- Email: [email protected]
Chromosomal changes in ovarian cancer
Current Diagnostic Pathology (2003) 9, 328 --332
c 2003 Published by Elsevier Ltd. doi:10.1016/S0968 - 6053(03)00050 - 4
REVIEW
Chromosomal changes in ovarian cancer D. Mayr and J. Diebold Pathologisches Institut der Ludwigs-Maximilians-Universitt Mˇnchen, Thalkirchner Strasse 36, D- 80337 Mˇnchen, Germany
KEYWORDS ovary; carcinoma; granulosa cell tumour; immunohistochemistry; fluorescence-in-situhybridization (FISH); comparative genomic hybridization (CGH)
Summary Ovarian neoplasms display a wide range of different phenotypic patterns. Recently, there has been much research that has identif|ed the molecular genetic aberrations which are present in ovarian neoplasms, and that has linked these to the phenotypic patterns. Invasive ovarian carcinomas show complex genetic changes. Chromosomal gains at 3q26, 8q24 and 20q13 apparently represent early lesions, whereas loss of material of chromosomes 4,13,16,18 and X is associated with tumour progression and poor prognosis.The maintargets ofchromosomalchanges areregulatorygenes ofcellproliferation and apoptosis (e.g. p16, cyclin D1,Rb, p53, myc and bcl-2), and members of the signalling cascade of tyrosine kinase receptors (e.g. HER/2neu, dab-2, K-ras, PI3-K and PTEN). Granulosa cell tumours usually contain numeric chromosomal aberrations (monosomy 22, trisomy12 and14). The genetic alterations of ovarian neoplasms which have been described to date correlate with some aspects ofthe biologicalbehaviour ofthese tumours.However, theydo not fully explain the spectrum of phenotypic variability.
c 2003 Published by Elsevier Ltd.
Ovarian cancer is the leading cause of death from gynaecological malignancy in Europe and the USA. Nearly 95% of cases occur sporadically and, to date, no single causative factor has been identif|ed. Ninety percent of malignant ovarian neoplasms are epithelial tumours. More than half of the women present with an advanced stage of disease and therefore have a poor prognosis.1,2 Multiple genetic changes appear to be involved in the genesis and progression of cancer which affect cell proliferation, cell death or angiogenesis (e.g. p53 and bcl-2).3,4 Currently, FIGO stage and residual tumour mass remain the most important prognostic factors. Data from patients in the Munich Tumour Register show that borderline tumours (low malignant potential) have the best prognosis.5 Amongst invasive carcinomas, the mucinous and endometrioid types have a more favourable course than serous carcinomas (10 -year survival rate of 41% and 33%, respectively, vs 23%). The prognosis of malignant germ cell and stromal tumours, mostly granulosa cell tumours, falls between non-invasive and invasive epithelial tumours with a 10 -year survival rate of 70%. Since the morphological patterns of ovarian neoplasms seem to correlate with prognosis, it Correspondence to: DM. Tel.: +49 (0) 89/5160 - 4671; Fax: +49 (0) 89/ 5160 - 4043; E-mail: [email protected]
is of interest whether there are consistent genetic alterations associated with these patterns.
PRECURSOR LESIONS About 5% of invasive carcinomas are caused by genetic predisposition, either due to the autosomal-dominant hereditary breast-ovarian-cancer syndrome or as part of hereditary non-polyposis colorectal cancer (HNPCC) syndrome. The morphology of ovarian cancer precursor lesions is poorly described. For decades, endometriosis, one of the most common gynaecological diseases, has been regarded as a putative precursor lesion for endometrioid carcinomas. In agreement with this notion, several authors demonstrated monoclonality of endometrioid foci.6 --10. In our own study, def|ned tissue fractions of 32 endometrioid cysts were lasermicrodissected (Fig. 1). The clonality status was analysed by use of the X-chromosome inactivation pattern. Thirty of the 32 samples assayed displayed a polyclonal restriction pattern, and the remaining two were monoclonal. However, no neoplastic development was identif|ed during followup in either of these two patients with monoclonal endometriotic tissue. Thus, our results show that there is no
CHROMOSOMALCHANGES IN OVARIAN CANCER
Figure 1 Ideogram of CGH f|ndings in 20 granulosa cell tumours (red bars are losses and green bars are gains).
conclusive evidence to classify endometriosis as a premalignant condition.11 The behaviour of borderline epithelial neoplasms (LMP = low malignant potential) is intermediate between clearly benign and obviously malignant tumours. Some authors do regard them as precursor lesions, but complex aberrations are not seen in borderline tumours, and the difference between the genetic aberrations seen in LMP and in invasive carcinomas is an indicator that the invasive tumours do not arise from pre-existing borderline lesions.
