Mapping of Chromosomal Imbalances in Pancreatic Carcinoma by Comparative Genomic Hybridization1

[CANCER RESEARCH 56. 3803-3807. August 15. 19%] Mapping of Chromosomal Imbalances in Pancreatic Carcinoma by Comparative Genomic Hybridization1

Sabina Solinas-Toldo, Christine Wallrapp, Friedericke MÃ¼ller-Pillasch,MartinBentz,2 Thomas Gress, and Peter Lichter3

Deutsches Krebsforschungszentrum, Abteilung Organisation komplexer Genome. Im Neuenheimer Feld 280, D-69120 Heidelberg Â¡S.5-7".. M. B., P. L], and Medizinische UniversitÃ¤tsklinik und Poliklinik. Abteilung innere Medizin l. D-89081 Ulm Â¡C.W.. F. M-P.. T. G.Â¡.Germain

ABSTRACT lyzed 27 cases of pancreatic adenocarcinoma by using CGH4 (10). This approach does not require metaphase preparation of the tumor To identify recurrent chromosomal imbalances in pancreatic adenocar- sample and thus circumvents the limitations of karyotypic analysis cinoma, 27 tumors were analyzed by using comparative genomic hybrid possibly influenced by short-term culturing of tumor cells. CGH is ization. In 23 cases chromosomal imbalances were found. Gains of chro based on the use of genomic DNA of tumor cells as a probe for mosomal material were much more frequent than losses. The most common overrepresentations were observed on chromosomes 16p (eight fluorescence in situ hybridization to normal metaphase chromosomes cases), 20q (seven cases), 22q (six cases), and 17q (five cases) and under- (11, 12). The probe is cohybridized with genomic DNA, isolated from representations on a subregion of chromosome 9p (eight cases). Distinct normal lymphocytes, and visualized with a different fluorochrome. high-level amplifications were found on Ip32-p34, 6q24, 7q22, 12pl3, and Comparison of the signal intensities from tumor and control DNA 22q. These data provide evidence for a number of new cytogenetically probes allows the detection of chromosomal imbalances. Gains or defined recurrent aberrations which are characteristic of pancreatic car losses of chromosomal material in the tumor are indicated by the cinoma. The overrepresented or underrepresented chromosomal regions increase and decrease of the ratio of the fluorescence signal intensi represent candidate regions for potential oncogenes and tumor suppressor ties, respectively. genes, respectively, possibly involved in pancreatic tumorigenesis.

