Characterization of Chromosome 9 in Human Ovarian Neoplasia Identities Frequent Genetic Imbalance on 9Q and Rare Alterations Involving 9P, Including CDKN21

[CANCER RESEARCH 55, 2150-2157, May 15. 1995] Characterization of Chromosome 9 in Human Ovarian Neoplasia Identities Frequent Genetic Imbalance on 9q and Rare Alterations Involving 9p, Including CDKN21

David C. Schultz, Lisa Vanderveer, Kenneth H. Buetow, Matthew P. Boente, Robert F. Ozols, Thomas C. Hamilton, and Andrew K. Godwin2

Departments of Medical Oncology [D. C. S., L V., R. F. O., T. C. H., A. K. C.l, Population Science Â¡K.H. B.Â¡.and Surgical Oncology [M. P. B./. Fox Chase Cancer Center, Philadelphia, PA 19111

ABSTRACT process resulting in an accumulation of genetic lesions in proto-onco- genes or tumor suppressor genes. In ovarian cancer, cytogenetic and loss We have examined 41 forms of ovarian cancer for genetic alterations on of heterozygosity studies have implicated many regions of the genome, chromosome 9 using a combination of five RFLP DNA probes and 15 suggesting that a large number of genes may be involved in ovarian simple tandem repeat polymorphisms. Genetic imbalance (i.e., loss of cancer development and/or progression (2-11). We have chosen to char heterozygosity, microsatellite instability, amplification) for 1 or more informative markers on chromosome 9 was observed in 66% (27 of 41) of acterize chromosome 9 in ovarian cancers, based on the clinical mani our tumor panel. Genetic imbalance was observed on 9q in 59% (24 of 41) festation of ovarian fibromas and cysts of the ovary in patients with of tumors informative for at least one locus. In contrast, only 13% (5 of 40) nevoid basal cell carcinoma syndrome (NBCCS) and the relationship of of informative tumors demonstrated a genetic alteration involving 9p. this disease with chromosome 9q. A candidate tumor suppressor gene for Furthermore, allelic loss on 9q was more common in late stage tumors this syndrome has previously been genetically mapped to 9q22.3-q31. In (63%, 17 of 27) and poorly differentiated tumors (75%, 15 of 20) as addition to this putative tumor suppressor gene, genes for multiple self- compared to benign and early stage tumors (30%, 3 of 10). Evaluation of healing squamos epitheliomata (ESSI), familial dysautonomia (DYS), 15 tumors showing limited regions of genetic imbalance has identified 2 Fukuyama-type congenital muscular dystrophy (FCMB), and tuberous candidate tumor suppressor regions on 9q and 1 on 9p. Interestingly, the regions defined to 9p21-p24, 9q31, and 9q32-q34 all overlap with several sclerosis (TSCI) have also been localized to chromosome 9q. Secondly, we have reported previously that rat ovarian surface epithelial cells known disease loci. In this aspect, the potential role of the CDKN2 gene at 9p21-p22 in ovarian carcinogenesis was assessed in an extended panel of spontaneously transformed in vitro possess deletions on chromosome 5, ovarian tumors, 11 human ovarian carcinoma cell lines, and 1 cervical in a region syntenic to human chromosome 9p where the IFN gene cluster tumor cell line. With the use of comparative multiplex PCR, homozygous resides. It was reported recently that an inhibitor (CDKN2) of cyclin deletions were detected in 16 of 115 ( 14% ) fresh tumors and 3 of 12 cancer D/cyclin-dependent kinase 4 complexes maps to 9p21 and is homozy- cell lines. For those tumors demonstrating allelic loss for markers on 9p no gously deleted in nearly 30% of ovarian cancer cell lines (12). somatic mutations were observed in the retained alÃeleof CDKN2, as In this report we describe the detailed deletion mapping of 41 determined by single-strand conformation polymorphism analysis, but a paired ovarian tumors and corresponding peripheral blood lympho mutation was observed in an additional cell line. Furthermore, CDKN2 cyte DNA samples for genetic alterations on chromosome 9, using a mRNA levels were similar in the 9 cancer cell lines that retain CDKN2, as total of 20 polymorphic DNA markers. In addition, gene dosage compared to normal human ovarian surface epithelial cell lines. Overall, our results suggest the potential involvement of a gene or genes on studies were performed on 115 fresh ovarian tumors, 11 ovarian chromosome 9q and de-emphasize a significant role for the CDKN2 gene carcinoma cell lines, and 1 cervical tumor cell line for deletions of the on 9p in the initiation and progression of ovarian cancer. CDKN2 gene.

