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Large-Scale Allelotype of Pancreaticobiliary Carcinoma Provides Quantitative Estimates of Genome-Wide Allelic Loss

Departments of ¹ Pathology, ² Oncology, ³ Surgery, ⁴ Medicine, and ⁵ Biomedical Engineering and ⁶ The Center for Inherited Disease Research, The Johns Hopkins Medical Institutions, Baltimore, Maryland

	ABSTRACT

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Studies of the allelotype of human cancers have provided valuable insights into those chromosomes targeted for genetic inactivation during tumorigenesis. We present the comprehensive allelotype of 82 xenografted pancreatic or biliary cancers using 386 microsatellite markers and spanning the entire genome at an average coverage of 10 cM. Allelic losses were nonrandomly distributed across the genome and most prevalent for chromosome arms 9p, 17p, and 18q (>60%), sites of the known tumor suppressor genes CDKN2A, TP53, and MADH4. Moderate rates of loss (at any one locus) were noted for chromosome arms 3p, 6q, 8p, 17q, 18p, 21q, and 22q (40–60%). A mapping of individual loci of allelic loss revealed 11 "hot spots" of loss of heterozygosity (>30%) in addition to loci near known tumor suppressor genes, corresponding to 3p, 4q, 5q, 6q, 8p, 12q, 14q, 21q, 22q, and the X chromosome. The average genomic fractional allelic loss was 15.3% of all tested markers for the 82 xenografted cancers, with allelic loss affecting as little as 1.5% to as much as 32.1% of tested loci, a remarkable 20-fold range. We determined the chromosome location (in cM) of each of the 386 markers used based on mapping data available from the National Center for Biotechnology Information, and we provide the first distance-based estimates of chromosome material lost in a human epithelial cancer. Specifically, we found that the cumulative size of allelic losses ranged from 58 to 1160 cM, with an average loss of 561.32 cM/tumor. We compared the genomic fractional allelic loss of each xenografted cancer with known clinicopathological features for each patient and found a significant correlation with smoking status (P < 0.01). These findings offer new loci for investigation of the genetic alterations common to pancreaticobiliary cancers and aid the understanding of mechanisms of allelic loss in human carcinogenesis.

	INTRODUCTION

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Structural alterations of chromosomes are a virtually ubiquitous feature of human epithelial neoplasia (1) . Gross chromosomal alterations may occur after a variety of events, including chromosomal translocations, rearrangements, amplifications, and deletions, usually leading to general aneuploidy. Telomere dysfunction is thought to be a major mechanism for the development of aneuploidy (2, 3, 4) , although other factors have also been shown to have a role (5) . In some instances, DNA mismatch repair defects without gross chromosomal changes are the underlying feature of epithelial neoplasia (6) , as seen in the hereditary nonpolyposis colorectal cancer syndrome (7) or medullary carcinomas of the pancreas (8) .

Most pancreatic and biliary ductal adenocarcinomas have a complex karyotype including numerical and structural abnormalities (9, 10, 11, 12) . Chromosome losses are the most common numeric abnormalities found in these studies, with losses of chromosomes 18, 13, 12, 17, and 6 among those reported frequently (9, 10, 11) . Molecular genetic studies have confirmed these karyotypic findings, with frequent allelic losses of chromosomes 1p, 9p, 17p, and 18q and moderate frequencies of loss of additional chromosomal loci (13) . Identification of these allelic losses contributed to identification of the mutations of tumor suppressor genes CDKN2A on 9p (14) , SMAD4 on 18q (15) , LKB1 on 19p (16) , and ACVR1B on 12q (17) as playing a role in pancreatic carcinogenesis.

Since the original report of the pancreatic cancer allelotype determined in seven cryostat-dissected neoplasms by Seymour et al. (18) and the subsequent larger manual analysis of 18 pancreatic cancer xenografts by Hahn et al. (13) , high-throughput automated techniques have been developed that allow analyses of severalfold more efficient coverage of the genome (19) . Using such techniques, the new data presented from a large series of pancreaticobiliary cancers extend and refine the known allelotype maps as well as better define the relationship of global measures of allelic loss to clinicopathological features.

	MATERIALS AND METHODS

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Xenografted Cancers.
The generation of xenografted tumors derived from pancreatic and biliary cancers and their utility for allelotyping studies have been described in detail previously (13) . For each xenografted pancreatic or biliary cancer, paired normal duodenal mucosa was also available for analysis. All pancreatic cancer xenografts previously determined to have a medullary phenotype were excluded from analysis (8) . Clinical and pathological data were obtained from the Johns Hopkins Hospital Surgical Pathology files. The Johns Hopkins Institutional Review Board approved the study.