MATERIALS AND METHODS INTRODUCTION Tumor Samples. Tumor tissue from patients with adenocarcinoma of the In Western countries, pancreatic cancer represents the fourth and pancreas (n = 27; 18 males and 9 females) was provided by the Department fifth most common cause of cancer-related death in men and women, of Viszeral and Transplantation Surgery, University of Bern (Bern. Switzer respectively, and the incidence appears to be increasing ( 1, 2). Prog land), Department of Surgery, University of Ulm (Ulm. Germany). Department nosis of the disease is dismal, with a 5-year survival rate of <0.4% of Pathology, University of Heidelberg (Heidelberg, Germany), and the Insti and a medial survival time following diagnosis of 4-6 months (2, 3). tute of Experimental Medicine (Budapest, Hungary) with written consent from Early tumors are very difficult to detect; surgical resection is per each patient and after approval by the local Ethics Committees. The majority formed only when the tumor is localized, whereas advanced tumors of samples derived from tumors of T, or T2 [according to the UICC classifi cation (13)|, which are usually the only ones resected surgically (Table I). extending further into other organs are not considered for resection. Blocks of approximately 0.5 g of pancreatic cancer tissue were flash frozen Â¡n The development of new treatment modalities, as well as diagnostic liquid nitrogen immediately after surgical removal. Tissue blocks were split and preventive approaches, require the understanding of the molecular into four pieces of the same size, and tissue fragments were trimmed to obtain mechanisms underlying the complex multistep process of tumorigen frozen histological sections covering the complete tissue block. Sections of esis in the pancreas. In comparison to other tumors, the knowledge approximately 5 p.m were fixed with acetone and stained with H&E according about the molecular biology of pancreatic cancer is very limited. to standard procedures. Based on microscopic evaluation, only tissue frag Multiple cytogenetic aberrations of primary tumors and of cell lines ments shown to contain more than 50% tumor cells were selected for DNA extraction. DNA preparation was performed according to a modified guani- derived from pancreatic carcinoma detected by using traditional cy togenetic techniques have been described (4-9). According to these dinium thiocyanate extraction protocol, followed by centrifugation in a cesium chloride gradient (14). studies, chromosomal aberrations of chromosome 1, loss of 6q, gain CGH. Probe preparation, hybridization, and image acquisition were per of chromosomes 7 and 20, and aberrations involving chromosomes 17 formed as described previously (15). Briefly, metaphase chromosomes were and 18 seem to be the most frequent rearrangements present in this prepared from female peripheral blood leukocytes according to standard pro tumor. However, it is difficult to obtain sufficient metaphase spreads cedures. Control DNA was isolated from peripheral blood lymphocytes of a of good quality from such tumor specimens for cytogenetic analysis. healthy male individual. One ju.gof tumor DNA and l /ig of control DNA were This could also result in the selection of analyzed clones which are not labeled using nick translation with biotin and digoxigenin, respectively. The representative of the tumor. Moreover, many aberrant chromosomal two labeled DNAs were combined with 80 (xg of human Cot-1 DNA and regions may not have been identified due to the highly complex coprecipitated in ethanol. The DNA was then resuspended in 12 fil of hybrid ization mixture (10% dextran sulfate, 50% formamide, and 2x SSC) and karyotypes of cultured cancer cells carrying both multiple numerical denatured at 75Â°Cfor 5 min. Probe and Cot-1 DNA were then allowed to and structural chromosomal abnormalities. To contribute to the iden reanneal at 37Â°Cfor 25 min and were hybridized to denatured normal female tification of the most common chromosomal abnormalities, we ana- chromosomes. Hybridization was allowed to proceed for 3 days in a moist chamber at 37Â°C.After posthybridization washes in 50% formamide/2x SSC Received 2/28/96; accepted 6/18/96. at 42Â°Cand in 0.1 X SSC at 60Â°C,biotinylated tumor DNA was detected with The costs of publication of this article were defrayed in pan by the payment of page avidin-FITC and digoxigenin-labeled control DNA with mouse antidigoxige- charges. This article must therefore be hereby marked advertisement in accordance with nin antibody conjugated to TRITC. The chromosomes were counterstained 18 U.S.C. Section 1734 solely to indicate this fact. ' This study was supported by Grant 01 GB9401 from Bundes Minister TÃ¼rBildung with 4.6-diamidino-2-phenylindole and embedded in antifade solution und Forschung-FÃ¶rderschwerpunkt Klinisch-Biomedizinische Forschung. (Vectashield). 2 Present address: Medizinische Klinik und Poliklinik V. UniversitÃ¤t Heidelberg. D-69115 Heidelberg. Germany. 3 To whom requests for reprints should be addressed. Phone: 49-6221-424619: Fax: J The abbreviations used are: CGH. comparative genomic hybridization; UICC. Inter 49-6221-424639. national Union Against Cancer; TRITC. telramethylrhodamine. 3803

