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High frequency of allelic imbalance at regions of chromosome arm 8p in ovarian carcinoma
Cancer Genetics and Cytogenetics 129 (2001) 23–29
High frequency of allelic imbalance at regions of chromosome arm 8p in ovarian carcinoma Ingrid Pribilla,1, Paul Speiserb,1, Jennifer Learyc, Sepp Leodolterb,d, Neville F. Hackere, Michael L. Friedlanderf, Daniel Birnbaumg, Robert Zeillingerb,*, Michael Krainera a
Department of Medicine I, Clinical Division of Oncology, University Hospital, Molecular Oncology Group, Währingergürtel 18-20, A-1090 Vienna, Austria b Department of Gynecology and Obstetrics, University Hospital, Molecular Oncology Group, Währingergürtel 18-20, A-1090 Vienna, Austria c Department of Medical Oncology, Westmead Hospital, Westmead, Australia d Ludwig Boltzmann Institute for Gynecologic Oncology and Reproductive Medicine, University Hospital, Molecular Oncology Group, Währingergürtel 18-20, A-1090 Vienna, Austria e Gynaecological Cancer Centre, Royal Women Hospital, Randwick, Australia f Department Medical Oncology, Prince of Wales Hospital, Randwick, Australia g Institut Paoli-Clamettes, U.119 Inserm, Marseille, France Received 17 October 2000; received in revised form 11 January 2001; accepted 15 January 2001
Abstract
Progressive genetic changes such as the inactivation of tumor suppressor genes (TSG) are thought to play an important role in the initiation and progression of ovarian cancer. Frequent nonrandom allelic imbalance (AI) at 8p11p21 and 8p22pter suggests the existence of TSGs that may be involved in the carcinogenesis of several human malignancies. We investigated 70 ovarian tumors with 11 highly polymorphic markers spanning 8p12p21 and 8p22pter to produce an AI map of 8p in epithelial ovarian cancer. Allelic imbalance was demonstrated in 54 tumors (77%), most frequently occurring at D8S136 (54%) and at D8S1992 (55%). Poorly differentiated and advanced stage cancers were more often affected by AI (G1G2 vs. G3; 20% vs. 66%; stage III vs. IIIIV, 36% vs. 54%, P.001; Kruskal-Wallis test) than well differentiated and early stage tumors. There was no relationship between histological subtype and AI. Smallest regions of overlap (SRO) were delineated by analyzing 38 tumors with partial AI. This study provides compelling evidence for the involvement of TSGs on the short arm of chromosome 8, at 8p12p21 and at 8p23 in the development and progression of epithelial ovarian cancer. © 2001 Elsevier Science Inc. All rights reserved.
1. Introduction The molecular basis of ovarian cancer, and the genetic events leading to the initiation and progression of this disease, are poorly understood. As in other human malignancies, progressive genetic changes including the activation of proto-oncogenes, as well as inactivation of tumor suppressor genes (TSG) and anti-metastatic genes are thought to occur. Sustained allelic imbalance (AI) of polymorphic markers at a certain chromosomal region suggests the exist-
* Corresponding author. Tel.: 43-1-40400-7831; fax: 43-1-404007832. E-mail address: [email protected] (R. Zeillinger). 1 These authors contributed equally to the work.
ence of a gene involved in carcinogenesis. Advances in localizing and mapping polymorphic markers has made it possible to conduct comprehensive studies on AI on very small amounts of tumor DNA isolated from tumor cell enriched samples. Nonrandom AI affecting the short arm of chromosome 8 has been demonstrated in a number of human malignancies including prostate, colon and breast cancer [1,2]. In ovarian cancer the short arm of chromosome 8 has not yet been extensively studied. Of the studies reported so far, several have investigated only a few polymorphic markers [3–6], while others have focused on limited chromosomal regions [7,8]. The aim of our study was to extend the investigations of AI on 8p to 70 sporadic ovarian tumors and correlate our results with published findings and with clinico-pathological features wherever possible.
