GYNECOLOGIC
ONCOLOGY
45, 299-302 (1992)
Ha-ras Polymorphisms in Epithelial Ovarian Cancer KATHY O’BRIANT, M.S.,* NICK CHRYSSON, M.D.,* VERDA HUNTER, M.D.,*? FRED TYSON,PH.D.,* MARY TANNER, M.B.A.,* LEE DALY, PA-C,* STEPHENL. GEORGE, PH.D.,$ ANDREW BERCHUCK, M.D.,? JOHN SOPER,M.D.,? WESLEY FOWLER, M.D.,§ DANIEL CLARKE-PEARSON,M.D.,? AND ROBERT C. BAST,JR., M.D.*,(( Departments of *Medicine, TObstetrics and Gynecology, $Community and Family Medicine, and lIMicrobiology and Immunology, Duke University Medical Center and The Duke Comprehensive Cancer Center, Durham, North Carolina 27710; and aDepartment of Obstetrics and Gynecology, University of North Carolina, Chapel Hill, North Carolina 27514
ReceivedSeptember 18. 1991 Unusual restriction fragment length polymorphisms (RFLPs) of the Ha-ras locus have been found in DNA from leukocytes and tumor tissue of cancer patients. To determine whether rare alleles would be observed frequently in patients with ovarian cancer, Ha-ras RFLPs were studied in DNA from 42 different ovarian epithelial tumors and from the peripheral blood leukocytes of 76 normal individuals. Four common, sevenintermediate, and seven rare alleles were detected overall. Similar fractions of rare alleles were found in DNA from ovarian cancers and from the peripheral blood of normai individuals. Thus, the frequency of unusual Ha-rus RFLPs did not distinguish patients with ovarian cancers from apparently healthy individuals. 0 1992 Academic Press, Inc.
INTRODUCTION
Epithelial ovarian cancer is the most common cause of gynecologic cancer deaths. Two-thirds of all casesare not diagnosed until the cancer is in an advanced stage. Aside from advanced age and nulliparity, few epidemiologic factors are associated with ovarian cancer. If multiple firstdegree relatives are affected, an individual has an increased cancer risk. Most cases appear, however, to be sporadic. Given the relatively low prevelance of ovarian cancer, potential screening strategies require a very high degree of sensitivity and specificity to achieve an adequate positive predictive value. Genetic markers might facilitate identification of a subset of women who would be at increased risk for ovarian cancer. Although Ha-ras and Ki-rus genes have been amplified or mutated in only 5-10% of ovarian cancer cases, Haras polymorphisms in the germ line might provide a useful marker for cancer risk. The Ha-rus gene is associated with repetitive 28-bp DNA sequences outside the coding region [15]. These repetitive sequences are called tandem
repeats. The number of tandem repeats 3’ to the coding region varies between individuals. Heterogeneity in the number of terminal repeats is reflected in fragment length polymorphisms revealed by Southern blotting after digestion of DNA with restriction endonucleases. Unusual restriction fragment length polymorphisms (RFLPs) of Harm appear to be increased in the germ line and tumor tissue from patients with a variety of malignancies including acute myelogenous leukemia and carcinomas of the lung, bladder, and breast [15-201. Thus, the presence of rare Ha-ras alleles might provide a useful marker for increased cancer risk and could be involved in the pathogenesis of at least some of these malignancies. Little is known about the importance of Ha-rus RFLPs in the germ line of ovarian cancer patients or the potential utility of RFLPs as markers for assessingovarian cancer risk. METHODS Subjects. Tumor tissue was obtained from 42 women undergoing operations for primary epithelial ovarian cancer at Duke University Medical Center and at the University of North Carolina Hospital between 1985 and 1989. Samples were promptly frozen and stored at - 80°C until DNA was extracted from them. Peripheral blood was obtained from a group of apparently healthy control women who had no history of ovarian cancer. Healthy donors were chosen by age and race to match the women from whom tumor tissue was obtained. The median age for cancer patients was 54 years and that for normal donors 50 years. Black patients constituted 25% of cancer patients and 23% of controls. The remainder of patients in each group were Caucasians. Family histories could not be obtained from all subjects. DNA extraction. DNA from frozen tissue samples was
