Genetic alterations in endometrial hyperplasia and cancer

Cancer Letters 175 (2002) 205–211 www.elsevier.com/locate/canlet

Genetic alterations in endometrial hyperplasia and cancer Gerhild Fabjani a,1, Elisabeth Kucera a,1, Eva Schuster a, Michael Minai-Pour b, Klaus Czerwenka c, Gerhard Sliutz a, Sepp Leodolter a,d, Angelika Reiner b, Robert Zeillinger a,* a

Department of Gynecology and Obstetrics, University of Vienna, Medical School, Waehringer Guertel 18-20, EBO 05 A-1090 Vienna, Austria b Department of Pathology, Langobardenstrasse 122, A-1220 Vienna, Austria c Department of Clinical Pathology, University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria d Ludwig-Boltzmann Institute for Gynecological Oncology and Reproductive Medicine, Waehringer Guertel 18-20, A-1090 Vienna, Austria Received 2 July 2001; received in revised form 15 July 2001; accepted 16 July 2001

Abstract Putative precursors of endometrial cancer such as complex endometrial hyperplasia with atypia have been described to be monoclonal and considered to be genetically related. In order to identify a genetic marker that could serve as a putative predictor of endometrial cancer we analyzed 14 endometrial hyperplasia and 29 endometrial cancer samples for instabilities and loss of heterozygosity (LOH) in microsatellite sequences. Deletions on the short arm of chromosome 8 were frequently detected in both endometrial hyperplasia and cancer samples, suggesting that these deletions are early events in the development of endometrial cancer. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Endometrial hyperplasia; Endometrial cancer; Microsatellite markers; Loss of heterozygosity; Polymerase chain reaction

1. Introduction Endometrial cancer is the most common pelvic gynecological malignancy. The vast majority of endometrial cancer arises from precursor lesions such as complex hyperplasia with atypia. Like in other malignancies, accumulation of genetic alterations leading to inactivation of tumor suppressor genes, activation of proto-oncogenes and inefficiency of the mismatch repair system are observed in endometrial cancer, too. Allelotyping studies of loss of heterozygosity (LOH) in endometrial cancer have revealed regions with loss, thus indicating the presence of putative * Corresponding author. Tel.: 143-1-40400-7831; fax: 143-140400-7832. E-mail address: [email protected] (R. Zeillinger). 1 G. Fabjani and E. Kucera contributed equally to this study.

tumor suppressor genes. Inactivation of the tumor suppressor gene p53 by deletion and point mutation occurs in 10–20% of advanced stage endometrial cancer but not in endometrial hyperplasia [1–3]. In previous studies, LOH on several chromosome arms, such as 1p, 3p, 8p, 9p, 10q, 14q, 16q, 17p, and 18q have been reported in endometrial cancer [3–5]. LOH, especially on the short arm of chromosome 8 (8p) has been shown to be frequent in many human tumors, including endometrial cancer [5]. Several critical regions within 8p were defined, suggesting that more than one tumor suppressor gene might be located there [6]. Deletions on 8p were reported to occur also in atypical breast ductal hyperplasia and at the hyperplasia/metaplasia stage of lung cancer, thus supporting the hypothesis that these losses are early genetic alterations in the development of cancer

0304-3835/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(01)00714-5

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[7,8]. Some atypical ductal hyperplasias shared their LOH pattern with more advanced lesions from the same breast, therefore putative precursors and cancers might be genetically related [9]. Recently, LOH on 8p in breast cancer was found to be associated with advanced tumor stage and aggressive histologic type [10,11]. Regarding mutations that occur in both endometrial hyperplasia and cancer, the activation mutation in codon 12 of the K-ras oncogene was found in hyperplasia as well as in cancer (10–37%) [12,13]. Concordant PTEN mutations and LOH on chromosome 10q were observed in atypical hyperplasia with synchronous endometrial cancer demonstrating that these genetic alterations can occur relatively early in endometrial tumor development [14,15]. Microsatellite sequences, occurring throughout the genome, were found to be unstable in 17–26% of sporadic endometrial cancer [16,17]. The majority of endometrial cancer displaying microsatellite instability was characterized by aberrant promotor methylation of the DNA mismatch repair gene hMLH1 [18]. Hypermethylation of the hMLH1 gene promotor is thought to be one of the underlying mechanisms responsible for the microsatellite instability in endometrial cancer, which was already detected in a few atypical endometrial hyperplasia samples coexisting with endometrial cancer [18]. In addition, microsatellite instability was seen in putative precursors of endometrial cancer, such as atypical hyperplasia, which retained some microsatellite alterations acquired in earlier stages [16]. By analyzing the patterns of microsatellite sequences in normal and corresponding tumor or hyperplastic tissue microsatellite instability as well as LOH can be detected easily. Well-differentiated endometrial cancer often coexists with endometrial hyperplasia, which is believed to be a putative precursor lesion [19,20]. As endometrial hyperplasia as well as endometrial cancer were described to be monoclonal, cells deriving from a common progenitor can be recognized by comparison of their non-random X-chromosome inactivation patterns, as it was observed in well differentiated endometrial cancer and their putative precursors [21,22]. Based on these findings and the clonality of putative precursors of endometrial cancer, we analyzed