Table 1 UICC classification i>fthe analyzed minor samples always from a male donor and test and control DNA were not always matched Tumorsample1234567891011121314IS161718192021222324252627UICCclassificationTlb, for sex, scoring of X chromosomal imbalances had to take the sex of the tumor patient into account. N,.MOT,, Nâ€ž.MOT2, Nx,M,T,b.N,.MoTâ€ž RESULTS Nâ€žMOT2. N,.MOT,. Of a total of 27 cases of pancreatic carcinoma analyzed with CGH, N|,MOT~2-N,,MOT2. 23 cases showed chromosomal imbalances. With the exception of Nx,MOT,. chromosomes 2 and 10. all chromosomes were involved in imbal N0.MOT2. ances. A schematic representation of the genetic imbalances obtained N0.MOT2, from the analysis of 10-12 metaphases for each tumor are schemat Nx,MOT,. Nx.MXT,. ically summarized in Fig. 1 and represent the aberrations observed for Nx,MOTV each chromosome in this study. The most frequently identified im Nx.M,T;, NX.MXTâ€ž balances were gains of chromosomal material on chromosome 16p in Nâ€ž,MÂ«T2. eight cases, with the commonly gained region of distal 16p; chromo Nx,MxT,. some 20 in seven cases, five of which were on 2()q only; chromosome Nâ€ž.MÂ«T2. Nx.MXT2. 22q (six cases); chromosome 17q (five cases); and chromosomes 18p Nx.MXT,. and 16q (four cases each). In two tumors the presence of an isochro- N,,M,T3. mosome i( 18p) was suggested by the overrepresentation of 18p and NX,M,â€¢^â€žMOT,. deletion of 18q. Significant loss of chromosomal material was found N,,MXTÃŽ. in the telomeric part of 9p in eight cases and in an interstitial portion N,,MXT;, Nâ€ž.MX of 13q and 6q in four cases each. For the chromosomal regions Ip32-pter and 19, the ratio profiles indicated overrepresentation in 8 and 11 tumors, respectively. These were, however, not scored (*, Fig. Digital Image Analysis. Gray level images of 15-20 metaphases were 1), since the data in these regions are potentially false positive (for taken separately for each fluorochrome using a fluorescence microscope detailed description, see "Materials and Methods"). Profiles of aber (Zeiss) equipped with selective single-bandpass filters and with a cooled rant chromosomes involved in at least five tumors, i.e., chromosomes charged coupled device camera (Photometries). The ratio of FITC:TRITC 9, 16, 17, and 20, are shown in Fig. la. In three cases for each of the fluorescence intensities along each individual chromosome was calculated chromosomes 9. 16, and 20. the profile deviated from the balanced using dedicated software described in more detail elsewhere (16). For each value in the same direction, but did not reach the diagnostic threshold. chromosome the ratio values obtained from 10 to 12 metaphase cells were averaged, and the resulting profile was plotted next to chromosomal ideo These instances are shown separately in Fig. 2. Distinct high copy number amplifications were observed in the following regions: Ip32- grams. Chromosomal imbalances were detected on the basis of the ratio profile deviating from the balanced value (FITC:TRITC = I). The values 1.25 and p34, 6q24, 7q22. 12pl3. each in one case, and 22q in two cases (Figs. 0.75 were used as diagnostic cutoff levels indicating overrepresentations and 1 and 2b). Chromosomal regions 7q22 and 12pl3 were also involved underrepresentalions. respectively. These thresholds for overrepresentation in the gain of chromosomal material in two other tumors and 22q in and underrepresentation correspond to the values expected for a trisomy or four other tumors. In four cases, no aberration could be detected, monosomy in 50% of the diploid cells. Chromosomal regions were scored as suggesting the presence of a balanced karyotype. Although a require Unbalanced regions when the ratio profile either reached or exceeded the ment for the tumor samples used was the presence of at least 50% diagnostic thresholds. The thresholds were thoroughly tested as reported pre cancer cells, there is the possibility that this fraction was too low for viously (16-18) and successfully applied in numerous CGH studies by a number of laboratories (e.g., see Refs. 19-21). Overrepresentations were CGH detection. considered as high-level amplifications when the ratio of the fluorescence exceeded the value of 2.0, or when the FITC fluorescence showed a strong DISCUSSION distinct signal detected by visual inspection and the corresponding ratio profile was diagnostic for overrepresentation. Although cytogenetic analyses of pancreatic adenocarcinoma have The use of female metaphase cells for hybridization did not allow us to revealed complex karyotypic changes, only a few recurrent aberra obtain CGH profiles for the Y chromosome. However, most of the Y chro tions have emerged from these studies (4-9). We analyzed 27 cases of mosome consists of a heterochromatin block containing clustered tandem pancreatic adenocarcinoma using CGH, a very efficient and valuable repeats, a feature common to the helerochromatic and centromeric regions. tool to detect chromosomal imbalances as demonstrated for a variety These areas often show very low signal intensity due to the high suppression by Cot-1 DNA. This results in gross ratio variations from only small variations of different tumors (e.g., Refs. 17-22). In the present study, a number of the fluorescence intensity (15. 16. 22). Accordingly, these regions were not of characteristic genetic changes were identified which were previ considered for CGH analysis. ously not recognized as recurrent aberration using cytogenetic tech Several other chromosomal regions have been recognized as being difficult niques. The newly described overrepresented or underrepresented for CGH analyses (15, 16, 22). Careful testing of our CGH protocol by areas define regions which contain putative proto-oncogenes and multiple hybridization experiments using normal test DNA versus normal tumor suppressor genes, respectively, presumably involved in the control DNA revealed that two of these regions occasionally show ratio values pathogenesis of pancreatic tumors. In the following text, we empha which would be diagnostic for imbalances: chromosomal region Ip32-pter and size those candidate genes which reside in the highly amplified chromosome 19. In contrast, none of the other regions that has been considered regions. difficult for CGH analyses showed a significant deviation of the ratio values. The most frequently found imbalance, gain of the distal region of To avoid the presentation of data with a small potential of being false positive, we did not score the CGH results regarding these two regions, except for the the short arm of chromosome 16, has not been described before as a high copy number amplifications (Fig. 1). recurrent genetic change in this tumor type. In contrast, gain of 20q, When CGH is performed with tumor DNA from a female patient and the second most frequent genetic imbalance detected using CGH, control DNA from a male donor, the ratio value for the balanced state of the confirms previous reports using chromosome banding analysis (5, 7). X chromosome is different (value 2). Since in this study the control DNA was Chromosome 22q was found to be overrepresented in six tumors of 3804