INVASIVE CARCINOMA Most epithelial tumours are derived from the ovarian surface epithelium, which is part of the mesothelium. Comparison with embryonic development of the Mullerian duct explains the different types of tumours: serous, mucinous, endometrioid or clear cell. Undifferentiated tumours are mostly serous carcinomas. The tumour may either develop from the surface and grow exophytically, or develop in an inclusion cyst, leading to a cystic tumour. Genetic analysis of tumour cells by fluorescence in-situ hybridization (FISH) shows highly variable changes when single cells are compared. In contrast, the pattern revealed by comparative genomic hybridization (CGH) analysis is relatively uniform.The reason for this paradox of genetic instability is unknown. The results of comprehensive CGH studies12--14 show that the most frequently
329
occurring CGH changes in grade 1 and grade 2 carcinomas are gains of 3q26 -qter, 8q24 -qter and 20q13-qter. In grade 3 carcinomas, gains of 1q22-q25, 1q41-qter and 7q32-qter and losses of 4q, 12q21-qter, 16q13-qter, 18q21qter and Xq12-q25 are also observed.Overall, gains predominate over losses. Serous carcinomas show gains rather than losses with twice the frequency seen in mucinous or endometrioid carcinomas. Tapper et al.15 showed an association between genetic alterations and tumour type (+11q13 in serous, +10 -q23-qter in endometrioid and +17qcen-q21 in mucinous tumours), but to date, no pathognomonic CGH pattern for a specif|c histological type of tumour has been described. Losses of genetic material on chromosomes 4, 16, 18 and X seem to be of prognostic signif|cance for tumour progression and prognosis. This points to the existence of tumour suppressor genes, but to date, the search for these has been unsuccessful. In the following text, the better characterized areas of gains on 3q, 8q and 20q will be described in more detail. On the long arm of chromosome 20, there is an amplicon with three independent subamplicons. They contain transcription factors and proteins that are important for organization of the cytoskeleton and the spindle apparatus15 (Table 1). Gains of 20q13 (similar to breast or colon cancer) are prognostically important in ovarian carcinomas. In very recent studies, we analysed two genes lying in this chromosomal region. We demonstrated that eukaryote translation factor 1 alpha 2 (EEF1A2) belongs to this amplicon. EEF1A2 is involved in translation and organization of the actin cytoskeleton.16 On the other hand, we analysed the gene CSE1L/CAS (chromosome segregation 1 like/cellular apoptosis susceptibility). It codes for a protein that is important for transportation of factors between the nucleus and the cytoplasm during the segregation of chromosomes. Overexpression of CSE1L/ CAS in ovarian carcinoma correlates with amplif|cation of 20q13 and shows a prognostic signif|cance17--19 (Table1). The myc oncogene is located on 8q24. Normally strong activation of myc, for example by growth factors, does not lead to proliferation but to apoptosis. In tumours, myc amplif|cation and increased proliferation are only linked if apoptosis is blocked, for example by overexpression of bcl-2. In ovarian carcinomas, strong bcl-2 expression is often seen (Table 2). A lot of proteins are important in cell-cycle regulation and apoptosis.The restriction point at the end of G1phase is under the control of the cyclinD1/CDK-complex. Activation leads to release of E2F transcription factors from binding of retinoblastoma (Rb) protein. This makes the progression of the cell cycle possible.20 p16INK4A inhibits this regulatory pathway. Loss of expression of p16, amplif|cation of cyclin D1 or loss of Rb all lead to increased proliferation, as does activation of myc. However, ongoing stimulation activates p19/ARF, which leads to apoptosis by inhibition of MDM2 and activation of p53. This explains why
330
CURRENT DIAGNOSTIC PATHOLOGY
Table 1 Genes in the 20q amplicon 20q11 20q11 20q11.2 20q11.2
AIB3 AIB4 E2F1 MMP9/24
Function unknown Function unknown E2F1-transcription factor Matrixmetalloproteinase 9 and 24
20q12 20q12 20q12
AIB1 SRC CSE1L/CAS
Co-activator of steroid receptor Homologue to v-src oncogene Seggregation of chromosome
20q13 20q13.1 20q13.1 20q13.2 20q13.2 20q13.2
EEF1A2 MYBL2 PTPN1 ZNF217 BTAK NABC1
Translation elongation factor Transcription factor Proteintyrosinephophatase Transcription factor Separation of centrosomes Function unknown
Table 2 Invasive ovarian carcinomas.Changes in regulation of cell proliferation and apoptosis, as well in ras-signal transduction Serous carcinomas
Mucinous carcinomas
Endometrioid carcinomas
Cell proliferation and apoptosis Loss of p16 (expression) CyclinD1 (expression) Loss of Rb (expression) Loss of p53 (expression) Myc (amplif|cation) Bcl-2 (expression)
60 --86% 46% 49% 57% 35% 60%
36% 4/4 cases 1/4 cases 13% 50% 28%
62--84% 1/4 cases 2/4 cases 23% 47% 87%
Signal transduction Her2/neu (expression) Loss of Dab-2 (expression) K-ras (mutation) PI3-K (amplif|cation) PTEN (mutation)
14--28% 93% 9% 58% 0%
10 --29% 6/6 cases 46%
19% 4/4 cases 23%
10%
23%
proliferation by functional p53 is lost. p53 mutation, mostly associated with p53 accumulation, is more often seen in serous carcinomas than in mucinous or endometrioid carcinomas. Loss of p16 is described in 36--86% of cases, increase of cyclin D1 in about 40% of cases and loss of Rb in approximately 50% of cases; none of these changes correlated with the histological tumour type (Table 2). Mitogenic stimuli, transmitted through tyrosine kinase receptors and the Ras-signalling pathway, activate cyclin D1. In ovarian carcinomas, components of this signalling chain are almost always affected by alterations, e.g. amplif|cation or overexpression of Her-2/neu, K-ras mutation and loss of expression of the ras-inhibitor Dab-2. The inhibitory effects of ras on apoptosis are mediated by activation of phosphatidylinositol-3-kinase (PI3K). The PI3K gene is an important element of the amplicon on chromosome 3q26. Loss of DAB-2 expression is revealed in almost all ovarian carcinomas. Mutation of K-ras is particularly seen in mucinous and endometrioid
tumours. PTEN mutation, which has a similar effect to amplif|cation and activation of PI3K, is typical for endometrioid carcinomas -- a f|nding which is identical to endometrioid carcinomas of the endometrium. Amplif|cation or overexpression of Her-2/neu (ERBB2) is less often described than in carcinomas of the breast. Remarkably, the ERBB2 gene on 17q21.1 is more often deleted than amplif|ed. Approximately 15--25% of ovarian carcinomas show ‘3+’ Her2/neu overexpression. Again, there is no correlation between Her2/neu overexpression and histological type of tumour.