299 C@90-8258192 $4.00 1992 by Academic Press, Inc. of reproduction in any form reserved.
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300
O’BRIANT
TABLE 1 Analysis of Ha-ras Alleles in DNA from Ovarian Cancers and from Healthy Donors Allele
No. in tumor DNA
%
No. in normal DNA
%
kb
al a2 a4 a3 al.1” al.2 aO.1” a1.25b al.3 a2.1b a2.3’ a3.2’ a4.1” al.4 a2.2b a2.01b a2.02’ 0.5
37 15 10 7 3 3 2 2 1 1 1 1 1 0 0 0 0 0
44.05 17.86 11.90 8.33 3.57 3.57 2.38 2.38 1.19 1.19 1.19 1.19 1.19 0 0 0 0 0
69 24 17 5 5 3 5 0 3 2 0 3 2 2 2 2 1 1
45.39 15.79 11.18 7.24 3.29 1.97 3.29 0 1.97 1.32 0 1.97 1.32 1.32 1.32 1.32 0.66 0.66
1.00 1.50 2.16 2.56 1.06 1.11 0.98 1.18 1.23 1.71 1.88 2.28 2.61 1.45 1.82 1.56 1.65 2.80
18 alleles
84
152
’ Krontiris intermediate allele. b Krontiris rare allele.
obtained by using guanidium isothiocyanate and cesium chloride gradients as described by Tyson et al. [4]. To extract DNA from peripheral blood samples, the leukocytes were first separated on a Ficoll-diatrizoate gradient (Organon Teknika, Durham, NC). Procedures detailed by Sambrook et al. [21] were used to lyse the cells and obtain DNA. DNA digest, gel electrophoresis, and Southern blotting. Five micrograms of DNA was digested with 4 U/pg
HpaII DNA restriction endonuclease at 37°C overnight. Digests were then incubated with 12 U/,ug MspI DNA restriction endonuclease at 37°C overnight. Digested DNA was separated electrophoretically at 40 TABLE 2 Distribution of Common, Intermediate, and Rare Ha-ras Alleles in DNA from Ovarian Cancers and Healthy Donors Alleles Common
Intermediate
Rare
Total
Ovarian cancers Healthy donors
69 121
10 21
5 10
84 152
Total
190
31
15
236
x2 = 0.23, 2 df, P = 0.89. Uncommon (rare plus intermediate) alleles were found in 18% of cancers and 20% of healthy donors. The 95% confidence limits for the 2% difference ranged from - 12 to + 8%. Note.
ET AL.
V for 20-24 hr on a 1% agarose gel as described by Sambrook et al. Southern transfers were performed overnight to a Gene Screen Plus hybridization transfer membrane (DuPont-NEN Research Products, Boston, MA). Probe Labeling and hybridization. A multiprime DNA labeling kit (Amersham Corp., Arlington Heights, IL) was used to label 25 ng of Ha-rus plasmid DNA with [32P]dCTP according to the manufacturer’s instructions. Protocols found in Sambrook et al. were used for membrane prehybridization and hybridization. The blot was hybridized for at least 6 hr in a 42°C waterbath. Following hybridization, washing was performed according to the manufacturer’s recommendation (DuPont-NEN Research Products). DNA from donors of known allotype were obtained from Dr. T. Krontiris for use as standards for alleles al, a2, a3, and a4. Statistical methods. A standard x2 test was used to test the difference in allelic frequencies between the ovarian cancer and the normal population. STPLAN, a statistical software package, was used to calculate the power of the statistical tests used. Confidence intervals (95%) were calculated for the difference in the percentage of uncommon (rare and intermediate) alleles. The power to detect a difference of 0.15 in the percentage of uncommon alleles is approximately 0.81 assuming that the overall percentage is approximately 20% using a two-sided test with P = 0.05. The power to detect a difference of 0.25 in the percentage of patients with at least one uncommon allele is also 0.81 assuming that the overall percentage is approximately 33% using a two-sided test with P = 0.05. RESULTS Our present study examined RFLPs in the tumor DNA from 42 ovarian cancer patients and in the peripheral blood leukocyte DNA from 76 matched controls (Table 1). Southern blot analysis revealed 69 common (82.1%), 10 intermediate (11.9%) and 5 rare (6.0%) alleles in the cancer population (Table 2). In DNA from a race- and age-matched control group, 121 common (79.6%), 21 intermediate (13.8%), and 10 rare alleles (6.6%) were found (Table 2). Several of these alleles may be seen in Fig. 1. A x2 test with two degrees of freedom indicated that no significant difference in allelic frequency was observed between DNA from ovarian cancer patients and healthy donors (x2 = 0.23, P = 0.89). On a per subject basis, there were 11 ovarian cancer patients (26%) and 28 controls (37%) with at least one uncommon allele. Again, the x2 test indicated no significant difference (x2 = 1.38, 1 df, P = 0.24). DISCUSSION Ha-rus restriction fragments could be divided into common, intermediate, and rare alleles depending upon their
