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[Cancer Research 62, 835-839, February 1, 2002]
© 2002 American Association for Cancer Research

Molecular Biology and Genetics

Interglandular Cytogenetic Heterogeneity Detected by Comparative Genomic Hybridization in Pancreatic Cancer

Tomohiko Harada1, Kiwamu Okita, Kei Shiraishi, Noriyoshi Kusano, Satoshi Kondoh and Kohsuke Sasaki

Department of Pathology [T. H., K. Sh., N. K., K. Sa.] and First Department of Internal Medicine [T. H., K. O., K. Sh., N. K., S. K.], Yamaguchi University School of Medicine, Yamaguchi 755-8505, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The aim of this study is to explore the mechanisms of intratumoral cytogenetic heterogeneity (ICH) in pancreatic cancer. Using comparative genomic hybridization (CGH) analysis, we investigated interglandular variation in 20 primary invasive ductal adenocarcinomas of the pancreas. Three or four adjacent neoplastic glands were individually microdissected from a tumor specimen. Extracted DNA from each gland was amplified by degenerate oligonucleotide primed-PCR, followed by CGH. In addition, DNA index (DI) was measured by laser scanning cytometry in each case. CGH profiles displayed a wide variety of differences between glands within the same tumor in all cases, i.e., interglandular cytogenetic heterogeneity was distinct in pancreatic cancers. In this study, genetic changes detected in all regions of a tumor were classified as "region-independent" alterations, whereas changes seen in at least one, but not all regions were designated as "region-dependent" alterations, which resulted in ICH. The degree of ICH, which was manifested as the ratio of these two types of alterations, correlated closely with DI (Spearman {rho} = 0.842; P = 0.0002). Therefore, DI might be a surrogate marker for ICH. These results suggest that with tumor progression, ICH and DNA aneuploidy result from the successive appearance of region-dependent alterations attributable to chromosomal instability in tumor cells. Our data support a concept of individual cell heterogeneity in pancreatic cancer.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although a tumor arises from a single progenitor cell, subpopulations of neoplastic cells with different genotypic and phenotypic characteristics appear within a tumor during its progression. Such successive changes are considered attributable to genetic instability inherent in cancer cells, resulting in ICH2 (1) . The study of ICH is important not only for understanding variations in the biological characteristics of tumors, but also for developing treatment strategies (2, 3, 4, 5) . ICH has been investigated by various molecular cytogenetic techniques in PCs as well as in other solid tumors (6, 7, 8, 9, 10) . However, only limited chromosomal regions or genes have been examined in these prior studies.

CGH is a useful technique that provides information about DNA copy number alterations across the entire genome of malignant tumors (11 , 12) . Although CGH analyses have been performed by several investigators in primary PCs, these studies focused on a single sample taken from each tumor and provided no information about ICH (13, 14, 15, 16) . Because considerable amounts of tissue are necessary to obtain enough DNA for standard CGH analysis, it is difficult to examine chromosomal imbalances in small specimens. However, DOP-PCR overcomes this difficulty because it allows amplification of the whole genome by PCR (17 , 18) . We previously showed that DOP-PCR makes it possible to perform CGH analysis using DNA from 15 copies of a diploid genome and that as few as 7 copies can be used (19) .

The aim of this study was to explore the mechanisms of ICH in PC. For that purpose, we used DOP-PCR CGH to investigate genetic variations between multiple, individually dissected neoplastic glands within a single tumor. In addition, we examined the relationship between ICH and nuclear DNA content of tumor cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tumor Specimens.
We studied 20 primary PCs that were obtained surgically or at autopsy at Yamaguchi University Hospital and its affiliated hospitals (Table 1)Citation . The neoplasms were from 12 men and 8 women whose mean age at collection was 63.5 years. None of the carcinomas had been treated by chemotherapy or radiation therapy prior to resection of the tumor. Fifteen tumors were located in the head of the pancreas, 4 in the body, and 1 in the tail. Histologically, 16 specimens were classified as moderately differentiated tubular adenocarcinomas, 3 were poorly differentiated, and 1 was well differentiated. According to the Union International Contre Cancer classification, 1 was classified as stage II, 5 as stage III, 3 as stage IVa, and 11 as stage IVb. All specimens were stored at -80°C until use. The study protocol was approved by the Institutional Review Board for Human Use at the Yamaguchi University School of Medicine in May 1995, and informed consent for this study was obtained from all patients and/or their families.


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  		Table 1 Total cases of PCs

 
Microdissection and DNA Extraction.
Under microscopic observation, three or four samples per tumor were carefully microdissected from surrounding stromal tissue with a disposable fine needle. Samples were taken from neoplastic glands that were topographically separate but adjacent to each other (Fig. 1)Citation . Each tissue fragment contained ~50–100 tumor cells. DNA was extracted by digestion in 400 µg/ml proteinase K solution for 12 h at 55°C.