0165-4608/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S0165-4608(01)00 4 1 9 - 8
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I. Pribill et al. / Cancer Genetics and Cytogenetics 129 (2001) 23–29
2. Materials and methods 2.1. Study population We investigated 23 (33%) early stage (FIGO Stages I and II) tumors, including 5 borderline tumors, and 47 (67%) advanced stage (FIGO Stages III and IV) tumors. The majority of tumors were serous cancers (64%), but in addition there were 6 clear cell carcinomas, 6 endometrioid adenocarcinomas, 5 mucinous, 3 mixed, 2 undifferentiated cancers, and 1 carcinosarcoma. In three cases, there was no information on the histological type. Of the invasive cancers, 5 tumors were well differentiated, 14 moderately and 38 poorly differentiated. The clear cell cancers were not graded. 2.2. Tissue samples Ovarian tumor biopsies were obtained from 70 patients undergoing initial staging or debulking laparotomy at several major teaching hospitals in Sydney and Brisbane (Australia). Prior to freezing sample mass was reduced by dissecting away normal tissue. Germline DNA was prepared from peripheral blood leukocytes (PBL). High molecular weight DNA was obtained by standard methods, involving
proteinase K digestion and phenol/chloroform extraction. None of the patients had undergone treatment prior to surgery or had a known family history of Lynch II, familial ovarian cancer or hereditary breast-ovarian cancer. All patients were staged according to the FIGO classification and histological classification was based on the typing criteria of the WHO. 2.3. Allelic imbalance analysis Eleven highly polymorphic microsatellite markers mapping to chromosome arm 8p were used to test for AI. Primer sequences for the following markers were chosen and PCR was done as previously published: LPL-F, D8S133 [9], D8S136 [10], D8S137 [11], D8S258, D8S259, D8S261, D8S283, D8S535 [12], D8S339 [13], D8S1992 [14]. Senseprimers were fluorescently labeled with Cy5 (MWG, Munich, Germany). 2.4. Quantitation of allelic imbalance PCR products were separated on a 6% PAGE (19:1 acrylamide:bis) containing 7 M urea using an automated laser fluorescent sequencer apparatus (ALF express DNA Sequencer, Pharmacia, Uppsala, Sweden), and their size deter-
Fig. 1. Computer representation of the analysis of the amplification products from the microsatellite locus D8S133 by automated polyacrylamide gel electrophoresis. Allelic peaks are indicated by small squares. M, size marker 100 bp; Patient one is an example of retention of both alleles: N1, normal DNA; T1, tumor DNA. Patient two is an example of LOH: N2, normal DNA; T2, tumor DNA.
I. Pribill et al. / Cancer Genetics and Cytogenetics 129 (2001) 23–29
mined using Fragment Manager Software (Pharmacia). A mixture of Cy5-labeled DNA-fragments (50–500 bp in length) was used as an external size marker. An imbalance factor was defined as the ratio of relative allelic peak intensities of the tumor DNA to corresponding PBL DNA samples. An imbalance factor 1.5 or 0.67 was considered significant (Fig. 1). 2.5. Statistical analysis To correlate histopathological parameters with AI on chromosome arm 8p, the Fisher’s exact test was applied. The Kruskal-Wallis test was used to assess correlation between the frequency of AI, tumor differentiation grade and disease status. The Chi square-test was applied to correlate the grade of differentiation and disease stage.
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(11%) and 11 advanced (23%) stage tumors had AI with all markers, indicating extensive loss of genetic material. Next, we analyzed each marker separately for the frequency of AI (Table 1). For region 8p12p22 we found AI most frequently occurring at D8S136 (54%). At 8p23, AI was most frequently found at D8S1992 (55%). Again, to explore possible differences between early stage and advanced stage tumors, we analyzed these groups separately (Table 1). At 8p12p22, in the early stage tumors, AI most frequently occurred at D8S259 (57%). In the advanced stage tumors, all markers apart from D8S259 and D8S283 showed AI in 50% or more of the samples. Three out of five borderline tumors had evidence of AI at 8p involving D8S1992 in one case, and D8S136 in two cases. 3.2. Delineation of smallest region of overlap on chromosome arm 8p
3. Results 3.1. Allelic imbalance on chromosome 8p We investigated 11 markers in 70 tumors. A heterozygous, and thus informative situation, was detected in 73% of all loci tested. The overall frequency of AI in 65 invasive ovarian carcinomas (5 borderline tumors excluded) was high, with 51 cases (78%) demonstrating AI with at least one marker, and 44 tumors (68%) showing AI with two or more markers. In 38 cases (58%), at least one marker showed retention of heterozygosity (partial AI), while in 13 tumors (20%) all informative markers spanning 8p showed AI, indicating a large deletion, or possibly loss of the entire chromosome. Interstitial retention of heterozygosity, which reveals AI at multiple loci in the same tumor, was found in 21 cases (32%). To investigate the relationship between disease stage and AI, we analyzed separately 18 invasive early stage (5 borderline tumors were excluded) and 47 advanced stage ovarian cancers. Sixty-one percent of early and 85% of advanced stage tumors showed evidence of AI. Two early
