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Clonality analysis of bilateral ovarian endometrial cysts3
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FERTILITY AND STERILITY威 VOL. 72, NO. 6, DECEMBER 1999 Copyright ©1999 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Clonality analysis of bilateral ovarian endometrial cysts Haruko Jimbo, M.D.,*† Yoshiaki Hitomi, M.D.,† Hiroyuki Yoshikawa, M.D.,* Testu Yano, M.D.,* Mikio Momoeda, M.D.,* Toshiharu Yasugi, M.D.,* Yuji Taketani, M.D.,* and Hiroyasu Esumi, M.D.† University of Tokyo, Tokyo; and National Cancer Center Research Institute, East, Kashiwa, Japan
Endometriosis is a common disease affecting women of reproductive age. Pathologically, it is defined as the presence of endometrioid glands and stroma outside the uterus. It is generally thought that endometriosis is benign. However, it has some characteristics similar to those found in malignancies. The disease can exhibit cytologic atypia in frequent association with DNA aneuploidy. In addition, cells from peritoneal endometriotic lesions express an invasive phenotype in vitro (1). In our previous report, we presented evidence for the monoclonal expansion of epithelial cells in an ovarian endometrial cyst (2). Prompted by this finding, we asked whether bilateral ovarian endometrial cysts arise from a single clone or two independent clones. Received April 13, 1999; revised and accepted July 7, 1999. Supported by a Grant-inAid from the Ministry of Health and Welfare for the Second Term Comprehensive 10-Year Strategy for Cancer Control. Reprint requests: Hiroyuki Yoshikawa, M.D., Department of Obstetrics and Gynecology, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 1138655, Japan (FAX: 81-33816-2017; E-mail: [email protected]). * Department of Obstetrics and Gynecology, University of Tokyo. † The Investigative Treatment Division, National Cancer Center Research Institute.
TISSUE SAMPLES The materials were obtained from three patients with bilateral ovarian endometrial cysts who underwent bilateral cystectomies plus hysterectomy or cystectomy plus salpingo-oophorectomy at the University of Tokyo Hospital. Before surgery, we obtained the patients’ informed consent for the use of surgical materials for research purposes according to the guidelines of the Institutional Review Board. In two of three cases, fresh tissue specimens were obtained from each cyst by the method reported previously (2). This method allowed us to isolate cell samples in which epithelial cells constituted more than 80% of the total cells. In the remaining case, formalin-fixed, paraffin-embedded tissue specimens were used. A singlelayered epithelium was microdissected from 10-mm-thick sections under a microscope (Olympus, Tokyo, Japan). Control samples in each case consisted of normal myometrial or ovarian tissues obtained from surgical specimens.
CLONALITY ASSAY
The opinions and commentary expressed in Editor’s Corner articles are solely those of the author. Publication does not imply endorsement by the Editor or American Society for Reproductive Medicine.
We adapted the protocol for the clonality analysis using the human androgen receptor gene as an X-linked polymorphic marker. Genomic DNA was extracted from the epithelial cells of endometrial cysts and control samples using the DNA Extractor WB kit (Wako, Osaka, Japan). One nanogram of genomic DNA from an endometrial cyst or a control sample was digested with 1.25 U of RsaI with and without 1 U of HpaII. The digested products were subjected to polymerase chain reaction (PCR) with primers (2, 3) AR1 5⬘-CCGAGGAGCTTTCCAGAATC-3⬘ and indodicarbocyanine (Cy5)-labeled AR2 5⬘-TACGATGGGCTTGGGGAGAA-3⬘ in 30 L of reaction mixture. PCR amplification and electrophoresis using an automatic sequencer (ALF, Pharmacia, Uppsala, Sweden) were performed as described previously (2). The results were analyzed using the Fragment Manager software package (Pharmacia).
0015-0282/99/$20.00 PII S0015-0282(99)00416-1
Clonality analysis was performed on six endometrial cysts and the corresponding controls from three patients (Fig. 1). Upon digestion with HpaII, both peaks were retained in the
1142
FIGURE 1 Clonality analysis of bilateral ovarian endometrial cysts. Without HpaII digestion, the PCR products derived from the human androgen receptor gene alleles were detected as discrete peaks in all samples from the left cyst (L), right cyst (R), and the normal control (N) in each case. As a control, normal myometrial and ovarian tissues were used in case 1 and in cases 2 and 3, respectively. Upon HpaII digestion, one of the peaks was significantly reduced in all six endometrial cysts, whereas both peaks were retained in the normal controls. (A and B), Cases 1 and 2; the methylation pattern was divergent between both cysts. (C), Case 3; the methylation pattern was consistent between bilateral cysts. (⫺) ⫽ without HpaII digestion; (⫹) ⫽ with HpaII digestion.