endometrial hyperplasias and endometrial cancers for genetic alterations to determine specific markers which might reveal putative precursors of subsequent cancer in endometrial hyperplasia. As in endometrial cancers frequent chromosomal gains were found on chromosomes 1q, 3p, and 8q when either comparative genomic hypbridization or LOH analysis were performed, we looked for genetic alterations using the microsatellite markers D1S518, D3S2387, and D8S1132 [16,23,24]. Since in several neoplasms at least three 8p regions (8p12–21, 8p21, and 8p22) have been identified harboring potentially tumor suppressor genes, we studied genetic alterations in more detail on the short arm of chromosome 8 using a set of microsatellite markers D8S131, D8S133, D8S258, and D8S1992 [6,10,11,25].

2. Materials and methods 2.1. Case selection In 14 cases of endometrial hyperplasia, genomic DNA from paired normal and hyperplastic endometrium was extracted from formalin-fixed, paraffinembedded specimens after hematoxylin and eosin (H&E)-staining of tissue sections. Histological characteristics of endometrial hyperplasia are summarized in Table 1. Additionally, formalin-fixed and paraffinembedded tissue samples from 29 patients with sporadic endometrial cancer and corresponding blood samples were analyzed. Histopathological diagnosis and clinical staging were classified according to the criteria of the International Federation of Gynecology and Obstetrics (Table 2).

Table 1 Histologic characteristics of endometrial hyperplasia Endometrial hyperplasia

Number of samples

Histological Type Simple endometrial hyperplasia without atypia 3 Complex endometrial hyperplasia without atypia 3 Simple endometrial hyperplasia with atypia 4 Complex endometrial hyperplasia with atypia 4 Total 14

G. Fabjani et al. / Cancer Letters 175 (2002) 205–211 Table 2 Histologic characteristics of endometrial cancer Endometrial Cancer Histological Type Endometrioid adenocarcinoma Serous adenocarcinoma Mucinous adenocarcinoma Grading G1 G2 G3 FIGO Stage I II III Total

Number of samples

25 3 1 14 10 5 21 2 6 29

2.2. DNA extraction Hyperplasia and cancer specimens were examined for contamination of normal tissue. Only samples consisting of more than 90% hyperplastic or tumor cells were analyzed. Areas of hyperplasia were identified microscopically on H&E stained slides. After detaching cover glasses in xylene over night, areas of interest (hyperplastic and normal tissue) were scratched out using a micropipette tip and washed with ethanol for three times. Tumor DNA was extracted from 10 mm thick paraffin sections. Samples were deparaffinized in 1.5 ml xylene for 2 min at room temperature for four times, and then washed with absolute ethanol for 2 min for three times. Afterwards, the samples from tumors, hyperplasia, and normal tissues were dried under vacuum for 50 min. Each dried pellet was resuspended in 250 ml solution, containing 12.5 mg glass beads (Glass Controlled Pore, 120–200 m mesh, Sigma), PCR amplification-buffer and proteinase K (0.6 mg/ml). Protein digestion was carried out in an ultrasonicator at 568C for 30 min. Proteinase K was inactivated by heating the samples at 958C for 10 min. The suspension was spun twice for 4 min to remove debris and glass beads. 2.3. Microsatellite markers and PCR analysis All specimens were screened for genetic alterations at seven microsatellite markers located on three chromosomes. Three markers exhibiting tetranucleotide