Ha-ras POLYMORPHISMS
FIG. 1. A representative Southern transfer probed with Ha-rus after digestion with HpaII and MspI. Positions for common alleles are indicated by al, a2, a3, and a4. DNA samples from different normal donors are indicated in lanes marked NB and FS, whereas DNA samples from different ovarian cancers are in lanes marked ICOV. Intermediate alleles were found in NBll (a1.2), FS 71 (aO.l), FS 84 (al.l), FS 96 (a4.1), FS 105 (al.l), and ICOV 75 (aO.1). Rare alleles were found in FS 71 (a2.02) and FS 100 (a2.01).
frequency in the general population. RFLPs at the Harus locus arise from variations in the length of the variable tandem repeats (VTR) 3’ of the region encoding Ha-ras [17]. Krontiris et al. [22] have suggested that the VTR may enhance the transforming potential of the Ha-r-as gene. Since Ha-ras genes are inherited in a Mendelian fashion, studies to determine whether a correlation exists between particular alleles and cancer risk have been performed in cancer families [19,23,24]. To date, however, no linkage has been detected between the Ha-ras genotype and occurrence of cancer in the families studied. Krontiris et al. [ 15,221were among the first investigators to suggest a link between RFLPs at the Ha-ras locus and susceptibility to certain cancers. After digestion of genomic DNA from 800 different donors with MspI and HpaII or with BamHI restriction endonucleases, they identified 32 distinct Ha-ras RFLPs [22]. Rare Ha-ras alleles occurred almost exclusively in cancer patients (P < O.OOl), representing a wide spectrum of cancer types. Several investigators have studied Ha-rus alleles in specific cancer types and found an association between rare Haras alleles and cancer development [16,18,19,20,25]. Other investigators, however, have not found an association between rare Ha-ras alleles and the development of cancer [16,26-321. Few studies have examined a possible link between rare Ha-rus alleles and ovarian cancer. The present study compared DNA from 42 epithelial ovarian tumors to peripheral blood mononuclear cell DNA from unaffected age-
IN OVARIAN
301
CANCER
and race-matched individuals to determine whether rare Ha-rus alleles would be found more frequently in ovarian tumors. No specific alleles were discovered. Interestingly, the normal population carried as many rare alleles as did the ovarian cancer population. These results may reflect peculiarities in the population studied within North Carolina. Other investigators have found variations in allelic distribution in different populations. For example, Honda et al. [32] found that a group of Japanese subjects had only 6 Ha-ras RFLP compared to 32 alleles in a Caucasian population [21]. Krontiris et al. [15] found two rare alleles in ovarian tumor DNA. Core11and Zoll [30] examined DNA from 23 patients with ovarian cancer. Their results showed a comparable distribution of Ha-ras alleles between ovarian tumor DNA and DNA from normal individuals. Our study extends these previous observations to a larger population of ovarian cancers. In conclusion, our study compared tumor DNA to leukocyte DNA from apparently healthy individuals. No statistically significant difference in the occurrence of rare Ha-r-us alleles was found between the two populations. The presence of a rare Ha-ras allele in an individual’s DNA does not indicate a predisposition to the development of ovarian cancer. ACKNOWLEDGMENTS This project was supported in part by Grant CA 39930 from the National Cancer Institute, Department of Health and Human Services. It is a pleasure to acknowledge the outstanding secretarial assistance of Jana Carey and Karen Cash. We are also grateful to Dr. Ted Krontiris for useful discussions, aiding in the establishment of the assay, and providing standard DNA samples.
