Chromosome 6 Abnormalities in Ovarian Surface Epithelial Tumors of Borderline Malignancy Suggest a Genetic Continuum in the Progression Model of Ovarian Neoplasms1

3404 Vol. 7, 3404–3409, November 2001 Clinical Cancer Research

Maria Grazia Tibiletti, Barbara Bernasconi, Conclusion: Alterations in the above-mentioned inter- Daniela Furlan, Paola Bressan, Roberta Cerutti, val are a common finding in advanced ovarian carcinomas but also in benign ovarian cysts, implying that some tumors Carla Facco, Massimo Franchi, Cristina Riva, of borderline malignancy may arise from benign tumors and Raffaella Cinquetti, Carlo Capella, and that malignant ones may evolve from tumors of borderline Roberto Taramelli2 malignancy. Genes located in 6q27 seem to be crucial for this Laboratorio di Anatomia Patologica Ospedale di Circolo, Fondazione mechanism of early events in ovarian tumorigenesis. Macchi, 21100 Varese [M. G. T., C. F., C. R.]; and Dipartimento di Scienze Cliniche e Biologiche [B. B., D. F., P. B., R. Ce., C. C.], INTRODUCTION Clinica Ostetrica e Ginecologica [M. F.], and Dipartimento di Biologia Strutturale e Funzionale [R. Ci., R. T.], Universita` Ovarian cancer represents the most lethal of the gyneco- dell’Insubria, 21100 Varese, Italy logical tumors, and its pathogenesis is poorly understood. More than 80% of malignant tumors of the ovary are of epithelial origin and arise from the surface epithelium (1). Ovarian surface ABSTRACT epithelial tumors are divided into three histological subtypes: Purpose: We used conventional cytogenetics, molecular benign cystoadenoma, TBM,3 and AC, which are related to a cytogenetics, and molecular genetic analyses to study the different biological behavior (1). The molecular events under- pattern of allelic loss on chromosome 6q in a cohort of lying the development of ovarian tumorigenesis are still largely borderline epithelial ovarian tumors. unknown. Although the clinical and pathological features of Experimental Design: Fifteen tumor samples were col- TBM are intermediate between benign and frankly malignant lected from patients undergoing surgery for ovarian tumors. tumors (1), it is not clear whether they represent a transitional The tumors of borderline malignancy, classified according form between benign tumors and invasive carcinomas, as a stage to the standard criteria, included 4 mucinous and 11 serous in multistep carcinogenesis (1–3), or alternatively, whether all tumors. Cytogenetic and molecular cytogenetic (with yeast three tumor types may be regarded as independent entities artificial chromosome clones from 6q26-27) studies were caused by different molecular mechanisms (1–3). performed on tumor areas contiguous to those used for Although a comparison of genetic abnormalities occurring histological examination ensuring the appropriate sampling. in ovarian ACs and TBMs should provide insight into their Moreover loss of heterozygosity analysis was performed relationships, little information is available regarding the genetic using PCR amplification of eight microsatellite markers aberrations of TBM. Because most ovarian neoplasms are diag- mapping on 6q27 (D6S193, D6S297), 6q26 (D6S305, D6S415, nosed at an advanced stage, many studies have focused on D6S441), 6q21 (D6S287), 6q16 (D6S311), and 6q14 invasive ovarian ACs, whereas very few reports have examined (D6S300). genetic alterations of TBM (4–7). Indeed, chromosomal abnor- Results: Deletions of this chromosome arm, in particu- malities have been reported in Ͼ300 ovarian carcinomas exam- lar of 6q24-27, were the most frequent lesions found in this ined cytogenetically (8), whereas the TBM karyotypes reported set of tumors. In a tumor with a normal karyotype the only in the literature are Ͻ40, and less than half of them demonstrate detectable alteration was a deletion of ϳ300 kb within the clonal abnormalities (9–16). Very recently, Wolf et al. (17), D6S149–D6S193 interval at band 6q27. This is, to date, the using comparative genomic hybridization, found chromosomal smallest deletion described for borderline tumors. imbalances in 3 of 10 TBM. At the molecular level, previous studies of TBM have identified several regions of LOH, including 7p, 7q, 9p, 11q (18), 3p (19), 17q (20), and Xq12 (21), but the contribution of chromosome 6q was not found to be of significance. This Received 3/16/01; revised 7/11/01; accepted 7/25/01. chromosome arm has always been associated, almost exclu- The costs of publication of this article were defrayed in part by the sively, with late-stage ovarian carcinomas (6, 22–27). More- payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to over, in the only report focusing on 6q LOH studies in border- indicate this fact. line versus ovarian carcinomas, higher frequencies of 6q losses 1 R. T. was supported by grants from the Associazione Italiana Ricerca Cancro (AIRC) and Ministero dell’Universita` e Ricerca Scientifica e Tecnologica (Cofin 98). 2 To whom requests for reprints should be addressed, at Dipartimento di Biologia Strutturale e Funzionale, Universita`dell’Insubria, Varese, It- 3 The abbreviations used are: TBM, tumor(s) of borderline malignancy; aly. Phone: 39-0332-421514; Fax: 39-0332-421500; E-mail: roberto. AC, adenocarcinoma; LOH, loss of heterozygosity; FISH, fluorescence [email protected]. in situ hybridization; YAC, yeast artificial chromosome.