GRANULOSA CELLTUMOURS Six percent of malignant ovarian tumours are granulosa cell tumours (GCT) of the ovary. GCTs, normally tumours of postmenopausal patients, are considered to
CHROMOSOMALCHANGES IN OVARIAN CANCER
331
Figure 2 Laser microdissection of an endometriotic lesion (magnif|ed100 ).
be a low-grade malignancy with a non-aggressive clinical course, discovered in stage I with a 10 -year survival rate of more than 90%. The juvenile type of GCT is a distinctive form that occurs almost exclusively in children and young adults. Ninety-f|ve percent of GCTs are unilateral. Tumour stage is the only established prognostic factor. Histological type, mitotic activity or patients age do not show a correlation with clinical outcome. Although p53 accumulation is found in more than 95% of cases, aberration of chromosome 17 is rare. This suggests that accumulation of p53 protein does not play such a pivotal role as in ovarian carcinomas. Approximately 80% of GCTs are diploid,21--24 but in our own studies, we could not f|nd a correlation between ploidy and survival. A high prevalence of trisomy12 and several other aberrations such as monosomy 22 and trisomy 14 by FISH have been reported.25--29 Our own FISH analysis showed monosomy 22 in 44% of cases, trisomy12 in 25% of cases and monosomy 17 in 5% of cases (1/20) (Fig.1). Two of three GCTs show genetic changes in CGH. Almost all chromosomes can be affected. The most frequently described changes are complete loss of chromosome 22 and complete gain of chromosome 12. In our own study, almost all chromosomes were involved (Fig. 2). Complete losses or gains were seen more often than partial losses or gains of chromosomes. As in other studies, loss of chromosome 22 was seen in 40% of cases. Numerous other chromosomes (4, 5, 13, 16, 17, 18, 19, 21 and X) showed a complete loss in 5--10% of cases. Gain of chromosome 12 was seen in 15% of cases. In addition to the changes described, we saw a gain of chromosome 14 in one in three cases. In more than 80% of cases, this was combined with monosomy 22. Further complete gains were seen for chromosomes 3, 7, 8, 9, 10, 12, 18 and 20. NF2 is a well-characterized tumour suppressor gene on chromosome 22. Regarding specif|c genes, however, mutations of NF2 in GCTs have not been reported to date. In other tumours, such as breast and colorectal carcinomas, NF2 does not appear to play a major role. Therefore, the existence of other tumour suppressor loci has been proposed. Several important genes have been identif|ed on chromosome 12 (e.g. KRAS 2, KRAG
and MDM2). On chromosome14, a number of important genes have been characterized that are involved in the regulation of cell proliferation and cell death: e.g. FOS, the major component of the activator protein-1 (AP-1) transcription factor complex; BCL2L2 (BCL2like2), a regulator molecule of apoptotic cell death; and TGF3, which controls cell proliferation and differentiation.
CONCLUSION Molecular pathological observations in epithelial tumours of the ovary show that there are considerable quantitative differences in genetic aberrations, but the targets of these changes are the same: the signal transduction machinery of tyrosine kinase receptors and central components of cell-cycle regulation and apoptosis. Differences in biological behaviour and aggressiveness are apparently due to the number of genetic alterations. However, this is no explanation for the wide range of different phenotypic patterns.Thus, the pivotal question of morphology has not been answered. The cytogenetic changes observed in GCTs differ from those in epithelial ovarian cancer.The demonstration of non-random chromosomal aberrations may eventually lead to the identif|cation of oncogenes and tumour suppressor genes that are pivotal for the development and progression of GCT.