MATERIALS AND METHODS INTRODUCTION Tumor Samples and Histology. One hundred fifteen ovarian tumors (58 Ovarian cancer is the most lethal gynecological malignancy and the common epithelial tumors, 3 mixed Miillerian tumors, 3 stromal tumors, 37 fifth most common cause of cancer related deaths in women in the benign tumors, 7 tumors of low malignant potential, and 7 tumors of some United States. By the end of 1994 it is estimated that 22,000 new cases other pathology) were collected from consenting patients undergoing surgery of ovarian cancer will have been diagnosed and that approximately for ovarian cancer at the American Oncological Hospital and the Lankenau 13,000 deaths will have resulted from this disease. These numbers Hospital in Philadelphia, or were obtained from the Gynecological Oncology translate to an average lifetime risk of 1.4% for women in the United Group/Cooperative Human Tissue Network ovarian tissue bank (Columbus, States (1). Furthermore, more than two-thirds of the women with OH) which is funded by the National Cancer Institute. A portion of the ovarian cancer are diagnosed with advanced disease, when existing debulked tumor mass was used immediately for DNA isolation, while the remaining tumor sample was either frozen in liquid nitrogen and stored at therapeutic measures are often ineffective. Thus, understanding the -70Â°C or frozen in isopentane. The histopathological classification was based genetic events that lead to initiation and progression of disease re on the typing criteria of the WHO. mains an important challenge in gynecological research and ulti Cell Lines. Ovarian cancer cell lines A2780, 1847, PEO1, and SKOV3 mately may enable the development of better approaches for earlier were derived from patients prior to treatment with chemotherapy (13-15). The diagnosis, when current therapeutic strategies have a high cure rate. OVCAR ovarian cancer cell lines -2, 3, 4, 8, and 10 were obtained from It is well accepted that cancer, particularly adult cancer, is a multistep patients who were refractory to platinum-based combination chemotherapy, while OVCAR-5 and OVCAR-7 are cell lines derived from ovarian cancer Received 11/17/94; accepted 3/13/95. patients prior to treatment. CVCAR-13 was derived from a cervical carcinoma. The costs of publication of this article were defrayed in part by the payment of page HOSE1 cell lines were derived from individuals who underwent prophylactic charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this faci. 1This work was supported by National Cancer Institute Grant CA60643 (A. K. G.) and ^ The abbreviations used are: HOSE, human ovarian surface epithelial; STRP, simple the Mary Smith Charitable Lead Trust. tandem repeat polymorphism; SET, standard SDS-EDTA-Tris buffer; LOU, loss of 2 To whom requests for reprints should be addressed, at Department of Medical heterozygosity; XPA. Xeroderma pigmentosum, complementation A; SSCP, single-strand Oncology, Fox Chase Cancer Center. Philadelphia. PA 19111. conformation polymorphism analysis. 2150

