Mutations of the Ki-ras oncogene in endometrial carcinoma Diane Ignar-Trowbridge, PhD,. John I. Risinger, MS: Georgette A. Dent, MD,c Matthew Kohler, MD/ Andrew Berchuck, MD/ John A. McLachlan, PhD,. and Jeff Boyd, Ph Db Research Triangle Park, Chapel Hill, and Durham, North Carolina OBJECTIVE: The purpose of this study was to assess the extent of involvement of the (as oncogene in endometrial carcinoma. STUDY DESIGN: Genomic deoxyribonucleic acid from 30 samples of endometrial carcinoma was examined for point mutations in codons 12, 13, and 61 from the Ha-ras, Ki-ras, and N-ras genes by means of the polymerase chain reaction, slot-blotting, and deoxyribonucleic acid sequencing procedures. RESULTS: An apparent somatic mutation of Ki-ras codon 12 in one of 10 paraffin-embedded tumors was confirmed by deoxyribonucleic acid sequence analysis. Two of 20 frozen endometrial carcinoma specimens were also shown to contain a point mutation in Ki-ras codon 12. No correlation between ras mutation and a number of histologic or clinical parameters was observed. CONCLUSIONS: These data suggest a potential role for Ki-ras codon 12 mutations in the development of some (10%) endometrial cancers. (AM J OSSTET GVNECOL 1992;167:227-32.)
Key words: Endometrial carcinoma, oncogene, ras, polymerase chain reaction Carcinoma of the uterine endometrium is the most frequently diagnosed gynecologic malignancy in the United States, but few data on the molecular genetic features of this tumor exist. We believe that a series of genetic alterations involving both protooncogenes and tumor suppressor genes plays a role in the development of endometrial carcinomas as it does in other human cancers.' The variable nature of these genetic events can be noted in the clinical observation of two distinct forms of endometrial carcinoma. e. 3 Type I tumors are frequently associated with a history of unopposed estrogen exposure or other hyperestrogenic condition such as obesity. They occur in perimenopausal women, are typically of low grade and good prognosis, and are often associated with or preceded by endometrial hyperplasia. In contrast, type II tumors, which have a poorer prognosis, appear unrelated to estrogenic stimulation, manifest at a later age (>60), and are generally of higher grade and/ or uncommon histologic subtype. The clustering of endometrial carcinoma in certain families· and the occurrence in patients with multiple primary tumors (especially those of the colon),·7 suggest genetic transmission of endometrial cancer risk. From the Laboratory of Reproductive and Developmental Toxicology" and the Laboratory of Molecular Carcinogenesis,' National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, the Department of Pathology, University of North Carolina School of Medicine, Chapel Hill,' and the Department of Obstetrics and Gynecology. Duke University Medical Center, Durham. d Received for publication October 23,1991; rev~~edJanuary 1,1992: accepted January 23,1992. Reprint requests: Jeff Boyd, PhD. National Institute of Environmental Health Sciences! NIH, P.O. Box 12233, Research Triangle Park, NC 27709.
6/1 !36672
The potential role of protooncogenes in the cause of endometrial carcinoma has only recently been addressed, unlike the etiologic factors of the more wellcharacterized female cancers of the breast, ovary, and uterine cervix. Amplification and/ or overexpression of the c-erbB2 protooncogene may occur in a subset of tumors, although the extent of this phenomenon is still unclear. s, 1O In addition, the inappropriate expression of colony stimulating factor-l and its receptor, the c-fms protooncogene, has been implicated as a potential autouine growth stimulatory pathway in endometrial carcinoma.'" .2 Activating point mutations in members of the ras gene family represent the most frequent oncogene abnormality in human tumors.· 3 Several recent reports suggest that ras mutations may playa role in some endometrial carcinomas; specifically, a total of 6 of 12 human endometrial carcinoma cell lines in two studies lo ." and 13 of 41 primary tumors in three studies"·'6 exhibit point mutations in Ki-ras codon 12. In the three previous studies of primary tumor specimens, however, a relatively small number of cases were examined in each study (10, 12, and 19, respectively), and a majority of the mutations (11 of 13) were found in endometrial carcinomas from Japan'·' '6; the remaining two were from Australia.' s Additional studies are needed to define the extent of involvement of the ras gene family in endometrial carcinomas from the United States, where the incidence of endometrial carcinoma is the highest in the world and where a majority of tumors are of the type I variety, in contrast to Japan, where the incidence of endometrial carcinoma is the lowest in the world and where a significantly higher fraction of tumors are of the type II classification. 17
227
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-
2
July 1992 Am J Obstet Gynecol
--:
3
4
F
:
5
6
7
&
-8
9
10
~I----------------~--------------------~..~----------------~
wild-type GGT (gly)
GTT (val)
Fig. 1. Somatic mutation of Ki-ras codon 12 as detected by oligonucleotide hybridization with polymerase chain reaction-amplified DNA. DNA samples from normal (N) and tumor (T) tissue from 10 cases of formalin-fixed, paraffin-embedded endometrial carcinoma were analyzed for all possible activating point mutations in codons 12, 13, and 61 of Ha-ras, Ki-ras, and N-ras genes. Tumor but not normal tissue DNA from case 7 exhibited positive hybridization with mutant probe GTT for Ki-ras codon 12.