PRACTICE POINTS *
*
*
*
The behaviour of borderline epithelial neoplasms (LMP=low malignant potential) is intermediate between clearly benign and obviously malignant tumours At present, there is no conclusive evidence to classify endometriosis as a premalignant condition GCTs are considered a low-grade malignancy with a non-aggressive clinical course with a 10 -year survival rate of more than 90% Currently,FIGO stage and residualtumour mass remain the most important prognostic factors for all ovarian cancer
332
CURRENT DIAGNOSTIC PATHOLOGY
RESEARCH DIRECTIONS *
The search for genetic aberrations, which are characteristic for a histological type of carcinoma or signif|cant for prognosis and survival
REFERENCES 1. Mayr D, Diebold J. Grading of ovarian carcinomas. Int J Gynecol Pathol 2000; 19: 348--353. 2. Mayr D, Pannekamp U, Baretton G B et al. Immunohistochemical analysis of drug resistance-associated proteins in ovarian carcinomas. Pathol Res Pract 2000; 196: 469--475. 3. Diebold J. Pha¨notyp -- Genotyp -- Korrelation bei Ovarialneoplasien. Verh Dtsch Ges Path 2001; 85: 153--160. 4. Suzuki S, Moore I I D, Ginzinger D et al. An approach to analysis of large-scale correlations between genome changes and clinical endpoints in ovarian cancer. Cancer Res 2000; 60: 5382--5385. 5. Tumorregister Mu¨nchen. Jahresbericht 1999. Schwerpunkt: Gyna¨kologische Tumoren. Zuckschwerdt Verlag, Germering/ Mu¨nchen, 2000. 6. Jimbo J, Hitomi Y, Yoshikawa H et al. Clonality analysis of bilateral ovarian endometriod cysts. Fertil Steril 1999; 72: 1142--1143. 7. Corner G W Jr, Hu C Y, Hertig A T. Ovarian carcinoma arising in endometrioisis. Am J Obstet Gynecol 1950; 59: 760--774. 8. Scully R E, Richardson G S, Barlow J F. The development of malignancy in endometriosis. Clin Obstet Gynecol 1966; 381--414. 9. Jiang X, Hitchcock A, Bryan E J et al. Microsatellite analysis of endometriosis reveals loss of heterozygosity at cancidate ovarian tumor suppressor gene loci. Cancer Res 1996; 56: 3534--3539. 10. Yano T, Jimbo H, Yoshikawa H, Tsusumi O, Taketani Y. Molecular analysis of clonality in ovarian endometrial cysts. Gynecol Obstet Invest 1999; 47 (Suppl 1): 41--46. 11. Mayr D, Amann G, Siefert C, Diebold J, Anderegg B. Does endometriosis really have premalignant potential? A clonal analysis of laser-microdissected tissue. FASEB J 2003; 10: 1096. 12. Kiechle M, Jacobson A, Schwarz-Boeger U, Hederich J, Pfisterer J, Arnold N. Comparative genomic hybridisation detects genetic imbalances in primary ovarian carcinomas as correlated with grade of differentation. Cancer 2001; 91: 534--540. 13. Suzuki S, Moore D H, Ginzinger D G et al. An approach to analysis of large-scale correlations between genome changes and clinical endpoints in ovarian cancer. Cancer Res 2000; 60: 5382--5385. 14. Tanner M M, Grenman S, Koul A et al. Frequent amplification of chromosomal region 20q12-q13 in ovarian cancer. Clin Cancer Res 2000; 6: 1833--1839.
15. Tapper J, Bu¨tzow R, Wahlstro¨m T, Seppa¨la¨ M, Knuutila S. Evidence for divergence of DNA copy number changes in serous, mucinous and endometrioid ovarian carcinomas. Br J Cancer 1997; 75: 1782--1787. 16. Anand N, Merthy S, Amann G et al. Protein elongation factor EEF1A2 is a putative oncogene in ovarian cancer. Nat Genet 2002; 31: 301--305 17. Jazaeri A A, Lu K, Schmandt R et al. Molecular determinants of tumor differentiation in papillary serous ovarian carcinoma. Mol Carcinog 2003; 36: 53--59. 18. Peiro G, Mayr D, Hillemanns P, Lo¨hrs U, Diebold J. Analysis of HER-2/neu amplification in endometrial carcinoma by chromogenic in situ hybridisation (CISH). Correlation with FISH, HER/ 2neu, p53 and ki67 protein expression, and outcome. Mod Pathol, in press, 2003. 19. Sonoda G, Palazzo J, du Manoir S et al. Comparative genomic hybridization detects frequent overrepresentation of chromosomal material from 3q26, 8q24 and 20q13 in human carcinomas. Gene Chromosome Cancer 1997; 20: 320--328. 20. Sherr C J. The Pezcoller lecture: cancer cell cycles revisited. Cancer Res 2000; 60: 3689--3695. 21. Mayr D, Kaltz-Wittmer C, Arbogast S, Amann G, Aust D E, Diebold J. Characteristic pattern of genetic aberrations in ovarian granulosa cell tumors. Mod Pathol 2001; 15: 951--957. 22. Halperin D, Visscher D W, Wallis T, Lawrence W D. Evaluation of chromosome 12 copy number in ovarian granulosa cell tumours using interphase cytogenetics. Int Gynecol Pathol 1995; 14: 319--323. 23. Gorski G K, McMorrow L E, Blumstein L, Faasse D, Donaldson M H. Trisomy 14 in two cases of granulosa cell tumor of the ovary. Cancer Genet Cytogenet 1992; 60: 202--205. 24. Suh K S, Silverberg S G, Rhame J G, Wilkinson D S. Granulosa cell tumor of the ovary. Arch Pathol Lab Med 1990; 114: 496--501. 25. Persons D L, Hartmann L C, Herath J F, Keeney G L, Jenkins R B. Fluorescence in situ hybridization analysis of trismoy 12 in ovarian tumors. Am J Clin Pathol 1994; 102: 775--779. 26. Shashi V, Golden W L, von Kap-Herr C, Andersen W A, Gaffey M J. Interphase fluorescence in situ hybridization for trisomy 12 on archival ovarian sex cord-stromal tumors. Gynecol Oncol 1994; 55: 349--354. 27. Cin P D, Qi H, Paulwels P, Backx C, van den Berghe H. Monosomy 22 in a forbothecoma. Cancer Genet Cytogenet 1997; 99: 129--131. 28. Speleman F, Dermaut B, de Potter C R et al. Monosomy 22 in a mixed germ cell-sex cord-stromal tumor of the ovary. Genes Chromosomes Cancer 1997; 19: 192--194. 29. Van den Berghe I, Dal Cin P, de Groef K, Michielssen P, van den Berghe H. Monosomy 22 and trisomy 14 may be early events in the tumorigenesis of adult granulosa cell tumor. Cancer Genet Cytogenet 1999; 112: 46--48.