REFERENCES 1. Tabin, C. J., Bradley, S. M., Bargmann, C. I., Weinberg, R. A., Papageorge, A. G., Scolnick, E. M., Dhar, R., Lowy, D. R., and Chang, E. H. Mechanism of activation of a human oncogene, Nuture 300, 143-149 (1982). 2. Bishop, J. M. Molecular themes in oncogenesis, Cell 64, 235-248 (1991). 3. Marshall, C. J. Tumor suppressor genes, Cell 64, 313-326 (1991). 4. Tyson, F. L., Boyer, C. M., Kaufman, R., O’Briant, K., Cram, G., Crews, J. R., Soper, J. T., Daly, L., Fowler, W. C., Jr., Haskill, J. S., and Bast, R. C., Jr. Overexpression and amplification of the HER-2/neu (c-erbB-2) proto-oncogene in epithelial ovarian tumors and cell lines, Am. J. Obstet. Gynecol. 165, MO-646 (1991). 5. Berchuck, A., Kamel, A., Whitaker, R., Kerns, B., Oh, G., Kinney, R., Soper, J. T., Dodge, R., Clarke-Pearson, D. L., Marks, P., McKenzie, S., Yin, S., and Bast, R. C., Jr. Overexpression of HER-2/neu is associated with poor survival in advanced epithelial ovarian cancer, Cancer Res. 50, 4087-4091 (1990). 6. Marks, J. R., Davidoff, A. M., Kerns, B. J., Humphrey, P. A., Pence, J. C., Dodge, R. K., Clarke-Pearson, D. L., Iglehart, J. D., Bast, R. C., Jr., and Berchuck, A. Overexpression and mutation
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of p.53 in epithelial ovarian cancer, Cancer Res. 51, 2979-2984 (1991). 7. Fieg, L. A., Bast, R. C., Jr., Knapp, R. C., and Cooper, G. M. Somatic activation of rask gene in a human ovarian carcinoma, Science 223 698-701 (1984). 8. van’t Veer, L. J., Hermens, R., Van den Berg-Bakker, L. A., Cheng, N. C., Fleuren, G. J., Bos, J. L., Cleton, F. J., and Schrier, P. I. rus oncogene activation in human ovarian carcinoma, Oncogene 2, 157-165 (1988). 9. Zhou, D. J., Gonzalez-Cadavid, N., Ahuja, H., Battifora, H., Moore, G. E., and Cline, M. J. A unique pattern of protooncogene abnormalities in ovarian adenocarcinomas, Cancer 62, 1573-1576 (1988). 10. Boltz, E. M., Kefford, R. F., Leary, J. A., Houghton, C. R., and Friedlander, M. L. Amplification of c-rus-ki oncogene in human ovarian tumours, ht. J. Cancer 43, 428-430 (1989). 11 Yokota, J., Tsunetsugu-Yokota, Y., Battifora, H., Le Fevre, C., and Cline, M. J. Alterations of myc, myb and ras protooncogenes in cancers are frequent and show clinical correlation, Science 231, 261-265 (1986). 12. Filmus, J., Trent, J. M., Pullano, R., and Buick, R. N. A cell line from a human ovarian carcinoma with amplification of the K-ras gene, Cancer Rex 46, 5179-5182 (1986). 13. Barbacid, M. ras genes, Annu. Rev. B&hem. 56,779-827 (1987). 14. Capon, D. J., Chen, E. Y., Levinson, A. D., Seeburg, P. H., and Goeddel, D. V. Complete nucleotide sequences of the T24 human bladder carcinoma oncogene and its normal homologue, Nature 302, 33-37 (1983). 15. Krontiris, T. G., DiMartino, N. A., Colb, M., and Parkinson, D. R. Unique allelic restriction fragments of the human Ha-ras locus in leukocyte and tumour DNA’s of cancer patients, Nature 313, 369-374 (1985). 16. Carter, G., Worwood, M., and Jacobs, A. The Ha-ras polymorphism in myelodysphasia and acute myeloid leukaemia, Leuk. Res. 12, 38.5-391 (1988). 17. Heighway, J., Thatcher, N., Cerny, T., and Hasleton, P. S. Genetic predisposition to human lung cancer, Br. J. Cancer 53, 453-457 (1986). 18. Sugimura, H., Caporaso, N. E., Modali, R. V., Hoover, R. N., Resau, J. H., Trump, B. F., Longergan, J. A., Krontiris, T. G., Mann, D. L., Weston, A., and Harris, C. C. Association of rare alleles of the Harvey ras protooncogene locus with lung cancer, Cancer Res. 50, 1857-1862 (1990). 19. Hayward, N. K., Keegan, R., Nancarrow, D. J., Little, M. H., Smith, P. J., Gardiner, R. A., Seymour, G. J., Kidson, C., and Lavin, M. F. c-Ha-ras-1 alleles in bladder cancer, Wilms’ tumour, and malignant melanoma, Hum. Genet. 78, 115-120 (1988).