codon
11 12 13 14
Normal
Tumor
CG T G TG GG C G T
GC T GT* G T G G C T A
A
codon
11 12 13 14
CTAG CTAG Fig. 2. Autoradiogram of sequencing gel for endometrial carcinoma case 7 in Fig. I. The slot-blotting data indicating a point mutation of Ki-Tas codon 12 was confirmed by DNA sequence analysis ; clone of polymerase chain reaction-amplified DNA from normal tissue contained wild-type sequence GGT (gly), and clone of polymerase chain reaction-amplified DNA from tumor tissue contained mutant sequence CTT (val). Asterisk, Mutant codon .
The primary goal of this study was to examine a sufficiently large sample of endometrial carcinomas from the United States so that the extent of involvement of the ras oncogene in tumors from this population could be assessed. We then wished to determine if the presence of ras mutations correlated with parameters such as histologic factors, grade, stage, and receptor status. Material and methods
Tumor samples and DNA isolation. Ten formalinfixed, paraffin-embedded endometrial carcinoma specimens from the year 1990 were retrieved at random from the surgical pathology archives at the North Carolina Memorial Hospital. Genomic deoxyribonucleic acid (DNA) was extracted and was purified with a modification of a procedure described by Goelz et al. 18 Histopathologic sections 10 fLm thick were cut and placed on untreated glass slides; 10 to 30 sections were cut for each tumor. One slide stained with hematoxylin and eosin stain was used to discriminate areas of tumor from
normal tissue, and the bottom of each unstained slide was marked accordingly. Normal and tumor tissue were scraped separately from the slides with a straight-edged scalpel blade, were minced into small pieces, and were collected in separate 15 ml polypropylene conical tubes. DNA was then extracted and purified as described,1 8 with the exception that samples were not sheared through a syringe needle (this step was found to decrease the size of genomic DNA without increasing the yield appreciably). Twenty fresh-frozen endometrial carcinoma samples , obtained between 1989 and 1991 at the Duke University Medical Center, were processed for genomic DNA by means of standard procedures. I" Polymerase chain reaction and slot-blotting procedures~ DNA was screened for ras mutations by means of polymerase chain reaction a mplification, slot-blotting, and hybridization with oligodeoxynucleotides, as described previously. 10, 20 A panel of 66 oligonucleotide probes that were specific for wild-type or mutant sequences at codons 12, 13, and 61 of the Ha-ras,
ras Mutations in endometrial carcinoma
VOIlllllC l li7 :-.lumber I
229
Fig. 3. Histologic photomicrograph of stained seClion from endometrial carcinoma case 7. T he junction of carcinoma with normal myometrium illustrates the distinction of "tumor" versus "normal" in separating paraffinized tissue for DNA extraction; glandular organization indicates moderately differentiated endometrioid adenocarci noma. (Hematoxylin and eosin. Original magnification x2UO.)
wild-type GGT(gly)
GTI(val)
TGT(cys)
1
8
15
1
8
15
2
9
16
2
9
16
J
10
17
J
10
17
4
11
18
4
11
18
5
12
19
5
12
6
13
20
6
13
20
7
14
C
7
14
C
,
•
19
Fig. 4. Mu tation of Ki-ras codon 12 in fresh-frozen endometrial carcinoma sa mples as detected by oligonucleotide hybridization with polymerase chain reaction - amplified DNA. Cases 5 and 8 exhibit mutations of wild-type GGT (g(y) to GTT (val) and TGT (').1) , respec tively.
Ki-ras, and N-ras genes was used to analyze DNA from normal and tumor tissue from each of the lO archival endometrial carcinoma cases. T he 20 fresh-frozen cases were analyzed only for mutations at Ki-ms codon 12. Before s[ot-blouing was performed , all sam ples were analyzed by agarose gel electrophoresis to e nsure success of the polymerase chain reaction; in addition , all experiments were carried out with positive controls (DNA fro m previously characterized endometrial carcinoma cell lines 10) and negative controls (plasmid DNA).
Sequence analysis. For cases determined to be harboring probable ras mutations, polymerase chain reaction amplified DNA fragm en ts were subcloned and sequenced with the dideoxy chain termination method. After amplification, pol ymerase chain reaction products were purified by centrifugaLion in a Centrico n- 30 microconcentrator (Amicon, Danvers, Mass.) and were subcloned into the SmaI site of pBluescript SKII' (Stratagene, La Jolla, Calif.). Clones were sequenced with polymerase chain reaction primers according to the manufacturer's protocol for Sequenase version 2.0. Se-
230
Ignar-Trowbridge et al.
July 1992 Am J Obstet Gynecol
Table I. Clinicopathologic parameters and ras mutations Steroid receptors Case No.
