Hyperplasia and Carcinoma Protooncogene in Human Endometrial ...

Mutation of the Ki-ras Protooncogene in Human Endometrial Hyperplasia and Carcinoma Hiroshi Sasaki, Hiroshi Nishii, Hiroyuki Takahashi, et al. Cancer Res 1993;53:1906-1910. Published online April 1, 1993.

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[CANCER RESEARCH 53. 1906-1910. April 15. 1993]

Mutation of the Ki-ra.v Protooncogene

in Human Endometrial Hyperplasia and

Carcinoma Hiroshi Sasaki, Hiroshi Nishii, Hiroyuki Takahashi, Akio Tada, Masakuni Furusato, Yoshiteru Terashima, Gene P. Slegai, Sheryl L. Parker, Matthew F. Kohler, Andrew Berchuck, and Jeff Boyd1 Gynecologic Palhobiology Section. Laboratory of Molecular Carcinogenesis. National Institute of Environmental Health Sciences. N1H. Research Triangle Park. North Carolina 27709 Â¡H.N.. H. T.. S. L P.. J. B.Â¡;Departments of Obstetrics and Gynecology Â¡H.S.. A. T.. Y. T.Â¡and Pathology Â¡M.F.]. Jikei University School of Medicine. Tokyo 105. Japan: Departments of Pathology. Cell Biology and Surgery, University of Alabama at Birmingham School of Medicine, Birmingham, Alabama 35294 [G. P. S.I: and Department of Obstetrics and Gynecology, Duke University School of Medicine. Durham, North Carolina 27710 Â¡M.F. K., A. B.I

ABSTRACT Previous studies have demonstrated that some human endometrial car cinomas contain an activating point mutation in codon 12 of the Ki-ras protooncogene. To examine the hypothesis that this mutation may occur at an earlier stage of neoplastic progression in the endometrium, we analyzed 89 samples of premalignant endometrial hyperplasia and an additional 84 samples of endometrial carcinoma for point mutations of Ki-ras codon 12. Mutations were found in all three types of endometrial hyperplasia, sim ple, complex, and atypical, with no clear evidence of a differential distri bution in any particular type. Furthermore, the overall incidence of Ki-ras mutations in the hyperplasia specimens (16%) was similar to the incidence detected in carcinomas (18%), indicating that ras mutation may represent an early event in a subset of endometrial carcinomas. When the tissue samples were segregated as to country of origin, the frequency of this mutation was approximately 2-fold higher in hyperplasia and carcinoma samples from Japan than from the United States, where the incidence, clinicopathological characteristics, and risk factors for endometrial car cinoma differ dramatically. There was no apparent correlation, however, between ras mutation and any pathological, histolÃ³gica!, or clinical pa rameter examined, except survival. The presence of a nix mutation was inversely associated with death from disease, suggesting that this mole cular feature may characterize a subset of endometrial carcinomas with a good prognosis.

propensity to advance to invasive carcinoma. The most recent classi fication scheme for these proliferations is based on the presence or absence of cytological atypia and includes simple (formerly cystic), complex (formerly adenomatous), and atypical hyperplasia (9). The primary goal of this study was to address the hypothesis that ras mutation may occur at a premalignant stage of endometrial tumori genesis. Colorectal carcinomas, for example, exhibit Ki-ras mutations at a premalignant stage between early and late adenoma (10). A secondary goal of this study was to further characterize the clinical significance of ras mutation in invasive endometrial cancers. In a previous study (8), we found no correlation between the presence of Ki-ras mutation and any clinical or histopathological feature ex amined, but another recent report describes a correlation between the presence of Ki-ras mutation and a less favorable clinical outcome in endometrial carcinoma (11). In addition, we previously reported an incidence of 10% for Ki-ras mutations in endometrial carcinomas obtained exclusively from the United States (8), while two reports by Enomoto et al. (6, 7) describe a significantly higher incidence of Ki-ras mutations in endometrial carcinomas obtained from Japan. In this study, we addressed these literature discrepancies by obtaining clinical and pathology data from all cases of hyperplasia and carci noma examined and utilized hyperplasia and carcinoma tissue samples from both the United States and Japan.

