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ORIGINAL RESEARCH |
From the Division of Genetics, Departments of Obstetrics and Gynecology and Pediatrics, Tufts–New England Medical Center, Boston, Massachusetts; and Department of Obstetrics and Gynecology, Yonsei University College of Medicine, Seoul, Korea.
Address reprint requests to: Kirby L. Johnson, PhD, Tufts–New England Medical Center, Box 394, Division of Genetics, Department of Pediatrics, 750 Washington Street, Boston, MA 02111; E-mail: kjohnson{at}tufts-nemc.org.
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ABSTRACT |
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METHODS: Cervical tissue was obtained from eight women who had at least one live-born son and who underwent radical hysterectomy after a diagnosis of cervical cancer. Control tissue was obtained from four women without cervical cancer who had at least one live-born son and from three women with cervical cancer and no male births. Tissue sections were analyzed with fluorescence in situ hybridization for the presence of fetal cells, defined by an X and Y chromosome. Immunolabeling was used to determine the phenotype of the presumed fetal cells.
RESULTS: Male cells were found in cervical tissue from all four patients for whom large sections (approximately 1.5 x 2 cm) were analyzed. Only one male cell was found in two of the four patients for whom small biopsy specimens (approximately 0.1 x 0.5 cm) were analyzed. No male cells were found in tissue specimens from controls, whether they were small or large sections. In immunolabeling studies, eight of 18 male cells from one patient were CD45-positive and nine of 37 male cells from two patients were cytokeratin-positive. No cells were positive for both markers.
CONCLUSION: Cervical cancer might be associated with microchimerism, possibly from fetomaternal cell trafficking. These results further expand the potential relationship between microchimerism and disease in women.
Microchimerism is defined by the presence of a low level of circulating cells transferred from one individual to another.1,2 This trafficking of cells takes place under a number of circumstances, including pregnancy, transfusion, and transplantation.1–5 During pregnancy, fetal cells cross the placenta and reach the maternal circulation and can ultimately infiltrate a variety of tissues.1 The recognition that fetal cells can pass into the maternal circulation and tissues has led to the question of whether persistent microchimerism is implicated in diseases associated with pregnancy or diseases predominantly found in women. For example, studies have implicated fetal cells in the development of preeclampsia6 and in the pathogenesis of autoimmune disorders, such as scleroderma,7–9 lupus,10 thyroiditis,11,12 primary biliary cirrhosis,13 and Sjögren syndrome.14 However, microchimerism has also been described in nonautoimmune disease, such as infectious hepatitis15 and nonautoimmune thyroid disorders.11 This suggests that microchimerism might be a result of disease and not involved in the pathogenesis of autoimmune disease.
Cervical cancer is the third most common type of cancer in women worldwide.16 It has been shown that certain risk factors, such as sexual practices and human papillomavirus (HPV) infection, can increase a woman’s chance of developing cervical cancer.17–20 It is thought that sexually transmitted HPVs might cause cells in the cervix to begin a series of changes that lead to cancer; the effects of these viruses on the development of cervical cancer are currently being studied.18–21 However, most women who are infected with HPV do not develop cervical cancer, and the virus is not present in all women who have this disease. For these reasons, it is believed that other factors, including those that weaken immune system function, act together with or in the absence of HPVs in the development of cervical cancer. These potential risk factors include cigarette smoking and human immunodeficiency virus infection. Furthermore, organ transplant recipients, who receive drugs that suppress the immune system to prevent rejection of the donor organ, are more likely than others to develop precancerous lesions of the cervix.22 Cervical cancer is an important women’s health concern, and its etiology is unknown but appears to be complex.
For this study, we hypothesized that fetal cell microchimerism might be involved in the pathogenesis or progression of cervical cancer, because this phenomenon has been shown to be associated with a number of diseases. In addition, it has been shown that high parity is a consistently identified factor likely to influence the risk of progression from HPV infection to invasive cervical cancer.23 Microchimerism in the cervix might develop from three different sources: the trafficking of fetal cells during pregnancy, the persistence of spermatocytes or semen leukocytes after sexual intercourse, or the horizontal transfer of cell-free deoxyribonucleic acid (DNA) after phagocytosis of the male nuclei. To investigate the potential role of persistent male DNA or intact cells in cervical cancer, we performed molecular cytogenetic analysis to determine whether cells bearing a Y chromosome were present within cervical tissue from affected women. To determine whether these cells were of hematopoietic origin or whether they had differentiated into cervical tissue, we performed immunolabeling with CD45 as a leukocyte marker and cytokeratin as a cervical cell marker.