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1995 American Association for Cancer Research. CHROMOSOME 9 ALTERATIONS IN OVARIAN NEOPLASIA oopherectomy with or without a strong family history of ovarian cancer. The followed by 35 cycles of denaturation at 94Â°Cfor 1 min, annealing at 55Â°C HOSE cell lines were immortalized with SV40 to enhance the growth potential for 1 min, elongation at 72Â°Cfor 1 min, and a final elongation step for 10 min of the cells. All tumor cells were maintained in RPMI 1640 (Bethesda Re at 72Â°C. search Laboratories, Gaithersburg, MD) containing 10% (v/v) PCS and 100 /ig of streptomycin, 100 units of penicillin, 0.3 mg of glutamine, and 0.3 unit of porcine insulin/ml. HOSE cells were maintained in MCDB105/199 (1:1) RESULTS medium (Sigma Chemical Co., St. Louis, MO) supplemented with 4% (v/v) FCS. All cells were grown at 37Â°Cin 5% CO2. Loss of Heterozygosity Analysis. Forty-one ovarian tumors (29 Southern and Microsatellite Analysis. Isolation of DNA from tumor and common epithelial tumors, 2 recurrent adenocarcinomas, 2 mixed matching blood samples, typing of RFLPs, and PCR typing of STRPs was as Mullerian tumors, 1 clear cell tumor, 1 teratoma, 1 Granulosa cell described previously (6). The following loci were evaluated: D9S324, 9pter- tumor, 1 Brenner tumor, 2 mucinous cystadenomas, and 2 thecomas) p22; IFN, 9p21-p22 (16); D9SI26, 9p21 (17); MCTI12, 9q21 (18); D9S201, were examined for genetic alterations on chromosome 9. DNA iso 9q21.3 (19); D9SI2, 9q22.3 (20); D9SI09, 9q31(21); D9SI27, 9q31 (22); lated from these tumors was analyzed with the use of 5 RFLP DNA D9S53, 9q31 (23); LA.MP92, 9q31 (24); D9S58, 9q22.3-q31 (25); D9S59, probes (all on 9q) and 15 STRP PCR-primer pairs (3 for 9p and 12 for 9q31 (25); HXB, 9q32-q34 (26); MCOA12, 9q32 (27); GSN, 9q33 (28); ASSI, 9q). Concordant results were observed between the two methods as 9q34.1 (25); ABLI, 9q34.1 (25); D9S64, 9q34 (25); MCTI36, 9q34.3 (29); and represented in a direct comparison between D9S29 (LAMP 92) and EFDI26.3, 9q34 (30). Allelic loss was interpreted by D. C. S., L. V., and D9S53 (Figs. 1 and 2). Loss of heterozygosity for at least one A. K. G. LOH was characterized by the absence or reduction in the signal informative marker on chromosome 9 was observed in 63% (26 of 41 ) intensity of one alÃelerelative to the other in tumor versus constitutional samples. DNA RFLPs or STRPs were classified as heterozygous with no loss, of our tumors (Table 1). Allelic loss was observed in 56% (23 of heterozygous with loss, or homozygous/uninformative. 41) of tumors informative for markers on 9q, in contrast to 13% Gene Dosage Analysis of CDKN2. CDKN2 was amplified in three seg (5 of 40) for informative 9p markers. It should be noted that for 2 of ments with the following primers: exon 1, 5'-GAAGAAAGAGGAGGGG- the tumors that show LOH for markers on 9p, LOH was concordant CTG-3' and 5'-GCGCTACCTGATTCCAATTC-3'; exon2, 5'GGAAATTG- on 9q. The other three cases that demonstrate allelic loss for 9p GAAACTGGAAGC-3' and 5'-TCTGAGCTTTGGAAGCTCT-3' (12); exon markers, tumors 83, 89, and 160 retain all informative markers for 9q 3, 5'-GGATGTTCCACACATCTTTG-3' and 5'-ATGAAAACTACGAAA- (Fig. 2). Of the 41 tumors studied, 6 were obtained from patients with GCGGG-3'. Amplification of exons was performed in a final volume of 50 Â¡u a family history of ovarian cancer or breast cancer. When we exam containing 50 ng of DNA template, 10 mM Tris-HCl (pH 8.3), 50 mM KC1, 1.5 ined 2 early stage and 4 late stage familial ovarian cancers for LOH mM MgCl2, 0.001% gelatin, 5% DMSO, 100 fiM each dNTP, 2.5 units of at these loci, we observed a higher frequency of alÃeleloss on Amplitaq DNA polymerase (Perkin Elmer Cetus), and 1 /XMeach primer. DNA was amplified with an initial denaturation step of 2 min at 95Â°C,followed by chromosome 9q (83%) and 9p (17%), as compared to the sporadic 35 cycles of 1 in at 95Â°C,1 min at 58Â°C,and 1 min at 72Â°C,with a 5-min final tumors (Table 1). extension at 72Â°C.PCR products were resolved on a 2% SeaKem agarose gel Loss of heterozygosity was characterized according to the his- (FMC, Rockland, ME). topathological subtypes of the tumors. LOH for informative markers Comparative Multiplex PCR. Deletions of the CDKN2 gene were ana on 9q was observed for 88% (7 of 8) and 67% (12 of 18) of the lyzed by PCR with primers flanking exon 1. A primer set was designed