We investigated 38 tumors with partial AI to delineate smallest regions of overlap (SRO) and divided the samples into advanced and early stage tumors (Fig. 2). One SRO emerged in the region of highest loss around marker D8S261. The centromeric breakpoint is defined by retention of marker LPL-F in several tumors (192B, 125C, 142D, 55A, 140B, 68B) whereas the telomeric breakpoint is only hold by one tumor (192B), so that the SRO around D8S261 may well include marker D8S1992. The possible SRO D8S259–D8S535 with breakpoints D8S339 and D8S283 is found deleted in early stage as well as in late stage cases. In contrast, SRO D8S136–D8S137, defined by retention of heterozygosity of markers D8S133 and D8S339, was observed only in the advanced tumors. 3.3. Correlation to histopathological parameters We correlated AI with grade of differentiation, stage and histological tumor type. The percentage of markers showing AI was significantly lower in the group of well to moder-
Table 1 Summary of allelic imbalance (AI) in tumor samples ALL
Stage I II
Stage III IV
Grade 1 2
Grade 3
Marker
Inform. patients # (%)
# AI
% AI
Inform. patients # (%)
# AI
(%) AI
Inform. patients # (%)
# AI
% AI
Inform. patients # (%)
# AI
% AI
Inform. patients # (%)
# AI
% AI
D8S1992 D8S261 LPL-F D8S258 D8S133 D8S136 D8S137 D8S339 D8S259 D8S535 D8S283
51 (73) 51 (73) 62 (89) 47 (67) 45 (64) 59 (84) 45 (64) 50 (71) 52 (74) 47 (67) 57 (81)
28 23 27 21 21 32 22 21 23 19 19
55 45 44 45 47 54 49 42 44 40 33
13 (72) 14 (77) 17 (94) 13 (72) 12 (66) 13 (72) 10 (56) 13 (72) 14 (77) 13 (72) 10 (3)
6 4 5 4 5 4 3 5 8 3 3
46 29 29 31 42 31 30 38 57 23 30
34 (72) 32 (68) 41 (22) 30 (64) 30 (64) 42 (89) 32 (68) 32 (68) 34 (72) 30 (64) 42 (89)
21 19 22 17 16 26 19 16 15 16 16
62 59 54 57 53 62 59 50 44 53 38
17 (81) 14 (67) 18 (86) 13 (62) 16 (76) 16 (76) 15 (71) 16 (76) 13 (62) 17 (81) 16 (76)
7 4 4 4 3 6 5 7 5 5 3
41 29 22 31 19 38 33 44 38 29 19
29 (76) 26 (68) 33 (87) 26 (61) 22 (58) 33 (87) 22 (58) 23 (61) 32 (84) 21 (55) 32 (84)
18 16 20 14 15 19 14 11 15 11 13
62 62 61 54 68 58 64 48 47 52 41
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Fig. 2. Map of allelic imbalane (AI) in human ovarian carcinomas. Only tumors with partial AI are illustrated: , AI; retention of heterozygosity. Analysis of shortest regions of overlap (SRO): —, SRO; ni, noninformative; nd, not done.
ately differentiated (G1G2) tumors (20%) than in the group of poorly differentiated (G3) tumors (66%) (P.001, Kruskal-Wallis test). A similar relationship existed for tumor stage and AI was significantly lower (36%) among early stage tumors as compared to advanced stage tumors (54%) (P.001, Kruskal-Wallis test). Differentiation grade was significantly associated with stage (G1G2 and G3 vs.
stage III and IIIIV; P.001, chi square-test). We found no association between AI and histological subtype. 4. Discussion In this investigation seventy-seven percent of invasive ovarian cancers showed AI at 8p. LOH was more com-
I. Pribill et al. / Cancer Genetics and Cytogenetics 129 (2001) 23–29
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Fig. 3. Integrational map comparing LOH data in ovarian cancer and homozygous deletions (HD) found in several other tumors. Bold markers represent anchor markers from genemap99 (http://www.ncbi.nlm.nih.gov/genemap99). Distances are given in centiRay (cR 3000) according to the Stanford G3 radiation hybrid map. The first column summarizes our own data whereas the following columns represent the results taken from the literature. X denotes marker analyzed in the individual study.
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monly found in poorly differentiated (G3) than in well differentiated (G1 & 2) tumors and more often in advanced (Stage III & IV) than in early stages (Stage I & II) of the disease. When tumors with partial LOH were analyzed for SROs three potential areas were identified. This corroborates the findings of other investigators, which suggest that the short arm of chromosome 8 may harbor several TSGs involved in the development and progression of ovarian cancer. The comparison and integration of data available from studies of genetic loss in tumors is often hampered by the use of different markers in each investigation. However, we have been able to put the markers used in our study and in two other similar investigations in context with the anchor markers of the genemap99 (http://www.ncbi.nlm. nih.gov/genemap99.org) (Fig. 3). This allows us to compare results from the three most comprehensive LOH studies in ovarian cancer published to date. The first of these studies to be published focused mainly on the telomeric region 8p22–p23 [7]. Fifty-three tumors were investigated using initially eight microsatellite markers. Twenty-seven cases (51%) showed AI and in 16, all markers were involved indicating loss of large parts of 8p. We found the overall frequency of AI on 8p in invasive tumors to be higher at 78%. This may reflect our use of more sensitive methods of computer-based quantitation of AI. Furthermore, we found that only 24% of tumors showing AI had all markers involved, significantly less then the 60% reported by Wright et al. [7]. This difference might be explained in part by differences in the patient cohorts, specifically the higher percentage of early stage tumors in our series. Wright et al. found AI correlated only with tumor stage but not with grade of tumor differentiation. In contrast, we found not only early stage, but also well differentiated tumors were significantly less frequently affected by AI than late stage and relatively undifferentiated tumors. This was no surprise, since the correlation between tumor differentiation and tumor stage is a very well described phenomenon in ovarian carcinoma [15]. To investigate SROs at 8p22–p23, Wright et al. analyzed nine tumors with additional eight markers and delineated three