ET AL. 20. Lidereau, R., Escot, C., Theillet, C., Champeme, M. H., Brunet, M., Gest, J., and Callahan, R. High frequency of rare alleles of the human c-Ha-ras-1 proto-oncogene in breast cancer patients, J. Natl. Cancer Inst. 77, 697-701 (1986). 21. Sambrook, J., Fritsch, E. F., and Maniatis, T. Molecular cloning: A laboratory manual. Cold Spring Harbor Press, New York 2nd ed., (1989). 22. Krontiris, T. G., DiMartino, N. A., Mitcheson, H. D., Lonergan, J. A., Begg, C., and Parkinson, D. R. Human hypervariable sequences in risk assessment: Rare Ha-ras alleles in cancer patients, Environ. Health Perspect. 76, 147-153 (1987). 23. Murayama, M., Mita, T., Suenaga, Y., and Nishio, K. The BAMHI restriction fragment length polymorphism detected in the c-Ha-rus locus of human skin tumors, J. Dermatol. 15, 141-148 (1988). 24. Hall, J. M., Huey, B., Morrow, J., Newman, B., Lee, M., Jones, E., Carter, C., Buehring, G. C., and King, M. C. Rare HRAS alleles and susceptibility to human breast cancer, Genomics 6, 188191 (1990). 25. Diedrich, U., Eckermann, O., and Schmidtke, J. Rare Ha-ras and c-mos alleles in patients with intracranial tumors, Neurology 38, 587-589 (1988). 26. Ceccherini-Nelli, L., De Re, V., Viel, A., Molaro, G., Zilli, L., Clemente, C., and Boiocchi, M. Ha-ras-1 restriction fragment length polymorphism and susceptibility to colon adenocarcinoma, Br. J. Cancer 56, l-5 (1987). 27. Wylhe, F. S., Wynford-Thomas, V., Lemoine, N. R., Williams, G. T., Williams, E. D., and Wynford-Thomas, D. Ha-ras restriction fragment length polymorphisms in colorectal cancer, Br. J. Cancer 57, 135-138 (1988). 28. Thein, S. L., Oscier, D. G., Flint, J., and Wainscoat, J. S. Ha-ras hypervariable alleles in myelodysplasia, Nature 321, 84-85 (1986). 29. Barkardottir, R. B., Johansson, 0. T., Arason, A., Gudnason, V., and Egilsson, V. Polymorphism of the c-Ha-ras-I protooncogene in sporadic and familial breast cancer, In!. J. Cancer 44, 251-255 (1989). 30. Corell, B., and Zoll, B. Comparison between the allelic frequency distribution of the Ha-rus-I locus in normal individuals and patients with lymphoma, breast, and ovarian cancer, Hum. Genet. 79,255259 (1988). 31. Ishikawa, J., Maeda, S., Takahashi, R., Kamidono, S., and Sugiyama, T. Lack of correlation between rare Ha-rus alleles and urothelial cancer in Japan, Int. J. Cancer 40, 474-478 (1987). 32. Honda, K., Ishizaki, K., Ikenaga, M., Toguchida, J., Inamoto, T., Tanaka, K., and Ozawa, K. Increased frequency of specific alleles of the c-Ha-ras gene in Japanese cancer patients, Hum. Genet. 79, 297-300 (1988).