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repeat sequences (D1S518, D3S2387, and D8S1132), and four markers containing dinucleotide repeat sequences (D8S131, D8S133, D8S258, and D8S1992) were studied in each case. Primer sequences of these markers were obtained from the Genome Database (The Johns Hopkins University, Baltimore, Maryland, USA). PCR was performed with an initial denaturation step of 1 min at 948C coupled to 35 cycles of 30 s at 948C, 30 s at 558C, and 30 s at 728C with a Gene-Amp PCR System 9600 (Perkin-Elmer, Foster City, CA). Amplification of 2 ml DNA-solution was performed in a 25-ml PCR reaction mix (10 mM Tris-HCl, pH 9.0, 1.5 mM MgCl2, 50 mM KCl, 0.1 % Triton-X100, 0.01 % ( v/v) stabilizer, 0.25 mM of each deoxynucleotide triphosphate, 0.1 mM each oligonucleotide primers, and 0.7 units Super Taq Polymerase (HT Biotechnology Ltd, Cambridge, UK)). The sense-primers were fluorescence-labeled with Cy5. Denaturated PCR products were electrophoresed in 8% polyacrylamide gels (ReproGel High Resolution, Amersham Pharmacia, Uppsala, Sweden) using an automated laser fluorescent sequencer (ALF express DNA Sequencer II, Pharmacia, Uppsala, Sweden). Electrophoretic mobility shifts of PCR products were analyzed for changes by comparing allelic patterns of tumor or hyperplastic tissues and matched normal control samples. Length of fragments were calculated using an external standard and the Fragment Managere Software (Pharmacia). The ratio of relative allelic peak intensities in the tumor or hyperplastic DNA to that in the corresponding normal DNA was calculated. An allelic loss was considered significant when this ratio was #0.67 or $1.5. Microsatellite instability was defined by the presence of at least two markers exhibiting novel allele bands in hyperplastic or tumor DNA, which were not present in the corresponding normal DNA.

3. Results Twenty-nine endometrial cancer and 14 endometrial hyperplasia samples were screened for genetic alterations in seven microsatellite markers. The results analyzed in the present study are summarized in Table 3. LOH for at least one microsatellite marker was observed in 8/14 (57%) endometrial hyperplasia

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map position, the percentage of informative cases and the frequency of LOH for each marker is listed in Table 4. Fig. 1 shows representative cases of LOH for each matched DNA pair. Furthermore, 4/8 endometrial hyperplasia samples exhibiting LOH and in 13/16 endometrial cancer samples exhibiting LOH are detected by the combined use of three markers D1S518, D3S2387, and D8S1992. A novel allele band was present in at least one of the seven markers in 6/29 (21%) endometrial cancer samples of which 3/29 (10%) demonstrated microsatellite instability in two or more markers. Three endometrial hyperplasia samples displayed a novel allele band in only one microsatellite sequence located on chromosome 8p.

Table 3 Summary of genetic alterations in endometrial hyperplasia and cancer samples a

Endometrial hyperplasia EH 2 EH 1 CH 2 CH 1 Total Endometrial cancer e.a. s.a. Others Total

LOH

MI 1 LOH

Number of samples

MI

3 4 3 4 14

1 1 0 0 2

1 1 3 2 7

0 0 0 1 1

25 3 1 29

4 0 0 4

12 1 1 14

1 1 0 2

a

MI, microsatellite instability; LOH, loss of heterozygosity; EH-, simple endometrial hyperplasia without atypia; EH 1 ,simple endometrial hyperplasia with atypia; CH 2 , complex endometrial hyperplasia without atypia; CH 1 , complex endometrial hyperplasia with atypia; e.a., endometrial adenocarcinoma; s.a., serous adenocarcinoma; others, mucinous adenocarcinoma.

4. Discussion Previous studies dealing with the detection of gene expression such as p16 protein-expression, bcl-2, keratinocyte growth factor receptor, Ki-67, CK-20 or p53-expression have suggested some additional or independent prognostic indicators in endometrial cancer [26–30]. However, no specific tumor marker has been established. LOH-data have already been reported for endometrial cancer, implicating chromosome arms 1p, 3p, and 8p [4,5]. Our findings concerning the detection of LOH in endometrial hyperplasia and endometrial cancer by the combined use of the microsatellite markers D1S518, D3S2387, and D8S1992, could be an additional useful information to identify patients at risk for endometrial cancer.

samples and in 16/29 (55%) of endometrial cancer samples. LOH occurred in eight endometrial hyperplasia sample: 4/8 in endometrial hyperplasia samples with atypia and 4/6 in hyperplasia samples without atypia. All endometrial hyperplasia samples exhibiting LOH were characterized by at least one loss on the short arm of chromosome 8. Among the 16 cases of endometrial cancer with LOH, seven had allelic losses with at least one of four detectable markers on the short arm of chromosome 8. Remarkably, microsatellite marker D8S131 was non-informative in most cases analyzed. Microsatellite markers used, their Table 4 Frequency of LOH in endometrial hyperplasia and cancer specimens a Marker