were found in the latter (6). Our group and others have docu- dom priming technique were used as probes. Preparations were mented and confirmed the association between 6q alterations mounted in antifade solution containing propidium or 4Ј-6- and ovarian carcinomas (6, 22–27), although we have recently diamidino-2phenylindole and observed with a Leica DMR flu- challenged this view by reporting 6q26-27 genetic lesions in orescence microscope under a single band filter for FITC or a ovarian benign neoplasms (28). We have now extended our triple band filter. analysis to 15 TBM to assess whether 6q deletions or rearrange- Interphasic FISH was carried out on formalin-fixed, paraf- ments are significantly present in this type of tumors. fin-embedded archival specimens of borderline ovarian tumors. The biotin-labeled ␣-satellite for chromosome 6 (purchased from Oncor) was used as probe and revealed with fluorescein- MATERIALS AND METHODS labeled streptavidin. One slide for each case was stained with Fifteen tumor samples were collected from patients under- H&E, which enabled identification of tumor cell areas of the going surgery for ovarian tumors at the Obstetric and Gynae- tissue section. In each case, 2-␮m sections were fastened on cological Clinic, Varese. Fresh specimens were collected under polylysine-coated slides and incubated at 60°C for 30 min. sterile conditions for histological, cytogenetic, and molecular Sections for hybridization were deparaffinized in xylene and an investigations. Cytogenetic and molecular studies were per- alcohol series, air dried, treated with proteinase K (0.25 mg/ml) formed on tumor areas contiguous to those used for histological at 45°C for 5–20 min, rinsed briefly in 2ϫ SSC, and dehydrated examination, ensuring appropriate sampling. The TBM, classi- in an ethanol series before air drying and hybridization. Probe fied according to WHO criteria (1), included 4 mucinous and 11 and target DNAs were denatured simultaneously at 70°C for 5 serous tumors. Among the serous borderline tumors, one case min. After an overnight incubation at 42°C, slides were washed displayed a fibromatous component (cystoadenofibroma) and in 50% formamide in 2ϫ SSC, followed by two 15-min washes two cases showed foci of stromal microinvasion. Histological in 0.1ϫ SSC at 37°C and a brief rinse at room temperature. examination revealed high degrees of architectural complexity Nuclei were counterstained with a propidium iodide/antifade associated with prominent cytological heterogeneity in the ma- solution. jority of the cases. In the remaining cases, pattern of growth and To ensure representative samples of each tumor and avoid cytological feature showed a lower extent of viability. the nuclear truncation, Ͼ100 cells from at least five to eight Cytogenetic Analysis. Chromosome analysis was car- different areas were evaluated. Only areas with well-preserved ried out in each case on direct preparations to avoid selection cellular and nuclear morphology were selected. In each case, biases produced by culturing. Tumor cell suspensions were nuclei of epithelial cells were counted into categories containing prepared by mincing small pieces of the tumor and incubating 1, 2, or ୑3 signals. Scoring was performed in random order and them for 48–72hat37°C with RPMI supplemented with 10% by three independent operators to avoid bias in analysis. The FCS, penicillin (100 IU/ml), streptomycin (0.2 mg/ml), L- results were evaluated using FISH data obtained with the same glutamine (0.23 mg/ml), insulin (1 ␮g/ml), cholera toxin, and probe on specimens of normal epithelial nuclei of cervical epidermal growth factor (Sigma Chemical Co.). After overnight epithelium (four cases), proliferative and postmenopausal endo- exposure to Colcemid (0.02 ␮g/ml), cells were harvested by metrium (two cases), and ovarian epithelium (one case) selected standard methods. The cells were suspended in 70% acetic acid, as controls (Table 2B). These control cases were carefully and the metaphase spread was performed on a warm plate at selected so that FISH evaluation was performed on nuclei show- 40°C. Karyotype evaluation was performed using Q- and G- ing dimensions similar to those of tumoral nuclei. An average of Wright banding techniques. Chromosome abnormalities were 17.5% (SD 3.9), 82.3% (SD 3.7), and 0.2% (SD 0.2) of epithe- described according to the recommendations of the Cancer lial nuclei in normal tissues had one, two, or three signals, Cytogenetics Supplement (29). In particular, only clonal abnor- respectively. Borderline specimens were considered showing malities were considered in the description of the tumor karyo- monosomic or trisomic cell lines when the average of nuclei type; more specifically, the same structural rearrangement or showing one or three signals, respectively, was greater than the chromosomal gain had to be present in at least two metaphases, mean ϩ3 SD of the controls. Cases with an average of Ͼ30% of whereas loss of a chromosome had to be detected in at least nuclei showing one spot were considered showing monosomic three metaphases. When different tumor cell populations were cell lines. Cases with an average of Ͼ1% of nuclei showing identified, the chromosome complement of each population was three spots were considered showing trisomic cell lines. described. A haploid cell line was defined when the chromo- DNA for LOH analysis was extracted from fresh tumor some number ranged from 10 to 34, whereas a diploid cell line tissue by QIAamp tissue kits, and the corresponding normal was defined when the chromosome number ranged from 35 DNA was obtained from peripheral blood samples by QIAamp to 57. blood kits (Qiagen, Hilden, Germany). FISH Analysis. FISH analysis on tumor metaphases was The LOH study was performed using PCR amplification of performed according to the method of Pinkel et al. (30) with eight microsatellite markers mapping on 6q27 (D6S193 and some modifications, as previously reported by Tibiletti et al. D6S297), 6q26 (D6S305, D6S415, and D6S441), 6q21 (26). A whole chromosome painting for chromosome 6 digoxi- (D6S287), 6q16 (D6S311), and 6q14 (D6S300). Primers were genin-labeled (purchased by Oncor) and YAC clones mapped in prepared with the forward primer end labeled with either 6- 6q26-27 (17IA12, 4HE8, and 74E9 from the Imperial Cancer carboxyfluorescein or hexachloro-6-carboxyfluorescein phos- Research Fund library; 962B4 from the Centre d’Etude du phoramidites and were purified by standard HPLC. Genomic Polymorphisme Humain library) labeled with biotinylated 16- DNA was amplified according standard protocols in a Perkin- dUTP (Boehringer Mannheim, Mannheim, Germany) by a ran- Elmer Gene Amp Thermal 2400. All reactions were performed