INTRODUCTION Endometrial carcinoma is the most frequently diagnosed gyneco logical malignancy in the United States, with approximately 33,000 new cases and 5,500 deaths annually (1). but studies on the charac terization of molecular genetic alterations in this tumor type have only recently begun to be reported. The most frequently altered genes in human cancers, the p53 tumor suppressor gene and the ras family of protooncogenes, also appear to be involved in the development in endometrial carcinoma. Several studies have now confirmed that the p53 gene is subject to inactivating point mutations in 10-20% of these tumors (2-4), while activating point mutations in codon 12 of the Ki-ras protooncogene are found in 10-30% of endometrial carcino mas (5-8). To more fully understand the process of endometrial tumorigenesis. however, it will be necessary to expand upon these observations by defining the temporal occurrence of these mutations in the multistep process of neoplastic progression, as well as by correlating the presence of such mutations with particular clinical or pathological features of the tumors that contain them. In this regard, many human tumor types are difficult to detect and seldom diagnosed in a premalignant form; the endometrium is a notable exception. Noninvasive, abnormal endometrial proliferations form a morphological continuum ranging from focal glandular crowd ing through various degrees of hyperplasia with progressively greater Received 10/22/92; accepted 2/11/93. The costs of publication of this article were defrayed in pan by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1To whom requests for reprints should be addressed, at NIEHS/NIH. P. O. Box 12233.

MATERIALS

AND METHODS

Tissue Samples and DNA Isolation. The tissue specimens utilized in this study consisted of both fresh frozen biopsy material (20 hyperplasias and 41 carcinomas) and formalin-fixed paraffin-embedded samples (69 hyperplasias and 43 carcinomas). The frozen tissues were collected directly from surgery during the period 1987-1991 and were selected for this study based on sub sequent histopathological evidence of a large tumor to normal tissue ratio in the tissue sample. The formalin-fixed, paraffin-embedded samples were obtained from surgical pathology archives from the period 1986-1991. Tumor (or hyperplastic) and normal tissue were separated in these samples as previously described (4, 8). High molecular weight, genomic DNA was isolated and purified from paraffin-embedded tissues as described (4, 8) and from frozen tissue using standard procedures (12). Clinical Data. Histological slides from all cases reported in this study, both Japanese and American, were examined by the same independent pathologist to confirm the original diagnosis regarding hyperplasia versus carcinoma, as well as the specific type of hyperplasia. All other clinical data were extracted directly from patient histories. Hemotoxylin and eosin-stained histolÃ³gica! sections were examined and photographed using an Olympus Vanox-S AH-2 photomicroscope and Kodak Extachrome 64T tungsten film. PCR and Slot-Blotting Procedures. DNA samples were analyzed for mu tations in Ki-ras codon 12 using PCR2 amplification, slot-blotting, and hy bridization with radiolabeled oligodeoxynucleotides as previously described (5, 13). A panel of seven oligonucleotide probes, specific for wild-type or activating mutant sequences at this codon. was utilized. Prior to blotting, all PCR products were analyzed by agarose gel electrophoresis and ethidium bromide staining to validate successful amplification of the 120-base pair

2 The abbreviation used is: PCR. polymerase chain reaction.

Research Triangle Park. NC 27709.

1906


ras MUTATION

IN ENDOMETRIAI.

codon 12 region. In addition, all experiments were carried oui with a positive DNA control sample [from previously characterized endometrial carcinoma cell lines (5)], a negative DNA control sample (from plasmid). and a negative control (water) for potential DNA contamination of PCR reactions.