SROs including the loci D8S550-D8S264, D8S549, and LPL. The most telomeric SRO found by us seems to be in the same region as the centromeric SRO in the series of Wright et al. The second LOH study on chromosome 8p in ovarian cancer published, analyzed 45 ovarian cancer cases with 6 highly polymorphic markers and found LOH of one of these markers in 23 of 40 (58%) tumors [8]. The highest loss showed marker D8S136 in 15 of 30 informative cases with lower rates at adjacent loci. This finding is in accordance with our results, where the same marker showed the highest loss particularly in the advanced tumor stages. As in the previous two studies discussed previously, there was a direct correlation between tumor aggressiveness and LOH at chromosome 8. The short arm of chromosome 8 has not been investigated solely in the context of ovarian cancer, but in a wide
variety of other human malignancies and AI was frequently displayed. Allelic imbalance at 8p was found as an early as well as a late event in carcinogenesis [1,2]. In the future, identification and cloning of putative 8p tumor suppressor genes will reveal whether one or several different genes are involved in prostate, breast and ovarian tumorigenesis and whether the timing of gene inactivation relates to tumor progression. To achieve this goal the identification of regions of homozygous deletions (HD) is a critical step, since the loss of the second allele usually covers a smaller chromosomal region. Fig. 3 shows an overview of areas of homozygous loss identified on the short arm of chromosome 8 in various cancer types. Most of the studies have been performed in prostate cancer [1,16–18]. Additional HDs were identified in pancreatic carcinoma [19] and oral cancer [2]. Interestingly, all SROs identified in ovarian cancer to date are in areas where HDs have been described in the literature at least in other tumor entities corroborating the significance of the LOH findings. This information, taken together with the progress of the Human Genome Project in generating sequence and mapping information (as illustrated in the use of the genemap99 in this article) will greatly facilitate the identification of candidate TSGs in ovarian cancer. A challenge for the future will be to develop clear criteria and innovative methods to quickly evaluate the high number of genes and ESTs created by random sequence efforts, which map to these LOH areas like, e.g., FEZ1 [20] as possible TSGs. Proof for the involvement of any of these candidate genes in tumorigenesis may lead to a better understanding of the biology and ultimately better diagnostic and treatment options for this common cancer.
Acknowledgments We would like to thank Eva Schuster and Carina Bednar for their help in this study and Dr. Suzanne Brill for a critical review of the manuscript. This work was supported by “Austrian Research Funds” Nr.: J01313-MED.
References [1] Van Alewjik DC, Van der Weiden MM, Eussen BJ, Van den AndelThijssen LD, Ehren-Van Eekelen CC, König JJ, Van Steenbrugge GJ, Dinjens WN, Trapman J. Identification of a homozygous deletion at 8p12–21 in a human prostate cancer xenograft. Genes Chromosom Cancer 1999;24:119–26. [2] Ishwad CS, Shuster M, Bockmühl U, Thakker N, Shah P, Toomes C, Dixon M, Ferrell RE, Gollin SM. Frequent allelic loss and homozygous deletion in chromosome band 8p23 in oral cancer. Int J Cancer 1999;80:25–31. [3] Emi M, Fujiwara Y, Nakajima T, Tsuchiya E, Tsuda H, Hirohashi S, Maeda Y, Tsuruta K, Miyaki M, Nakamura Y. Frequent loss of heterozygosity for loci on chromosome 8p in hepatocellular carcinoma, colorectal cancer, and lung cancer. Cancer Res 1992;52:5368–72. [4] Cliby W, Ritland S, Hartmann L, Dodson M, Halling KC, Keeney G, Podratz KC, Jenkins RB. Human epithelial ovarian cancer allelotype. Cancer Res 1993;53:2393–8.
I. Pribill et al. / Cancer Genetics and Cytogenetics 129 (2001) 23–29 [5] Osborne RJ, Leech V. Polymerase chain allelotyping of human ovarian cancer. Br J Cancer 1994;69:429–38. [6] Tapper J, Sarantaus L, Vahteristo P, Nevanlinna H, Hemmer S, Seppala M, Knuutila S, Butzow R. Genetic changes in inherited and sporadic ovarian carcinomas by comparative genomic hybridization: extensive similarity except for a difference at chromosome 2q24-q32. Cancer Res 1998;58:2715–19. [7] Wright K, Wilson PJ, Kerr J, Do K, Hurst D, Khoo S-K, Ward B, Chenevix-Trench G. Frequent loss of heterozygosity and three critical regions on the short arm of chromosome 8 in ovarian adenocarcinoma. Oncogene 1998;17:1185–8. [8] Brown MR, Chuaqui R, Vocke CD, Berchuck A, Middleton LP, Emmert- Buck MR, Kohn EC. Allelic loss on chromosome arm 8p: analysis of sporadic epithelial ovarian tumors. Gynecol Oncol 1999;74: 98–102. [9] Rogaev E, Rogaeva E, Lukiw WJ, Vaula G, Liang Y, Hancock R, McLachlan DC, St. George-Hyslop PH. An informative microsatellite repeat polymorphism in the human neurofilament light peptide (NEFL) gene. Hum Mol Genet 1992;1:781. [10] Oshima J, Yu CE, Boehnke M, Weber JL, Edelhoff S, Wagner MJ, Wells DE, Wood S, Disteche CM, Martin GM, Schellenberg GD. Integrated mapping analysis of the Werner syndrome region of chromosome 8. Genomics 1994;23:100–13. [11] Tomfohrde J, Wood S, Schertzer M, Wagner MJ, Wells DE, Parrish J, Sadler LA, Blanton SH, Daiger SP, Wang Z, Wilkie PJ, Weber JL. Human chromosome 8 linkage map based on short tandem repeat polymorphisms: effect of genotyping errors. Genomics 1992;14:144–52. [12] Gyapay G, Morissette J, Vignal A, Dib C, Fizames C, Millasseau P, Marc S, Bernardi G, Lathrop M, Weissenbach J. The 1993–94 Genethon human genetic linkage map. Nature Genet 1994;7:246–339.