from undifferentiated and serous papillary adenocarcinomas, respectively, the 3' untranslated region of the human XPA gene (5'-GATTTTTTAGT- as compared to 12% (3 of 26) for both subtypes at informative 9p TCAGTGACCTG-3' and S'-AACTTGCTTTTAAGCCATAAC-S') and used markers (Table 1). Furthermore, Table 1 also illustrates that only one as the control locus. PCR was performed in a 5-/J.1reaction volume containing of four endometrioid adenocarcinomas and the only mucinous adeno- 30 ng of DNA; 10 mM Tris-HCl (pH 8.3); 50 mM KC1; 1.5 mM MgCU; 0.001% carcinoma demonstrate loss of heterozygosity for an informative gelatin; 5% DMSO; 0.4 JJ.Meach primer; 16 JJ.MdGTP, dCTP. and TTP; 2 JAM dATP, and 1.25 fiCi [a15S]dATP. Thermal cycling was performed as de marker on chromosome 9q. Table 2 demonstrates that allelic loss on scribed above, except that the number of amplification cycles was reduced 9q was more common in late stage tumors (63%; 17 of 27) and high from 30 to 27. After amplification, an aliquot of amplified constitutional DNA grade (III) tumors (75%; 15 of 20). was loaded alongside varying amounts of amplified tumor DNA. On the Retention of heterozygosity at all informative loci on chromosome resulting autoradiograph, a tumor lane was chosen in which the signal of the 9 was observed in 15 of the 41 tumors evaluated. Of the 23 tumors that retained alÃeleof the control locus was similar to that in the normal lane, and demonstrate LOH for 9q markers, 9 of these tumors showed allelic the intensity of the exon l CDKN2 signal in the two lanes was compared. loss for all informative markers on 9q and retention of heterozygosity Deletion was scored if the CDKN2 signal was less than 20% of the signal from for 9p markers. Fifteen of the tumors evaluated revealed limited the control alÃeles,as determined by densitometry. regions of genetic imbalance. The pattern of genetic imbalance for RNA PCR Amplification OÃ•CDKN2. Total cellular RNA was obtained by these tumors is illustrated in Fig. 2. Analyses of tumors 57, 60, 81, and a one-step guanidinium isothiocyanate/phenol/chloroform extraction proce 167 reveal large interstitial deletions with telomeric retention. Tumor dure. First strand cDNA synthesis was carried out by denaturing 1 fig of total RNA at 70Â°Cfor 10 min in 12 /Â¿Iof diethylpyrocarbonate-treated water with 82 revealed allelic loss for all informative markers distal to D9SI09. 1 fiM poly T12MN (M = A, G, C; N = A, G, C, or T). The denatured RNA Tumors 78, 79, 93, 120, 142, and 173 reveal deletions telomeric to the was chilled on ice for 2 min and incubated for 10 min at room temperature and HXB (hexabrachion) locus and retention of heterozygosity for inform then 60 min at 42Â°Cin a final volume of 20 /Â¿I,containing 50 mM Tris-HCl (pH ative proximal markers. In addition to the telomeric loss, tumor 78 8.3), 75 mM KC1, 3 mM MgCl,, 10 mM DTT, 0.5 mM each dNTP, and 200 units also revealed the amplification of an alÃelefor the marker MCOAI2 of Superscript RNase H- Reverse Transcriptase (Bethesda Research Labora (D9SI6), as represented in Fig. 3A. Of particular interest is tumor 66, tories, Gaithersburg, MD). The reaction was terminated by heating to 95Â°Cfor a stage I mixed Mullerian tumor. This tumor does not show LOH for 2 min and cooling on ice. Amplification by PCR was performed in a final any of the loci typed on chromosome 9, but microsatellite instability volume of 50 fil containing 5 /il of the reverse transcribed product, 10 mM was observed for the markers D9S109, D9S127, and D9S53, exclu Tris-HCl (pH 8.3), 50 mM KC1, 1.5 mM MgCl,, 0.001% gelatin, 5% DMSO, sively (Fig. 3ÃŸ).The mutated alÃeleswere subcloned and sequenced 200 Â¿Â¿MeachdNTP, 2.0 units of Amplitaq DNA polymerase (Perkin Elmer Cetus), and 1 /XM each primer (CDKN2: 5'-ACGTAGATATAAGCCTTC- to verify a 2-bp addition in the lower alÃelesfor D9SI09 and D9S53, CCCC-3' and 5:-TAACCCCCCTGAGCTTCC-3'; XPA: S'-GATTTTTTA- and a 4-bp deletion in the lower alÃelefor D9SÃŒ27.Furthermore, GTTCAGTGACCTG-3' and 5'-AACTTGCTTTTAAGCCATAAC-3'). The genome wide allelotyping of this tumor with more than 60 different amplified PCR products were resolved on a 2% SeaKem agarose gel. Ampli dinucleotide and tetranucleotide repeat polymorphisms revealed no fication was carried out with an initial denaturation step of 2 min at 94Â°C, other genetic alterations (Fig. 3C and data not shown), suggesting 2151