c 2003 Published by Elsevier Ltd. doi:10.1016/S0968 - 6053(03)00050 - 4
REVIEW
Chromosomal changes in ovarian cancer D. Mayr and J. Diebold Pathologisches Institut der Ludwigs-Maximilians-Universitt Mˇnchen, Thalkirchner Strasse 36, D- 80337 Mˇnchen, Germany
KEYWORDS ovary; carcinoma; granulosa cell tumour; immunohistochemistry; fluorescence-in-situhybridization (FISH); comparative genomic hybridization (CGH)
Summary Ovarian neoplasms display a wide range of different phenotypic patterns. Recently, there has been much research that has identif|ed the molecular genetic aberrations which are present in ovarian neoplasms, and that has linked these to the phenotypic patterns. Invasive ovarian carcinomas show complex genetic changes. Chromosomal gains at 3q26, 8q24 and 20q13 apparently represent early lesions, whereas loss of material of chromosomes 4,13,16,18 and X is associated with tumour progression and poor prognosis.The maintargets ofchromosomalchanges areregulatorygenes ofcellproliferation and apoptosis (e.g. p16, cyclin D1,Rb, p53, myc and bcl-2), and members of the signalling cascade of tyrosine kinase receptors (e.g. HER/2neu, dab-2, K-ras, PI3-K and PTEN). Granulosa cell tumours usually contain numeric chromosomal aberrations (monosomy 22, trisomy12 and14). The genetic alterations of ovarian neoplasms which have been described to date correlate with some aspects ofthe biologicalbehaviour ofthese tumours.However, theydo not fully explain the spectrum of phenotypic variability.
c 2003 Published by Elsevier Ltd.
Ovarian cancer is the leading cause of death from gynaecological malignancy in Europe and the USA. Nearly 95% of cases occur sporadically and, to date, no single causative factor has been identif|ed. Ninety percent of malignant ovarian neoplasms are epithelial tumours. More than half of the women present with an advanced stage of disease and therefore have a poor prognosis.1,2 Multiple genetic changes appear to be involved in the genesis and progression of cancer which affect cell proliferation, cell death or angiogenesis (e.g. p53 and bcl-2).3,4 Currently, FIGO stage and residual tumour mass remain the most important prognostic factors. Data from patients in the Munich Tumour Register show that borderline tumours (low malignant potential) have the best prognosis.5 Amongst invasive carcinomas, the mucinous and endometrioid types have a more favourable course than serous carcinomas (10 -year survival rate of 41% and 33%, respectively, vs 23%). The prognosis of malignant germ cell and stromal tumours, mostly granulosa cell tumours, falls between non-invasive and invasive epithelial tumours with a 10 -year survival rate of 70%. Since the morphological patterns of ovarian neoplasms seem to correlate with prognosis, it Correspondence to: DM. Tel.: +49 (0) 89/5160 - 4671; Fax: +49 (0) 89/ 5160 - 4043; E-mail: [email protected]
is of interest whether there are consistent genetic alterations associated with these patterns.
PRECURSOR LESIONS About 5% of invasive carcinomas are caused by genetic predisposition, either due to the autosomal-dominant hereditary breast-ovarian-cancer syndrome or as part of hereditary non-polyposis colorectal cancer (HNPCC) syndrome. The morphology of ovarian cancer precursor lesions is poorly described. For decades, endometriosis, one of the most common gynaecological diseases, has been regarded as a putative precursor lesion for endometrioid carcinomas. In agreement with this notion, several authors demonstrated monoclonality of endometrioid foci.6 --10. In our own study, def|ned tissue fractions of 32 endometrioid cysts were lasermicrodissected (Fig. 1). The clonality status was analysed by use of the X-chromosome inactivation pattern. Thirty of the 32 samples assayed displayed a polyclonal restriction pattern, and the remaining two were monoclonal. However, no neoplastic development was identif|ed during followup in either of these two patients with monoclonal endometriotic tissue. Thus, our results show that there is no
CHROMOSOMALCHANGES IN OVARIAN CANCER
Figure 1 Ideogram of CGH f|ndings in 20 granulosa cell tumours (red bars are losses and green bars are gains).
conclusive evidence to classify endometriosis as a premalignant condition.11 The behaviour of borderline epithelial neoplasms (LMP = low malignant potential) is intermediate between clearly benign and obviously malignant tumours. Some authors do regard them as precursor lesions, but complex aberrations are not seen in borderline tumours, and the difference between the genetic aberrations seen in LMP and in invasive carcinomas is an indicator that the invasive tumours do not arise from pre-existing borderline lesions.