TUMORIGENESIS

Table I Sitnnnan i>fendotnrÃ-riitl bypgtpiata examined89204227551639341641725225" CasesTotalHistologySimple-ComplexAtypicalCountry

caaes t'tatninetl ras14266303II1106T4Ki-ra.v

RESULTS Incidence of Ki-ras Codon 12 Mutations in I nclonu-Ã¬i Â¡alHyperplasia. Previous studies on primary tissue samples of human en dometrial carcinoma have shown that mutations of the ras protoon cogene family are confined primarily to codon 12 of the Ki-ra.Ã§gene (6-8, 11). These studies have also shown that the PCR amplification, slot-blotting, and oligonucleotide hybridization procedure is a highly sensitive method for the detection of ms point mutations and that these mutations may be confirmed by direct sequence analysis of the PCR products. A representative example of results obtained using this procedure for endometrial carcinoma samples is shown in Fig. 1. An analysis of premalignant hyperplastic endometrial tissue revealed the presence of activating point mutations in Ki-ra.v codon 12 in a total of 14 of 89 (16%) specimens examined (Tables I and 2). Mutations of the Ki-ras gene were found in all three histopathological types of endometrial hyperplasia, simple, complex, and atypical, with no clear evidence of a preferential distribution in any particular type. Of these, cases from the United States accounted for 3 of 55 (5%) mutations and those from Japan accounted for 11 of 34 mutations (32%). A further separation of the United States cases by race revealed that all of the mutations were found in white as opposed to black patients. A con sideration of the individual histolÃ³gica! subtypes by country of origin revealed no significant trend in regard to the presence of ras mutation. Of the cases harboring a ras mutation, micrographs of representative histolÃ³gica! sections, one from each of the three types of endometrial hyperplasia, are shown in Fig. 2. Obtaining clinical outcome data on premalignant lesions of this type is problematic, since years and in some cases decades of clinical observation would be required to accurately document the occurrence of progression to carcinoma in these patients. During the time period of this study, however, at least two of the hyperplasia cases with mutant Ki-ra.s genes, sample num bers 37 (United States) and 81 (Japan), showed clinical progression to carcinoma. Incidence of Ki-ras Codon 12 Mutations in Endometrial Car cinoma. Activating point mutations in Ki-ras codon 12 were detected in 15 of 84 (18%) endometrial carcinoma specimens (Tables 3 and 4). The tumors containing ras mutations were all well or moderately differentiated adenocarcinomas and of typical glandular or glandular wild-type GGT(gly)

originUnited of StatesBlackWhiteJapanHistology

originSimpleA"JComplexAJAtypicalAJNo. by country of

mutant'' Percentage of cases examined that contain 12.Table Country of origin: A. United States:J. Japan.Mutant

codon

12Ki-raj/12Sample 2 Details of eniiiwu'lrial hyperplasia fuses with Ki-ras codon (glycine)"29 (valine)34 (arginine)37 acid)56 acid)57 valine)64 (valine)68 (valine)70 acid)71 (valine)73 acid)74

GGT GGT CGT GAT (aspartic GAT (aspartic GTT{ GTT GTT GAT (aspartic GTT GAT (aspartic

(valine)75 GTT acid)80 acid)81

GAT (aspartic GAT (aspartic GAT (aspartic acid)Country*AAAJJJJJJJJJJJHistologySimpleAtypicalAtypicalComplexComplexC " Wild-type Ki-ras codon 12 is GGT (glycine). h Country of origin: A, United States; J. Japan. ' Patient's age at time of diagnosis.

mixed with squamous histology. We examined 20 tumors that were poorly differentiated and/or of a histological subtype with an unfa vorable clinical prognosis (clear cell; serous papillary), and none of these tumors contained ras mutations. There was also a correlation between the presence of a ras mutation and clinical status: without

ACT (KF)

GAT (asp)

f-'ig. 1. Detection of activating point mutations in Ki-ra.v codon 12 using the PCR amplification, slotblotting, and oligonucleotide hybridization proce dure. The wild-type codon GGT (glycine) is detected in all 45 endometrial carcinoma samples shown in this experiment. Point mutations lo AGT (serine) and GAT (aspanic acid) are evident in one tumor sample each, and mutation to GTT (valine) is evident in three tumors.