ONCOLOGY
45, 299-302 (1992)
Ha-ras Polymorphisms in Epithelial Ovarian Cancer KATHY O’BRIANT, M.S.,* NICK CHRYSSON, M.D.,* VERDA HUNTER, M.D.,*? FRED TYSON,PH.D.,* MARY TANNER, M.B.A.,* LEE DALY, PA-C,* STEPHENL. GEORGE, PH.D.,$ ANDREW BERCHUCK, M.D.,? JOHN SOPER,M.D.,? WESLEY FOWLER, M.D.,§ DANIEL CLARKE-PEARSON,M.D.,? AND ROBERT C. BAST,JR., M.D.*,(( Departments of *Medicine, TObstetrics and Gynecology, $Community and Family Medicine, and lIMicrobiology and Immunology, Duke University Medical Center and The Duke Comprehensive Cancer Center, Durham, North Carolina 27710; and aDepartment of Obstetrics and Gynecology, University of North Carolina, Chapel Hill, North Carolina 27514
ReceivedSeptember 18. 1991 Unusual restriction fragment length polymorphisms (RFLPs) of the Ha-ras locus have been found in DNA from leukocytes and tumor tissue of cancer patients. To determine whether rare alleles would be observed frequently in patients with ovarian cancer, Ha-ras RFLPs were studied in DNA from 42 different ovarian epithelial tumors and from the peripheral blood leukocytes of 76 normal individuals. Four common, sevenintermediate, and seven rare alleles were detected overall. Similar fractions of rare alleles were found in DNA from ovarian cancers and from the peripheral blood of normai individuals. Thus, the frequency of unusual Ha-rus RFLPs did not distinguish patients with ovarian cancers from apparently healthy individuals. 0 1992 Academic Press, Inc.
INTRODUCTION
Epithelial ovarian cancer is the most common cause of gynecologic cancer deaths. Two-thirds of all casesare not diagnosed until the cancer is in an advanced stage. Aside from advanced age and nulliparity, few epidemiologic factors are associated with ovarian cancer. If multiple firstdegree relatives are affected, an individual has an increased cancer risk. Most cases appear, however, to be sporadic. Given the relatively low prevelance of ovarian cancer, potential screening strategies require a very high degree of sensitivity and specificity to achieve an adequate positive predictive value. Genetic markers might facilitate identification of a subset of women who would be at increased risk for ovarian cancer. Although Ha-ras and Ki-rus genes have been amplified or mutated in only 5-10% of ovarian cancer cases, Haras polymorphisms in the germ line might provide a useful marker for cancer risk. The Ha-rus gene is associated with repetitive 28-bp DNA sequences outside the coding region [15]. These repetitive sequences are called tandem
repeats. The number of tandem repeats 3’ to the coding region varies between individuals. Heterogeneity in the number of terminal repeats is reflected in fragment length polymorphisms revealed by Southern blotting after digestion of DNA with restriction endonucleases. Unusual restriction fragment length polymorphisms (RFLPs) of Harm appear to be increased in the germ line and tumor tissue from patients with a variety of malignancies including acute myelogenous leukemia and carcinomas of the lung, bladder, and breast [15-201. Thus, the presence of rare Ha-ras alleles might provide a useful marker for increased cancer risk and could be involved in the pathogenesis of at least some of these malignancies. Little is known about the importance of Ha-rus RFLPs in the germ line of ovarian cancer patients or the potential utility of RFLPs as markers for assessingovarian cancer risk. METHODS Subjects. Tumor tissue was obtained from 42 women undergoing operations for primary epithelial ovarian cancer at Duke University Medical Center and at the University of North Carolina Hospital between 1985 and 1989. Samples were promptly frozen and stored at - 80°C until DNA was extracted from them. Peripheral blood was obtained from a group of apparently healthy control women who had no history of ovarian cancer. Healthy donors were chosen by age and race to match the women from whom tumor tissue was obtained. The median age for cancer patients was 54 years and that for normal donors 50 years. Black patients constituted 25% of cancer patients and 23% of controls. The remainder of patients in each group were Caucasians. Family histories could not be obtained from all subjects. DNA extraction. DNA from frozen tissue samples was