INVASIVE CARCINOMA Most epithelial tumours are derived from the ovarian surface epithelium, which is part of the mesothelium. Comparison with embryonic development of the Mullerian duct explains the different types of tumours: serous, mucinous, endometrioid or clear cell. Undifferentiated tumours are mostly serous carcinomas. The tumour may either develop from the surface and grow exophytically, or develop in an inclusion cyst, leading to a cystic tumour. Genetic analysis of tumour cells by fluorescence in-situ hybridization (FISH) shows highly variable changes when single cells are compared. In contrast, the pattern revealed by comparative genomic hybridization (CGH) analysis is relatively uniform.The reason for this paradox of genetic instability is unknown. The results of comprehensive CGH studies12--14 show that the most frequently
329
occurring CGH changes in grade 1 and grade 2 carcinomas are gains of 3q26 -qter, 8q24 -qter and 20q13-qter. In grade 3 carcinomas, gains of 1q22-q25, 1q41-qter and 7q32-qter and losses of 4q, 12q21-qter, 16q13-qter, 18q21qter and Xq12-q25 are also observed.Overall, gains predominate over losses. Serous carcinomas show gains rather than losses with twice the frequency seen in mucinous or endometrioid carcinomas. Tapper et al.15 showed an association between genetic alterations and tumour type (+11q13 in serous, +10 -q23-qter in endometrioid and +17qcen-q21 in mucinous tumours), but to date, no pathognomonic CGH pattern for a specif|c histological type of tumour has been described. Losses of genetic material on chromosomes 4, 16, 18 and X seem to be of prognostic signif|cance for tumour progression and prognosis. This points to the existence of tumour suppressor genes, but to date, the search for these has been unsuccessful. In the following text, the better characterized areas of gains on 3q, 8q and 20q will be described in more detail. On the long arm of chromosome 20, there is an amplicon with three independent subamplicons. They contain transcription factors and proteins that are important for organization of the cytoskeleton and the spindle apparatus15 (Table 1). Gains of 20q13 (similar to breast or colon cancer) are prognostically important in ovarian carcinomas. In very recent studies, we analysed two genes lying in this chromosomal region. We demonstrated that eukaryote translation factor 1 alpha 2 (EEF1A2) belongs to this amplicon. EEF1A2 is involved in translation and organization of the actin cytoskeleton.16 On the other hand, we analysed the gene CSE1L/CAS (chromosome segregation 1 like/cellular apoptosis susceptibility). It codes for a protein that is important for transportation of factors between the nucleus and the cytoplasm during the segregation of chromosomes. Overexpression of CSE1L/ CAS in ovarian carcinoma correlates with amplif|cation of 20q13 and shows a prognostic signif|cance17--19 (Table1). The myc oncogene is located on 8q24. Normally strong activation of myc, for example by growth factors, does not lead to proliferation but to apoptosis. In tumours, myc amplif|cation and increased proliferation are only linked if apoptosis is blocked, for example by overexpression of bcl-2. In ovarian carcinomas, strong bcl-2 expression is often seen (Table 2). A lot of proteins are important in cell-cycle regulation and apoptosis.The restriction point at the end of G1phase is under the control of the cyclinD1/CDK-complex. Activation leads to release of E2F transcription factors from binding of retinoblastoma (Rb) protein. This makes the progression of the cell cycle possible.20 p16INK4A inhibits this regulatory pathway. Loss of expression of p16, amplif|cation of cyclin D1 or loss of Rb all lead to increased proliferation, as does activation of myc. However, ongoing stimulation activates p19/ARF, which leads to apoptosis by inhibition of MDM2 and activation of p53. This explains why
330
CURRENT DIAGNOSTIC PATHOLOGY
Table 1 Genes in the 20q amplicon 20q11 20q11 20q11.2 20q11.2
AIB3 AIB4 E2F1 MMP9/24
Function unknown Function unknown E2F1-transcription factor Matrixmetalloproteinase 9 and 24
20q12 20q12 20q12
AIB1 SRC CSE1L/CAS
Co-activator of steroid receptor Homologue to v-src oncogene Seggregation of chromosome
20q13 20q13.1 20q13.1 20q13.2 20q13.2 20q13.2
EEF1A2 MYBL2 PTPN1 ZNF217 BTAK NABC1
Translation elongation factor Transcription factor Proteintyrosinephophatase Transcription factor Separation of centrosomes Function unknown
Table 2 Invasive ovarian carcinomas.Changes in regulation of cell proliferation and apoptosis, as well in ras-signal transduction Serous carcinomas
Mucinous carcinomas
Endometrioid carcinomas
Cell proliferation and apoptosis Loss of p16 (expression) CyclinD1 (expression) Loss of Rb (expression) Loss of p53 (expression) Myc (amplif|cation) Bcl-2 (expression)
60 --86% 46% 49% 57% 35% 60%
36% 4/4 cases 1/4 cases 13% 50% 28%
62--84% 1/4 cases 2/4 cases 23% 47% 87%
Signal transduction Her2/neu (expression) Loss of Dab-2 (expression) K-ras (mutation) PI3-K (amplif|cation) PTEN (mutation)
14--28% 93% 9% 58% 0%
10 --29% 6/6 cases 46%
19% 4/4 cases 23%
10%
23%
proliferation by functional p53 is lost. p53 mutation, mostly associated with p53 accumulation, is more often seen in serous carcinomas than in mucinous or endometrioid carcinomas. Loss of p16 is described in 36--86% of cases, increase of cyclin D1 in about 40% of cases and loss of Rb in approximately 50% of cases; none of these changes correlated with the histological tumour type (Table 2). Mitogenic stimuli, transmitted through tyrosine kinase receptors and the Ras-signalling pathway, activate cyclin D1. In ovarian carcinomas, components of this signalling chain are almost always affected by alterations, e.g. amplif|cation or overexpression of Her-2/neu, K-ras mutation and loss of expression of the ras-inhibitor Dab-2. The inhibitory effects of ras on apoptosis are mediated by activation of phosphatidylinositol-3-kinase (PI3K). The PI3K gene is an important element of the amplicon on chromosome 3q26. Loss of DAB-2 expression is revealed in almost all ovarian carcinomas. Mutation of K-ras is particularly seen in mucinous and endometrioid
tumours. PTEN mutation, which has a similar effect to amplif|cation and activation of PI3K, is typical for endometrioid carcinomas -- a f|nding which is identical to endometrioid carcinomas of the endometrium. Amplif|cation or overexpression of Her-2/neu (ERBB2) is less often described than in carcinomas of the breast. Remarkably, the ERBB2 gene on 17q21.1 is more often deleted than amplif|ed. Approximately 15--25% of ovarian carcinomas show ‘3+’ Her2/neu overexpression. Again, there is no correlation between Her2/neu overexpression and histological type of tumour.