GTT (val)

ras MUTATION

IN ENDOMETRIAL

TUMORIGENESIS

i

Fig. 2. HistolÃ³gica! photomicrographs of hematoxylin and eosin-stained sections from endometrial hyperplasia cases with Ki-rav mutations, a. simple hyperplasia. Glands are cystically dilated, with occasional outpouching. and are surrounded by an abundant stroma (sample 68, X 40). b, complex hyperplasia. Glands are markedly crowded with little intervening stroma. and epithelial stratification is evident. Cytological atypia, however, is not present (sample 57. x 80). c. atypical hyperplasia. Glands are complex and crowded and exhibit cytological atypia. Characteristic nuclear dyspolarity and pleomorphism are evident (sample 37, x 80).

exception, all of the patients whose tumors contained this mutation showed no evidence of disease or recurrence following treatment, with a follow-up of up to five years. In contrast, approximately onefourth (22/84) of the patients in this study died of their disease, and these tumors were uniformly free of the ras mutation. As for the hyperplasia specimens, there was also a marked difference in the incidence of ras mutation and the country of origin of the tumor sample. Tumors from Japan showed a 2-fold higher incidence of this mutation than did tumors from the United States (23 versus 12%). Also similar to the hyperplasia results was the occurrence of ras mutation in carcinomas from white patients only. There was no ap parent correlation between ra.v mutation and any of the other param eters studied, including surgical stage, myometrial invasion, metasta sis, or age at diagnosis.

Table 3 Summary of emltitnetriiil carcinoma cases examined CasesTotalHistologyEndometrioid

ra\151230780121244439604471500505IO examined845913122947856111713212228325291936206022241

adenocarcinomaSquamous"Other*Grade'123Stage''I11III

IVInvasion''0123Metastasis'PositiveNegativeAge* and

DISCUSSION The data presented herein support the hypothesis that activating point mutations in codon 12 of the K\-ras protooncogene occur at a premalignant stage of neoplastic progression in the human endometrium. The percentage of hyperplasias found to harbor ras mutations (16%) was similar to the total percentage of carcinomas (18%) con taining this mutation, suggesting that in the subset of endometrial carcinomas in which ras mutation occurs, the mutation is present prior to the transition from hyperplasia to invasive carcinoma. These mu tations were evident in simple (cystic) hyperplasia. complex (adenom-

(yrl61Status"NEDAWDDODCountry

originUnited of StatesBlackWhiteJapanNo.

atous) hyperplasia. and atypical hyperplasia, indicating that the mu " Includes adenosquamous carcinomas and endometrial adenocarcinomas with squatation may occur in the earliest clinically detectable stage of abnormal mous metaplasia. '' Includes clear cell, serous papillary, and mucinous carcinomas. endometrial proliferation. ' International Federation of Gynecology and Obstetrics grade: 1, well differentiated; These data are consistent with those from other studies in which ras 2. moderately differentiated; 3. poorly differentiated. '' Surgical stage. mutations have been shown to occur in premalignant conditions as *'Myomelrial wall invasion: 0, none; I. inner one-third; 2. middle one-third; 3, outer sociated with a number of malignancies, including chemically induced one-third. tumors of rodent skin and liver (14) and in humans, leukemia (15), ' Metastasis was scored positive if endometrial carcinoma was found in the udnexu. keratoacanthoma (16), thyroid carcinoma (17). and colorectal carci lymph nodes, or peritoneum. * Age at initial surgery. noma (10). Interestingly, carcinoma of the endometrium appears to '' NED. alive with no evidence of disease (for four of these cases, the patient died of share a number of additional molecular genetic features with that of other causes with no evidence of endometrial carcinoma at autopsy); AWD. alive with the colon (18), including mutation of the p53 gene as a relatively late disease: DOD. dead of disease. 1908


MlTAliON

IN I.MX>Mi:iR]\]