D9S126 D9S53 81 86 88 89 93 94 95 1 3 4 5679 58 60 66 71 NTNTNTTNTNTTNTNTNTNTNTTNTNTNTNT NTNTNTNTT

D9S59 80 81 82 83 86 88 89 90 91 92 93 94 95 1 3 4 567 NTNTNTNTNTNTTNTNTNTTNTNTTNTNTNTNTNTTNTNTNT

Â»

ASS 58 83 86 88 89 90 91 92 93 94 95 1 3 4 567 NTNTNTNTTNTNTNTTNTNTTNTNTNTNTNTTNTNTNT

B. LAMP 92 55 58 59 60 66 71 82 N T Om N T T N T Om N T NT N T Om N T

Fig. 1. Representative autoradiograms demonstrating alÃeleloss on chromosome 9q at (A) D9S12fi, D9S53, D9S59, ASS and (B) LAMP 92 (D9S29). DNA isolated from peripheral blood lymphocytes and ovarian tumor tissue were assayed as described in "Materials and Methods." Allelic loss was scored by either the absence or reduction in signal intensity of one alÃelerelative to the other in tumor versus constitutional samples. /V, constitutional DNA isolated from peripheral blood lymphocytes; T, tumor DNA; Om, omentum DNA. regional genetic instability in this area of chromosome 9 for this approximately 1.5 cM, which is bounded by, but does not include, the tumor. anonymous markers D9SÃŒ27and D9S53 (Fig. 2). In our panel of Deletion Mapping of Minimum Regions of Overlap. The pat ovarian tumors, 39% (16 of 41) show genetic imbalance for inform terns of genetic imbalance observed in these tumors define two ative markers localized to 9q31. A third candidate tumor suppressor candidate tumor suppressor regions on chromosome 9q and one on region has been identified on 9p, and this region extends distal to chromosome 9p. As illustrated in Fig. 2, a region at 9q32-q34 is the marker D9S126 but proximal to INFA, as defined by tumors 81 defined by a minimal region of imbalance spanning the markers HXB and 83. and ASSI (arginosuccinate synthetase) and a genetic distance of Mutational Analysis of CDKN2. On the basis of the recent approximately 23 cM. Analysis of our tumors reveals that 53% (21 of observation that an inhibitor (CDKN2) of the cyclin-dependent 40) show allelic loss in the this region. Of the 23 tumors in which kinase 4 mapped to 9p21 and was homozygously deleted in ap allelic loss was observed on 9q, 6 tumors (78, 79, 93, 120, 142, and proximately 50% of cell lines derived from various tumor types 173) exclusively demonstrated LOH for markers at 9q32-q34. If the and nearly 30% of ovarian cancer cell lines evaluated, we exam microsatellite instability data is used as a mapping tool, a second ined the gene dosage of CDKN2 in the 41 initial ovarian neo region at 9q31 is defined by a minimal region of instability of plasms, as well as an additional 74 ovarian tumors by comparative 2152

OVARIAN TUMORS

UPN57 UPN 60 UPN78 UPN79UPN81 UPN8I UPN83 UPN W UPN 93 UPN 120 UPN 142 UPN160 UPN IÃ•7 UPN173 Locus D9S324 9 P (2/23) i (3/28)

(3/30)

(2/16)

(7/26)

(6/13)

(6/29)

(7/24)

(12/35)

(11/19)

(7/27)

(10/30) (10/24) (6/12)

(7/15) (13/34)

(10/25)

(10/18)

T i (8/15) i (1/6) Fig. 2. Genetic imbalance patterns in ovarian tumors for markers on chromosome 9. DNA isolated from ovarian tumor tissue and corresponding peripheral blood were typed with DNA RFLP probes and STRPs as described in "Materials and Methods." All informative loci are shown. D, retention of constitutional heterozygosity; â€¢.loss of constitutional heterozygosity; blank spaces, homozygous/uninformative; arrows, increase or decrease of 2 base pairs in one alÃete;â€¢,amplification of an alÃele.It is assumed that alÃelesbetween loci showing allelic loss are lost. , regions that are retained; open areas, regions of alleile loss. , regions of chromosome 9 that are uncertain in tumors with LOH. Numbers in parentheses, allelic loss per informative cases for that particular polymorphism. Thick black bars, minimum regions of overlap showing genetic imbalance. multiplex PCR. In addition 11 ovarian cancer cell lines and 1 somatic mutation in the retained alÃeleofCDKN2 as determined by cervical cancer cell line were also analyzed for CDKN2 deletions. SSCP. However, a nonsense mutation was discovered in exon 2 of As determined by comparative multiplex PCR, homozygous dele OVCAR10 (G to A at nucleotide position 324). resulting in a tions of the CDKN2 gene were observed in 14% (16 of 115) of termination of translation at codon 102. Furthermore, as expected, fresh ovarian tumors (Fig. 4) and 3 of 12 cancer cell lines (Fig. expression of CDKN2 was absent in the 3 cell lines showing 5A ). Furthermore, of the 5 fresh tumors demonstrating LOH on 9p, homozygous deletion of CDKN2. while the 9 cancer cell lines that 2 demonstrated reduction to hemizygosity for CDKN2. For those retain CDKN2 were similar to the mRNA levels observed in 6 tumors showing allelic loss for markers on 9p, none possessed a normal human surface epithelial cell lines (Fig. 55).