Table 1 Karyotypes of borderline ovarian tumors Case no. Karyotype 1 36-52,XX,ϩ3[5],ϩ4[4],ϩ5[5],del(6)(q26-qter)[7],ish(wcp6x1)[5]Ϫ10[3],ϩ12[6],Ϫ15[4],Ϫ17[4][cp10] 2 39-41,XX,Ϫ6[3],ish(wcp6x1)[8]Ϫ7[3],Ϫ16[3],Ϫ21[3][cp5] 3 46,XX,del(6)(q25-qter)ishdel(YAC756b12-)[15] 4 23-34,X,ϩX[3],ϩ2[2],ϩ5[2],ϩ11[2],ϩ13[2],ϩ19[2][cp3]a 40-46,XX,Ϫ6[3][cp4]a 5 42-47,XX,Ϫ8[3],ϩ1-5mar[cp7] 6 36-49,XX,ishdel(6)(q27)(YAC 17ia12-;YAC 4he8-)ϩmar[1][cp9] 7 35-47,XX,del(6)(q24-qter)[5],Ϫ7[3],Ϫ8[5],Ϫ11[3],Ϫ13[3],Ϫ14[3],Ϫ15[4],Ϫ16[3],Ϫ22[3],[cp10] 8 46,XX,ishdel(6)(q27)(YAC30cd7-;YAC 4he8-)[20] 9 37-48,XX,del(6)(q27-qter)[4],Ϫ17[4],Ϫ19[3][cp6],ish nunc(wcp6x1)[179] 10 44-47,XX,del(6)(q26-qter)[7],ϩ14[2][cp8] 11 42-45,XX,ϪX[3],ϩ1[2],del(6)(q24-qter)[4],ϩ1-2mar[cp5] 12 10-31,X,ϩ1[2],Ϫ3[3],ϩ13[2],Ϫ17[3],Ϫ19[3],ϩ22[3][cp4]a 38-44,X,ϪX[3],Ϫ15[3],Ϫ17[4],Ϫ18[3],Ϫ20[4][cp6]a 13 27-34,X,ϩ1[2],ϩ2[3],ϩ3[3],ϩ5[2],ϩ9[2],ϩ11[2],ϩ12[3],ϩ13[2],ϩ14[2],ϩ15[2],ϩ18[3][cp3]a 35-47,X,ϪX[4],del(6)(q24-qter)[3],Ϫ6[3],Ϫ14[3],Ϫ15[5],Ϫ16[5],Ϫ17[4],Ϫ18[3],Ϫ19[3],Ϫ20[4],Ϫ21[3],Ϫ22[3][cp12]a 14 14-29,X,ϩ1[3],ϩ2[2],Ϫ9[4],ϩ10[2],Ϫ10[3],Ϫ13[3],Ϫ14[3],ϩ17[2],ϩ20[2],ϩ21[2],Ϫ22[4][cp6]a 46,XX,del(6)(q26-qter)[4]a 35-47,XX,del(4)(p14-pter)[2],del(6)(q26-qter)[3],Ϫ6[4],Ϫ10[4],Ϫ11[3],Ϫ14[4],Ϫ15[6],Ϫ18[5],Ϫ19[6],Ϫ21[3],Ϫ22[3][cp10]a 15 36-46,XX,ϪX[2],del(6)(q24-qter)[4],Ϫ10[2],Ϫ17[3],Ϫ20[4],[cp16] a Different cell lines in the same case.