Genotyping of Pancreatic Cancers.
Genotyping was performed using a modification of the CHLC version 9 marker set (386 microsatellite markers; average spacing, 10 cM). Detailed information can be found online.⁷ The approximate location of each marker, expressed as cM, was determined using human genome recombination map data derived from the National Center for Biotechnology Information website available as of April 2004.⁸ Genomic DNA was obtained from pancreatic cancer xenograft tissues, matched normal tissues and pancreatic cancer cell lines using the DNeasy Tissue Kit (Qiagen, Valencia, CA) and diluted to 20 ng/µl in a 96-well deep-well plate. Four blind duplicates as well as 4 positive and 2 negative control samples were included with each batch of 86 samples. PCR was performed using 40 ng of genomic DNA in a final reaction volume of 5 µl. Forward primers were labeled with fluorescent dyes FAM and HEX (obtained from Life Technologies, Inc., Carlsbad, CA) and NED (obtained from Applied Biosystems, Foster City, CA) for detection on an ABI 377 DNA analyzer. Reverse primers were obtained from MWG (High Point, NC). Primer concentrations ranged from 0.035 to 0.170 µm each. Two to three loci were routinely amplified together in multiplex PCR. An average of eight PCR products were pooled together using a Hydra 96 Microdispenser unit (Matrix Technologies, Hudson, NH), and PAGE was performed on the ABI 377 XL DNA analyzers. Gels were prepared using Cambrex Long Ranger XL Single Packs (East Rutherford, NJ). The size standard used for fragment analysis consisted of 21 PCR products labeled with a fluorescent dye (ROX) and generated by PCR amplification of the pUC18 plasmid DNA-amplified fragments. ABI Genescan software was used for image analysis of each gel containing fluorescence-labeled PCR products. The inherent error rate for the genome scan, based on paired genotypes from these blind duplicates, was 0.25%. The overall missing data rate was 6.4%.

A subset of 30 microsatellite markers spanning 17 chromosomes with varying frequencies of allelic loss was selected for independent verification of allelic status. PCR products incorporating [-³²P]deoxycytidine were separated on a 6.0% polyacrylamide-8 M urea gel, and autoradiography was performed. Determinations of heterozygosity and the presence or absence of allelic loss were scored by three of the authors (C. A. I-D., M. S. v. d. H., and S. E. K.). Loss of heterozygosity (LOH) was defined as the complete loss of one of the two bands in xenograft DNA as compared with the matched informative normal DNA. A comparison of manual and automated determinations of LOH indicated a concordance rate of >99%.

Statistics.
Summary data are expressed as the mean ± SD unless otherwise indicated. When evaluating distributions of fractional allelic loss in relation to gender, location, tumor differentiation, and smoking history, a one-tailed Student’s t test was used. When evaluating distributions in relation to age, tumor diameter, and survival, Wilcoxon’s rank-sum test was used. Probability values of 0.05 or less were considered significant.

	RESULTS AND DISCUSSION

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General Features of the Pancreatic Cancer Allelotype.
We analyzed a total of 82 primary cancers representing 73 xenografted pancreatic cancers and 9 xenografted biliary cancers. These 82 pancreatic or biliary cancers were analyzed using 386 microsatellite markers spanning the 22 autosomes and the X chromosome, resulting in a total of 63,304 individual loci analyzed. The average coverage was 16.7 markers/chromosome, and the average spacing of markers was approximately 10 cM. The allelotype determined for 18 of these xenografts at 283 loci has been reported previously (13) .

Among the 82 pancreatic or biliary cancer xenografts having matched normal DNA samples, a total of 31,652 determinations of heterozygosity were made, of which 21,203 (67%) were informative. The average informativeness ranged from 0.24 (DXS1068) to 0.90 (D3S2406). LOH was found in 5,398 informative determinations, indicating an average allelic loss of 25.4% of informative markers for the 82 xenografted tumors analyzed (Fig. 1) . Rare allelic shifts that extended or shortened the normal allele size by 2–5 bp were also found in 65 xenografted cancers, involving 1–9 individual allele(s)/case. Thus, among this subset of 65 tumors, an average of <0.01% loci/case had minimal shifts, similar to previous reports indicating that microsatellite instability is infrequent among pancreaticobiliary cancers that lack the severe form of the mutator phenotype (18) .

