Somatic genetic alterations (loh) in benign, borderline and invasive ovarian tumours: Intratumoral molecular heterogeneity
1999; Wiley; Volume: 82; Issue: 6 Linguagem: Inglês
10.1002/(sici)1097-0215(19990909)82
ISSN1097-0215
AutoresI. B. Zborovskaya, Alexander V. Gasparian, Appolon Karseladze, Irina Elcheva, Е. А. Трофимова, Keltouma Driouch, Martine Trassard, Alexander Tatosyan, Rosette Lidereau,
Tópico(s)RNA modifications and cancer
ResumoInternational Journal of CancerVolume 82, Issue 6 p. 822-826 Human CancerFree Access Somatic genetic alterations (loh) in benign, borderline and invasive ovarian tumours: Intratumoral molecular heterogeneity Irina Zborovskaya, Irina Zborovskaya Laboratory of Oncogene Regulation, Cancer Research Centre, Moscow, RussiaSearch for more papers by this authorAlexander Gasparian, Alexander Gasparian Laboratory of Oncogene Regulation, Cancer Research Centre, Moscow, RussiaSearch for more papers by this authorAppolon Karseladze, Appolon Karseladze Laboratory of Oncogene Regulation, Cancer Research Centre, Moscow, RussiaSearch for more papers by this authorIrina Elcheva, Irina Elcheva Laboratory of Oncogene Regulation, Cancer Research Centre, Moscow, RussiaSearch for more papers by this authorElena Trofimova, Elena Trofimova Laboratory of Oncogene Regulation, Cancer Research Centre, Moscow, RussiaSearch for more papers by this authorKeltouma Driouch, Keltouma Driouch Oncogénétique, Centre René Huguenin, St-Cloud, FranceSearch for more papers by this authorMartine Trassard, Martine Trassard Anatomo-Pathologie, Centre René Huguenin, St-Cloud, FranceSearch for more papers by this authorAlexander Tatosyan, Alexander Tatosyan Laboratory of Oncogene Regulation, Cancer Research Centre, Moscow, RussiaSearch for more papers by this authorRosette Lidereau, Corresponding Author Rosette Lidereau lidereau@infobiogen.fr Oncogénétique, Centre René Huguenin, St-Cloud, FranceOncogénétique, Centre René Huguenin, F-92211 St-Cloud, France. Fax: +33-1-47 11 16 96.Search for more papers by this author Irina Zborovskaya, Irina Zborovskaya Laboratory of Oncogene Regulation, Cancer Research Centre, Moscow, RussiaSearch for more papers by this authorAlexander Gasparian, Alexander Gasparian Laboratory of Oncogene Regulation, Cancer Research Centre, Moscow, RussiaSearch for more papers by this authorAppolon Karseladze, Appolon Karseladze Laboratory of Oncogene Regulation, Cancer Research Centre, Moscow, RussiaSearch for more papers by this authorIrina Elcheva, Irina Elcheva Laboratory of Oncogene Regulation, Cancer Research Centre, Moscow, RussiaSearch for more papers by this authorElena Trofimova, Elena Trofimova Laboratory of Oncogene Regulation, Cancer Research Centre, Moscow, RussiaSearch for more papers by this authorKeltouma Driouch, Keltouma Driouch Oncogénétique, Centre René Huguenin, St-Cloud, FranceSearch for more papers by this authorMartine Trassard, Martine Trassard Anatomo-Pathologie, Centre René Huguenin, St-Cloud, FranceSearch for more papers by this authorAlexander Tatosyan, Alexander Tatosyan Laboratory of Oncogene Regulation, Cancer Research Centre, Moscow, RussiaSearch for more papers by this authorRosette Lidereau, Corresponding Author Rosette Lidereau lidereau@infobiogen.fr Oncogénétique, Centre René Huguenin, St-Cloud, FranceOncogénétique, Centre René Huguenin, F-92211 St-Cloud, France. Fax: +33-1-47 11 16 96.Search for more papers by this author First published: 10 November 1999 https://doi.org/10.1002/(SICI)1097-0215(19990909)82:6 3.0.CO;2-ICitations: 23AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Loss of heterozygosity (LOH) affects a number of chromosome regions in ovarian cancer, pointing to the possible involvement of tumour-suppressor genes in ovarian tumorigenesis. We performed comparative