in duplicate, and losses were always confirmed by extra repe- titions. PCR products were analyzed by an automated laser fluo- rescent sequencer apparatus (ABI Prism 310 Genetic Analyzer Applied Biosystems, Milan, Italy), and fragment sizing analysis was performed with GeneScan 672 (version 1.2) software (Ap- plied Biosystems). An imbalance factor was defined as the ratio of relative allelic peak height in the tumor DNA to relative allelic peak height in the corresponding normal DNA. The formula used for the calculation was: N1:N2/T1:T2, where T1 and N1 are the height values for the smaller allele and T2 and N2 are the height values for the larger allele of the tumor and normal samples, respectively. LOH was scored in informative cases (heterozy- gous individuals) when the imbalance factor was Ͻ0.5 or Ͼ1.5.

RESULTS The tumoral karyotype was established in 15 TBM (Table 1). In 11 cases, only a diploid cell line was detected, whereas in Fig. 1 FISH analysis with chromosome 6 ␣-satellite and YAC clone the remaining 4 tumors, a haploid cell line was detected in 4HE8 as probes on TBM case 8 metaphase. The loss of YAC clone addition to the diploid one. 4HE8 region in the interval defined by markers D6S193 and D6S149 was observed on one chromosome 6 homologue. Two cases showed a simple karyotype, in which the sole chromosome anomaly was 6q deletion (Table 1, cases 3 and 8). A composite karyotype was found in the remaining 13 TBM, in which structural chromosome rearrangements were observed, lomere order), both mapped to 6q27 (28, 31). Fig. 1 depicts the including a 6q deletion (in 9 cases), chromosome markers of loss of one YAC clone (clone 4HE8) in one of the chromosome unknown origin (in 3 cases), and a 4p deletion (in 1 case). A 6 homologues from the same case. Clone 4HE8 is located within variety of aneuploidies were identified, the more frequent being the genomic region bordered by clones 17IA12 and 74E9. Fig. monosomies of chromosomes 6, 15, and 17. FISH with YACs 2 shows the locations of the relevant YAC clones encompassing mapping in 6q as probes was performed on tumor metaphases of the chromosomal region defined by markers D6S149 and 6 TBM (Table 1, cases 1, 2, 3, 6, 8, and 9). These experiments D6S193. The latter region, in particular the smaller interval confirmed, at a molecular level, the presence of deletion span- bounded by markers D6S193 and D6S297, is considered by ning the 6q24 to 6q27 regions. Interestingly, in one case (Table several authors the region of minimal deletion in ovarian carci- 1, case 8) with normal karyotype, FISH analysis identified a nomas (25). FISH with WCP 6 as probe on direct preparations small molecular 6q deletion whose breakpoints fall within YAC confirmed monosomy for chromosome 6 in case 2 and revealed clones 17IA12 and 74E9 (positioned in the centromere to te- the same monosomy in cases 1 and 9.

Fig. 2 Top, YAC contig spanning the D6S193–D6S149 interval in 6q27. Middle, long- range restriction map of the same genomic interval. CEN, centromere; TEL, telomere. Bottom, subterminal and termi- nal bands of the long arm of chromosome 6.