Jimbo. Clonality analysis. Fertil Steril 1999.
corresponding controls, but all six cysts showed a single peak, suggesting that the epithelial cells in each ovarian endometrial cyst were monoclonal in origin. Subsequently, we asked whether both cysts in each woman arose from a single clone or two independent clones. In two cases (case 1, case 2), the methylation pattern of the human androgen receptor gene was divergent; that is, the retained peak was different between the bilateral endometrial cysts, indicating that the bilateral cysts in these patients originated from different initiated clones (Fig. 1A, 1B). The remaining case (case 3) had the same methylation pattern in
FERTILITY & STERILITY威
bilateral endometrial cysts, which gave no information for determining whether they originated from a single clone or from different clones (Fig. 1C). Based on the clonality analysis used in this study, when bilateral cysts arise from a single clone, the methylation patterns of the human androgen receptor gene are supposed to be identical. On the other hand, when bilateral cysts arise from two independent clones, the predicted methylation pattern is identical in half and different in the other half. The different patterns between the cysts indicate that the cysts arose from independent clones, whereas the identical pattern gives us no information on the origin of the cysts. In the present study, all six endometrial cysts from three patients were monoclonal, in keeping with our recent work (2), and two of the three patients demonstrated a divergent methylation pattern between the cysts. Although the number of cases studied was small because of the difficulty in collecting sufficient materials for the assay, the present data are compatible with the notion that bilateral endometrial cysts in each individual are principally derived from different precursor cells. The data presented here increase the plausibility that multiple endometriotic lesions might arise independently from different clones. The present finding is in sharp contrast to the fact that bilateral ovarian carcinomas arise as a unifocal neoplasia (4). The result presented here seems to have implications for the treatment of endometriosis. That is, unlike recurrences of carcinoma, some “recurrent” endometriotic lesions may be de novo lesions that developed independently of the surgically removed lesions. To minimize the recurrence of endometriosis, the prevention of new disease should be sought in addition to complete eradication of already existing lesions. References 1. Gaetje R, Kotzian S, Herrmann G, Baumann R, Starzinski-Powitz A. Invasiveness of endometriotic cells in vitro. Lancet 1995;346:1463– 4. 2. Jimbo H, Hitomi Y, Yoshikawa H, Yano T, Momoeda M, Sakamoto A, et al. Evidence for monoclonal expansion of epithelial cells in ovarian endometrial cysts. Am J Pathol 1997;150:1173– 8. 3. Mutter GL, Chaponot ML, Fletcher JA. A polymerase chain reaction assay for non-random X chromosome inactivation identifies monoclonal endometrial cancers and precancers. Am J Pathol 1995;146:501– 8. 4. Park TW, Felix JC, Wright TC Jr. X chromosome inactivation and microsatellite instability in early and advanced bilateral ovarian carcinomas. Cancer Res 1995;55:4793– 6.
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FERTILITY AND STERILITY威 VOL. 72, NO. 6, DECEMBER 1999 Copyright ©1999 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Clonality analysis of bilateral ovarian endometrial cysts Haruko Jimbo, M.D.,*† Yoshiaki Hitomi, M.D.,† Hiroyuki Yoshikawa, M.D.,* Testu Yano, M.D.,* Mikio Momoeda, M.D.,* Toshiharu Yasugi, M.D.,* Yuji Taketani, M.D.,* and Hiroyasu Esumi, M.D.† University of Tokyo, Tokyo; and National Cancer Center Research Institute, East, Kashiwa, Japan
Endometriosis is a common disease affecting women of reproductive age. Pathologically, it is defined as the presence of endometrioid glands and stroma outside the uterus. It is generally thought that endometriosis is benign. However, it has some characteristics similar to those found in malignancies. The disease can exhibit cytologic atypia in frequent association with DNA aneuploidy. In addition, cells from peritoneal endometriotic lesions express an invasive phenotype in vitro (1). In our previous report, we presented evidence for the monoclonal expansion of epithelial cells in an ovarian endometrial cyst (2). Prompted by this finding, we asked whether bilateral ovarian endometrial cysts arise from a single clone or two independent clones. Received April 13, 1999; revised and accepted July 7, 1999. Supported by a Grant-inAid from the Ministry of Health and Welfare for the Second Term Comprehensive 10-Year Strategy for Cancer Control. Reprint requests: Hiroyuki Yoshikawa, M.D., Department of Obstetrics and Gynecology, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 1138655, Japan (FAX: 81-33816-2017; E-mail: [email protected]). * Department of Obstetrics and Gynecology, University of Tokyo. † The Investigative Treatment Division, National Cancer Center Research Institute.
TISSUE SAMPLES The materials were obtained from three patients with bilateral ovarian endometrial cysts who underwent bilateral cystectomies plus hysterectomy or cystectomy plus salpingo-oophorectomy at the University of Tokyo Hospital. Before surgery, we obtained the patients’ informed consent for the use of surgical materials for research purposes according to the guidelines of the Institutional Review Board. In two of three cases, fresh tissue specimens were obtained from each cyst by the method reported previously (2). This method allowed us to isolate cell samples in which epithelial cells constituted more than 80% of the total cells. In the remaining case, formalin-fixed, paraffin-embedded tissue specimens were used. A singlelayered epithelium was microdissected from 10-mm-thick sections under a microscope (Olympus, Tokyo, Japan). Control samples in each case consisted of normal myometrial or ovarian tissues obtained from surgical specimens.