Archival I
2 3 4 5 6 7 8 9 10 Frozen I
2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19
20
Histologic factors
FICO grade
Surgical stage
Uterine invasion
Estrogen
EA MA EA EA AA EA EA EA AS EA
2
NA IA IC
2 2 2 2
IB IB
2 0 2 2 I 2 I I 2 I
+ + + + + + + + +
EA EA PC EA EA EA EA EA AS EA EA EA EA AS CC EA EA EA EA EA
I 3 2 2 3 I
IlIA IVB IlIA IlIA
I I
2 2 I
2 2 2 2 2 2 I
3 3 2 2 2 2 2
II
IA IC IC IA
mc
IlIA IC IB
IlIC IC IVB IC IA IC IVB IC
IB
IC IlIA IB
3 3 2 I 2 I
3 I 3 2 3 3 0 3 I 3 I 3 I I
+ +
I NA
NA
+ +
Ki-ras/12
+ + + + + + + + +
wt wt wt wt wt wt mut wt wt wt
+
+
+ + +
+ +
+ +
+ + NA
+
Progesterone
+ + + +
wt wt wt wt mut wt wt mut wt wt wt wt wt wt wt wt wt wt wt wt
FICO, International Federation of Gynecology and Obstetrics; EA, endometrioid adenocarcinoma; MA, mucinous adenocarcinoma; AA, adenoacanthoma; AS, adenosquamous carcinoma; PC, papillary carcinoma; CC, clear cell carcinoma. For uterine invasion: 0, none; 1, inner third; 2, middle third; 3, outer third. For steroid receptors: Estrogen/progesterone; +, >10 fmol/mg. For Ki-ras: wt, wild-type; mut, mutant. NA, not available.
quencing reactions were subjected to electrophoresis in 7 moll L urea-6% polyacrylamide gels at 65 W constant power, were fixed in 10% methanol-lO% acetic acid, were dried. and were exposed to Kodak X-Omat AR x-ray film for 1 to 3 days. Results
All 10 of the formalin-fixed, paraffin-embedded tissue specimens analyzed yielded both normal and tumor DNA of sufficient quality and quantity for subsequent genetic analysis. The paraffin blocks invariably contained enough tumor and normal tissue so that from 30 to several hundred micrograms of genomic DNA was isolated from all cases. For each sample, the DNA migrated on ethidium bromide-stained agarose gels as a single high-molecular-weight band (>23 kb) with a smear of degraded, lower-molecular-weight DNA. Although the tumors described in this paper were all GTT (Gly-,> Val) transversion in codon 12 of Ki-ras was confirmed by sequencing of the polymerase chain reaction product; only the wild-type sequence was evident in DNA from the normal tissue of this case (Fig. 2). This tumor was classified with respect to histologic factors as a moderately differentiated endometrioid adenocarcinoma; a hematoxylin and eosin stained photomicrograph of the tumor and myometrial border is shown in Fig. 3. Twenty additional specimens of fresh-frozen endometrial carcinoma were examined for mutations in Kims codon 12. Two of these tumors, a moderately differentiated endometrioid adenocarcinoma and a
Volume 167 Number 1
poorly differentiated endometrioid adenocarcinoma, exhibited activating point mutations: One contained the same GGT--+GTT transversion described above, and the other contained a GGT--+TGT (Gly--+Cys) transversion in the first nucleotide of this codon (Fig. 4). These mutations were also heterozygous in nature; the tumors contained both a mutant and wild-type allele. The mutations and histopathologic data for the 30 tumors described in this report are summarized in Table I in which no correlation between Ki-ras mutation and any of the clinical, pathologic, or histologic criteria was reported.
Comment These data indicate that somatic point mutation of a ras oncogene occurs at a significant but relatively low (10%) frequency in endometrial carcinomas in the United States. Two previous studies 14 . 16 showed that ras mutation occurs in endometrial carcinomas from Japan at about a threefold higher frequency than that observed here. These data suggest that ras mutation may be a more frequent occurrence in the type II tumors more prevalent in Japan than in the type I estrogenrelated tumors that are more prevalent in the United States. The higher incidence of ras mutation that we had observed in endometrial carcinoma celilines 'o may reflect spontaneous mutation and! or clonal selection of mutant ras genes during establishment and culture of these cells. All the existing data, however, implicate Kiras codon 12 as the most frequently altered ras site in endometrial carcinoma. The ras family of protooncogenes encodes a unique form of GTPase molecule that appears to playa central role in cell proliferation as a result of its regulation of signal transduction. Point mutations in codons 12, 13, or 61 appear to inactivate this GTPase activity, which leaves the mutant protein in a constitutively activated state. 2l A large number and variety of human tumors contain point-mutated ras oncogenes,l3 which suggest a generalized role for ras in the proliferation of diverse tissues. Other solid tumors that contain mutations of Ki-ras at significant frequencies include carcinomas of the lung (20% to 30%), pancreas (70% to 90%), thyroid (20% to 60%), and colon (40% to 50%).13 Endometrial carcinomas, like those of the lung, pancreas, and colon, exhibit a predominance of Ki-ras codon 12 mutations; thus a similar role for Ki-ras in the regulation of cell proliferation in these tissues or perhaps similar molecular pathways of neoplastic development or progression in these tissues. Mutated ras genes have been strongly implicated in experimental chemical carcinogenesis 22 ; this suggests that ras mutations in endometrial tumors may arise from chemical (including hormonal) mutagenesis. Al-