Interphasic FISH analysis on paraffin-embedded sections, using the ␣-satellite probe for chromosome 6, was possible in Table 2 FISH analysis of chromosome 6 number in epithelial cells six cases (Table 1, cases 1–7). Table 2A shows the results of Data are percentage of total number of nuclei with 1, 2, or Ն3 spot evaluation. With the appropriate controls (Table 2b), a signals/nucleus, as a mean value obtained from three independent op- monosomic cell line for chromosome 6 was identified in all but erators. The cutoff values were 30% for monosomy and 1% for trisomy. one case in addition to a disomic cell line. A trisomic cell line Nuclei (%) with number of was identified in three cases (cases 2, 3, and 4). signals DNAs of 15 TBM were also examined for 6q LOH analysis No. of Ն at eight polymorphic markers scattered along 6q at the following Case no. nuclei 12 3 positions: D6S193, D6S297 (6q27), D6S305, D6S415, D6S441 A. TBM (6q26), D6S311 (6q16), D6S287 (6q21), and D6S300 (6q14). 1 181 33.9 64.2 1.9 Microdissection was performed in samples in which the epithe- 2 144 35.8 63.1 1.1 lial components appeared to be well isolable from tumoral 3 143 50.9 49.1 0.0 stroma or surrounding normal tissues; nevertheless, the percent- 4 165 36.6 62.0 1.4 6 136 26.0 73.1 0.9 age of tumoral cells increased to 70% in only four cases. Allelic 7 215 37.9 61.6 0.5 imbalance at all analyzed loci was observed in two of these four cases, suggesting a complete 6q loss, whereas no LOH and LOH B. Normal controls at a single D6S193 locus was detected in the two remaining 1 102 16.1 83.9 0.0 cases, respectively. In the other 11 TBM samples showing a 2 95 16.2 83.5 0.3 percentage of tumoral cells ranging from 10% to 60%, absence 3 117 16.6 83.3 0.1 4 84 18.6 81.4 0.0 of allelic imbalance or an inconclusive LOH pattern at noncon- 5 160 25.2 74.8 0.0 tiguous loci were detected (data not shown). 6 107 16.9 82.6 0.5 LOH analysis thus was able to demonstrate loss on 6q in 2 7 105 12.6 87.0 0.4 cases, whereas cytogenetic approach revealed 6q loss in 13 of 15 Mean value 17.5 72.1 0.2 cases. SD 3.9 3.7 0.2

DISCUSSION ular relevance with respect to another important and unresolved The present study was designed to determine whether issue that relates to the question of whether ovarian cystadeno- ovarian TBM display a pattern of allelic LOH and 6q deletions mas, borderline tumors, and carcinomas represent distinct dis- similar to that of invasive carcinomas. Different technical ap- ease entities or are part of a disease continuum. Our results proaches were used (conventional cytogenetics, interphasic showing that 6q abnormalities are present in benign (28), bor- FISH, and LOH analysis) to avoid the bias of intratumor heter- derline (this report), and malignant ovarian neoplasms (26) ogeneity that characterize the neoplasms of borderline malig- suggest that some TBM may arise from benign tumors and that nancy. malignant tumors may evolve from tumors of borderline malig- Our data demonstrate that a variety of chromosome 6 nancy. In particular, our findings are compatible with the con- anomalies, either as cytogenetic and molecular deletion of 6q cept that higher grade components might arise from the ele- regions or as numeric anomalies (monosomy or trisomy) are ments of borderline malignancy as a result of clonal expansion present in ovarian TBM. Cytogenetic results demonstrated the and that genes located on 6q may play a part in the early events heterogeneity of TBM, showing clones with a 6q deletion, of ovarian tumorigenesis. It is now established, as recently clones with monosomy of chromosome 6 (Table 1, cases 1, 2, 9, reviewed by Scully (37), that a portion (although not the ma- 13, and 14), and clones with a haploid chromosome comple- jority) of ovarian cancers do not arise de novo but within or ment. These results are clearly in contrast to the few data contiguous to benign epithelial tumors. Although the vast ma- reported in previous studies, which indicate that 6q alterations jority of tumors with a de novo origin are mucinous in nature, it are a marker of more advanced stages of ovarian tumorigenesis has been reported that a subset of serous carcinomas arise in (6). Cytogenetic deletions detected in our cases ranged from benign (and borderline) serous epithelial tumors (37). We be- 6q24-qter to 6q27-qter. Of