Table I Relationship between loss of heterozygosity on chromosome 9 and histopathological subtype in sporadic versus familial ovarian cancer

Histopathological subtype No. LOH on 9 LOH" on 9p LOH" on 9q LOH" at 9q3l LOH" at 9q32-q34 SporadicTumorsSerous adenocarcinoma'Undifferentiatedpapillary adenocarcinomaEndometrioid adenocarcinomaMucinous adenocarcinomaMixed tumorClearMulla KIM cellTeratomaMucinous

cystadenomaThecomaFamilial

(51%)2/22/2(VII/I5/6 (54%)2/22/20/1I/I5/6 (34%)1/22/20/1I/I4/6

TumorsSerous adenocarcinoma'Undifferentiatedpapillary adenocarcinomaGranulosa cellBrenner tumorOverall (83%)26/41 (17%)5/40 (83%)23/41 (67%)16/41 (83%)21/40

(%)166412112235221164110/165/61/41/11/21/1I/I1/2on.21/41(63%)2/160160/40/10/10/1I/I1/20/24/34(12%)on1/2(VI0/11/6(13%)8/165/61/41/11/21/11/10/20/219/35(56%)3/16V61/4I/I1/2I/II/I0/2on12/35(39%)8/165/6(V4I/I0/1I/II/I0/20/216/34(47%)2/22/2(VII/I5/6(53%) " LOH with any informative marker on chromosomal arm out of the total number of informative tumors in that histopathological subtype. The number of cases of LOH affecting informative markers within the chromosomal region 9q31 or 9q32-q34 out of those tumors with any LOH on the long arm of chromosome 9. ' Serous papillary adenocarcinomas include serous papillary cystadenocarcinomas, serous carcinomas, and papillary carcinomas.

2153

Table 2 Relationship befa'een loss of heterozygosity on chromosome 9 and Â¡umorstage and differentiation grade at9q311/5 at9q32-<]341/5 on92/5 on9p1/5 on9q1/5 TumorstageBenignStage (40%)1/4 (20%)1/3 (20%)1/4 (20%)1/4 (20%)1/4 IStage (25%)1/19 (33%)0/11/14 (25%)1/18 (25%)1/16 (25%)1/17 IIStage IIIStage /14(64%)9 (7%)1/13 /14(57%)9/13(69%)1/5 /14(43%)6 /13(54%)9 IVTumor /13(69%)2/5 (8%)1/5 /13(46%)1/5 /13(69%)1/5

differentiationBenignMixedrWell (40%)1/2 (20%)0/10/41 (20%)1/2 (20%)1/2 (20%)0/10/45 (50%)0/47/10(70%)16 (50%)0/46 (50%)0/43 (I)Moderatelydifferentiated (II)Poorly differentiated /10(10%)3/20(15%)LOHÂ° /IO(60%)15 /IO(30%)1 /10(50%)15/20(75%) differentiated (III)LOH /20 (80%)LOH" W (75%)LOH'' 1 /20 (55%)LOH* ' LOH for any informative marker on that chromosomal arm out of the total number of informative case in that tumor stage or differentiation grade. LOH affecting informative markers within the chromosomal region 9q31 or 9q32-q34 out of those tumors with any LOH on the long arm of chromosome 9. Mixed tumor differentiation includes tumors that have been classified as moderate to poorly differentiated.

DISCUSSION A. MCOA12 LAMP 92 It is becoming increasing clearer that multiple genetic alterations are required for the initiation and progression of cancer. In ovarian NT NT cancer much attention has been focused on chromosome 17 as a consequence of many studies involving the familial breast/ovarian cancer susceptibility gene, BRCA1, on 17q21 (31). However, the potential role of this gene in the development of sporadic ovarian cancers remains to be seen (32). Moreover, numerous additional genes and chromosomal regions have been implicated in ovarian tumorigen- esis(2-ll). We have chosen to characterize chromosome 9 for genetic alter ations in a panel of human ovarian tumors, based on the development of ovarian fibromas and cysts in the ovary in patients with the B. D9S127 autosomal dominant NBCCS and the relationship of this disease to chromosome 9q (33). Second, we have reported previously that rat NT ovarian surface epithelial cells spontaneously transformed in vitro have deletions on chromosome 5, in a region syntenic to human chromosome 9p where the IFN gene cluster resides (34). In this study we demonstrate that 59% (24 of 41) of our tumors display genetic imbalance (i.e., loss of heterozygosity, microsatellite instability, amplification) on the long arm of chromosome 9 (Table 1 and Fig. 2). This result is in agreement with LOH results reported by Cliby et al. (54%; Ref. 10) and Osborne and Leech (48%; Ref. 11) for 9q in large allelotyping studies of common epithelial ovarian tumors. Moreover, the high frequency of alÃeleloss for markers on 9q suggests that this alteration is a nonrandom phenomenon. Furthermore, allelic loss on chromosome 9q was observed in 75% (15 of 20) of high grade (III) tumors, and 63% (17 of 27) of late stage (III and IV) tumors (Table 2), which is similar to the values reported by Dodson et al. c D9S103 ASS (35). It should be noted that in a previous study (6) in which chro NT NT mosome 17 alterations were evaluated, all tumors that showed 9q alterations were also observed to have chromosome 17 changes. Thus, similar to other molecular abnormalities suggested previously (10, 36-38), acquisition of genetic alterations on chromosome 9q may be instrumental in acquiring the aggressive tumor phenotype. II In order to better define the regions on chromosome 9q in which a candidate growth or tumor suppressor gene(s) may reside, we have created a high density deletion map of 9q using 17 highly polymor phic markers. Limited regions of genetic imbalance were observed in 15 of the tumors evaluated. The pattern of genetic imbalance in these tumors identifies two potential subchromosomal regions that may Fig. 3. Additional genetic alterations in tumors 66 and 78. (A) Amplification of an alÃelefor MCOA12 (D9SI6) in tumor 78. Arrow, amplified alÃele.(B) Microsatellite harbor candidate genes. Our frequent observation of LOH for markers instability observed in tumor 66. The locus names are shown above Ihe (Â»responding set at 9q32-q34 (53%; 21 of 40) is suggestive of a candidate tumor of alÃeles.Arrows, alleles that have either gained 2 base pairs (D9SI09 and D9S53) or lost 4 base pairs (D95/27). (C) Representative heterozygous STRPs for tumor 66 demon suppressor locus on chromosome 9q. Moreover, nearly 100% (23 of strating retention of heterozygosity flanking the region of instability. N, constitutional 24) of the tumors that show genetic imbalance for any informative 9q DNA isolated from peripheral blood lymphocytes; T, tumor DNA. 2154