analysis of allelic loss at 6 frequently affected chromosome regions in a panel of 53 benign, borderline and malignant ovarian tumours. Precursor lesions could provide evidence that an accumulation of genetic events is required for normal ovarian epithelium to generate malignant tumours. LOH on chromosome 1p was relatively common in benign, borderline and malignant tumours, while at 11p and 7q it was observed not only in invasive but also in borderline tumours. Moreover, 17q and 18q were affected mainly in advanced malignant tumours and revealed a high frequency of clonal intratumoral heterogeneity. We encountered different spectra of genetic alterations in primary tumours and their metastasis, which may be the results of intratumoral heterogeneity leading to dissemination in only some sub-clones. Int. J. Cancer 82:822–826, 1999. © 1999 Wiley-Liss, Inc. Exciting progress has been made in the identification of specific genetic abnormalities associated with ovarian malignancies, especially hereditary forms, but over 95% of ovarian cancers are sporadic. Somatic genetic events are involved both in sporadic and in familial ovarian tumorigenesis. The majority of hereditary ovarian cancers are linked to the BRCA1 locus on chromosome 17q (Easton et al., 1993; Foulkes et al., 1993; Narod et al., 1995; Takahashi et al., 1995), while some are linked to the BRCA2 locus on 13q (Takahashi et al., 1996). Other loci may be associated with sporadic ovarian cancer. Studies of loss of heterozygosity (LOH) in ovarian cancer have revealed a number of regions with high frequencies of LOH, pointing to possible involvement of tumour-suppressor genes (Jacobs and Lancaster, 1996). LOH has been observed on almost all chromosome arms, at various frequencies. Allelic deletions in 25 to 40% of tumours have been found on chromosome arms 5q, 6p, 6q, 7q, 9q, 13q and Xp, and in more than 60% of cases on 11p, 11q 17p, 17q, 18q and 22q (Sato et al., 1991; Gallion et al., 1995; King et al., 1995; Kerr et al., 1996). The involvement of tumour-suppressor genes (TSGs) located in these deleted regions in the development of sporadic ovarian cancer is unclear. The high rate of LOH on chromosome 17q may be due to TSGs other than BRCAI, located on the same chromosome arm (Tangir et al., 1996). Few pre-invasive epithelial ovarian cancers have so far been studied. Since some benign and borderline tumours may represent early steps in ovarian carcinogenesis, analysis of precursor lesions could provide evidence that an accumulation of genetic events is required in order for normal ovarian epithelium to generate benign, borderline, malignant or metastatic tumours (Gallion et al., 1995; Iwabuchi et al., 1995; Chenevix-Trench et al., 1997). We studied ovarian tumours of different histological grades (from benign to malignant) for LOH on 6 chromosome arms frequently affected in ovarian cancer. DNA isolated from different tumour regions with various degrees of differentiation and anaplasia (53 paraffin-embedded primary epithelial ovarian tumours, and paired metastases from 9 of these patients) were tested for LOH at 10 polymorphic markers located on 6 chromosome arms (1p, 7q, 8p, 11p, 17q and 18q), in relation to histological and clinical characteristics. LOH was observed in some benign and early malignant tumours. LOH on chromosome 1p was relatively common in benign, borderline and malignant tumours, while LOH on 17q and 18q was observed mostly in advanced malignant tumours. Intratumoral genetic heterogeneity was linked to these chromosome arms. MATERIAL AND METHODS Clinical specimens and DNA extraction We examined 53 formalin-fixed, paraffin-embedded primary epithelial ovarian tumours (3–8 paraffin blocks for each tumour) and the corresponding normal tissues (corpus uteri