299 C@90-8258192 $4.00 1992 by Academic Press, Inc. of reproduction in any form reserved.
Copyright 0 All
rights
300
O’BRIANT
TABLE 1 Analysis of Ha-ras Alleles in DNA from Ovarian Cancers and from Healthy Donors Allele
No. in tumor DNA
%
No. in normal DNA
%
kb
al a2 a4 a3 al.1” al.2 aO.1” a1.25b al.3 a2.1b a2.3’ a3.2’ a4.1” al.4 a2.2b a2.01b a2.02’ 0.5
37 15 10 7 3 3 2 2 1 1 1 1 1 0 0 0 0 0
44.05 17.86 11.90 8.33 3.57 3.57 2.38 2.38 1.19 1.19 1.19 1.19 1.19 0 0 0 0 0
69 24 17 5 5 3 5 0 3 2 0 3 2 2 2 2 1 1
45.39 15.79 11.18 7.24 3.29 1.97 3.29 0 1.97 1.32 0 1.97 1.32 1.32 1.32 1.32 0.66 0.66
1.00 1.50 2.16 2.56 1.06 1.11 0.98 1.18 1.23 1.71 1.88 2.28 2.61 1.45 1.82 1.56 1.65 2.80
18 alleles
84
152
’ Krontiris intermediate allele. b Krontiris rare allele.
obtained by using guanidium isothiocyanate and cesium chloride gradients as described by Tyson et al. [4]. To extract DNA from peripheral blood samples, the leukocytes were first separated on a Ficoll-diatrizoate gradient (Organon Teknika, Durham, NC). Procedures detailed by Sambrook et al. [21] were used to lyse the cells and obtain DNA. DNA digest, gel electrophoresis, and Southern blotting. Five micrograms of DNA was digested with 4 U/pg
HpaII DNA restriction endonuclease at 37°C overnight. Digests were then incubated with 12 U/,ug MspI DNA restriction endonuclease at 37°C overnight. Digested DNA was separated electrophoretically at 40 TABLE 2 Distribution of Common, Intermediate, and Rare Ha-ras Alleles in DNA from Ovarian Cancers and Healthy Donors Alleles Common
Intermediate
Rare
Total
Ovarian cancers Healthy donors
69 121
10 21
5 10
84 152
Total
190
31
15
236
x2 = 0.23, 2 df, P = 0.89. Uncommon (rare plus intermediate) alleles were found in 18% of cancers and 20% of healthy donors. The 95% confidence limits for the 2% difference ranged from - 12 to + 8%. Note.
ET AL.
V for 20-24 hr on a 1% agarose gel as described by Sambrook et al. Southern transfers were performed overnight to a Gene Screen Plus hybridization transfer membrane (DuPont-NEN Research Products, Boston, MA). Probe Labeling and hybridization. A multiprime DNA labeling kit (Amersham Corp., Arlington Heights, IL) was used to label 25 ng of Ha-rus plasmid DNA with [32P]dCTP according to the manufacturer’s instructions. Protocols found in Sambrook et al. were used for membrane prehybridization and hybridization. The blot was hybridized for at least 6 hr in a 42°C waterbath. Following hybridization, washing was performed according to the manufacturer’s recommendation (DuPont-NEN Research Products). DNA from donors of known allotype were obtained from Dr. T. Krontiris for use as standards for alleles al, a2, a3, and a4. Statistical methods. A standard x2 test was used to test the difference in allelic frequencies between the ovarian cancer and the normal population. STPLAN, a statistical software package, was used to calculate the power of the statistical tests used. Confidence intervals (95%) were calculated for the difference in the percentage of uncommon (rare and intermediate) alleles. The power to detect a difference of 0.15 in the percentage of uncommon alleles is approximately 0.81 assuming that the overall percentage is approximately 20% using a two-sided test with P = 0.05. The power to detect a difference of 0.25 in the percentage of patients with at least one uncommon allele is also 0.81 assuming that the overall percentage is approximately 33% using a two-sided test with P = 0.05. RESULTS Our present study examined RFLPs in the tumor DNA from 42 ovarian cancer patients and in the peripheral blood leukocyte DNA from 76 matched controls (Table 1). Southern blot analysis revealed 69 common (82.1%), 10 intermediate (11.9%) and 5 rare (6.0%) alleles in the cancer population (Table 2). In DNA from a race- and age-matched control group, 121 common (79.6%), 21 intermediate (13.8%), and 10 rare alleles (6.6%) were found (Table 2). Several of these alleles may be seen in Fig. 1. A x2 test with two degrees of freedom indicated that no significant difference in allelic frequency was observed between DNA from ovarian cancer patients and healthy donors (x2 = 0.23, P = 0.89). On a per subject basis, there were 11 ovarian cancer patients (26%) and 28 controls (37%) with at least one uncommon allele. Again, the x2 test indicated no significant difference (x2 = 1.38, 1 df, P = 0.24). DISCUSSION Ha-rus restriction fragments could be divided into common, intermediate, and rare alleles depending upon their