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[13] Thomas W, Drayna D. A polymorphic dinucleotide repeat at the D8S339 locus. Hum Mol Genet 1993;2:828. [14] MacGrogan D, Levy A, Bova GS, Isaacs WB, Bookstein R. Structure and methylation-associated silencing of a gene within a homozygously deleted region of human chromosome band 8p22. Genomics 1996;35:55–65. [15] Shimizu Y, Kamoi S, Amada S, Hasumi K, Akiyama F, Silverberg SG. Towards the development of a universal grading system for ovarian epithelial carcinoma. I. Prognostic significance of histopathologic features–problems involved in the architectural grading system. Gynecol Oncol 1998;70:2–12. [16] Prasad MA, Trybus TM, Wojno KJ, Macoska JA. Homozygous and frequent deletion of proximal 8p sequences in human prostate cancers: identification of a potential tumor suppressor gene site. Genes Chromosom Cancer 1998;23:255–62. [17] Bova GS, MacGrogan D, Levy A, Sokhom SP, Bookstein R, Isaacs WB. Physical mapping of chromosome 8p22 markers and their homozygous deletion in a metastatic prostate cancer. Genomics 1996; 35:46–54. [18] Kagan J, Stein J, Babaian RJ, Joe Y-S, Pisters LL, Glassman AB, von Eschenbach AC, Troncoso P. Homozygous deletions at 8p22 and 8p21 in prostate cancer implicate these regions as the sites for candidate tumor suppressor genes. Oncogene 1995;11:2121–6. [19] Levy A, Uyen- Chi D, Bookstein R. High-density screen of human tumor cell lines for homozygous deletions of loci on chromosome arm 8p. Genes Chromosomes Cancer 1999;24:42–7. [20] Ishii H, Baffa R, Numata SI, Murakumo Y, Rattan S, Inoue H, Mori M, Fidanza V, Alder H, Croce CM. The FEZ1 gene at chromosome 8p22 encodes a leucine-zipper protein, and its expression is altered in multiple human tumors. Proc Natl Acad Sci USA 1999;96:3928–33.
High frequency of allelic imbalance at regions of chromosome arm 8p in ovarian carcinoma Ingrid Pribilla,1, Paul Speiserb,1, Jennifer Learyc, Sepp Leodolterb,d, Neville F. Hackere, Michael L. Friedlanderf, Daniel Birnbaumg, Robert Zeillingerb,*, Michael Krainera a
Department of Medicine I, Clinical Division of Oncology, University Hospital, Molecular Oncology Group, Währingergürtel 18-20, A-1090 Vienna, Austria b Department of Gynecology and Obstetrics, University Hospital, Molecular Oncology Group, Währingergürtel 18-20, A-1090 Vienna, Austria c Department of Medical Oncology, Westmead Hospital, Westmead, Australia d Ludwig Boltzmann Institute for Gynecologic Oncology and Reproductive Medicine, University Hospital, Molecular Oncology Group, Währingergürtel 18-20, A-1090 Vienna, Austria e Gynaecological Cancer Centre, Royal Women Hospital, Randwick, Australia f Department Medical Oncology, Prince of Wales Hospital, Randwick, Australia g Institut Paoli-Clamettes, U.119 Inserm, Marseille, France Received 17 October 2000; received in revised form 11 January 2001; accepted 15 January 2001
Abstract
Progressive genetic changes such as the inactivation of tumor suppressor genes (TSG) are thought to play an important role in the initiation and progression of ovarian cancer. Frequent nonrandom allelic imbalance (AI) at 8p11p21 and 8p22pter suggests the existence of TSGs that may be involved in the carcinogenesis of several human malignancies. We investigated 70 ovarian tumors with 11 highly polymorphic markers spanning 8p12p21 and 8p22pter to produce an AI map of 8p in epithelial ovarian cancer. Allelic imbalance was demonstrated in 54 tumors (77%), most frequently occurring at D8S136 (54%) and at D8S1992 (55%). Poorly differentiated and advanced stage cancers were more often affected by AI (G1G2 vs. G3; 20% vs. 66%; stage III vs. IIIIV, 36% vs. 54%, P.001; Kruskal-Wallis test) than well differentiated and early stage tumors. There was no relationship between histological subtype and AI. Smallest regions of overlap (SRO) were delineated by analyzing 38 tumors with partial AI. This study provides compelling evidence for the involvement of TSGs on the short arm of chromosome 8, at 8p12p21 and at 8p23 in the development and progression of epithelial ovarian cancer. © 2001 Elsevier Science Inc. All rights reserved.