or Fallopian tube), also 9 metastatic nodes from the same patients. The specimens were selected from the archives of the Pathology Department of the Cancer Research Centre in Moscow. None of the patients had received chemotherapy or radiotherapy before surgery. The FIGO staging, histopathology and differentiation status were reviewed in standardized fashion. The clinical and histopathological characteristics of the 53 sporadic ovarian tumours are presented in Table I. Table I. CHARACTERISTICS OF THE 53 OVARIAN TUMOURS INCLUDED IN THIS STUDY Histological types of Benign Borderline Malignant ovarian tumours Serous 11 5 12 Mucinous 5 5 4 Mesonephroid 3 Endometrioid 1 2 Mixed 1 2 Undifferentiated 2 Total 18 10 25 In each case, normal samples and foci of neoplastic tissues with various degrees of differentiation and anaplasia were identified independently by 2 histopathologists on haematoxylin-eosin-stained tissue sections to focus on intratumoral heterogeneity. Manual microdissection of selected areas was used to avoid contamination by normal tissues and stromal elements. The 3 to 6 slides of 10-μm sections were de-waxed in toluol and dehydrated in absolute ethanol. The DNA in each area was extracted and purified as described by Farabegoli et al. (1996). Oligonucleotide primers and polymerase chain reaction (PCR) and detection of LOH The markers used and their chromosomal locations are listed in Table II. The markers tested were chosen inside or as close as possible to LOH regions already described in ovarian cancer (Iwabuchi et al., 1995). Microsatellite markers were detected by PCR amplification with standard methods (Driouch et al., 1998). Products were loaded on 6% acrylamide gel containing 7.5 M urea. DNA was then transferred to nylon membrane filters. Probes (CA repeat oligonucleotide or specific primer) were labeled with 32P-dCTP by terminal deoxynucleotidyl transferase (Driouch et al., 1998). Table II. POLYMORPHIC MARKERS USED IN THIS STUDY Microsatellite types Informative Frequency of Markers for Chromosomal tumours/tested heterozygosity allelotyping location tumours (%) D1S160 1p36.2 Ca repeat 29/49 59 MYCL1 1p32 Tetranucleotide 34/46 73 repeat D1S162 1p22.3 Ca repeat 48/52 92 D7S522 7q31.1 Ca repeat 46/54 85 D8S261 8p21.1 Ca repeat 29/39 74 D11S860 11p15.1 Ca repeat 33/42 79 D17S800 17q21.2 Ca repeat 36/50 72 D17S855 17q21.3 Ca repeat 42/51 82 D17S932 17q21.3 Ca repeat 42/51 82 D18S50 18q23 Ca repeat 35/48 73 Tumour DNA samples from each patient were run in adjacent tracks, together with corresponding normal-tissue DNA. LOH was considered to be present when the relative intensity of the 2 alleles in tumour DNA differed from the relative intensity in normal DNA as described (Driouch et al., 1998). Statistical analysis Differences in LOH distribution between the groups of samples were tested by Fisher's exact test; differences between 2 groups were judged significant at a confidence level greater than 95% (p < 0.05). RESULTS In all, 187 tumour DNA samples from various parts of 53 paraffin-embedded primary epithelial ovarian tumours were tested in comparison with normal DNA from each of the 53 patients. Different microdissected tumour DNA samples (3 to 9) from different paraffin blocks were assayed in each case of the 53 cases. LOH was detected by the use of one (7q, 8p, 11p, 18q) or 3 different markers (1p, 17q). When 3 markers were used for the same locus, loss of at least one marker was taken into account. The data were analyzed at 2 levels. The first involved identification of cases bearing deletions of definite markers in at least one DNA sample from a specific tumour (Table III). The second involved intratumour heterogeneity, detected as genetic alterations using the LOH-pattern discrepancies within a single tumour, by studying the various DNA samples isolated from a tumour. Table III. LOH FREQUENCIES IN EPITHELIAL OVARIAN TUMOURS Histology 1p 7q 8p 11p 17q 18q Malignant Serous 6/1211 Number of tumours with LOH/number of informative tumours. 