GRANULOSA CELLTUMOURS Six percent of malignant ovarian tumours are granulosa cell tumours (GCT) of the ovary. GCTs, normally tumours of postmenopausal patients, are considered to
CHROMOSOMALCHANGES IN OVARIAN CANCER
331
Figure 2 Laser microdissection of an endometriotic lesion (magnif|ed100 ).
be a low-grade malignancy with a non-aggressive clinical course, discovered in stage I with a 10 -year survival rate of more than 90%. The juvenile type of GCT is a distinctive form that occurs almost exclusively in children and young adults. Ninety-f|ve percent of GCTs are unilateral. Tumour stage is the only established prognostic factor. Histological type, mitotic activity or patients age do not show a correlation with clinical outcome. Although p53 accumulation is found in more than 95% of cases, aberration of chromosome 17 is rare. This suggests that accumulation of p53 protein does not play such a pivotal role as in ovarian carcinomas. Approximately 80% of GCTs are diploid,21--24 but in our own studies, we could not f|nd a correlation between ploidy and survival. A high prevalence of trisomy12 and several other aberrations such as monosomy 22 and trisomy 14 by FISH have been reported.25--29 Our own FISH analysis showed monosomy 22 in 44% of cases, trisomy12 in 25% of cases and monosomy 17 in 5% of cases (1/20) (Fig.1). Two of three GCTs show genetic changes in CGH. Almost all chromosomes can be affected. The most frequently described changes are complete loss of chromosome 22 and complete gain of chromosome 12. In our own study, almost all chromosomes were involved (Fig. 2). Complete losses or gains were seen more often than partial losses or gains of chromosomes. As in other studies, loss of chromosome 22 was seen in 40% of cases. Numerous other chromosomes (4, 5, 13, 16, 17, 18, 19, 21 and X) showed a complete loss in 5--10% of cases. Gain of chromosome 12 was seen in 15% of cases. In addition to the changes described, we saw a gain of chromosome 14 in one in three cases. In more than 80% of cases, this was combined with monosomy 22. Further complete gains were seen for chromosomes 3, 7, 8, 9, 10, 12, 18 and 20. NF2 is a well-characterized tumour suppressor gene on chromosome 22. Regarding specif|c genes, however, mutations of NF2 in GCTs have not been reported to date. In other tumours, such as breast and colorectal carcinomas, NF2 does not appear to play a major role. Therefore, the existence of other tumour suppressor loci has been proposed. Several important genes have been identif|ed on chromosome 12 (e.g. KRAS 2, KRAG
and MDM2). On chromosome14, a number of important genes have been characterized that are involved in the regulation of cell proliferation and cell death: e.g. FOS, the major component of the activator protein-1 (AP-1) transcription factor complex; BCL2L2 (BCL2like2), a regulator molecule of apoptotic cell death; and TGF3, which controls cell proliferation and differentiation.
CONCLUSION Molecular pathological observations in epithelial tumours of the ovary show that there are considerable quantitative differences in genetic aberrations, but the targets of these changes are the same: the signal transduction machinery of tyrosine kinase receptors and central components of cell-cycle regulation and apoptosis. Differences in biological behaviour and aggressiveness are apparently due to the number of genetic alterations. However, this is no explanation for the wide range of different phenotypic patterns.Thus, the pivotal question of morphology has not been answered. The cytogenetic changes observed in GCTs differ from those in epithelial ovarian cancer.The demonstration of non-random chromosomal aberrations may eventually lead to the identif|cation of oncogenes and tumour suppressor genes that are pivotal for the development and progression of GCT.
