC4 genes. Point mutational Ki-ras activation is a feature of ductalcancer (reviewed in Refs. 23 and 24) and occurs at precancerous stages, within le-sions termed pancreatic intraepithelial neoplasia (PIN).25, 26 They also seem to beearly events in about 35% of papilla of Vater and 50% of IPMT.27–31 Ki-ras muta-tions have also been found in SCT.32 Adenomatous polyposis coli (APC) gene muta-tions are infrequent events in papilla of Vater cancers in spite of a high frequency ofchromosome 5q LOH7,33 and are exceedingly rare in ductal cancer.15,34–36) Biallelicinactivation of the p53 gene in which one copy of the gene on chromosome 17p islost and the remaining copy is inactivated by a point mutation is found in about 70%of ductal and papilla of Vater cancers.3,19–21,27 The occurrence of intragenic 1–2-basepair microdeletions is a peculiarity of ductal cancers in comparison with othertumor types.19, 21 In papilla of Vater tumors and IPMT, p53 alterations are believedto accompany the adenoma-carcinoma transition.27,29 The gene coding for the cy-clin-dependent kinase inhibitor p16INK4a is frequently inactivated in ductal cancerand in its precursor lesion either by homozygous deletion (48%) or by intragenic mu-tation (34%).16,25 It has recently been shown that the gene is transcriptionally si-lenced by methylation in an additional 16% of ductal cancers.37 IPMT shows LOHof the p16INK4a locus on chromosome 9p21 in 60% of cases,12 whereas papilla ofVater cancer rarely shows LOH at 9p21 (Scarpa, unpublished data).

The loss of one copy of chromosome 18q occurs at high frequency in ductal can-cer.6,10 The DPC4 gene is located on this chromosome and is inactivated in about50% of cases by either intragenic mutation or homozygous deletion.38 This gene isthought to belong to the transforming growth factor-β (TGF-β) superfamily signal-ing pathway, and data exist about additional genetic anomalies disrupting the TGF-β pathway.39 Both IPMT and papilla of Vater tumors show LOH at 18q in 40% ofcases, but no data are available for DPC4 mutations. The BRCA2 gene was found tobe mutated in the germ-line of 7% of pancreatic cancer patients.40 No data are avail-able for IPMT and papilla of Vater tumors.

Widespread alterations at simple repeat sequences (microsatellite instability) oc-cur in about 20% of papilla of Vater cancers, where it is an early phenomenon and isassociated with a good prognosis.3 The TGF-β signaling pathway is hampered inproportion of this subgroup of papilla of Vater tumors by mutation of the TGF-β–type II receptor (Scarpa, unpublished). At variance, microsatellite instability is a rareevent in ductal tumors and seems to be associated with peculiar clinicopathologicalcharacteristics.41

No or limited data are available on molecular abnormalities of acinar cell,89 se-rous cystic,32,42 mucinous cystic and solid pseudopapillary tumors (SPT).42 The lat-


ter is a low grade malignancy primarily affecting prepuberal and young women,which characteristically show progesterone receptor immunostaining.96 Nogenome-wide analysis for chromosomal anomalies in IPMT, papilla of Vater tumorsand MCT has been reported to date.

In summary, the limited information about genetic anomalies in tumors other thanductal cancer suggests that IPMT seem to contain chromosomal anomalies similarto those found in ductal cancers, although at a lower frequency,12 whereas papilla ofVater cancers show genetic anomalies more closely resembling those of gastrointes-tinal malignancies.3,7,28

Chromosomal Losses with No Identified Genes

In ductal cancers there is an additional collection of chromosomal arms for whichlosses have been seen in at least 50% of cases, including chromosomes 1p, 3p, 6p,6q, 8p, 10q, 12q, 21q, and 22q.1 A high frequency of chromosome 7q31–32 LOHhas been detected in the highly cellular subgroup of ductal cancers, and 5q LOH hasbeen observed in papilla of Vater cancers (see below).6,7 Although no target gene hasbeen conclusively identified for the aforementioned chromosomal deletions, the fol-lowing genes have been excluded: p18 cyclin-dependent kinase inhibitor for chro-mosome 1p; the retinoblastoma (Rb) for 13q; and neurofibromatosis type 2Schwannomin (SCH) for chromosome 22q.10 However, data from our group suggestthat the fragile histidine triad (FHIT) gene at chromosome 3p14.2 might be a targetfor chromosome 3p deletion.43

Our DNA fingerprinting studies of cryostat-enriched pancreatic and papilla ofVater cancers using AP-PCR detected nonrandom anomalies that were further char-acterized by allelotyping of the chromosome from which the AP-PCR bands werederived. Chromosome 7q LOH is a frequent event (80%) in cryostat-enrichable com-mon pancreatic ductal carcinomas; the smallest common deleted region described byour cases was between 7q31.1 and 7q32 markers.6 Pax-4 located in this chromosom-al region is not the target of this deletion (Scarpa, unpublished). Similarly, 75% ofpapilla of Vater cancers showed LOH at chromosome 5q, with two smallest commondeleted regions at 5q13.3–q14 and at 5q23–q31, which do not include the APC genelocus and are similar to those found in gastric cancer.7

In the course of allelotyping studies of cryostat-enriched primary pancreatic can-cers, we found allelic losses at chromosome 3p14.2 in about 60% of cases and thatthey occur much more rarely on chromosome 3 outside this region. The FHIT geneis located at this site and has been proposed as a candidate tumor suppressor gene,due to its frequent alteration in different cancer types.44 Further studies have shownthat the FHIT gene is expressed in the terminal ductules and centrocinar cells of nor-mal pancreas and that 60% of primary pancreatic ductal cancers exhibit various al-terations affecting FHIT gene expression, often represented by FHIT gene intragenicdeletions.43