View larger version (16K): [in this window] [in a new window]   Fig. 1. Allelic status of individual pancreatic cancers. Fluorescence-labeled PCR products were separated electrophoretically on 6% polyacrylamide gels as described in "Materials and Methods," and sizes of amplified products were determined by Genescan software. The resulting scans are shown in graphical format, with longer amplified products presented on the right. In A–C, the amplified products of the xenografted cancer DNA are located above that of the matched normal DNA analyzed for the same case. A, example of an informative allelic pattern (two bands in the normal DNA) with loss of heterozygosity of the matched xenografted tumor (only the larger DNA product remains). B, example of an informative allelic pattern in the normal DNA without loss of heterozygosity in the matched xenografted tumor. C, example of a noninformative allelic pattern. Both the normal and xenografted tumor DNA are homozygous for the marker shown.

The vast majority of markers showing LOH occurred within areas of contiguous loss of whole chromosomes or chromosomal arms. Specifically, 1355 markers with LOH (33.5%) occurred within a region of multimarker LOH (3 or more contiguous markers with LOH), and an additional 1958 markers with LOH (54.6%) were adjacent to a second marker with LOH and flanked on either one or both sides by noninformative markers. The remaining markers with LOH occurred singly, being flanked by two informative markers without LOH (49 markers, 1.2% of all LOH calls), two noninformative markers (129 markers, 3.2% of all calls), or a pattern intermediate to these. Single marker LOH was thus reasonably uncommon relative to contiguous marker LOH.

As a general estimate of allelic loss related to each chromosomal arm, we determined its prevalence as the number of cancers with at least one marker showing LOH for a chromosomal arm divided by all cases that were informative for that same arm (20) . The highest prevalence (>60%) was noted for chromosomal arms 9p (84%), 17p (81%), and 18q (80%). Moderate prevalence of allelic loss (40–60%) was noted for chromosomal arms 3p (43%), 6q (54%), 8p (51%), 17q (41%), 18p (41%), 21p (48%), and 21q (46%). In contrast, allelic losses were a rare event for chromosomal arms 14p (0%), 15p (0%), and 16q (8%), similar to previous findings (Ref. 13 ; Fig. 2 ). The prevalence rates of multimarker, double marker, and single marker LOH patterns described above correlated with the prevalence of allelic loss determined for each chromosomal arm.

View larger version (18K): [in this window] [in a new window]   Fig. 2. Allelotype of pancreaticobiliary cancers. The percentage of all 82 xenografted pancreaticobiliary cancers with at least 1 site of allelic loss/chromosome arm is depicted.

View larger version (86K): [in this window] [in a new window]   Fig. 3. Genomic fractional allelic loss (G-FAL) determined in 82 pancreatic and biliary cancers analyzed by 386 microsatellite markers. The identity of each carcinoma analyzed is listed on the left, and the identity of each chromosome is shown at the top. Carcinomas were sorted by increasing genomic fractional allelic loss from top to bottom, as were chromosomes, based on increasing prevalence of loss of heterozygosity (LOH) from left to right. The nine carcinomas listed in bold indicate those that originated within the distal common bile duct. Shaded boxes indicate the total number of markers having LOH per chromosome for each carcinoma. White, no informative markers with LOH in the corresponding tumor; lightest gray, total of 1–5 informative markers with LOH in the corresponding tumor; medium gray, total of 5–10 informative markers with LOH in the corresponding tumor; dark gray, total of >10 informative markers with LOH in the corresponding tumor.

View larger version (31K): [in this window] [in a new window]   Fig. 4. Loss of heterozygosity (LOH) maps determined in 82 pancreatic and biliary cancers. Schematic maps of each chromosome depict the proportion of tumors having LOH at each of 386 markers. Data are plotted from pter to qter, based on chromosome location as determined from recombination map data (see "Materials and Methods" for details). The approximate location of the centromere is depicted by a black circle within each schematic chromosome. The prevalence of LOH as a fraction of all informative cases for each marker is represented by the width of each chromosome, normalized to a baseline representing the set of informative markers (shown below each chromosome). For illustrative purposes, a standardized chromosome at bottom right (gridded) has 0% LOH (corresponding to 100% width) at the q arm terminus and 100% LOH (0% width) at the p arm terminus. Hot spots were defined as those loci having 30% LOH among all informative tumors for that marker. Arrowheads indicate the sites of both known (9p, 17p, and 18q) and novel (3p, 4q, 5q, 6q, 8p, 12q, 14q, 21q, 22q, and Xq) hot spots of LOH.

Patterns of Allelic Loss Related to Individual Cancers.
The comprehensiveness of this data set also allowed a determination of the relationship of the genomic fractional allelic loss [G-FAL (defined as the number of markers with LOH divided by all informative markers/carcinoma)] to the overall allelotype for each cancer analyzed. Our data indicate a wide spectrum of allelic loss among pancreatic and biliary cancers. For example, in individual tumors, the G-FAL ranged from 124 of 386 markers (32%; sample PX171) to 6 of 386 markers (1.5%; sample PX147) with an average of 59 markers (15%; median of 56 markers) lost per cancer. The G-FAL was also determined in units of cumulative cM of chromosomal material lost and ranged in size from 58 cM (PX147) to 1160 cM (PX171) [average for all cancers, 561 cM; median, 520 cM].