ras Mutations in endometrial carcinoma
231
ternatively, because cyclical cell proliferation is a prominent and somewhat unique feature of the mammalian endometrium, spontaneous DNA aberrations would be subject to a greater probability of conversion to permanent mutations in this tissue. Estrogen may thus act indirectly to stimulate tumorigenesis, by increasing the proliferative index of endometrial tissue. 23 The Ki-ras mutations reported in this paper whether they were spontaneous or chemically induced, did not appear to be associated with any histopathologic or clinical criteria. This suggests a sporadic induction and random distribution in endometrial carcinoma. To extend these findings it will be necessary to examine a larger number of tumors, especially those of the more uncommon histologic (type II) variants. In particular, it would be instructive to compare a larger number of tumor samples from the United States with those from Japan, where estrogen-related risk factors and the incidence of endometrial carcinoma are much lower than those in the United States.' 17 That comparison might indicate a dramatically different ratio of type II to type I tumors. In addition, it would be of interest to analyze premalignant hyperplastic lesions of the endometrium for ras mutations to determine if that mutation is an early event in the development of some endometrial cancers as it is in colon carcinoma. REFERENCES 1. Boyd JA, Barrett Je. Genetic and cellular basis of multistep carcinogenesis. Pharmacol Ther 1990;46:469-86. 2. Deligdisch L, Holinka CF. Endometrial carcinoma: two diseases? Cancer Detect Prev 1987;10:237-46. 3. Kurman RJ, Norris HJ. Endometrial carcinoma. In: Kurman RJ, ed. Blaustein's pathology of the female genital t,act. 3rd ed. New York: Springer-Verlag, 1987. 4. Boltenberg A, Furgyik S. Kullander S. Familial cancer aggregation in cases of adenocarcinoma corporis uteri. Acta Obstet Gynecol Scand 1990;69:249-58. 5. Lynch HT, Lanspa S, Smyrk T, Boman B, Watson P, Lynch J. Hereditary nonpolyposis colorectal cancer (Lynch syndromes I & II). Genetics, pathology, natural history, and cancer contro\. Part 1. Cancer Genet Cytogenet 1991;53: 143-60. 6. Vasen HFA. Offerhaus GJA, den Hartog Jager FCA, et a\. The tumor spectrum in hereditary non-polyposis colorectal cancer: a study of 24 kindreds in the Netherlands. Int J Cancer 1990:46:31-4. 7. Schwartz Z, Ohel G, Birkenfeld A, Anteby SO, Schenker JG. Second primary malignancy in endometrial carcinoma patients. Gynecol Oncol 1985;22:40-5. 8. Berchuck A, Rodriquez G, Kinney RB, Soper JT, Dodge RK, Clarke-Pearson DL, Bast RC JR. Overexpression of HER-21neu in endometrial cancer is associated with advanced stage disease. AMJ OBSTET GYNECOL 1991; 164: 1521. 9. Brumm C, Riviere A, Wilckens C, Loning T. Immunohistochemical investigation and Northern blot analysis of c-erbB-2 expression in normal, premalignant and malignant tissues of the corpus and cervix uteri. Virchows Arch [A] 1990;417:477-84. 10. Boyd J, Risinger J1. Analysis of oncogene alterations in human endometrial carcinoma: prevalence of ras mutations. Mol Carcinog 1991;4:189-95.
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11. Kacinski BM, Chambers SK, Stanley ER, et al. The cytokine CSF-I (M-CSF) expressed by endometrial carcinomas in vivo and in vitm, may also be a circulating tumor marker of neoplastic disease activity in endometrial carcinoma patients. Int J Radiat Oncol BioI Phys 1990; 19:619-26. 12. Baiocchi G, Kavanagh Jj, Talpaz M, Wharton JT, Gutterman JU, Kurzrock R. Expression of the macrophage colony-stimulating factor and its receptor in gynecologic malignancies. Cancer 1991 ;67:990-6. 13. Bos JL. Ras oncogenes in human cancer: a review. Cancer Res 1989;49:4682-9. 14. Enomoto T, Inoue M, Perantoni AO, Terakawa N, Tanizawa 0, Rice JM. K-ras activation in neoplasms of the human female reproductive tract. Cancer Res 1990;50:6139-45. 15. Lester DR, Cauchi MN. Point mutations at codon 12 of c-K-ras in human endometrial carcinomas. Cancer Lett 1990;51 :7-10. 16. Enomoto T, Inoue M, Pera11l0ni AO, Buzard GS, Miki H, Tanizawa 0, Rice JM. K-ras activation in premalignant and malignant epithelial lesions of the human uterus. Cancer Res 1991;51:5308-14.