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1995 American Association for Cancer Research. CHROMOSOME 9 ALTERATIONS IN OVARIAN NEOPLASIA 59 81 89 96 97 123 131 132 T,N T2T3T,NT2T,T,N T2T3T,N T2T,T,NT2T3T,NT2T3T,NT2T,T,NT2T3 Fig. 4. Comparative multiplex PCR analysis of CDKN2 (pl6) in ovarian cancers. N, 6-ng amplified normal DNA; 7",, 12-ng amplified tumor DNA; T2, 6-ng amplified tumor DNA; 73, 3-ng amplified pi 6 exon 1 tumor DNA. Tumors 81 and 89 demonstrate homozygous deletion of CDKN2, confirmed by Southern blotting (data not shown). XPA

marker include alterations for 9q32-q34 markers, suggesting that this genetically altered in ovarian tumors, it would be additional support region may in fact be closer to the gene of interest. Several candidate for the concept that genes responsible for maintaining the integrity of genes that have been mapped to this subchromosomal region include the genome are important in the initiation or progression of human the c-Abelson proto-oncogene (c-ABL), hexabrachion (HXB), gelso- cancers. lin (GSN), and Xeroderma pigmentosum, complementation group A In addition to the potential tumor suppressor locus at 9q32-q34, a (XPA). Although considered by many to be a proto-oncogene, over- second candidate locus on 9q can be defined by several tumors. expression of c-ABL has been shown to inhibit growth by causing cell Tumors 57, 60, and 82 demonstrate allelic loss distal to the markers cycle arrest. Overexpression of a dominant negative c-ABL disrupts D9S127, D9S201, and D9S109, respectively. Tumor 160 demonstrates cell cycle control and enhances phenotypic transformation (39). Fur a large deletion extending proximal to D9S53, and no mutation was thermore, hexabrachion/tenascin is a extracellular glycoprotein that observed in CDKN2. In addition to the partial deletions observed in has been proposed as a candidate tumor suppressor gene. It has been these 4 tumors, tumor 66 exhibits microsatellite instability for the reported that HXB can support epithelial tumor growth in serum-free closely linked markers D9S109, D9S127, and D9S53, as shown in Fig. medium and is abnormally expressed in common epithelial tumors 3ÃŸ.It is important to note that no other genetic alterations were (40, 41). However, deletion mapping in our tumor panel has placed observed for more than 60 different di- and tetranucleotide repeat this gene outside the minimal region of deletion observed for tumors polymorphisms typed genome wide in this tumor (data not shown). In 78, 79, 93, 120, 142, and 173. An interesting candidate gene in this general, utilizing these 4 tumors, we have identified a minimal region region may be the gene for Xeroderma pigmentosum, complementa of imbalance, spanning markers D9S127 and D9S53 at 9q31 and a tion group A (XPA ), the gene product of which participates in the distance of approximately 1.5 cM. Furthermore, this region of chro process of nucleotide excision repair of mutagen-damaged DNA. mosome 9 was genetically altered in 41% (17 of 41) of the tumors Recently, mutations in the DNA mismatch repair enzymes hMSH2, examined. With regard to tumor 66, it is noteworthy that reports hMLHl, PMS1, and PMS2 have been implicated in a subset of suggest that approximately 8% of human cancers examined demon sporadic colorectal cancers and most tumors developing from hered strate a pattern of microsatellite instability that differs from that itary nonpolyposis colorectal cancer (42-44). If the XPA gene is responsible for the replication error-positive phenotype observed in

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lOObpâ€”