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MATERIALS AND METHODS |
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summarizes the pregnancy histories, diagnoses, and type of surgery performed. No patients or controls had a history of other potential sources of microchimerism (ie, blood transfusion, organ transplantation, or a twin sibling). The mean ages of the patients and controls were 60 years (range, 49–75) and 51 years (range, 39–75), respectively, which are not significantly different (P = .148 by Student t test). Human papillomavirus status was unknown in all patients and controls.
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-satellite regions of the X and Y chromosomes. The X chromosome probe (DXZ1) was labeled with cyanine-3 (red), and the Y chromosome probe (PHY 10) was labeled with fluorescein isothiocyanate (green). Purified, unlabeled DNA was obtained from Genzyme Genetics (Framingham, MA) and was labeled by nick translation, as described previously.25 For detection of cytokeratin-positive cells, fluorescence in situ hybridization and immunofluorescence staining were performed sequentially. Fluorescence in situ hybridization was performed as described previously26 with X and Y chromosome probes. The antibody used was AE1/AE3 anticytokeratin (Chemicon, Temecula, CA). After fluorescence in situ hybridization, the tissue sections were washed in phosphate-buffered saline, incubated with 10% goat serum for 20 minutes, rinsed in phosphate-buffered saline, and then incubated with primary antibody for 1 hour. After three washes with phosphate-buffered saline for 1 minute each, the sections were incubated with secondary antibody (phycoerythrin-labeled goat antimouse immunoglobulin) for 30 minutes. The sections were then washed three times in phosphate-buffered saline for 5 minutes each.
In all cases, the tissue sections were stained with 0.03 µg/mL 4',6-diamidino-2-phenylindole and mounted with Fluoroguard (BioRad, Hercules, CA), and chromosome signals and immunoperoxidase or fluorescence staining were visualized simultaneously. Isotypic control mouse immunoglobulin G1 antibody was used as a negative control for all experiments.
Scoring was performed by observers who were blind to the reproductive history of the patients and controls. Tissue specimens were included for analysis if the following criteria were met: There was minimal loss of cells from the tissue section during the hybridization procedure, more than 75% of nuclei contained fluorescence in situ hybridization signals, and the signals representing the X and Y chromosomes were of equal intensity. When these criteria were satisfied, three slides from each patient and control were analyzed and scored; the exception was patient 1, for whom only one section was suitable for analysis (Table 2). The number of cells considered to be male within the tissue sections was determined by counting those nuclei that had two different-colored fluorescence in situ hybridization signals, representing both the X and Y chromosomes, and that had an intact nuclear border as indicated by 4',6-diamidino-2-phenylindole staining. For immunolabeling, the cells were observed through single-band pass red and dual-band pass filters with a fluorescent microscope, because both the phycoerythrin-labeled secondary antibody and the AEC substrate were visible through these filters.
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RESULTS |
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shows examples of male cells that were observed in the tissue specimens from the patients with cervical cancer. Male cells were found in cervical tissue from all four patients for whom large tissue sections (approximately 1.5 x 2 cm) were analyzed (Table 2
). The number of male cells was fairly consistent in all sections that were analyzed from each patient. In some cases, the male cells appeared as clusters (patients 3 and 4). For patient 1, no male cells were observed in the noncervical tissue that was analyzed. Only one male cell was found in two of the four patients for whom small sections (approximately 0.1 x 0.5 cm) were analyzed. No male cells were found in the tissue specimens from the controls, whether the sections were small or large, even though two of them had male children, and all of them had a history of sexual activity with presumed exposure to semen.
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). Figures 2 and 3 show examples of CD45 and cytokeratin staining, respectively. The clusters of three and six cells that were CD45- and cytokeratin-positive, respectively (Table 3), were found in slide 2 from patient 4 (Table 2). No cells were found to be positive for both markers. No immunolabeling was obtained with isotypic control mouse immunoglobulin G1.
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DISCUSSION |
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To further investigate the origin of the male microchimeric cells, we combined fluorescence in situ hybridization and immunolabeling for the simultaneous detection of sex chromosomes and protein markers (CD45 and cytokeratin). We observed that 44% (eight of 18) of the microchimeric cells were positive for CD45, a common leukocyte marker, and 24% (nine of 37) of the microchimeric cells were positive for cytokeratin. No cells were positive for both CD45 and cytokeratin. The finding of CD45-positive cells suggests the migration of leukocytes from either the maternal circulation or semen into the cervical tissue. The finding of cytokeratin-positive cells suggests the presence of epithelial cells. Because cervical tissue consists of squamous and columnar cell layers that are represented by epithelial and endothelial cells, respectively, these results suggest that circulating precursor cells, possibly fetal in origin, might migrate into the squamous cell layer of cervical tissue and differentiate.