2/11 5/6 5/10 11/12 4/10 Mucinous 0/4 3/4 2/2 2/3 4/4 1/2 Mesonephroid 3/3 2/3 1/2 1/1 2/3 1/2 Endometrioid 0/2 2/2 0/1 2/2 2/2 1/2 Mixed 2/2 2/2 0/1 1/1 2/2 2/2 Undifferentiated 2/2 0/2 2/2 Total11 Number of tumours with LOH/number of informative tumours. 13/25 11/24 8/12 11/17 23/25 9/18 (52.0%) (45.8%) (66.6%) (64.7%) (92.0%) (50.0%) Borderline Serous 1/5 2/4 2/5 3/4 2/4 0/3 Mucinous 1/5 2/4 0/2 0/1 1/5 1/4 Total11 Number of tumours with LOH/number of informative tumours. 2/10 4/8 2/7 3/5 3/9 1/7 (20.0%) (50.0%) (28.6%) (60%) (33.3%) (14.3%) Benign Serous 1/9 3/10 1/6 1/8 0/11 0/6 Mucinous 0/5 1/3 1/2 1/3 2/5 1/3 Endometrioid 1/1 0/1 0/1 0/1 Mixed 1/1 0/1 0/1 Total11 Number of tumours with LOH/number of informative tumours. 3/16 4/15 2/10 2/11 2/16 1/10 (18.7%) (26.6%) (20.0%) (18.2%) (12.5%) (10.0%) 1 Number of tumours with LOH/number of informative tumours. LOH was infrequent in benign tumours of various origins. Less than 25% of benign tumours bore LOH at at least one marker (Table III), and only 2 of the 18 benign cystadenomas bore 2 different deleted regions. One of these cases showed LOH at markers D11S860 and D17S855, the other at markers D17S855 and D18S50. In the latter case, marker D17S855 was deleted in the 5 DNA samples from different regions of the same benign tumour, while marker D18S50 was lost in only one of the 5 samples. The frequency of LOH in borderline tumours was either lower than that in malignant tumours (1p, 8p, 17q and 18q) or similar (7q and 11p). Parts of 8p, 11p and 17q were frequently deleted in malignant tumours (66.6, 64.7 and 92.0% respectively). A lower frequency of LOH was found on chromosome arms 1p, 7q and 18q. LOH was found on at least 3 different chromosome arms in 19 (76%) of the 25 malignant tumours. The frequency of LOH on chromosome arms 8p, 11p and 17q was similar in early and advanced malignant tumours, while 7q and 18q LOH appear to be more frequent in stages III–IV. We also found a stage-dependent tendency towards 1p LOH in malignant tumours. We then investigated the relationship between LOH and the histological type of the malignant tumours. 1p LOH was more frequent in serous than in mucinous tumours (6/12 and 0/4 respectively). In contrast, marker D7S522 was more frequently deleted in mucinous (3/4) than in serous (2/11) malignant tumours. LOH was more frequent in more aggressive tumours, such as mesonephroid and undifferentiated forms. Two tumours of mixed histological type bore LOH in most of the loci tested (Table III). Finally, we examined the pattern of LOH in various histological types of malignant and benign tumours. No significant difference in the pattern of LOH was found, but some interesting features emerged (Table III, IV). Independently of malignancy, 1p LOH was more frequent in serous than in mucinous tumours (8/26 and 1/14 respectively) (Table IV). In contrast, chromosome 7q was more frequently deleted in mucinous than in serous tumours (Table IV). Table IV. FREQUENCY OF LOH IN SEROUS AND IN MUCINOUS EPITHELIAL OVARIAN TUMOURS Histology 1p 7q 8p 11p 17q 18q Serous Cancer 6/1211 Number of tumours with LOH/number of informative tumours. 2/11 5/6 5/10 11/12 4/10 Borderline 1/5 2/4 1/5 3/4 2/4 0/3 Benign 1/9 3/10 1/6 1/8 0/11 0/6 Total11 Number of tumours with LOH/number of informative tumours. 8/26 7/25 8/17 9/22 13/27 4/9 (30.8%) (28%) (47.1%) (40.9%) (48.1%) (44.4%) Mucinous Cancer 0/4 3/4 2/2 2/3 4/4 1/2 Borderline 1/5 2/4 0/2 0/1 1/5 1/4 Benign 0/5 1/3 1/3 1/3 2/5 1/3 Total11 Number of tumours with LOH/number of informative tumours. 