July 1992 Am J Obstet Gynecol
17. Parazzini F, La Vecchia C, Bocciolone L, Franceschi S. The epidemiology of endometrial cancer. Gynecol Oncol 1991;41:1-16. 18. Goelz SE, Hamilton SR, Vogel stein B. Purification of DNA from formaldehyde fixed and paraffin embedded human tissue. Biochem Biophys Res Commun 1985; 130: 118-26. 19. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning, a laboratory manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 1989. 20. de Vries MV, Bogaard ME, van den Elst H, van Boom JH, van der Eb AJ, BosJL. A dot-blot screening procedure for mutated ras oncogenes using synthetic oligodeoxynucleotides. Gene 1986;50:313-20. 21. Barbacid M. Ras genes. Annu Rev Biochem 1987;56:779827. 22. Sukumar S. Ras oncogenes in chemical carcinogenesis. Curr Top Microbial Iml1lunol 1989; 148:93-114. 23. Henderson BE, Ross R, Bernstein L. Estrogens as a cause of human cancer: the Richard and Hinda Rosenthal Foundation Award Lecture. Cancer Res 1988;48:246-53.
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Key words: Endometrial carcinoma, oncogene, ras, polymerase chain reaction Carcinoma of the uterine endometrium is the most frequently diagnosed gynecologic malignancy in the United States, but few data on the molecular genetic features of this tumor exist. We believe that a series of genetic alterations involving both protooncogenes and tumor suppressor genes plays a role in the development of endometrial carcinomas as it does in other human cancers.' The variable nature of these genetic events can be noted in the clinical observation of two distinct forms of endometrial carcinoma. e. 3 Type I tumors are frequently associated with a history of unopposed estrogen exposure or other hyperestrogenic condition such as obesity. They occur in perimenopausal women, are typically of low grade and good prognosis, and are often associated with or preceded by endometrial hyperplasia. In contrast, type II tumors, which have a poorer prognosis, appear unrelated to estrogenic stimulation, manifest at a later age (>60), and are generally of higher grade and/ or uncommon histologic subtype. The clustering of endometrial carcinoma in certain families· and the occurrence in patients with multiple primary tumors (especially those of the colon),·7 suggest genetic transmission of endometrial cancer risk. From the Laboratory of Reproductive and Developmental Toxicology" and the Laboratory of Molecular Carcinogenesis,' National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, the Department of Pathology, University of North Carolina School of Medicine, Chapel Hill,' and the Department of Obstetrics and Gynecology. Duke University Medical Center, Durham. d Received for publication October 23,1991; rev~~edJanuary 1,1992: accepted January 23,1992. Reprint requests: Jeff Boyd, PhD. National Institute of Environmental Health Sciences! NIH, P.O. Box 12233, Research Triangle Park, NC 27709.
6/1 !36672
The potential role of protooncogenes in the cause of endometrial carcinoma has only recently been addressed, unlike the etiologic factors of the more wellcharacterized female cancers of the breast, ovary, and uterine cervix. Amplification and/ or overexpression of the c-erbB2 protooncogene may occur in a subset of tumors, although the extent of this phenomenon is still unclear. s, 1O In addition, the inappropriate expression of colony stimulating factor-l and its receptor, the c-fms protooncogene, has been implicated as a potential autouine growth stimulatory pathway in endometrial carcinoma.'" .2 Activating point mutations in members of the ras gene family represent the most frequent oncogene abnormality in human tumors.· 3 Several recent reports suggest that ras mutations may playa role in some endometrial carcinomas; specifically, a total of 6 of 12 human endometrial carcinoma cell lines in two studies lo ." and 13 of 41 primary tumors in three studies"·'6 exhibit point mutations in Ki-ras codon 12. In the three previous studies of primary tumor specimens, however, a relatively small number of cases were examined in each study (10, 12, and 19, respectively), and a majority of the mutations (11 of 13) were found in endometrial carcinomas from Japan'·' '6; the remaining two were from Australia.' s Additional studies are needed to define the extent of involvement of the ras gene family in endometrial carcinomas from the United States, where the incidence of endometrial carcinoma is the highest in the world and where a majority of tumors are of the type I variety, in contrast to Japan, where the incidence of endometrial carcinoma is the lowest in the world and where a significantly higher fraction of tumors are of the type II classification. 17
227
228
Ignar-Trowbridge et al.
-
2
July 1992 Am J Obstet Gynecol
--:
3
4
F
:
5
6
7
&
-8
9
10
~I----------------~--------------------~..~----------------~
wild-type GGT (gly)
GTT (val)
Fig. 1. Somatic mutation of Ki-ras codon 12 as detected by oligonucleotide hybridization with polymerase chain reaction-amplified DNA. DNA samples from normal (N) and tumor (T) tissue from 10 cases of formalin-fixed, paraffin-embedded endometrial carcinoma were analyzed for all possible activating point mutations in codons 12, 13, and 61 of Ha-ras, Ki-ras, and N-ras genes. Tumor but not normal tissue DNA from case 7 exhibited positive hybridization with mutant probe GTT for Ki-ras codon 12.
codon
11 12 13 14
Normal
Tumor
CG T G TG GG C G T
GC T GT* G T G G C T A
A
codon
11 12 13 14
CTAG CTAG Fig. 2. Autoradiogram of sequencing gel for endometrial carcinoma case 7 in Fig. I. The slot-blotting data indicating a point mutation of Ki-Tas codon 12 was confirmed by DNA sequence analysis ; clone of polymerase chain reaction-amplified DNA from normal tissue contained wild-type sequence GGT (gly), and clone of polymerase chain reaction-amplified DNA from tumor tissue contained mutant sequence CTT (val). Asterisk, Mutant codon .