note, molecular cytogenetic analysis lieve that the data presented here, based on the genetic contri- of case 8 (Table 1) revealed the smallest deletion detected in our bution of chromosome 6 to the pathogenesis of tumors of cohort, spanning at least the genomic interval corresponding to borderline malignancy, might offer experimental evidence at YAC clones 4HE8 and 30CD7, which are part of a YAC contig least for the above-mentioned subset of serous tumors. bounded in centromere-to-telomere order by YAC clones Clearly, in addition to genes located on 6q, other genes 17IA12 and 74E9, respectively (28). This interval is estimated mapping to different chromosomal arms, such as 17q (20), Xq to be 300 kb in size; it is therefore considered the region of (21), 7p, 7q, 9p, 11q (18), and 3p (19), might be crucial in minimal deletion for these type of tumors. Interestingly, a few ovarian carcinogenesis; more importantly, these genetic lesions genes have already been positioned in such intervals, including are shared between TBM and invasive carcinomas, strengthen- (from the centromere to the telomere) RNASE6PL which is the ing the concept of a common molecular origin of these two human homologue of the Rh/T2/S-glycoprotein RNase family entities (18–21, 38). (32, 33); FOP, a gene coding for a protein with leucine repeats Additionally, it is worth noting that most of the deletions or folding into a ␣ helix (34); STRL22, which codes for a new rearrangements in our TBM cohort map to or include the 6q27 G-protein-coupled receptor related to chemokine receptors (35), region, where an SV40 immortalization gene (SEN6) has re- the human homologue of the Candida elegans unc-93;4 and cently been mapped (39). This gene has been localized to the TCP10, the human homologue of the mouse t-complex re- broad D6S133–D6S281 interval, which includes the smaller sponder gene (36). These genes could potentially be considered genomic region defined by YACs 4HE8 and 30CD7. Interest- candidates for harboring inactivating mutations in the remaining ingly, we have recently found that the RNASE6PL gene, local- allele, and case 8 could be very instructive in this respect. As ized in this region (32, 33), induces senescence on transfection mentioned above, very few molecular data are available in the into immortal cell lines and might therefore fulfill the functions literature on 6q assessment in TBM, and although LOH was of the above-mentioned senescence gene (40). Furthermore, the detected at several 6q loci, it was not considered of particular region corresponding to the previously mentioned YAC clones relevance (6). For example, in a study analyzing 46 TBM and 20 has also been found to be deleted in benign cysts of the ovary invasive carcinomas (6), 6q LOH was identified with a fre- (28). These intriguing findings raise an important and still quency of 11% in TBM compared with 29% in AC, and the controversial point as to whether an immortalization step is a conclusion was that allelic loss at 6q may not be involved in the prerequisite for ovarian tumorigenesis. development of borderline ovarian tumors. We report data different from those reported in the literature and envisage that some of the reasons for the failure to REFERENCES detect, by molecular means, a more significant frequency of 6q 1. Scully, R. E. World Health Organization. International Histological lesions could be ascribed to either the diffuse intratumor heter- Classification of Tumors. Berlin: Springer Verlag, 1999. ogeneity in neoplasms of borderline malignancy or (less likely) 2. Gallion, H. H., Pieretti, M., DePriest, P. D., and van Nagell, J. R. The to contamination by normal stromal cells intermingled with molecular basis of ovarian cancer. Cancer (Phila.), 76: 1992–1997, tumoral cells. Indeed, the LOH data that we have obtained 1995. reflect such caveats. 3. McCluskey, L. L., and Dubeau, L. Biology of ovarian cancer. Curr. Opin. Oncol., 9: 465–470, 1997. The involvement of 6q abnormalities in TBM is of partic- 4. Dodson, M. K., Hartmann, L. C., Cliby, W. A., DeLacey, K. A., Keeney, G. L., Ritland, S. R., Su, J. Q., Podratz, K. C., and Jenkins, R. B. Comparison of loss of heterozygosity patterns in invasive low- grade and high-grade epithelial ovarian carcinomas. Cancer Res., 53: 4 F. Acquati and R. Taramelli, unpublished data. 4456–4460, 1993.