PRACTICE POINTS *
*
*
*
The behaviour of borderline epithelial neoplasms (LMP=low malignant potential) is intermediate between clearly benign and obviously malignant tumours At present, there is no conclusive evidence to classify endometriosis as a premalignant condition GCTs are considered a low-grade malignancy with a non-aggressive clinical course with a 10 -year survival rate of more than 90% Currently,FIGO stage and residualtumour mass remain the most important prognostic factors for all ovarian cancer
332
CURRENT DIAGNOSTIC PATHOLOGY
RESEARCH DIRECTIONS *
The search for genetic aberrations, which are characteristic for a histological type of carcinoma or signif|cant for prognosis and survival
REFERENCES 1. Mayr D, Diebold J. Grading of ovarian carcinomas. Int J Gynecol Pathol 2000; 19: 348--353. 2. Mayr D, Pannekamp U, Baretton G B et al. Immunohistochemical analysis of drug resistance-associated proteins in ovarian carcinomas. Pathol Res Pract 2000; 196: 469--475. 3. Diebold J. Pha¨notyp -- Genotyp -- Korrelation bei Ovarialneoplasien. Verh Dtsch Ges Path 2001; 85: 153--160. 4. Suzuki S, Moore I I D, Ginzinger D et al. An approach to analysis of large-scale correlations between genome changes and clinical endpoints in ovarian cancer. Cancer Res 2000; 60: 5382--5385. 5. Tumorregister Mu¨nchen. Jahresbericht 1999. Schwerpunkt: Gyna¨kologische Tumoren. Zuckschwerdt Verlag, Germering/ Mu¨nchen, 2000. 6. Jimbo J, Hitomi Y, Yoshikawa H et al. Clonality analysis of bilateral ovarian endometriod cysts. Fertil Steril 1999; 72: 1142--1143. 7. Corner G W Jr, Hu C Y, Hertig A T. Ovarian carcinoma arising in endometrioisis. Am J Obstet Gynecol 1950; 59: 760--774. 8. Scully R E, Richardson G S, Barlow J F. The development of malignancy in endometriosis. Clin Obstet Gynecol 1966; 381--414. 9. Jiang X, Hitchcock A, Bryan E J et al. Microsatellite analysis of endometriosis reveals loss of heterozygosity at cancidate ovarian tumor suppressor gene loci. Cancer Res 1996; 56: 3534--3539. 10. Yano T, Jimbo H, Yoshikawa H, Tsusumi O, Taketani Y. Molecular analysis of clonality in ovarian endometrial cysts. Gynecol Obstet Invest 1999; 47 (Suppl 1): 41--46. 11. Mayr D, Amann G, Siefert C, Diebold J, Anderegg B. Does endometriosis really have premalignant potential? A clonal analysis of laser-microdissected tissue. FASEB J 2003; 10: 1096. 12. Kiechle M, Jacobson A, Schwarz-Boeger U, Hederich J, Pfisterer J, Arnold N. Comparative genomic hybridisation detects genetic imbalances in primary ovarian carcinomas as correlated with grade of differentation. Cancer 2001; 91: 534--540. 13. Suzuki S, Moore D H, Ginzinger D G et al. An approach to analysis of large-scale correlations between genome changes and clinical endpoints in ovarian cancer. Cancer Res 2000; 60: 5382--5385. 14. Tanner M M, Grenman S, Koul A et al. Frequent amplification of chromosomal region 20q12-q13 in ovarian cancer. Clin Cancer Res 2000; 6: 1833--1839.
15. Tapper J, Bu¨tzow R, Wahlstro¨m T, Seppa¨la¨ M, Knuutila S. Evidence for divergence of DNA copy number changes in serous, mucinous and endometrioid ovarian carcinomas. Br J Cancer 1997; 75: 1782--1787. 16. Anand N, Merthy S, Amann G et al. Protein elongation factor EEF1A2 is a putative oncogene in ovarian cancer. Nat Genet 2002; 31: 301--305 17. Jazaeri A A, Lu K, Schmandt R et al. Molecular determinants of tumor differentiation in papillary serous ovarian carcinoma. Mol Carcinog 2003; 36: 53--59. 18. Peiro G, Mayr D, Hillemanns P, Lo¨hrs U, Diebold J. Analysis of HER-2/neu amplification in endometrial carcinoma by chromogenic in situ hybridisation (CISH). Correlation with FISH, HER/ 2neu, p53 and ki67 protein expression, and outcome. Mod Pathol, in press, 2003. 19. Sonoda G, Palazzo J, du Manoir S et al. Comparative genomic hybridization detects frequent overrepresentation of chromosomal material from 3q26, 8q24 and 20q13 in human carcinomas. Gene Chromosome Cancer 1997; 20: 320--328. 20. Sherr C J. The Pezcoller lecture: cancer cell cycles revisited. Cancer Res 2000; 60: 3689--3695. 21. Mayr D, Kaltz-Wittmer C, Arbogast S, Amann G, Aust D E, Diebold J. Characteristic pattern of genetic aberrations in ovarian granulosa cell tumors. Mod Pathol 2001; 15: 951--957. 22. Halperin D, Visscher D W, Wallis T, Lawrence W D. Evaluation of chromosome 12 copy number in ovarian granulosa cell tumours using interphase cytogenetics. Int Gynecol Pathol 1995; 14: 319--323. 23. Gorski G K, McMorrow L E, Blumstein L, Faasse D, Donaldson M H. Trisomy 14 in two cases of granulosa cell tumor of the ovary. Cancer Genet Cytogenet 1992; 60: 202--205. 24. Suh K S, Silverberg S G, Rhame J G, Wilkinson D S. Granulosa cell tumor of the ovary. Arch Pathol Lab Med 1990; 114: 496--501. 25. Persons D L, Hartmann L C, Herath J F, Keeney G L, Jenkins R B. Fluorescence in situ hybridization analysis of trismoy 12 in ovarian tumors. Am J Clin Pathol 1994; 102: 775--779. 26. Shashi V, Golden W L, von Kap-Herr C, Andersen W A, Gaffey M J. Interphase fluorescence in situ hybridization for trisomy 12 on archival ovarian sex cord-stromal tumors. Gynecol Oncol 1994; 55: 349--354. 27. Cin P D, Qi H, Paulwels P, Backx C, van den Berghe H. Monosomy 22 in a forbothecoma. Cancer Genet Cytogenet 1997; 99: 129--131. 28. Speleman F, Dermaut B, de Potter C R et al. Monosomy 22 in a mixed germ cell-sex cord-stromal tumor of the ovary. Genes Chromosomes Cancer 1997; 19: 192--194. 29. Van den Berghe I, Dal Cin P, de Groef K, Michielssen P, van den Berghe H. Monosomy 22 and trisomy 14 may be early events in the tumorigenesis of adult granulosa cell tumor. Cancer Genet Cytogenet 1999; 112: 46--48.