1. Introduction The molecular basis of ovarian cancer, and the genetic events leading to the initiation and progression of this disease, are poorly understood. As in other human malignancies, progressive genetic changes including the activation of proto-oncogenes, as well as inactivation of tumor suppressor genes (TSG) and anti-metastatic genes are thought to occur. Sustained allelic imbalance (AI) of polymorphic markers at a certain chromosomal region suggests the exist-
* Corresponding author. Tel.: 43-1-40400-7831; fax: 43-1-404007832. E-mail address: [email protected] (R. Zeillinger). 1 These authors contributed equally to the work.
ence of a gene involved in carcinogenesis. Advances in localizing and mapping polymorphic markers has made it possible to conduct comprehensive studies on AI on very small amounts of tumor DNA isolated from tumor cell enriched samples. Nonrandom AI affecting the short arm of chromosome 8 has been demonstrated in a number of human malignancies including prostate, colon and breast cancer [1,2]. In ovarian cancer the short arm of chromosome 8 has not yet been extensively studied. Of the studies reported so far, several have investigated only a few polymorphic markers [3–6], while others have focused on limited chromosomal regions [7,8]. The aim of our study was to extend the investigations of AI on 8p to 70 sporadic ovarian tumors and correlate our results with published findings and with clinico-pathological features wherever possible.
0165-4608/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S0165-4608(01)00 4 1 9 - 8
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I. Pribill et al. / Cancer Genetics and Cytogenetics 129 (2001) 23–29
2. Materials and methods 2.1. Study population We investigated 23 (33%) early stage (FIGO Stages I and II) tumors, including 5 borderline tumors, and 47 (67%) advanced stage (FIGO Stages III and IV) tumors. The majority of tumors were serous cancers (64%), but in addition there were 6 clear cell carcinomas, 6 endometrioid adenocarcinomas, 5 mucinous, 3 mixed, 2 undifferentiated cancers, and 1 carcinosarcoma. In three cases, there was no information on the histological type. Of the invasive cancers, 5 tumors were well differentiated, 14 moderately and 38 poorly differentiated. The clear cell cancers were not graded. 2.2. Tissue samples Ovarian tumor biopsies were obtained from 70 patients undergoing initial staging or debulking laparotomy at several major teaching hospitals in Sydney and Brisbane (Australia). Prior to freezing sample mass was reduced by dissecting away normal tissue. Germline DNA was prepared from peripheral blood leukocytes (PBL). High molecular weight DNA was obtained by standard methods, involving
proteinase K digestion and phenol/chloroform extraction. None of the patients had undergone treatment prior to surgery or had a known family history of Lynch II, familial ovarian cancer or hereditary breast-ovarian cancer. All patients were staged according to the FIGO classification and histological classification was based on the typing criteria of the WHO. 2.3. Allelic imbalance analysis Eleven highly polymorphic microsatellite markers mapping to chromosome arm 8p were used to test for AI. Primer sequences for the following markers were chosen and PCR was done as previously published: LPL-F, D8S133 [9], D8S136 [10], D8S137 [11], D8S258, D8S259, D8S261, D8S283, D8S535 [12], D8S339 [13], D8S1992 [14]. Senseprimers were fluorescently labeled with Cy5 (MWG, Munich, Germany). 2.4. Quantitation of allelic imbalance PCR products were separated on a 6% PAGE (19:1 acrylamide:bis) containing 7 M urea using an automated laser fluorescent sequencer apparatus (ALF express DNA Sequencer, Pharmacia, Uppsala, Sweden), and their size deter-
Fig. 1. Computer representation of the analysis of the amplification products from the microsatellite locus D8S133 by automated polyacrylamide gel electrophoresis. Allelic peaks are indicated by small squares. M, size marker 100 bp; Patient one is an example of retention of both alleles: N1, normal DNA; T1, tumor DNA. Patient two is an example of LOH: N2, normal DNA; T2, tumor DNA.
I. Pribill et al. / Cancer Genetics and Cytogenetics 129 (2001) 23–29
mined using Fragment Manager Software (Pharmacia). A mixture of Cy5-labeled DNA-fragments (50–500 bp in length) was used as an external size marker. An imbalance factor was defined as the ratio of relative allelic peak intensities of the tumor DNA to corresponding PBL DNA samples. An imbalance factor 1.5 or 0.67 was considered significant (Fig. 1). 2.5. Statistical analysis To correlate histopathological parameters with AI on chromosome arm 8p, the Fisher’s exact test was applied. The Kruskal-Wallis test was used to assess correlation between the frequency of AI, tumor differentiation grade and disease status. The Chi square-test was applied to correlate the grade of differentiation and disease stage.