1/14 6/11 3/6 3/7 7/14 3/9 (7.1%) (54.5%) (50%) (42.8%) (50%) (33.3%) 1 Number of tumours with LOH/number of informative tumours. The main problem with ovarian tumours is their extreme structural diversity, meaning that LOH at some chromosome locations could occur in specific histological foci or cell sub-clones. By studying multiple regions of a given tumour for LOH at various loci, we found tumour areas with and without LOH in the same sample. We detected intratumoral clonal heterogeneity by analyzing genomic alterations in different parts of a given tumour. Loss of the D17S855 and D18S50 markers in different DNA samples from a single tumour is represented in Figure 1. The various tumour areas differed in their grade of anaplasia and cell differentiation. LOH was observed only in poorly differentiated areas of this tumour (lanes 1, 4 and 5 for D17S855 and lanes 4 and 5 for D18S50). Figure 2 shows slides from tumour regions corresponding to DNA samples 5 and 6. Figure 1Open in figure viewerPowerPoint LOH analysis of 2 microsatellite markers on chromosomes 17q and 18q in DNA extracted from different regions of one ovarian serous carcinoma (2864). Lanes 1, 4, 5, DNA from poorly differentiated areas; lane 2, DNA from normal tissue; lane 3, DNA from metastasis to omentum; lane 6, DNA from well-differentiated areas of the same tumour. Figure 2Open in figure viewerPowerPoint Various microscopic patterns in the ovarian serous carcinoma 2864. (a) Well-differentiated papillary structure corresponding to lane 6 DNA sample in Figure 1. (b) Poorly differentiated solid structure corresponding to lane 5 in Figure 1. Scale bars = 100 μm. The distribution of intratumoral heterogeneity using the LOH study is shown in Table V. We observed only 2 cases of intratumoral heterogeneity among the 16 cases bearing LOH at the marker on 11p, and both were malignant. Markers located on 1p, 7q and 8p also showed a low rate of intratumoral heterogeneity. In contrast, more than half the tumours bore 17q and 18q LOH in only certain areas. Moreover, in mixed-type epithelial ovarian cancer, distinct spectra of LOH were found in different histological areas. Table V. INTRATUMORAL HETEROGENEITY DETECTED BY LOH ANALYSIS Markers Tumours with Borderline and LOH showing Malignant tumours benign tumours genetic with LOH showing with LOH Tumours heterogeneity genetic heterogeneity showing genetic with LOH (%) (%) heterogeneity D1S160 12/29 4/12 (33) 4/11 (36.4) 0/1 MYCL1 11/34 4/11 (36) 3/10 (30.0) 1/1 D1S162 10/48 3/10 (30) 2/9 (22.2) 1/1 D7S522 19/47 6/19 (32) 4/11 (36.4) 2/8 D8S261 12/29 3/12 (25) 3/8 (37.5) 0/4 D11S860 16/33 2/16 (12) 2/11 (18.2) 0/5 D17S800 17/36 10/17 (59)11 Significantly different LOH intratumoral heterogeneity compared with the D11S860 marker (p < 0.02; Fisher's test). 8/15 (53.3) 2/2 D17S855 19/42 11/19 (58)11 Significantly different LOH intratumoral heterogeneity compared with the D11S860 marker (p < 0.02; Fisher's test). 9/17 (52.9) 2/2 D18S50 11/35 5/11 (45) 4/9 (44.4) 1/2 1 Significantly different LOH intratumoral heterogeneity compared with the D11S860 marker (p < 0.02; Fisher's test). The pattern of LOH was also studied in 9 primary tumours and metastatic nodes (Table VI). In 4 of the 9 cases, the spectrum of LOH in the metastasis was identical to that in the primary tumour (Group A). These cases did not show intratumoral heterogeneity with any of the markers tested. In the other 5 cases, the LOH patterns were not completely identical in the primary tumour and the metastatic node (Group B). In 3 cases, we observed deletions in the metastatic nodes that were absent from the corresponding primary tumour (serous cancers 11225 and 2864 for marker D7S522; and mesonephroid cancer 1496 for D18S50). It is interesting to note that