D1S518 D3S2387 D8S1132 D8S131 D8S133 D8S258 D8S1992 a

Chromosomal location

1pter-1qter 3pter 8q22.1 8p21 8p21.3 8p22 8p22

Informative cases

EH

EC

EH (n ¼ 14)

EC (n ¼ 29)

LOH/informative (%)

LOH/informative (%)

10/13 12/14 13/13 6/14 12/14 10/14 10/14

25/29 23/29 26/29 15/29 23/29 20/29 24/29

2/10 (20) 1/12 (8) 0/13 (0) 1/6 (17) 4/12 (33) 3/10 (30) 2/10 (20)

7/25 (28) 3/23 (13) 1/26 (4) 3/15 (20) 2/23(9) 2/20 (10) 5/24 (21)

EH, endometrial hyperplasia; EC, endometrial cancer; LOH, Loss of heterozygosity.

G. Fabjani et al. / Cancer Letters 175 (2002) 205–211

Fig. 1. Representative electropherograms showing LOH in endometrial hyperplasia (A) and carcinoma (B). Genomic DNA from paired normal and hyperplasia or tumor tissue were subjected to PCR using the microsatellite marker D1S518. Alleles that are lost in hyperplastic or tumor cells are indicated by arrowheads.

To our knowledge this is the first report showing LOH on chromosome 8p in endometrial hyperplasia. Like in lung and breast cancer, our data confirm that deletions on 8p are early events in the development of cancer [7,8]. Putative precursors of endometrial cancer might be revealed easier by molecular screening of these microsatellite markers in endometrial hyperplasia obtained from specimens of dilatation and curettage. As LOH was mostly present in complex hyerplasia irrespective of atypia, this might help the pathologist to confirm diagnosis of this entity relevant for further therapy e.g. hysterectomy or progesterone treatment. Interestingly, hyperplasia with LOH showed at least one loss on chromosome 8p indicating that possible tumor suppressor gene(s) lying in that region might be involved in the development of endometrial cancer.

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As it was shown in previous studies specific genetic alterations are shared by putative precursor lesions and the corresponding carcinomas, indicating that in fact these findings are early events in the development of the individual cancers but need not to be common features for putative precancerous lesions. Our results deserve special attention because our genetic findings of endometrial alterations in hyperplasias and carcinomas were obtained from different patients with varying lesions. LOH on 8p seems to be such a common genetic feature indicating putative precancers. However, it must be emphasized that our number of endometrial hyperplasias are small and thus these preliminary results merit further investigation. Based on observed patterns of LOH on chromosome 8p no common regions of deletions within the used microsatellite markers can be detected indicating that the chosen 8p markers are independent targets of LOH. Hence our data are consistent with the existence of distinct regions of loss on 8p and suggest the existence of at least four tumor suppressor genes on 8p [6,10,11,25]. Additional effort is still necessary to describe more exact locations of losses on the short arm of chromosome 8, which might lead to identification of tumor suppressor gene locations. In contrast to a previous finding of a higher frequency of LOH in serous endometrial cancers, the frequency of detected LOH in sporadic endometrial cancer was similar in serous cancer and in endometrial adenocarcinoma in this study [31]. Perhaps this discrepancy can be explained by the number and kind of used microsatellite markers. We conclude that the microsatellite markers D1S518, D3S2387, and D8S1992 can serve as possible genetic markers to identify endometrial hyperplasia as presumed precursors of subsequent endometrial cancer. As loss on chromosome arm 8p was frequently found in endometrial hyperplasia as well as in endometrial cancer, this finding supports the hypothesis that chromosome 8 contains putative tumor suppressor gene(s) and that this loss occurs early during endometrial tumorigenesis. References [1] T. Enomoto, M. Fujita, M. Inoue, J.M. Rice, R. Nakajima, O. Tanizawa, T. Nomura, Alterations of the p53 tumor suppressor gene and its association with activation of the c-K-ras-2

210

[2]

[3]

[4]

[5]

[6]

[7]

[8]

[9]

[10]

[11]

[12]

[13]

[14]