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(11%) and 11 advanced (23%) stage tumors had AI with all markers, indicating extensive loss of genetic material. Next, we analyzed each marker separately for the frequency of AI (Table 1). For region 8p12p22 we found AI most frequently occurring at D8S136 (54%). At 8p23, AI was most frequently found at D8S1992 (55%). Again, to explore possible differences between early stage and advanced stage tumors, we analyzed these groups separately (Table 1). At 8p12p22, in the early stage tumors, AI most frequently occurred at D8S259 (57%). In the advanced stage tumors, all markers apart from D8S259 and D8S283 showed AI in 50% or more of the samples. Three out of five borderline tumors had evidence of AI at 8p involving D8S1992 in one case, and D8S136 in two cases. 3.2. Delineation of smallest region of overlap on chromosome arm 8p
3. Results 3.1. Allelic imbalance on chromosome 8p We investigated 11 markers in 70 tumors. A heterozygous, and thus informative situation, was detected in 73% of all loci tested. The overall frequency of AI in 65 invasive ovarian carcinomas (5 borderline tumors excluded) was high, with 51 cases (78%) demonstrating AI with at least one marker, and 44 tumors (68%) showing AI with two or more markers. In 38 cases (58%), at least one marker showed retention of heterozygosity (partial AI), while in 13 tumors (20%) all informative markers spanning 8p showed AI, indicating a large deletion, or possibly loss of the entire chromosome. Interstitial retention of heterozygosity, which reveals AI at multiple loci in the same tumor, was found in 21 cases (32%). To investigate the relationship between disease stage and AI, we analyzed separately 18 invasive early stage (5 borderline tumors were excluded) and 47 advanced stage ovarian cancers. Sixty-one percent of early and 85% of advanced stage tumors showed evidence of AI. Two early
We investigated 38 tumors with partial AI to delineate smallest regions of overlap (SRO) and divided the samples into advanced and early stage tumors (Fig. 2). One SRO emerged in the region of highest loss around marker D8S261. The centromeric breakpoint is defined by retention of marker LPL-F in several tumors (192B, 125C, 142D, 55A, 140B, 68B) whereas the telomeric breakpoint is only hold by one tumor (192B), so that the SRO around D8S261 may well include marker D8S1992. The possible SRO D8S259–D8S535 with breakpoints D8S339 and D8S283 is found deleted in early stage as well as in late stage cases. In contrast, SRO D8S136–D8S137, defined by retention of heterozygosity of markers D8S133 and D8S339, was observed only in the advanced tumors. 3.3. Correlation to histopathological parameters We correlated AI with grade of differentiation, stage and histological tumor type. The percentage of markers showing AI was significantly lower in the group of well to moder-
Table 1 Summary of allelic imbalance (AI) in tumor samples ALL
Stage I II
Stage III IV
Grade 1 2
Grade 3
Marker
Inform. patients # (%)
# AI
% AI
Inform. patients # (%)
# AI
(%) AI
Inform. patients # (%)
# AI
% AI
Inform. patients # (%)
# AI
% AI
Inform. patients # (%)
# AI
% AI
D8S1992 D8S261 LPL-F D8S258 D8S133 D8S136 D8S137 D8S339 D8S259 D8S535 D8S283
51 (73) 51 (73) 62 (89) 47 (67) 45 (64) 59 (84) 45 (64) 50 (71) 52 (74) 47 (67) 57 (81)
28 23 27 21 21 32 22 21 23 19 19
55 45 44 45 47 54 49 42 44 40 33
13 (72) 14 (77) 17 (94) 13 (72) 12 (66) 13 (72) 10 (56) 13 (72) 14 (77) 13 (72) 10 (3)
6 4 5 4 5 4 3 5 8 3 3
46 29 29 31 42 31 30 38 57 23 30
34 (72) 32 (68) 41 (22) 30 (64) 30 (64) 42 (89) 32 (68) 32 (68) 34 (72) 30 (64) 42 (89)
21 19 22 17 16 26 19 16 15 16 16
62 59 54 57 53 62 59 50 44 53 38
17 (81) 14 (67) 18 (86) 13 (62) 16 (76) 16 (76) 15 (71) 16 (76) 13 (62) 17 (81) 16 (76)
7 4 4 4 3 6 5 7 5 5 3
41 29 22 31 19 38 33 44 38 29 19
29 (76) 26 (68) 33 (87) 26 (61) 22 (58) 33 (87) 22 (58) 23 (61) 32 (84) 21 (55) 32 (84)
18 16 20 14 15 19 14 11 15 11 13
62 62 61 54 68 58 64 48 47 52 41
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Fig. 2. Map of allelic imbalane (AI) in human ovarian carcinomas. Only tumors with partial AI are illustrated: , AI; retention of heterozygosity. Analysis of shortest regions of overlap (SRO): —, SRO; ni, noninformative; nd, not done.
ately differentiated (G1G2) tumors (20%) than in the group of poorly differentiated (G3) tumors (66%) (P.001, Kruskal-Wallis test). A similar relationship existed for tumor stage and AI was significantly lower (36%) among early stage tumors as compared to advanced stage tumors (54%) (P.001, Kruskal-Wallis test). Differentiation grade was significantly associated with stage (G1G2 and G3 vs.
stage III and IIIIV; P.001, chi square-test). We found no association between AI and histological subtype. 4. Discussion In this investigation seventy-seven percent of invasive ovarian cancers showed AI at 8p. LOH was more com-
I. Pribill et al. / Cancer Genetics and Cytogenetics 129 (2001) 23–29
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Fig. 3. Integrational map comparing LOH data in ovarian cancer and homozygous deletions (HD) found in several other tumors. Bold markers represent anchor markers from genemap99 (http://www.ncbi.nlm.nih.gov/genemap99). Distances are given in centiRay (cR 3000) according to the Stanford G3 radiation hybrid map. The first column summarizes our own data whereas the following columns represent the results taken from the literature. X denotes marker analyzed in the individual study.