CLONALITY ASSAY
The opinions and commentary expressed in Editor’s Corner articles are solely those of the author. Publication does not imply endorsement by the Editor or American Society for Reproductive Medicine.
We adapted the protocol for the clonality analysis using the human androgen receptor gene as an X-linked polymorphic marker. Genomic DNA was extracted from the epithelial cells of endometrial cysts and control samples using the DNA Extractor WB kit (Wako, Osaka, Japan). One nanogram of genomic DNA from an endometrial cyst or a control sample was digested with 1.25 U of RsaI with and without 1 U of HpaII. The digested products were subjected to polymerase chain reaction (PCR) with primers (2, 3) AR1 5⬘-CCGAGGAGCTTTCCAGAATC-3⬘ and indodicarbocyanine (Cy5)-labeled AR2 5⬘-TACGATGGGCTTGGGGAGAA-3⬘ in 30 L of reaction mixture. PCR amplification and electrophoresis using an automatic sequencer (ALF, Pharmacia, Uppsala, Sweden) were performed as described previously (2). The results were analyzed using the Fragment Manager software package (Pharmacia).
0015-0282/99/$20.00 PII S0015-0282(99)00416-1
Clonality analysis was performed on six endometrial cysts and the corresponding controls from three patients (Fig. 1). Upon digestion with HpaII, both peaks were retained in the
1142
FIGURE 1 Clonality analysis of bilateral ovarian endometrial cysts. Without HpaII digestion, the PCR products derived from the human androgen receptor gene alleles were detected as discrete peaks in all samples from the left cyst (L), right cyst (R), and the normal control (N) in each case. As a control, normal myometrial and ovarian tissues were used in case 1 and in cases 2 and 3, respectively. Upon HpaII digestion, one of the peaks was significantly reduced in all six endometrial cysts, whereas both peaks were retained in the normal controls. (A and B), Cases 1 and 2; the methylation pattern was divergent between both cysts. (C), Case 3; the methylation pattern was consistent between bilateral cysts. (⫺) ⫽ without HpaII digestion; (⫹) ⫽ with HpaII digestion.
Jimbo. Clonality analysis. Fertil Steril 1999.
corresponding controls, but all six cysts showed a single peak, suggesting that the epithelial cells in each ovarian endometrial cyst were monoclonal in origin. Subsequently, we asked whether both cysts in each woman arose from a single clone or two independent clones. In two cases (case 1, case 2), the methylation pattern of the human androgen receptor gene was divergent; that is, the retained peak was different between the bilateral endometrial cysts, indicating that the bilateral cysts in these patients originated from different initiated clones (Fig. 1A, 1B). The remaining case (case 3) had the same methylation pattern in
FERTILITY & STERILITY威
bilateral endometrial cysts, which gave no information for determining whether they originated from a single clone or from different clones (Fig. 1C). Based on the clonality analysis used in this study, when bilateral cysts arise from a single clone, the methylation patterns of the human androgen receptor gene are supposed to be identical. On the other hand, when bilateral cysts arise from two independent clones, the predicted methylation pattern is identical in half and different in the other half. The different patterns between the cysts indicate that the cysts arose from independent clones, whereas the identical pattern gives us no information on the origin of the cysts. In the present study, all six endometrial cysts from three patients were monoclonal, in keeping with our recent work (2), and two of the three patients demonstrated a divergent methylation pattern between the cysts. Although the number of cases studied was small because of the difficulty in collecting sufficient materials for the assay, the present data are compatible with the notion that bilateral endometrial cysts in each individual are principally derived from different precursor cells. The data presented here increase the plausibility that multiple endometriotic lesions might arise independently from different clones. The present finding is in sharp contrast to the fact that bilateral ovarian carcinomas arise as a unifocal neoplasia (4). The result presented here seems to have implications for the treatment of endometriosis. That is, unlike recurrences of carcinoma, some “recurrent” endometriotic lesions may be de novo lesions that developed independently of the surgically removed lesions. To minimize the recurrence of endometriosis, the prevention of new disease should be sought in addition to complete eradication of already existing lesions. References 1. Gaetje R, Kotzian S, Herrmann G, Baumann R, Starzinski-Powitz A. Invasiveness of endometriotic cells in vitro. Lancet 1995;346:1463– 4. 2. Jimbo H, Hitomi Y, Yoshikawa H, Yano T, Momoeda M, Sakamoto A, et al. Evidence for monoclonal expansion of epithelial cells in ovarian endometrial cysts. Am J Pathol 1997;150:1173– 8. 3. Mutter GL, Chaponot ML, Fletcher JA. A polymerase chain reaction assay for non-random X chromosome inactivation identifies monoclonal endometrial cancers and precancers. Am J Pathol 1995;146:501– 8. 4. Park TW, Felix JC, Wright TC Jr. X chromosome inactivation and microsatellite instability in early and advanced bilateral ovarian carcinomas. Cancer Res 1995;55:4793– 6.
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