no loss of markers on 1p (cases 10657 and 1496) or 18q (case 2864) was detected in metastases, while some areas of the corresponding primary tumours had lost these markers (Fig. 1, case 2864, lanes 1, 3–5) but more differentiated regions had not (lane 6). Table VI. CHROMOSOMAL DELETIONS IN MALIGNANT TUMOURS AND PAIRED METASTASES11 Group A, group with identical spectrum of deletions in tumour and its paired metastasis; Group B, group with differences in spectrum of deletions in tumour and its paired metastasis. Case Histology Sample22 Type of samples: T, primary tumour; M, node metastasis. 1p33 I, informative cases; NI, non-informative cases; LOH, loss of heterozygosity on this chromosome arm; LOH (1/2), LOH affected 1 of the 2 different DNA samples from the same tumour tested for a specific region. Boldtype cases with differences between tumours and metastatic samples. 7q 8p 11p 17q 18q Group A 9697 Serous T I I NI I LOH I M I I NI I LOH I 8097 Serous T I LOH NI LOH LOH LOH M I LOH NI LOH LOH LOH 10083 Serous T LOH NI LOH LOH LOH M LOH NI LOH LOH LOH 56069 Mesonephroid T LOH I LOH LOH NI M LOH I LOH LOH NI Group B 11225 Serous T LOH (1/2) I NI I LOH (1/2) NI M LOH LOH NI I LOH NI 2864 Serous T I I I NI LOH (4/6) LOH (2/5) M I LOH I NI LOH I 9305 Mucinous T I LOH (3/9) LOH (6/9) I LOH (9/11) NI M I LOH I I LOH NI 1496 Mesonephroid T LOH (2/4) LOH (3/4) LOH (1/4) LOH LOH (3/4) I M I LOH LOH LOH LOH LOH 10657 Mesonephroid T LOH (1/3) LOH LOH I I M I LOH LOH I I 1 Group A, group with identical spectrum of deletions in tumour and its paired metastasis; Group B, group with differences in spectrum of deletions in tumour and its paired metastasis. 2 Type of samples: T, primary tumour; M, node metastasis. 3 I, informative cases; NI, non-informative cases; LOH, loss of heterozygosity on this chromosome arm; LOH (1/2), LOH affected 1 of the 2 different DNA samples from the same tumour tested for a specific region. Boldtype cases with differences between tumours and metastatic samples. DISCUSSION The identification of primary, genetic abnormalities and secondary, growth-modulating changes is crucial to the understanding of tumour genesis and progression. Chromosome losses, deletions and unbalanced translocations, all entailing a loss of chromosomal material, are frequent in ovarian tumours (Jacobs and Lancaster, 1996). In view of the numerous histological types of ovarian cancer, it would not be surprising if different sets of genetic events were associated with different pathways of tumour development. Evidence of this is emerging from studies of LOH at particular loci and its link to tumour grade and histological type (Papp et al., 1996; Lu et al., 1997). This study of allelic loss in benign, borderline and malignant tumours suggests that an accumulation of genetic events is associated with progression from benign adenomas via borderline to invasive malignancy. Moreover, somatic genetic alterations seem to be specific to some histological tumour types: 1p LOH was more frequent in serous than in mucinous tumours, while chromosome 7q was more frequently deleted in mucinous tumours (Table IV). The observed LOH at D11S860 and D7S522 in borderline cystadenomas and in stage-II invasive tumours raises the possibility that these somatic genetic events occur relatively early in ovarian neoplastic development. This is in keeping with data on allelic imbalance on chromosome 11 and clinicopathological data on ovarian tumours (Gabra et al., 1995). In our study, a high frequency of LOH at D11S922 (11p15.5) was found in benign and borderline tumours, suggesting that a 18.6-Mb interval within 11p15 houses a gene involved early in ovarian carcinogenesis. Now, the interval has been narrowed down to 4 cM on 11p15.1, and this region may harbor tumour-suppressor