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I. Pribill et al. / Cancer Genetics and Cytogenetics 129 (2001) 23–29
monly found in poorly differentiated (G3) than in well differentiated (G1 & 2) tumors and more often in advanced (Stage III & IV) than in early stages (Stage I & II) of the disease. When tumors with partial LOH were analyzed for SROs three potential areas were identified. This corroborates the findings of other investigators, which suggest that the short arm of chromosome 8 may harbor several TSGs involved in the development and progression of ovarian cancer. The comparison and integration of data available from studies of genetic loss in tumors is often hampered by the use of different markers in each investigation. However, we have been able to put the markers used in our study and in two other similar investigations in context with the anchor markers of the genemap99 (http://www.ncbi.nlm. nih.gov/genemap99.org) (Fig. 3). This allows us to compare results from the three most comprehensive LOH studies in ovarian cancer published to date. The first of these studies to be published focused mainly on the telomeric region 8p22–p23 [7]. Fifty-three tumors were investigated using initially eight microsatellite markers. Twenty-seven cases (51%) showed AI and in 16, all markers were involved indicating loss of large parts of 8p. We found the overall frequency of AI on 8p in invasive tumors to be higher at 78%. This may reflect our use of more sensitive methods of computer-based quantitation of AI. Furthermore, we found that only 24% of tumors showing AI had all markers involved, significantly less then the 60% reported by Wright et al. [7]. This difference might be explained in part by differences in the patient cohorts, specifically the higher percentage of early stage tumors in our series. Wright et al. found AI correlated only with tumor stage but not with grade of tumor differentiation. In contrast, we found not only early stage, but also well differentiated tumors were significantly less frequently affected by AI than late stage and relatively undifferentiated tumors. This was no surprise, since the correlation between tumor differentiation and tumor stage is a very well described phenomenon in ovarian carcinoma [15]. To investigate SROs at 8p22–p23, Wright et al. analyzed nine tumors with additional eight markers and delineated three SROs including the loci D8S550-D8S264, D8S549, and LPL. The most telomeric SRO found by us seems to be in the same region as the centromeric SRO in the series of Wright et al. The second LOH study on chromosome 8p in ovarian cancer published, analyzed 45 ovarian cancer cases with 6 highly polymorphic markers and found LOH of one of these markers in 23 of 40 (58%) tumors [8]. The highest loss showed marker D8S136 in 15 of 30 informative cases with lower rates at adjacent loci. This finding is in accordance with our results, where the same marker showed the highest loss particularly in the advanced tumor stages. As in the previous two studies discussed previously, there was a direct correlation between tumor aggressiveness and LOH at chromosome 8. The short arm of chromosome 8 has not been investigated solely in the context of ovarian cancer, but in a wide
variety of other human malignancies and AI was frequently displayed. Allelic imbalance at 8p was found as an early as well as a late event in carcinogenesis [1,2]. In the future, identification and cloning of putative 8p tumor suppressor genes will reveal whether one or several different genes are involved in prostate, breast and ovarian tumorigenesis and whether the timing of gene inactivation relates to tumor progression. To achieve this goal the identification of regions of homozygous deletions (HD) is a critical step, since the loss of the second allele usually covers a smaller chromosomal region. Fig. 3 shows an overview of areas of homozygous loss identified on the short arm of chromosome 8 in various cancer types. Most of the studies have been performed in prostate cancer [1,16–18]. Additional HDs were identified in pancreatic carcinoma [19] and oral cancer [2]. Interestingly, all SROs identified in ovarian cancer to date are in areas where HDs have been described in the literature at least in other tumor entities corroborating the significance of the LOH findings. This information, taken together with the progress of the Human Genome Project in generating sequence and mapping information (as illustrated in the use of the genemap99 in this article) will greatly facilitate the identification of candidate TSGs in ovarian cancer. A challenge for the future will be to develop clear criteria and innovative methods to quickly evaluate the high number of genes and ESTs created by random sequence efforts, which map to these LOH areas like, e.g., FEZ1 [20] as possible TSGs. Proof for the involvement of any of these candidate genes in tumorigenesis may lead to a better understanding of the biology and ultimately better diagnostic and treatment options for this common cancer.
Acknowledgments We would like to thank Eva Schuster and Carina Bednar for their help in this study and Dr. Suzanne Brill for a critical review of the manuscript. This work was supported by “Austrian Research Funds” Nr.: J01313-MED.
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