The primary goal of this study was to examine a sufficiently large sample of endometrial carcinomas from the United States so that the extent of involvement of the ras oncogene in tumors from this population could be assessed. We then wished to determine if the presence of ras mutations correlated with parameters such as histologic factors, grade, stage, and receptor status. Material and methods
Tumor samples and DNA isolation. Ten formalinfixed, paraffin-embedded endometrial carcinoma specimens from the year 1990 were retrieved at random from the surgical pathology archives at the North Carolina Memorial Hospital. Genomic deoxyribonucleic acid (DNA) was extracted and was purified with a modification of a procedure described by Goelz et al. 18 Histopathologic sections 10 fLm thick were cut and placed on untreated glass slides; 10 to 30 sections were cut for each tumor. One slide stained with hematoxylin and eosin stain was used to discriminate areas of tumor from
normal tissue, and the bottom of each unstained slide was marked accordingly. Normal and tumor tissue were scraped separately from the slides with a straight-edged scalpel blade, were minced into small pieces, and were collected in separate 15 ml polypropylene conical tubes. DNA was then extracted and purified as described,1 8 with the exception that samples were not sheared through a syringe needle (this step was found to decrease the size of genomic DNA without increasing the yield appreciably). Twenty fresh-frozen endometrial carcinoma samples , obtained between 1989 and 1991 at the Duke University Medical Center, were processed for genomic DNA by means of standard procedures. I" Polymerase chain reaction and slot-blotting procedures~ DNA was screened for ras mutations by means of polymerase chain reaction a mplification, slot-blotting, and hybridization with oligodeoxynucleotides, as described previously. 10, 20 A panel of 66 oligonucleotide probes that were specific for wild-type or mutant sequences at codons 12, 13, and 61 of the Ha-ras,
ras Mutations in endometrial carcinoma
VOIlllllC l li7 :-.lumber I
229
Fig. 3. Histologic photomicrograph of stained seClion from endometrial carcinoma case 7. T he junction of carcinoma with normal myometrium illustrates the distinction of "tumor" versus "normal" in separating paraffinized tissue for DNA extraction; glandular organization indicates moderately differentiated endometrioid adenocarci noma. (Hematoxylin and eosin. Original magnification x2UO.)
wild-type GGT(gly)
GTI(val)
TGT(cys)
1
8
15
1
8
15
2
9
16
2
9
16
J
10
17
J
10
17
4
11
18
4
11
18
5
12
19
5
12
6
13
20
6
13
20
7
14
C
7
14
C
,
•
19
Fig. 4. Mu tation of Ki-ras codon 12 in fresh-frozen endometrial carcinoma sa mples as detected by oligonucleotide hybridization with polymerase chain reaction - amplified DNA. Cases 5 and 8 exhibit mutations of wild-type GGT (g(y) to GTT (val) and TGT (').1) , respec tively.
Ki-ras, and N-ras genes was used to analyze DNA from normal and tumor tissue from each of the lO archival endometrial carcinoma cases. T he 20 fresh-frozen cases were analyzed only for mutations at Ki-ms codon 12. Before s[ot-blouing was performed , all sam ples were analyzed by agarose gel electrophoresis to e nsure success of the polymerase chain reaction; in addition , all experiments were carried out with positive controls (DNA fro m previously characterized endometrial carcinoma cell lines 10) and negative controls (plasmid DNA).
Sequence analysis. For cases determined to be harboring probable ras mutations, polymerase chain reaction amplified DNA fragm en ts were subcloned and sequenced with the dideoxy chain termination method. After amplification, pol ymerase chain reaction products were purified by centrifugaLion in a Centrico n- 30 microconcentrator (Amicon, Danvers, Mass.) and were subcloned into the SmaI site of pBluescript SKII' (Stratagene, La Jolla, Calif.). Clones were sequenced with polymerase chain reaction primers according to the manufacturer's protocol for Sequenase version 2.0. Se-
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Ignar-Trowbridge et al.
July 1992 Am J Obstet Gynecol
Table I. Clinicopathologic parameters and ras mutations Steroid receptors Case No.
Archival I
2 3 4 5 6 7 8 9 10 Frozen I
2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19
20
Histologic factors
FICO grade
Surgical stage
Uterine invasion
Estrogen
EA MA EA EA AA EA EA EA AS EA
2
NA IA IC
2 2 2 2
IB IB
2 0 2 2 I 2 I I 2 I
+ + + + + + + + +
EA EA PC EA EA EA EA EA AS EA EA EA EA AS CC EA EA EA EA EA
I 3 2 2 3 I
IlIA IVB IlIA IlIA
I I
2 2 I
2 2 2 2 2 2 I
3 3 2 2 2 2 2
II
IA IC IC IA
mc
IlIA IC IB
IlIC IC IVB IC IA IC IVB IC
IB
IC IlIA IB
3 3 2 I 2 I
3 I 3 2 3 3 0 3 I 3 I 3 I I
+ +
I NA
NA
+ +
Ki-ras/12
+ + + + + + + + +
wt wt wt wt wt wt mut wt wt wt
+
+
+ + +
+ +
+ +
+ + NA
+
Progesterone
+ + + +
wt wt wt wt mut wt wt mut wt wt wt wt wt wt wt wt wt wt wt wt
FICO, International Federation of Gynecology and Obstetrics; EA, endometrioid adenocarcinoma; MA, mucinous adenocarcinoma; AA, adenoacanthoma; AS, adenosquamous carcinoma; PC, papillary carcinoma; CC, clear cell carcinoma. For uterine invasion: 0, none; 1, inner third; 2, middle third; 3, outer third. For steroid receptors: Estrogen/progesterone; +, >10 fmol/mg. For Ki-ras: wt, wild-type; mut, mutant. NA, not available.