genes with a role in high-grade epithelial ovarian cancer (Lu et al., 1997). The low rate of intratumoral heterogeneity for marker D11S860 and the lack of any apparent link with the grade of anaplasia also supports this possibility. In contrast to Zheng et al. (1991), Gabra et al. (1995) found no link between LOH in the 11p15.5 region and advanced disease or poor prognosis. The alterations on chromosome arm 7q were found in the entire spectrum of ovarian tumours, from benign to malignant. We found frequent LOH at D7S522 in borderline and stage-II malignant tumours, in keeping with early events described in breast tumorigenesis (Champème et al., 1995); however, this does not fit with data from Kerr et al. (1996), who found that D7S522 LOH was significantly associated with advanced-stage tumours. We detected a relatively high level of LOH in the sub-telomeric (D1S162 and MYCL) and telomeric regions (D1S160) of chromosome 1 in advanced ovarian cancer, while loss of these loci was infrequent in benign and early-stage borderline tumours. The high frequency of LOH in the locus containing BRCAI observed in invasive tumours is consistent with earlier reports (Foulkes et al., 1993; Sato et al., 1991; Pieretti et al., 1995; Weitzel et al., 1994). Jacobs and Lancaster (1996) even reported allelic deletion on 17q in a benign ovarian tumour. The results of this analysis suggest that LOH at 17 q21.2 is common in undifferentiated tumour areas but infrequent in highly differentiated malignant tumours and in benign tumours. We observed a low level of LOH in this region, also in the DCC locus on 18q in benign and early-stage borderline tumours, suggesting that these events may not be critical for the development of tumours with low malignant potential, in agreement with other data (Wertheim et al., 1996). Nevertheless, 17q and 18q LOH was highly frequent in high-stage malignant invasive ovarian tumours. Benign and borderline tumours bearing LOH at specific loci might be precursors of invasive disease: in other words, 17q and 18q LOH in benign tumours might be a risk factor for malignancy. By analyzing different regions of the same tumour we observed intratumoral heterogeneity in some chromosomal regions, particularly those characteristic of high-grade tumour cells. LOH on 17q and 18q revealed a high frequency of genetic somatic heterogeneity, predominantly in advanced stage. We found only one case of this in a stage-II tumour. We also encountered a different spectrum of genetic alterations in primary tumours and in their node metastases; this could imply that intratumoral heterogeneity leads to dissemination of certain sub-clones only. This phenomenon was particularly marked in malignant tumours (particularly in 2 of the 3 mesonephreoid tumours tested). Interestingly, no additional LOH was observed in the metastases when no intratumoral genetic heterogeneity was detected in the primary tumour. In contrast, the absence of specific genetic alteration in metastases relative to the primary tumour tends to confirm that metastatic dissemination may occur early in the course of the disease. The picture currently emerging is that different tumour-suppressor genes are involved in ovarian cancer: genes whose inactivation leads to the transformation of normal cells (presumably on 7p or 11p), and other genes whose inactivation is responsible for disease progression. Our study has clearly indicated that clonal intratumoral heterogeneity might lead to errors in molecular analysis of cancer biopsy specimens. Acknowledgements This study was supported by INSERM (Project 94 E005, "Sporadic ovarian cancer: analysis of genes involved in ovary tumorigenesis," headed by Dr. A. Tavitian, U.248 INSERM). 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