* * *

'Mil

oo* II II

10 11 12

14 15 16 17 18

19 20 21 22

Fig. 1. Summary of genetic imbalances detected in 27 pancreatic adenocarcinomas. Vertical lines on the left, chromosome ideograms indicate loss of genetic material: vertical lines on the right, gain of genetic material. Bold lines, high-level amplifications. Numbers above each line, case analyzed. The distal part of chromosome l p and chromosome 19 are labeled (*), since in these two regions only high copy number amplifications were scored. In this illustration, low copy number imbalances of these regions were neglected for reasons which are outlined in detail in "Materials and Methods."

our study. Its commonly overrepresented region is characterized in region are known to be frequently involved in allelic loss in pancreatic part by two high copy number amplifications, defining 22ql2-ql3 as cell lines and xenografts (24). Caldas et al. (24) proposed the the critical region. This region harbors three proto-oncogenes (23): CDKN2A gene as a functional target for the frequent loss of chromo PDGFB, ECGF1, and EWSR1. some 9p. Recently, this observation has been confirmed (25, 26), the The distal part of chromosome 9p confined to the region latter reference reporting the occurrence of mutations in the CDKN2A 9p21â€”>9pterwas found frequently lost (30% of the cases). Notably, in gene in 34% of primary pancreatic tumors. Thus, our CGH results are three additional cases the ratio profiles of this region were clearly in agreement with the current hypothesis of an important role for shifted toward the threshold for underrepresentation (Fig. 2a). Al CDKN2A or CDKN2B in the development or progression of pancre though they do not reach the diagnostic value, they are suggestive for atic carcinoma. the existence of a loss of the corresponding chromosomal material in Chromosome arm 6q appears to be an area recurrently lost in cancer a smaller portion of tumor cells. Whereas previous karyotypic anal of the pancreas (5, 9). In the present study, in only four cases could yses of pancreatic tumors did not reveal 9p deletions, two tumor loss of 6q be diagnosed with CGH (Fig. 3a), whereas in 12 cases the suppressor genes, CDKN2A (pl6) and CDKN2B (pl5), located in this CGH profile suggested loss of chromosomal material in this region. 3805