11 Ml )K!5.f gene in human endometrial carcinoma. Mol. Carcinog.. 5: 250-253. 1992. 5. Boyd. J.. and Risinger. J. I. Analysis of oncogene alterations in human endometrial carcinoma: prevalence of rus mutations. Mol. Carcinog.. 4: 189-195. 1991. 6. Enomoto. T.. Inoue. M.. Perantoni. A. O.. Terakawa. N.. Tanizawa, O., and Rice, J. M. K-rrt.v activation in neoplasms of the human Iemale reproductive tract. Cancer Res.. 50: 6139-6145, 1990. 7. E-nomoio. T.. Inoue. M.. Perantoni, A. O.. Bu/unl. G. S.. Miki, H.. Tanizawa. O., and Rice. J. M. K-/UV activation in premalignant and malignant epithelial lesions of the human uterus. Cancer Res., 51: 5308-5314. 1991. 8. Ignar-Trow bridge. D.. Risinger. J. I.. Dent. G. A.. Kohler. M. F.. Berchuck. A.. McLachlan. J. A., and Boyd. J. Mutations of the Ki-ru.i oncogene in endometrial carcinoma. Am. J. Obstet. Gynecol.. 167: 227-232. 1992. 9. Kurman. R. J.. and Norris, H. J. Endometrial carcinoma. In: R. J. Kurman (ed.). Blaustein's Pathology of the Female Genital Tract. Ed. 3. pp. 338-372. New York: Springer-Verlag. 1987. K). Vogelstein. B.. Fearon. E. R.. Hamilton. S. R.. Kern. S. E.. Preisinger. A. C. Lcppert. M.. Nakamura. Y. White. R.. Smits. A. M. M., and Bos. J. L. Genetic alterations during colorectal-tumor development. N. Engl. J. Med.. MV: 525-532. 1988. 11. Mizuuchi. H.. Nasim. S.. Kudo. R., Silvcrbcrg. S. G.. Greenhouse. S., and Garren, C. T. Clinical implications of K-rÂ«.vmutations in malignant epithelial tumors of the endometrium. Cancer Res.. 52: 2777-2781. 1992. 12. Samhrwk. J.. Fritsch. E. F.. and Maniatis. T. Molecular Cloning: A Laboratory

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ras MUTATION

IN ENDOMETRIAL

Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press. 1989. 13. de Vries, M. V.. Bogaard, M. E., van den Elst, H., van Boom, J. H., van der Eb, A. J.. and Bos, J. L. A dot-blot screening procedure for mutated ras oncogenes using synthetic oligodeoxynucleotides. Gene (Amst.), 50: 313-320, 1986. 14. Balmain, A., and Brown, K. Oncogene activation in chemical carcinogenesis. Adv. Cancer Res., 51: 147-182, 1988. 15. Liu, E., Hjelle, B.. Morgan. R., Hecht. F., and Bishop, J. M. Mutations of the Kirsten-ras proto-oncogene in human preleukemia. Nature (Lond.), 330: 186-188. 1987. 16. Leon. J., Ramino. H., Steinberg. J. J.. and Pellicer. A. \\-ras activation in benign and self-regressing skin tumors (keratoacanthomas) in both humans and an animal model system. Mol. Cell. Biol.. Â«:786-793, 1988.

TUMORIGENESIS

17. Lemoinc. N. R.. Mayall. E. S.. Wyllie, F. S., Williams, E. D., Goyns. M., Stringer, B.. and Wynford-Thomas, D. High frequency of ras oncogene mutation in all stages of human thyroid tumorigenesis. Oncogene, 4: 159-164, 1989. 18. Fearon. E. R., and Vogelstein, B. Agenetic model for colorectal carcinogenesis. Cell, 61: 759-767, 1990. 19. Silverberg, S. G., Sasano, N.. and Yajima, A. Endometrial carcinoma in Miyagi Prefecture. Japan: histopathologic analysis of a cancer-based series and comparison with cases in American women. Cancer (Phila.). 49: 1504-1510. 1982. 20. Wynder. E. L.. and Hirayama. T. Comparative epidemiology of cancer of the United States and Japan. Prev. Med.. 6: 567-594, 1977. 21. Deligdisch, L.. and Holinka, C. F. Endometrial carcinoma: two diseases? Cancer Detect. Prev.. 10: 237-246. 1987.

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