5. Wertheim, I., Muto, M. G., Welch, W. R., Bell, D. A., Berkowitz, 24. Orphanos, V., McGown, G., Hey, Y., Thorncroft, M., Santibanez- R. S., and Mok, S. C. P53 gene mutation in human borderline epithelial Koref, M., Russel, S. E. H., Hickey, I., Atkinson, R. J., and Boyle, J. M. ovarian tumors. J. Natl. Cancer. Inst. (Bethesda), 86: 1549–1551, 1994. Allelic imbalance of chromosome 6q in ovarian tumors. Br. J. Cancer, 6. Rodabaugh, K. J., Blanchard, G., Welch, W. R., Bell, D. A., Berkow- 71: 666–669, 1995. itz, R. S., and Mok, S. C. Detailed deletion mapping of chromosome 6q 25. Cooke, I. E., Shelling, A. N., Le Meuth, V. G., Mark, F., Charnock, in borderline epithelial ovarian tumors. Cancer Res., 55: 2169–2172, L., and Ganesan, T. S. Allele loss on chromosome arm 6q and fine 1995. mapping of the region at 6q27 in epithelial ovarian cancer. Genes 7. Tangir, J., Loughridge, N. S., Berkowitz, R. S., Muto, M. G., Bell, Chromosomes Cancer, 15: 223–233, 1996. D. A., Welch, W. R., and Mok, S. C. Frequent microsatellite instability 26. Tibiletti, M. G., Bernasconi, B., Furlan, D., Riva, C., Trubia, M., in epithelial borderline ovarian tumors. Cancer Res., 56: 2501–2505, Buraggi, G., Franchi, M., Bolis, P., Mariani, A., Frigerio, L., Capella, 1996. C., and Taramelli, R. Early involvement of 6q in surface epithelial 8. Mitelman, F., Catalog of Chromosome Aberrations in Cancer, 7th ed. ovarian tumors. Cancer Res., 56: 4493–4498, 1996. New York: Wiley-Liss Inc., 1994. 27. Shridhar, V., Staub, J., Huntley, B., Cliby, W., Jenkins, R., Pass, 9. Roberts, C. G., and Tattersall, M. H. N. Cytogenetic study of solid H. I., Hartmann, L., and Smith, D. I. A novel region of deletion on ovarian tumors. Cancer Genet. Cytogenet., 48: 243–253, 1990. chromosome 6q23.3 spanning less than 500Kb in high grade invasive 10. Tharapel, S. A., Qumsiyeh, M. B., Photopulos, G. B. Numerical epithelial ovarian cancer. Oncogene, 18: 3913–3918, 1999. chromosome abnormalities associated with early clinical stages of 28. Tibiletti, M. G., Trubia, M., Ponti, E., Sessa, L., Acquati, F., Furlan, gynaecologic tumors. Cancer Genet. Cytogenet., 55: 89–96, 1991. D., Bernasconi, B., Fichera, M., Mihalich, A., Ziegler, A., Volz, A., 11. Yang-Feng, T. L., Li, S. B., Leung, W. Y., Carcangiu, M. L., and Facco, C., Riva, C., Cremonesi, L., Ferrari, M., and Taramelli, R. Schwartz, P. E. Trisomy 12 and K-ras-2 amplification in human ovarian Physical map of the D6S149–D6S193 region on chromosome 6q27 and tumors. Int. J. Cancer, 48: 678–684, 1991. its involvement in benign surface epithelial ovarian tumors. Oncogene, 12. Jenkins, R. B., Bartelt, D., Jr., Stalboerger, P., Persons, D., Dahl, 16: 1639–1642, 1998. R. J., Podratz, K., Keeney, G., and Hartmann, L. Cytogenetic studies of 29. Mitelman, F. (ed.). An International System for Human Cytogenetic epithelial ovarian carcinoma. Cancer Genet. Cytogenet., 71: 76–86, Nomenclature. Basel: S. Karger, 1995. 1993. 30. Pinkel, D., Straumet, T., and Gray, J. W. Cytogenetic analysis using 13. Thompson, F. H., Emerson, J., Alberts, D., Liu, Y., Guan, X-Y., quantitative high-sensitivity fluorescence hybridization. Proc. Natl. Burgess, A., Fox, S., Taetle, R., Weinstein, R., Makar, R., Powel, D., Acad. Sci. USA, 63: 2934–2938, 1986. and Trent, J. Clonal chromosome abnormalities in 54 cases of ovarian 31. Hauptschein, R. S., Gamberi, B., Rao, P. H., Frigeri, F., Scotto, L., carcinoma. Cancer Genet. Cytogenet., 73: 33–45, 1994. Venkatraj, V. S., Gaidano, G., Rutner, T., Edwards, Y. H., Chaganti, 14. Izutsu, T., Kudo, T., Shoji, T., and Nishiya, I. Comparative cyto- R. S., and Dalla Favera, R. Cloning and mapping of human chromosome genetic studies of benign, borderline, and malignant epithelial ovarian 6q26–q27 deleted in B-cell non-Hodgkin lymphoma and multiple tu- tumors. J. Obstet. Gynaecol. Res., 22: 541–549, 1996. mor types. Genomics, 50: 170–186, 1998. 15. Pejovic, T., Helm, S., Mandahl, N., Baldetorp, B., Elmfors, B., 32. Trubia, M., Sessa, L., and Taramelli, R. Mammalian Rh/T2/S- Floderus, U. M., Furgyik, S., Helm, G., Himmelman, A., Willen, H., and glycoprotein ribonuclease family genes cloning of a human member Mitelman, F. Karyotypic characteristics of borderline malignant tumors located in a region of chromosome 6(6q27) frequently deleted in human of the ovary: trisomy 12, trisomy 7, and r(1) as nonrandom features. malignancies. Genomics, 42: 342–344, 1997. Cancer Genet. Cytogenet., 4: 58–68, 1996. 