G. Fabjani et al. / Cancer Letters 175 (2002) 205–211 protooncogene in premalignant and malignant lesions of the human uterine endometrium, Cancer Res. 53 (1993) 1883– 1888. M.F. Kohler, H. Nishii, P.A. Humphrey, H. Saski, J. Marks, R.C. Bast, D.L. Clarke-Pearson, J. Boyd, A. Berchuck, Mutation of the p53 tumor-suppressor gene is not a feature of endometrial hyperplasias, Am. J. Obstet. Gynecol. 169 (1993) 690–694. A. Okamoto, Y. Sameshima, Y. Yamada, S. Teshima, Y. Terashima, M. Terada, J. Yokota, Allelic loss on chromosome 17p and p53 mutations in human endometrial carcinoma of the uterus, Cancer Res. 51 (1991) 5632–5635. M.F. Arlt, T.J. Herzog, D.G. Mutch, D.J. Gersell, H. Liu, P.J. Goodfellow, Frequent deletion of chromosome 1p sequences in an aggressive histologic subtype of endometrial cancer, Hum. Mol. Genet. 5 (1996) 1017–1021. T. Fujino, J.I. Risinger, N.K. Collins, F.S. Liu, H. Nishii, H. Takahashi, E.M. Westphal, J.C. Barrett, H. Sasaki, M.F. Kohler, A. Berchuck, J. Boyd, Allelotype of endometrial carcinoma, Cancer Res. 54 (1994) 4294–4298. Y. Fujiwara, M. Emi, H. Ohata, Y. Kato, T. Nakajima, T. Mori, Y. Nakamura, Evidence for the presence of two tumor suppressor genes on chromosome 8p for colorectal carcinoma, Cancer Res. 53 (1993) 1172–1174. M. Amari, A. Suzuki, T. Moriya, K. Yoshinaga, G. Amano, H. Sasano, N. Ohuchi, S. Satomi, A. Horii, L.O.H. analyses, of premalignant and malignant lesions of human breast: frequent LOH in 8p, 16q, and 17q in atypical ductal hyperplasia, Oncol. Rep. 6 (1999) 1277–1280. I.I. Wistuba, C. Behrens, A.K. Virmani, S. Milchgrub, S. Syed, S. Lam, B. Mackay, J.D. Minna, A.F. Gazdar, Allelic losses at chromosome 8p21-23 are early and frequent events in the pathogenesis of lung cancer, Cancer Res. 59 (1999) 1973– 1979. P. O’Connell, V. Pekkel, S.A. Fuqua, C.K. Osborne, G.M. Clark, D.C. Allred, Analysis of loss of heterozygosity in 399 premalignant breast lesions at 15 genetic loci, J. Natl. Cancer Inst. 90 (1998) 697–703. M.L. Yaremko, C. Kutza, J. Lyzak, R. Mick, W.M. Recant, C.A. Westbrook, Loss of heterozygosity from the short arm of chromosome 8 is associated with invasive behavior in breast cancer, Genes Chromosomes Cancer 16 (1996) 189–195. T. Yokota, M. Yoshimoto, F. Akiyama, G. Sakamoto, F. Kasumi, Y. Nakamura, M. Emi, Localization of a tumor suppressor gene associated with the progression of human breast carcinoma within a 1-cM interval of 8p22-p23.1, Cancer 85 (1999) 447–452. T. Enomoto, M. Inoue, A.O. Perantoni, G.S. Buzard, H. Miki, O. Tanizawa, J.M. Rice, K-ras activation in premalignant and malignant epithelial lesions of the human uterus, Cancer Res. 51 (1991) 5308–5314. H. Sasaki, H. Nishii, H. Takahashi, A. Tada, M. Furusato, Y. Terashima, G.P. Siegal, S.L. Parker, M.F. Kohler, A. Berchuck, J. Boyd, Mutation of the Ki-ras protooncogene in human endometrial hyperplasia and carcinoma, Cancer Res. 53 (1993) 1906–1910. R.L. Levine, C.B. Cargile, M.S. Blazes, B. van Rees, R.J.