PERIAMPULLARY CANCERS

The term “periampullary carcinoma” has been diffusely used to define cancers in-volving the region of the papilla (ampulla) of Vater. This has rendered it extremelydifficult to compare the results of the different clinicopathologic and few molecular


studies. In fact, under this definition a heterogeneous group of neoplasms is includ-ed, which originate from either the proper ampulla or periampullary anatomicalstructures, i.e., the pancreatic head, the duodenum and the extraduodenal bile duct.On the other hand, the comparable histopathologic characteristics of the epithelialtumors arising in this region and the advanced stage at which most cases are diag-nosed prevent a precise definition of the structure of origin. The exact site of origincan only be determined in small lesions. Only the study of a well-selected series ofother types of periampullary tumors may help in understanding the common and/ordistinct features in the pathogenesis of this heterogeneous group of neoplasms.

We have studied a series of cancers of unequivocal ampullary origin for geneticalterations including: i) mutations of Ras-family, APC, p53 and p16INK4 genes, ii)AP-PCR and partial or complete allelotyping of different chromosomes (see FIG. 3);iii) presence of a “RER+ phenotype,” as defined by the presence of DNA replicationerrors (RER) at simple repeat sequences (SRS) interspersed throughout the genome.

Ras mutations occur in about 35% of cases and usually affect Ki-ras, but rarelyN-ras.27, 28 They are a relatively early phenomena associated with adenomas and aproportion of cancers having an adenomatous phase in their pathogenesis. Ras mu-tated tumors included three of four cases which mainly involved the intraduodenalbile duct, suggesting that a proportion of Ras-mutated cancers might correspond tothose originating from the epithelium of the bile duct component of the ampulla.28

Interestingly, biliary tract cancers show a relatively high frequency of Ki-ras muta-tions,45–47 but with peculiar differences according to their location. Motojima etal.45–47 reported a frequency of 9%, 0% and 41% in cancers of proximal, middle anddistal bile duct, respectively.

APC gene mutations are not frequent in ampullary cancer pathogenesis.33 Inter-estingly, all APC mutated cases also contained a Ras gene mutation and were foundin tumors with an adenomatous component. This might suggest that APC mutations,like Ras gene mutations, are restricted to a proportion of cancers arising on an ade-nomatous precursor. In contrast with the low frequency of APC gene mutations, fre-quent allelic losses at chromosome 5q (75%) were found, as suggested by AP-PCRfingerprinting.7 Allelotyping of chromosome 5 located the previously mentionedtwo smallest common deleted regions at 5q13.3–q14 and at 5q23–q31, which corre-spond to those found in gastric tumors and do not include the APC locus. In addition,the presence of 5q LOH in 6 of 8 adenomas and in 3 of 4 early-stage cancers suggeststhat such a phenomenon occurs at early stages of neoplastic progression of the am-pullary epithelium.

Inactivation of tumor suppressor gene p53 by mutation of one allele and loss ofthe normal one is a frequent phenomenon and is mainly associated with high-gradeadvanced-stage disease, irrespective of the presence of adenomatous componentsand of Ras, APC, chromosome 5q or RER abnormalities.3,27 Neither losses nor mu-tations of the p16 gene were found (Scarpa, unpublished). Finally a RER+ pheno-type was detected in 20% of cases and was associated with long survival of patients.3

A proportion of these tumors also showed mutations in polyadenine tracts of TGF-β-RII, Bax and hMSH3 genes (Scarpa, unpublished).

In conclusion, our data suggest that papilla of Vater tumors arise from two differ-ent molecular pathogenetic pathways, characterized by either chromosomal instabil-ity (CIN) or microsatellite instability (MIN) with different clinicoprognostic


features (FIG. 4). We have also demonstrated the existence of these two alternativepathogenetic pathways in duodenal cancer.48 This scenario parallels that of the com-mon type sporadic colorectal and gastric cancers.49,50 The decision as to whether pa-pilla of Vater and duodenal adenocarcinoma should be viewed as a gastrointestinalcancer or as a peripancreatic cancer is unclear.51–53 This is not a trivial question,since it involves the debate about the opportunity and type of a surgical and/orchemotherapic treatment.51–53 Our findings suggest that both papilla of Vater andduodenal cancers should be considered as gastrointestinal malignancies.

AKNOWLEDGMENTS

Most of the results of our study have been supported by grants from the Associaz-ione Italiana Ricerca Cancro (AIRC), Milan, Italy; Consorzio Studi Universitari andBanca Popolare di Verona, Verona, Italy.

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FIGURE 4. Schematic representation of the molecular pathogenesis of papilla of Vaterand duodenal cancer. Two molecular pathways lead to papilla of Vater and duodenal cancer,and they may be termed “chromosome instability” (CIN) and “microsatellite instability”(MIN) pathways.55,56 The more common sporadic cancers (80%) belong to the first and arecharacterized at the molecular level by allelic losses in an average of at least 25% of ran-domly chosen DNA sequences. In these cases, the early genetic events include chromosome5q allelic losses and less frequently APC and Ras gene mutations. p53 gene inactivation bymutation of one allele and loss of the second together with chromosome 18q LOH charac-terize progression of malignancy. A proportion of about 20% of cancers show a MIN phe-notype, characterized by widespread genomic mutations and by rare chromosomal losses. Inthese cancers, the impaired function of DNA repair genes also causes the mutational activa-tion or inactivation of functional genes,56 such as the TGF-β–type II receptor (TGF-β-RII),Bax and hMSH3 genes.


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