quencing reactions were subjected to electrophoresis in 7 moll L urea-6% polyacrylamide gels at 65 W constant power, were fixed in 10% methanol-lO% acetic acid, were dried. and were exposed to Kodak X-Omat AR x-ray film for 1 to 3 days. Results
All 10 of the formalin-fixed, paraffin-embedded tissue specimens analyzed yielded both normal and tumor DNA of sufficient quality and quantity for subsequent genetic analysis. The paraffin blocks invariably contained enough tumor and normal tissue so that from 30 to several hundred micrograms of genomic DNA was isolated from all cases. For each sample, the DNA migrated on ethidium bromide-stained agarose gels as a single high-molecular-weight band (>23 kb) with a smear of degraded, lower-molecular-weight DNA. Although the tumors described in this paper were all GTT (Gly-,> Val) transversion in codon 12 of Ki-ras was confirmed by sequencing of the polymerase chain reaction product; only the wild-type sequence was evident in DNA from the normal tissue of this case (Fig. 2). This tumor was classified with respect to histologic factors as a moderately differentiated endometrioid adenocarcinoma; a hematoxylin and eosin stained photomicrograph of the tumor and myometrial border is shown in Fig. 3. Twenty additional specimens of fresh-frozen endometrial carcinoma were examined for mutations in Kims codon 12. Two of these tumors, a moderately differentiated endometrioid adenocarcinoma and a
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poorly differentiated endometrioid adenocarcinoma, exhibited activating point mutations: One contained the same GGT--+GTT transversion described above, and the other contained a GGT--+TGT (Gly--+Cys) transversion in the first nucleotide of this codon (Fig. 4). These mutations were also heterozygous in nature; the tumors contained both a mutant and wild-type allele. The mutations and histopathologic data for the 30 tumors described in this report are summarized in Table I in which no correlation between Ki-ras mutation and any of the clinical, pathologic, or histologic criteria was reported.
Comment These data indicate that somatic point mutation of a ras oncogene occurs at a significant but relatively low (10%) frequency in endometrial carcinomas in the United States. Two previous studies 14 . 16 showed that ras mutation occurs in endometrial carcinomas from Japan at about a threefold higher frequency than that observed here. These data suggest that ras mutation may be a more frequent occurrence in the type II tumors more prevalent in Japan than in the type I estrogenrelated tumors that are more prevalent in the United States. The higher incidence of ras mutation that we had observed in endometrial carcinoma celilines 'o may reflect spontaneous mutation and! or clonal selection of mutant ras genes during establishment and culture of these cells. All the existing data, however, implicate Kiras codon 12 as the most frequently altered ras site in endometrial carcinoma. The ras family of protooncogenes encodes a unique form of GTPase molecule that appears to playa central role in cell proliferation as a result of its regulation of signal transduction. Point mutations in codons 12, 13, or 61 appear to inactivate this GTPase activity, which leaves the mutant protein in a constitutively activated state. 2l A large number and variety of human tumors contain point-mutated ras oncogenes,l3 which suggest a generalized role for ras in the proliferation of diverse tissues. Other solid tumors that contain mutations of Ki-ras at significant frequencies include carcinomas of the lung (20% to 30%), pancreas (70% to 90%), thyroid (20% to 60%), and colon (40% to 50%).13 Endometrial carcinomas, like those of the lung, pancreas, and colon, exhibit a predominance of Ki-ras codon 12 mutations; thus a similar role for Ki-ras in the regulation of cell proliferation in these tissues or perhaps similar molecular pathways of neoplastic development or progression in these tissues. Mutated ras genes have been strongly implicated in experimental chemical carcinogenesis 22 ; this suggests that ras mutations in endometrial tumors may arise from chemical (including hormonal) mutagenesis. Al-
ras Mutations in endometrial carcinoma