Fig. 5. Analysis of CDKN2 in ovarian carcinoma cell lines. (A) Ethidium bromide-stained agarose gels of PCR amplified exon 1 (lop), 2 (middle), and 3 (bottom) in 11 ovarian carcinoma cell lines, and 1 cervical carcinoma cell line. Homozygous deletions of the 350-bp exon 1 and 5(X)-bp exon 2 PCR products were observed in OVCAR-5, PEO-1. and SKOV3. The 200-bp exon 3 PCR product is homozygously deleted in OVCAR-5 and PEO-1. (B) Reverse transcripta.se PCR amplification of CDKN2 (pl6) mRNA in human ovarian surface epithelial cell lines and ovarian cancer cell lines (top). The human Xeroderma pigmentosum complementation group A gene (XPA) mRNA served as a control (bottom). M.W., molecular weight. 2155

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1995 American Association for Cancer Research. CHROMOSOME 9 ALTERATIONS IN OVARIAN NEOPLASIA colorectal tumors (45). However, the phenotype observed in tumor 66 our panel of ovarian tumors adds to the growing list of human may be the first reported example of regional chromosomal instability neoplasms in which the dysfunction of 9q genes have been implicated. observed in epithelial tumors of the ovary. It has been reported that greater than 50% of bladder tumors show It was originally reported that the gene predisposing to NBCCS and LOH for chromosome 9 markers (53-56). Cairns et al. (53) have sporadic basal cell carcinomas maps to chromosome 9q22.3-q31 and identified a common region of deletion on 9q, mapping proximal to is believed to be a tumor suppressor gene, as would be predicted by 9q22.3 in a large cohort of bladder tumors. Deletion mapping studies genetic linkage and somatic loss of the wild-type alÃele(33, 46-48). in small cell lung cancers have identified a similar region of allelic This autosomal dominant disorder predisposes to basal cell carcino loss at 9q22.3 (57). More recently, it was suggested that a candidate mas of the skin and other neoplasms, including ovarian fibromas. tumor suppressor gene on 9q for a subset of superficial bladder tumors Furthermore, we have observed a higher than expected incidence of resides in the 9q34.1-q34.3 (58). Furthermore, a subgroup of non- Hodgkin's lymphoma has been characterized cytogenetically to have basal cell carcinomas in breast and ovarian cancer syndrome families which are not linked to BRCAÃŒon chromosome 17q.4 However, consistent deletion involving 9q31-32 (59). On the basis of our recent studies of large kindreds affected with NBCCS revealed re- observation of frequent imbalance for markers distal to 9q22.3, it combinants that have refined the localization of the NBCCS gene appears that we have identified two regions which could harbor novel distal to D9SI2 but proximal to D9S109 (49, 50). Thus, our data gene(s) important in the pathogenesis of ovarian cancer. have identified the locus of a possible novel gene in this region of chromosome 9q. ACKNOWLEDGMENTS A third region of imbalance has been identified on the short arm of chromosome 9. Previous studies have identified 9pl3-p22 as a fre We thank Drs. Norman Rosenblum, Michael Hogan, and Hernando Salazar quent site of chromosomal loss in leukemias, gliomas, melanomas, for their support in collecting the samples used in this study, and Rikki Maxwell from the Cooperative Human Tissue Network. lung carcinomas, bladder carcinomas, and malignant mesotheliomas. Overall, 13% of the tumors evaluated showed allelic loss for a 9p marker. Although this finding is much lower than the 30% LOH REFERENCES reported for 9p markers in large allelotyping studies of ovarian tumors 1.Boente, M. P., Godwin, A. K.. and Hogan, W. M. Screening, imaging, and early (9-11, 51), it appears that genetic alterations of genes on 9p play a diagnosis of ovarian cancer. Clin. Obstet. Gynecol., 37: 377-391, 1994. Ehlcn, T., and Dubcau, L. Loss of heterozygosity on chromosomal segments 3p, 6q less frequent role in the initiation and/or progression of ovarian and lip in human ovarian carcinomas. Oncogene, 5: 219-223, 1990. cancer. Eccles, D. M.. RÃ¼ssel,S. E. H., Hailes, N. E., and the T. A. O. C. G. Group It has been reported recently that an inhibitor (CDKN2) of cyclin- (Atkinson, R., Bell, D. W., Gruber, L., Hickey, I., Kelley, K., Kitchner. H., Leonard. R.. Lessells, A.. Lowry, S., Miller, !.. 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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1995 American Association for Cancer Research. Characterization of Chromosome 9 in Human Ovarian Neoplasia Identifies Frequent Genetic Imbalance on 9q and Rare Alterations Involving 9p, Including CDKN2

David C. Schultz, Lisa Vanderveer, Kenneth H. Buetow, et al.

Cancer Res 1995;55:2150-2157.

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