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  		Fig. 1. Invasive ductal adenocarcinoma of the pancreas (Table 1Citation , case 1). Each of four neoplastic glands (a–d) was microdissected individually. H&E staining; magnification, x50.

 
DOP-PCR.
DOP-PCR was performed using universal primer 6-MW (5'-CCGACTCGAGNNNNNNATGTGG-3'; N-G, C, A, and T) on a thermocycler (ASTEC, Fukuoka, Japan) with slight modifications as described previously (17, 18, 19, 20) . Briefly, a 1-µl aliquot of microdissected DNA was pretreated with 1 unit of topoisomerase-I (Promega, Madison, WI) for 30 min at 37°C. The topoisomerase-I pretreatment was followed by five treatment cycles (1 min at 94°C, 2 min at 30°C, and 2 min at 37°C) using 20 units of Thermosequenase (Amersham, Cleveland, OH). Preamplification was followed by one cycle at 95°C for 10 min; 45 µl of 1x PCR buffer containing 2.5 units of Taq DNA polymerase (TaKaRa, Shiga, Japan) was then added. This was followed by 35 cycles at 94°C for 1 min, 56°C for 1 min, and 72°C for 3 min, with a final extension at 72°C for 5 min.

CGH and Digital Image Analysis.
CGH analysis was carried out as described previously (19) . DNA from tumor tissues and reference normal cells was labeled with SpectrumGreen-dUTP and SpectrumRed-dUTP (Vysis Inc., Downers Grove, IL), respectively, by nick translation. Each labeled DNA sample (200 ng) and 10 µg of Cot-1 DNA (Life Technologies, Inc., Gaithersburg, MD) were dissolved in 10 µl of hybridization buffer and cohybridized onto normal denatured metaphase chromosomes for 48 h at 37°C. The specimens were mounted in an antifade solution containing 0.15 mg/ml 4',6-diamino-2-phenylindole as a counterstain. Images were captured with an Olympus BX 50 epifluorescence microscope equipped with a x100 UplanApo objective and a CCD camera (SenSys 1400; Photometrics Ltd., Tucson, AZ). The digital image analysis system (QUIPS XL; Vysis Inc.), developed specifically for CGH, was used in this experiment. Increases and decreases in DNA sequence copy number were defined by tumor:reference ratios of >1.2 and <0.8, respectively. High-level copy number increases in subregions (amplifications) were defined by a tumor:reference ratio of >1.4. In the present study, CGH analysis was performed at least two times per sample to obtain reliable results. Chromosomes 1p and 19 were excluded from CGH analysis because of the well-known uncertainty of these loci (12 , 19) .

Laser Scanning Cytometry.
Laser scanning cytometry was carried out as described previously (1 , 21 , 22) . Briefly, touch preparations of frozen specimens were made and fixed in ethanol at room temperature. The cells were stained in propidium iodide (50 µg/ml; Sigma Chemical Co., St. Louis, MO) solution containing 0.1% RNase (Sigma Chemical Co.). DI was measured by a laser scanning cytometer (LSC101; Olympus, Tokyo, Japan).

HI.
Referring to a report of Georgiades et al. (23) , we introduced HI into assessment of ICH in this study. The proportion of the number of "region-dependent" alterations to the number of total alterations per sample (HI) was calculated as follows:

where RIA is the total number of "region-independent" alterations detected in a tumor, RDA is the total number of region-dependent alterations detected in a tumor, and n is the number of samples from a tumor.

Statistical Analysis.
Statistical analysis was performed with a statistical software package (StatView). Fisher’s exact test was used to examine the difference in the frequencies of genetic alterations between two groups. Correlation between two indices was evaluated by Spearman’s rank correlation test. Differences at P < 0.05 were considered statistically significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Recurrent Genetic Alterations in Overall Cases.
We analyzed 71 samples taken from 20 cases (Table 2)Citation . In all cases, CGH profiles differed by various degrees between adjacent neoplastic glands within a tumor. All samples showed genetic alterations at 6–25 chromosomal loci. As shown in Fig. 2Citation , the highest frequency of gains was seen on 8q, followed by 3q, 20q, 7p, 1q, and 13q. The highest frequency of losses was seen on 17p, followed by 9p, 18q, 8p, 6q, 6p, 9q, and 22q.


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  		Table 2 Genetic alterations detected by DOP-PCR CGH

 


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  		Fig. 2. Frequencies of genetic alterations on individual chromosome arms in 20 cases. Thick lines, amplification; thin lines, region-independent alterations; hatched lines, region-dependent alterations. Chromosomes 1p and 19 were excluded from CGH analysis because of the well-known uncertainty of these loci.

 
Two Types of Genetic Alterations.
In the present study, genetic changes detected in all regions of a tumor were classified as region-independent alterations, and these were found more frequently in DNA copy number decreases than in increases (Fig. 2)Citation . Changes seen in at least one, but not all regions were designated as region-dependent alterations, and they represented interglandular differences in the CGH profile (Fig. 3)Citation . Region-dependent alterations seen in at least two regions of a tumor were located in the exactly same areas of the chromosome and, therefore, were likely to affect the same genes. As shown in Fig. 4Citation , recurrent genetic alterations in this study were identified as region-independent changes with a various probability.