Ha-ras POLYMORPHISMS
FIG. 1. A representative Southern transfer probed with Ha-rus after digestion with HpaII and MspI. Positions for common alleles are indicated by al, a2, a3, and a4. DNA samples from different normal donors are indicated in lanes marked NB and FS, whereas DNA samples from different ovarian cancers are in lanes marked ICOV. Intermediate alleles were found in NBll (a1.2), FS 71 (aO.l), FS 84 (al.l), FS 96 (a4.1), FS 105 (al.l), and ICOV 75 (aO.1). Rare alleles were found in FS 71 (a2.02) and FS 100 (a2.01).
frequency in the general population. RFLPs at the Harus locus arise from variations in the length of the variable tandem repeats (VTR) 3’ of the region encoding Ha-ras [17]. Krontiris et al. [22] have suggested that the VTR may enhance the transforming potential of the Ha-r-as gene. Since Ha-ras genes are inherited in a Mendelian fashion, studies to determine whether a correlation exists between particular alleles and cancer risk have been performed in cancer families [19,23,24]. To date, however, no linkage has been detected between the Ha-ras genotype and occurrence of cancer in the families studied. Krontiris et al. [ 15,221were among the first investigators to suggest a link between RFLPs at the Ha-ras locus and susceptibility to certain cancers. After digestion of genomic DNA from 800 different donors with MspI and HpaII or with BamHI restriction endonucleases, they identified 32 distinct Ha-ras RFLPs [22]. Rare Ha-ras alleles occurred almost exclusively in cancer patients (P < O.OOl), representing a wide spectrum of cancer types. Several investigators have studied Ha-rus alleles in specific cancer types and found an association between rare Haras alleles and cancer development [16,18,19,20,25]. Other investigators, however, have not found an association between rare Ha-ras alleles and the development of cancer [16,26-321. Few studies have examined a possible link between rare Ha-rus alleles and ovarian cancer. The present study compared DNA from 42 epithelial ovarian tumors to peripheral blood mononuclear cell DNA from unaffected age-
IN OVARIAN
301
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and race-matched individuals to determine whether rare Ha-rus alleles would be found more frequently in ovarian tumors. No specific alleles were discovered. Interestingly, the normal population carried as many rare alleles as did the ovarian cancer population. These results may reflect peculiarities in the population studied within North Carolina. Other investigators have found variations in allelic distribution in different populations. For example, Honda et al. [32] found that a group of Japanese subjects had only 6 Ha-ras RFLP compared to 32 alleles in a Caucasian population [21]. Krontiris et al. [15] found two rare alleles in ovarian tumor DNA. Core11and Zoll [30] examined DNA from 23 patients with ovarian cancer. Their results showed a comparable distribution of Ha-ras alleles between ovarian tumor DNA and DNA from normal individuals. Our study extends these previous observations to a larger population of ovarian cancers. In conclusion, our study compared tumor DNA to leukocyte DNA from apparently healthy individuals. No statistically significant difference in the occurrence of rare Ha-r-us alleles was found between the two populations. The presence of a rare Ha-ras allele in an individual’s DNA does not indicate a predisposition to the development of ovarian cancer. ACKNOWLEDGMENTS This project was supported in part by Grant CA 39930 from the National Cancer Institute, Department of Health and Human Services. It is a pleasure to acknowledge the outstanding secretarial assistance of Jana Carey and Karen Cash. We are also grateful to Dr. Ted Krontiris for useful discussions, aiding in the establishment of the assay, and providing standard DNA samples.
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