Two specific features of the pancreaticobiliary cancer allelotype were noted. First, although G-FAL was distributed across a spectrum, a special importance of certain chromosomes could be seen. As shown in Fig. 3 , allelic losses (of any length) involving chromosomes 9, 17, 18, or 6 occurred in the majority of cases analyzed. Even in those cancers with G-FAL < 5%, at least one of these chromosomes had allelic loss. For one of these cases (PX147), allelic losses of chromosomes 6 and 18 were the sole loci of LOH noted. Second, we noted a less deterministic pattern. With increasing G-FAL, there was a tendency toward allelic losses of increasing length and involvement of an increasing number of chromosomes. For example, among those cases with a G-FAL < 10%, the average number of chromosomes showing allelic loss of any length was 7.2, and the average cumulative amount of material lost was 243 cM. In contrast, among those cases with G-FAL > 20%, the average number of chromosomes affected reached 14.5, with an average loss of 942 cM. No specific differences between pancreatic and biliary cancers were found.

In recent years, chromosome instability has been recognized as a predominant mechanism underlying aneuploidy in solid human tumors (1) . Our data support this claim. However, we also show that within the spectrum of chromosome instability-positive pancreaticobiliary cancers, a 20-fold range of G-FAL values exists. At one end of this spectrum, pancreatic cancers were remarkable for minimal allelic losses (PX147; G-FAL = 1.55%), whereas at the opposite end of the spectrum, widespread allelic losses affecting the majority of chromosomes were found (PX179; G-FAL = 32%). Similar findings have been reported for other human tumor types, including lung carcinomas (24) , colorectal carcinomas (20) , and endocrine tumors of the pancreas (25) . One possible explanation offered is that structural alterations of chromosomes may not act as the major mechanism of genetic instability in all microsatellite instability-negative cases (26) . Alternatively, the fundamental property of chromosome instability may be the same for all cases, but some tumors may "fortuitously" acquire key changes early in carcinogenesis. This study also represents one of the first estimates of the actual cumulative lengths of allelic loss in a human cancer as determined from the human genome data. Several other large-scale allelotypes of human tumors have also been reported, but the data presented have not included LOH in relation to physical distances (24 , 25 , 27, 28, 29, 30, 31) .

Relationship of Allelic Loss to Clinicopathological Characteristics.
The ages and genders were similar among patients with primary pancreatic ductal adenocarcinomas and primary distal common bile duct carcinoma. When compared with the G-FAL determined for each case, no significant differences were found among the G-FAL values for each tumor type with respect to patient age, gender, lymph node status, tumor diameter, tumor location, or overall survival. A trend was noted for increasing G-FAL to be associated with worse tumor differentiation, but this trend did not reach statistical significance. However, we identified a significant difference in G-FAL in relation to a history of smoking (P = 0.01), with greater G-FAL noted in smokers (16.7 ± 5.6%) than in nonsmokers (13.2 ± 4.8%).

High frequencies of LOH were also found in lung cancers obtained from smokers as compared with nonsmokers (32) . Cigarette smoking is a known major risk factor for the development of several tumor types, including pancreatic cancer (33 , 34) . Cigarette smoke contains hundreds of toxic chemicals, including the carcinogens benzo()pyrene, nitrosamine, and nitrosamine 4-(methylnitrosamino)-1- (3-pyridyl)-1-butanone (34) . Metabolites of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone have been shown to bind DNA and induce activating point mutations in the TP53 or K-RAS2 genes (35 , 36) as well as stimulate the formation of mitogenic metabolites that stimulate DNA synthesis and proliferation of pancreatic cancer cells (37) . Thus, our finding of increased G-FAL in the pancreatic cancers from smoking patients suggests that one or more carcinogens may potentiate genetic instability in this tumor type.

	FOOTNOTES

 
Grant support: NIH Specialized Programs of Research Excellence in Gastrointestinal Cancer Grant CA 62924.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Scott E. Kern, Department of Oncology, Room 461, Cancer Research Building, 1650 Orleans Street, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231. Phone: (410) 614-3316; Fax: (410) 614-9705; E-mail: sk{at}jhmi.edu

⁷ www.cidr.jhmi.edu.

⁸ Build 34, http://www.ncbi.nlm.nih.gov/mapview/.

Received 9/ 2/03. Revised 11/ 5/03. Accepted 11/10/03.

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