33. Acquati, F., Nucci, C., Bianchi, M., Gorletta, T., and Taramelli, R. 16. Deger, R. B., Faruqi, S. A., and Noumoff, J. S. Karyotypic analysis Molecular cloning, tissue distribution and chromosomal localization of of 32 malignant epithelial ovarian tumors. Cancer Genet. Cytogenet., the human homologoue of the R2/th/stylar ribonuclease gene family. 96: 166–173, 1997. Methods Mol. Biol, 160: 87–101, 2001. 17. Wolf, N. G., Abdul-Karim, F. W., Farver, C., Schreck, E., Du 34. Papovici, C., Zhang, B., Gregoire, M., Jonveaux, P., Lafage-Pochi- Manoir, S., and Schwartz, S. Analysis of ovarian borderline tumors taloff, Birbaum, D., and Pebusque, M. The t(6;8)(q27;p11) translocation using comparative genomic hybridization and fluorescence in situ hy- in a stem cell myeloproliferative disorder fused a novel gene, FOP, to bridization. Genes Chromosomes Cancer, 25: 307–315, 1999. fibroblast growth factor receptor 1. Blood, 93: 1381–1389, 1999. 18. Watson, R. H., Neville, P. J., Roy, W. J., Hitchcock, A., and 35. Liao, F., Lee, H., and Farber, J. Cloning of STRL22, a new human Campbell, I. G. Loss of heterozygosity on chromosomes 7p, 7q, 9p and gene encoding a G-protein coupled receptor related to chemokine re- 11q is an early event in ovarian tumorigenesis. Oncogene, 17: 207–212, ceptor and located on chromosome 6q27. Genomics, 40: 175–180, 1997. 1998. 36. Islam, S., Pilder, S., Decker, C., Cebra-Thomas, J., and Silver, L. 19. Lounis, H., Mes-Masson, A. M., Dion, F., Edward, W., Bradley, C., Human homologues of two testes-expressed loci on mouse chromosome Seymour, R. J., Provencher, D., and Tonin, P. N. Mapping of chromo- 17 map to opposite arms of chromosome 6. Hum. Mol. Genet., 2: some 3p deletions in human epithelial ovarian tumors. Oncogene, 17: 2075–2079, 1993. 2359–2365, 1998. 37. Scully, R. E. Influence of the origin of the ovarian cancer on 20. Wertheim, I., Tangir, J., Muto, M. G., Welch, W. R., Berkowitz, efficacy of screening. Lancet, 355: 1028–1029, 2000. R. S., Chen, W. Y., and Mok, S. C. Loss of heterozygosity of chromo- 38. Haas, C. J., Diebold, J., Hirschmann, A., Rohrbach, H., Schmid, S., some 17 in human borderline and invasive epithelial ovarian tumors. and Lo¨hrs, U. Microsatellite analysis in serous tumors of the ovary. Int. Oncogene, 12: 2147–2153, 1996. J. Gynecol. Pathol., 18: 158–162, 1999. 21. Edelson, M. I., Lau, C. C., Colitti, C. V., Welch, W. R., Bell, D. A., 39. Banga, S., Kim, S., Hubbard, K., Dasgupta, T., Jha, K., Patsalis, P., Berkowitz, R. S., and Mok, S. C. A one centimorgan deletion unit on Hauptschein, R., Gamberi, B., Dalla Favera, R., Kraemer, P., and Ozer chromosome Xq12 is commonly lost in borderline and invasive epithe- H. SEN 6, a locus for SV40-mediated immortalization of human cells, lial ovarian tumors. Oncogene, 16: 197–202, 1998. maps to 6q26-27. Oncogene, 14: 313–321, 1997. 22. Saito, S., Saito, H., Koi, S., Sagae, S., Kudo, R., Saito, J., Noda, K., 40. Acquati, F., Morelli, C., Cinquett, R., Bianchi, M., Porrini, D., and Nakamura, Y. Fine-scale deletion mapping of the distal long arm of Varesco, L., Gismondi, V., Rocchetti, R., Talevi, S., Possati, L., Mag- chromosome 6 in 70 human ovarian cancers. Cancer Res., 52: 5815– nanini, C., Tibiletti, M. G., Bernasconi, B., Daidone, M. G., Shridhar, 5817, 1992. V., Smith, D., Negrini, M., Barbanti-Brodano, G., and Taramelli R. 23. Foulkes, W. D., Ragoussis, J., Stamp, G. W. H., Allan, G. J., and Cloning and characterization of a senescence inducing and class II Trowsdale, J. Frequent loss of heterozygosity on chromosome 6 in tumor suppressor gene in ovarian carcinoma at chromosome region human ovarian carcinoma. Br. J. Cancer, 67: 551–559, 1993. 6q27. Oncogene, 20: 980–988, 2001.

Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2001 American Association for Cancer Research. Chromosome 6 Abnormalities in Ovarian Surface Epithelial Tumors of Borderline Malignancy Suggest a Genetic Continuum in the Progression Model of Ovarian Neoplasms

Maria Grazia Tibiletti, Barbara Bernasconi, Daniela Furlan, et al.

Clin Cancer Res 2001;7:3404-3409.

Updated version Access the most recent version of this article at: http://clincancerres.aacrjournals.org/content/7/11/3404

Cited articles This article cites 36 articles, 6 of which you can access for free at: http://clincancerres.aacrjournals.org/content/7/11/3404.full#ref-list-1

Citing articles This article has been cited by 2 HighWire-hosted articles. Access the articles at: http://clincancerres.aacrjournals.org/content/7/11/3404.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/7/11/3404. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.