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  		Fig. 3. Representative CGH profiles of both region-independent and -dependent alterations in case 9. Amplification of 12p12-pter was detected in all regions of a tumor, whereas gains of 7p, 12q24-qter, and 14q31 were in parts of a tumor. Ch, chromosome number.

 


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  		Fig. 4. Recurrent genetic alterations classified by type in 20 cases. Alterations were identified as region-independent alterations with a various probability.

 
HI and DNA Ploidy.
HI was calculated in each case to assess the degree of ICH and ranged from 3.13 to 14.58, with an average of 7.10 (Table 1)Citation . There was no relationship of HI with clinicopathological parameters (age, sex, tumor portion, histology, stage, and TNM classification). DNA ploidy pattern was diploid (DI = 1.00) in 6 cases (30%) and aneuploid (DI > 1.00) in 14 cases (70%; Table 1Citation ). There were no significant differences in the frequencies of any genetic alteration between diploid and aneuploid tumors. DI correlated closely with HI (Spearman {rho} = 0.842; P = 0.0002; Fig. 5Citation ).



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  		Fig. 5. High degree of correlation between DI and HI (Spearman {rho} = 0.842; P = 0.0002).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In four previously reported CGH analyses of primary PCs, the results showed that frequent changes were gains of 8q and 20q and losses of 6q, 8p, 9p, 17p, and 18q (13, 14, 15, 16) . These alterations were presumed representative of the entire tumor. However, there were large discrepancies between reports in the overall frequencies of these aberrations. ICH may explain the differences not only between study results, but also between samples.

Gains of 8q, 3q, 20q, 7p, 1q, and 13q and losses of 17p, 9p, 18q, 8p, 6q, 6p, 9q, and 22q were detected as recurrent alterations in our series. Previous cytogenetic approaches indicated that these are usually nonrandom changes in pancreatic carcinogenesis (24, 25, 26, 27, 28) . Indeed, these changes were frequently found as region-independent alterations in the present study. However, even recurrent changes in our study were not always region-independent alterations (Fig. 4)Citation . These results might indicate a multiplicity of genetic background in PC.

CGH profiles displayed a wide variety of differences between adjacent neoplastic glands in all cases. To our knowledge, this is the first report describing a distinct difference in genetic alterations between neoplastic glands within a tumor, i.e., interglandular cytogenetic heterogeneity. In addition, there was a significant correlation between DI and HI, and DI might be a surrogate marker for ICH.

Because malignant tumors intrinsically exhibit genetic instability, successive genetic alterations accumulate with tumor progression, although they may differ in each cell of a tumor. It is well known that the total number of genetic alterations increases with tumor progression in various tumors (29) . Chromosomal instability in cancer cells leads to disruption of both cell cycle regulation and chromosomal segregation mechanisms, resulting in DNA aneuploidy (30, 31, 32, 33) . These previous findings support our present findings that the appearance of region-dependent alterations resulting in ICH is associated with DI. These regional genetic differences may explain morphological variations between neoplastic glands in a tumor. In addition, it is likely that biological characteristics of a tumor are affected by region-dependent alterations, which occur at the late stage in pancreatic carcinogenesis. These insights, when applied to ICH between adjacent tumor cells, lead to a concept of individual cell heterogeneity in a tumor. Biological behavior may be different between cells as well as between glands in the same tumor.

The present observations revealed that interglandular cytogenetic heterogeneity was distinct in PCs. In addition, our data suggest that with tumor progression, the development of ICH and the increase of nuclear DNA content result from the successive appearance of region-dependent alterations attributable to chromosomal instability in tumor cells.


   ACKNOWLEDGMENTS
 
We thank Drs. Shinji Noshima and Tadahiko Enoki (Department of Surgery I, Yamaguchi University School of Medicine) and Drs. Tomio Ueno and Kohtaro Yamamoto (Department of Surgery II, Yamaguchi University School of Medicine) for providing tumor specimens from PCs. In addition, we thank Drs. Tomoyuki Murakami and Kenji Umayahara for technical assistance.


   FOOTNOTES
 
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.

1 To whom requests for reprints should be addressed, at Department of Pathology, Yamaguchi University School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi 755-8505, Japan. Phone: 81-836-22-2222; Fax: 81-836-22-2223; E-mail: tharada{at}po.cc.yamaguchi-u.ac.jp Back

2 The abbreviations used are: ICH, intratumoral cytogenetic heterogeneity; CGH, comparative genomic hybridization; PC, pancreatic cancer; DOP-PCR, degenerate oligonucleotide primed-PCR; DI, DNA index; HI, heterogeneity index. Back

Received 7/27/01. Accepted 12/ 3/01.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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