case25 23 19 16 18 22 12 13 17

I 1 JJ jd TI m il 15 17 20 22 25 5 16 21 24

Fig. 2. a, average ratio profiles of the chromo somes (Chrom.) most frequently affected by loss (Chrom. 9) and by gain (Chrom. 16.17, and 20) of chromosomal material. The ratios of FITC:TR1TC fluorescence are plotted along the chromosome ideograms. The intensity ratio of a balanced copy number calculated from the mode of the intensity Kill ININN*IVWIÂ«IN ratio (value of 1.0. centra! line), and the threshold for overrepresentations (value of 1.25, right line) and underrepresentaiions lvalue of 0.75. left line) are shown (see Ref. 16). Centromeric regions are known io be difficult in CGH analyses (see "Ma terials and Methods"). Accordingly, these regions were not considered and appear as shaded bttxcs. Ratio profiles suggesting a copy number change of the respective chromosomal region but not o reaching the diagnostic threshold are listed sepa k Â£ ID rÂ» rately on the righi (under bold lines), b. average U E E ratio profiles of six high copy number amplifica e O tions in five genomic regions found in four differ ent tumor samples. Below each profile a repre sentative example is shown.

e a se 26 15 2Â« 15 22

but the ratio values dici not exceed the diagnostic threshold (Fig. 3e). high-level amplification on 7q22 is close to the proto-oncogenes The high frequency of ratio values below the diagnostic threshold WNT2 and MET: the latter encodes the receptor for hepatocyte growth might be due to the loss of material in a smaller portion of cells. Since factor scatter factor and is located on 7q31. MET has recently been in the corresponding 12 cases many other chromosomal imbalances found to be up-regulated in the majority of pancreatic ductal adeno- could be diagnosed using CGH, this might indicate that loss of 6q is carcinoma and overexpressed in some human pancreatic cancer cell a late event. lines, suggesting a possible role for this receptor in the growth and A remarkable finding of the present CGH study is the occurrence of behavior of pancreatic cancer (27). The overrepresentation of 12p six high copy number amplifications in 4 of the 27 tumors. Previous coincides with the localization of the proto-oncogene RAS ( 12pl2.1) cytogenetic studies have not revealed double minute chromosomes or and of the cyclin CCND2 gene (12pl3; Ref. 23). About 90% of the homogeneously staining regions, which both are chromosomal equiv pancreatic carcinoma cases contain a point mutation at the 12th codon alents of such amplifications. Since high copy number amplicons are of the RAS oncogene (28, 29). Overrepresentation of 12p in two more usually confined to DNA segments ^ 1 MB, they provide a good basis cases of our study might be considered as additional support for a role for the identification of candidate proto-oncogenes. In the amplified of the RAS gene in pancreatic tumorigenesis. region Ip32-p34, such candidate genes are the malignant TFSÃŒgene Surgical resection of pancreatic adenocarcinomas is usually per ( Ipter-p22.1 ) and RAB3B, a member of the RAS oncogene family formed only when tumors are localized and restricted to a confined (Ip32-p31). The high copy number amplification on 6q24 coincides area (see above). Accordingly, tumor samples available for genetic with the localization of the MYB and MASI oncogenes, the latter studies are derived from similar tumor stages greatly impeding cor known to be associated with tumor-specific rearrangements. The relation analyses of tumor stages or clinical course of the disease with 3806

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1996 American Association for Cancer Research. Mapping of Chromosomal Imbalances in Pancreatic Carcinoma by Comparative Genomic Hybridization

Sabina Solinas-Toldo, Christine Wallrapp, Friedericke Müller-Pillasch, et al.

Cancer Res 1996;56:3803-3807.

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