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ternatively, because cyclical cell proliferation is a prominent and somewhat unique feature of the mammalian endometrium, spontaneous DNA aberrations would be subject to a greater probability of conversion to permanent mutations in this tissue. Estrogen may thus act indirectly to stimulate tumorigenesis, by increasing the proliferative index of endometrial tissue. 23 The Ki-ras mutations reported in this paper whether they were spontaneous or chemically induced, did not appear to be associated with any histopathologic or clinical criteria. This suggests a sporadic induction and random distribution in endometrial carcinoma. To extend these findings it will be necessary to examine a larger number of tumors, especially those of the more uncommon histologic (type II) variants. In particular, it would be instructive to compare a larger number of tumor samples from the United States with those from Japan, where estrogen-related risk factors and the incidence of endometrial carcinoma are much lower than those in the United States.' 17 That comparison might indicate a dramatically different ratio of type II to type I tumors. In addition, it would be of interest to analyze premalignant hyperplastic lesions of the endometrium for ras mutations to determine if that mutation is an early event in the development of some endometrial cancers as it is in colon carcinoma. REFERENCES 1. Boyd JA, Barrett Je. Genetic and cellular basis of multistep carcinogenesis. Pharmacol Ther 1990;46:469-86. 2. Deligdisch L, Holinka CF. Endometrial carcinoma: two diseases? Cancer Detect Prev 1987;10:237-46. 3. Kurman RJ, Norris HJ. Endometrial carcinoma. In: Kurman RJ, ed. Blaustein's pathology of the female genital t,act. 3rd ed. New York: Springer-Verlag, 1987. 4. Boltenberg A, Furgyik S. Kullander S. Familial cancer aggregation in cases of adenocarcinoma corporis uteri. Acta Obstet Gynecol Scand 1990;69:249-58. 5. Lynch HT, Lanspa S, Smyrk T, Boman B, Watson P, Lynch J. Hereditary nonpolyposis colorectal cancer (Lynch syndromes I & II). Genetics, pathology, natural history, and cancer contro\. Part 1. Cancer Genet Cytogenet 1991;53: 143-60. 6. Vasen HFA. Offerhaus GJA, den Hartog Jager FCA, et a\. The tumor spectrum in hereditary non-polyposis colorectal cancer: a study of 24 kindreds in the Netherlands. Int J Cancer 1990:46:31-4. 7. Schwartz Z, Ohel G, Birkenfeld A, Anteby SO, Schenker JG. Second primary malignancy in endometrial carcinoma patients. Gynecol Oncol 1985;22:40-5. 8. Berchuck A, Rodriquez G, Kinney RB, Soper JT, Dodge RK, Clarke-Pearson DL, Bast RC JR. Overexpression of HER-21neu in endometrial cancer is associated with advanced stage disease. AMJ OBSTET GYNECOL 1991; 164: 1521. 9. Brumm C, Riviere A, Wilckens C, Loning T. Immunohistochemical investigation and Northern blot analysis of c-erbB-2 expression in normal, premalignant and malignant tissues of the corpus and cervix uteri. Virchows Arch [A] 1990;417:477-84. 10. Boyd J, Risinger J1. Analysis of oncogene alterations in human endometrial carcinoma: prevalence of ras mutations. Mol Carcinog 1991;4:189-95.
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11. Kacinski BM, Chambers SK, Stanley ER, et al. The cytokine CSF-I (M-CSF) expressed by endometrial carcinomas in vivo and in vitm, may also be a circulating tumor marker of neoplastic disease activity in endometrial carcinoma patients. Int J Radiat Oncol BioI Phys 1990; 19:619-26. 12. Baiocchi G, Kavanagh Jj, Talpaz M, Wharton JT, Gutterman JU, Kurzrock R. Expression of the macrophage colony-stimulating factor and its receptor in gynecologic malignancies. Cancer 1991 ;67:990-6. 13. Bos JL. Ras oncogenes in human cancer: a review. Cancer Res 1989;49:4682-9. 14. Enomoto T, Inoue M, Perantoni AO, Terakawa N, Tanizawa 0, Rice JM. K-ras activation in neoplasms of the human female reproductive tract. Cancer Res 1990;50:6139-45. 15. Lester DR, Cauchi MN. Point mutations at codon 12 of c-K-ras in human endometrial carcinomas. Cancer Lett 1990;51 :7-10. 16. Enomoto T, Inoue M, Pera11l0ni AO, Buzard GS, Miki H, Tanizawa 0, Rice JM. K-ras activation in premalignant and malignant epithelial lesions of the human uterus. Cancer Res 1991;51:5308-14.
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17. Parazzini F, La Vecchia C, Bocciolone L, Franceschi S. The epidemiology of endometrial cancer. Gynecol Oncol 1991;41:1-16. 18. Goelz SE, Hamilton SR, Vogel stein B. Purification of DNA from formaldehyde fixed and paraffin embedded human tissue. Biochem Biophys Res Commun 1985; 130: 118-26. 19. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning, a laboratory manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 1989. 20. de Vries MV, Bogaard ME, van den Elst H, van Boom JH, van der Eb AJ, BosJL. A dot-blot screening procedure for mutated ras oncogenes using synthetic oligodeoxynucleotides. Gene 1986;50:313-20. 21. Barbacid M. Ras genes. Annu Rev Biochem 1987;56:779827. 22. Sukumar S. Ras oncogenes in chemical carcinogenesis. Curr Top Microbial Iml1lunol 1989; 148:93-114. 23. Henderson BE, Ross R, Bernstein L. Estrogens as a cause of human cancer: the Richard and Hinda Rosenthal Foundation Award Lecture. Cancer Res 1988;48:246-53.
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