[15]

[16]

[17]

[18]

[19]

[20] [21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

Kurman, L.H. Ellenson, PTEN mutations and microsatellite instability in complex atypical hyperplasia, a precursor lesion to uterine endometrioid carcinoma, Cancer Res. 58 (1998) 3254–3258. K. Yoshinaga, H. Sasano, T. Furukawa, H. Yamakawa, M. Yuki, S. Sato, A. Yajima, A. Horii, The PTEN, BAX, and IGFIIR genes are mutated in endometrial atypical hyperplasia, Jpn. J. Cancer Res. 89 (1998) 985–990. G.L. Mutter, K.A. Boynton, W.C. Faquin, R.E. Ruiz, A.S. Jovanovic, Allelotype mapping of unstable microsatellites establishes direct lineage continuity between endometrial precancers and cancer, Cancer Res. 56 (1996) 4483–4486. J.I. Risinger, A. Berchuck, M.F. Kohler, P. Watson, H.T. Lynch, J. Boyd, Genetic instability of microsatellites in endometrial carcinoma, Cancer Res. 53 (1993) 5100–5103. M. Esteller, L. Catasus, X. Matias-Guiu, G.L. Mutter, J. Prat, S.B. Baylin, J.G. Herman, hMLH1 promoter hypermethylation is an early event in human endometrial tumorigenesis, Am. J. Pathol. 155 (1999) 1767–1772. R.J. Kurman, P.F. Kaminski, H.J. Norris, The behavior of endometrial hyperplasia. A long-term study of ‘untreated’ hyperplasia in 170 patients, Cancer 56 (1985) 403–412. A.I. Sherman, S. Brown, The precursors of endometrial carcinoma, Am. J. Obstet. Gynecol. 135 (1979) 947–956. G.L. Mutter, M.L. Chaponot, J.A. Fletcher, A. polymerase, chain reaction assay for non-random X chromosome inactivation identifies monoclonal endometrial cancers and precancers, Am. J. Pathol. 146 (1995) 501–508. A.S. Jovanovic, K.A. Boynton, G.L. Mutter, Uteri of women with endometrial carcinoma contain a histopathological spectrum of monoclonal putative precancers, some with microsatellite instability, Cancer Res. 56 (1996) 1917–1921. A. Suzuki, S. Fukushige, S. Nagase, N. Ohuchi, S. Satomi, A. Horii, Frequent gains on chromosome arms 1q and/or 8q in human endometrial cancer, Hum. Genet. 100 (1997) 629–636. A.M.F. Kersemaekers, J. Hermans, G.J. Fleuren, M.J. van de Vijver, Loss of heterozygosity for defined regions on chromosomes 3, 11 and 17 in carcinomas of the uterine cervix, Br. J. Cancer 77 (1998) 192–200. F. Kerangueven, L. Essioux, A. Dib, T. Noguchi, F. Allione, J. Geneix, M. Longy, R. Lidereau, F. Eisinger, M.J. Pe´ busque, J. Jacquemier, C. BonaI¨ti-Pellie´ , H. Sobol, D. Birnbaum, Loss of heterozygosity and linkage analysis in breast carcinoma: indication for a putative third susceptibility gene on the short arm of chromosome 8, Oncogene 10 (1995) 1023–1026. L.B. Mora, J.I. Diaz, A.B. Cantor, S.V. Nicosia, Differential diagnosis of endometrial hyperplasia and carcinoma by computerized image cytometry of cell proliferation, apoptosis and Bcl-2 expression, Ann. Clin. Lab. Sci. 29 (1999) 308–315. H.B. Salvesen, S. Das, L.A. Akslen, Loss of nuclear p16 protein expression is not associated with promoter methylation but defines a subgroup of aggressive endometrial carcinomas with poor prognosis, Clin. Cancer Res. 6 (2000) 153–159. H.B. Salvesen, O.E. Iversen, L.A. Akslen, Prognostic significance of angiogenesis and Ki-67, p53, and p21 expression: a population-based endometrial carcinoma study, J. Clin. Oncol. 17 (1999) 1380–1382.

G. Fabjani et al. / Cancer Letters 175 (2002) 205–211 [29] V. Visco, E. Carico, C. Marchese, M.R. Torrisi, L. Frati, A. Vecchione, R. Muraro, Expression of keratinocyte growth factor receptor compared with that of epidermal growth factor receptor and erbB-2 in endometrial adenocarcinoma, Int. J. Oncol. 15 (1999) 431–435. [30] R. Zemer, A. Fishman, J. Bernheim, S. Zimlichman, O.

211

Markowicz, M. Altaras, A. Klein, Expression of cytokeratin20 in endometrial carcinoma, Gynecol. Oncol. 70 (1998) 410– 413. [31] D. Tritz, M. Pieretti, S. Turner, D. Powell, Loss of heterozygosity in usual and special variant carcinomas of the endometrium, Hum. Pathol. 28 (1997) 607–612.