The presence of persistent microchimeric cells in the cervix might play a role in carcinogenesis. For example, these cells might induce the alteration of the woman’s immune system, or their presence might be a result of a suppressed immune system. These cells might make the cervical tissue more susceptible to HPV infection or provide a suitable environment for tumor growth. Unfortunately, HPV status in the patients and controls presented here was unknown and therefore could not be considered as a variable. Nevertheless, the incorporation of spermatocytes into cervical tissue might provide beneficial conditions for HPV infection, although the absence of these cells argues against this. In addition, the horizontal transfer of cell-free DNA after phagocytosis of the spermatocytes might be an alternative route for the exposure of cells of the cervix to foreign DNA.27 However, if this horizontal transfer were to occur, we would detect cells with two X chromosome signals along with a Y signal (which would be necessary for detection with our assay); this was not observed. Finally, the presence of microchimeric cells might also be a response to carcinogenesis, in that fetal precursor cells might be capable of differentiation in an attempt to repopulate or repair the affected tissue. Further study is necessary to understand the role of persistent microchimeric cells in the progression of cervical cancer.
The results presented here suggest that microchimerism might be a relatively common phenomenon in women affected with cervical cancer. Although microchimerism has been hypothesized to be involved in the pathogenesis of autoimmune disease,7–14 these data, along with other studies that demonstrate microchimerism in nonautoimmune disease,11,15 suggest that microchimerism might be involved in a wide range of diseases that predominantly affect women during and after pregnancy. More work is needed to determine the genetic origin of the microchimeric cells through the use of polymerase chain reaction of polymorphic short tandem repeat sequences, and to further understand the role of microchimerism that might potentially result from fetomaternal cell trafficking or sexual intercourse. This future work should include an analysis of a larger number of patients and controls and the incorporation of an independent, non-Y chromosomal marker of spermatocytes to verify exclusion of sperm in the tissue being analyzed. However, as demonstrated in this study, the observation of male cells in cervical tissue further expands the potential relationship between microchimerism and disease in women.
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Footnotes |
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doi:10.1016/S0029-7844(03)00615-X
Received January 13, 2003. Received in revised form April 17, 2003. Accepted May 2, 2003.
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REFERENCES |
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2. Artlett CM. Microchimerism in health and disease. Curr Mol Med 2002;2:525–35.[Medline]
3. Carter AS, Bunce M, Cerundolo L, Welsh KI, Morris PJ, Fuggle SV. Detection of microchimerism after allogeneic blood transfusion using nested polymerase chain reaction amplification with sequence-specific primers (PCR-SSP): A cautionary tale. Blood 1998;92:683–9.
4. Tajik N, Singal D, Pourmand G, Ebrahimi-Rad M, Radjabzadeh M, Tavasoli P, et al. Prospective study of microchimerism in renal allograft recipients: Association between HLA-DR matching, microchimerism and acute rejection. Clin Transplant 2001;15:192–8.[Medline]
5. Noris M, Cugini D, Casiraghi F, Azzollini N, De Deus Viera Moraes L, Mister M, et al. Thymic microchimerism correlates with the outcome of tolerance-inducing protocols for solid organ transplantation. J Am Soc Nephrol 2001;12:2815–26.
6. Holzgreve W, Ghezzi F, Di Naro E, Ganshirt D, Maymon E, Hahn S. Disturbed fetomaternal cell traffic in preeclampsia. Obstet Gynecol 1998;91:669–72.[Abstract]
7. Nelson JL, Furst DE, Maloney S, Gooley T, Evans PC, Smith A, et al. Microchimerism and HLA-compatible relationships of pregnancy in scleroderma. Lancet 1998;351: 559–62.[Medline]
8. Artlett CM, Smith JB, Jimenez SA. Identification of fetal DNA and cells in skin lesions from women with systemic sclerosis. N Engl J Med 1998;338:1186–91.
9. Johnson KL, Nelson JL, Furst DE, McSweeney PA, Roberts DJ, Zhen DK, et al. Fetal cell microchimerism in tissue from multiple sites in women with systemic sclerosis. Arthritis Rheum 2001;44:1848–54.[Medline]
10. Johnson KL, McAlindon TE, Mulcahy E, Bianchi DW. Microchimerism in a female patient with systemic lupus erythematosus. Arthritis Rheum 2001;44:2107–11.[Medline]
11. Srivatsa B, Srivatsa S, Johnson KL, Samura O, Lee SL, Bianchi DW. Microchimerism of presumed fetal origin in thyroid specimens from women: A case-control study. Lancet 2001;358:2034–8.[Medline]
12. Klintschar M, Schwaiger P, Mannweiler S, Regauer S, Kleiber M. Evidence of fetal microchimerism in Hashimoto’s thyroiditis. J Clin Endocrinol Metab 2001;86:2494–8.
13. Fanning PA, Jonsson JR, Clouston AD, Edwards-Smith C, Balderson GA, Macdonald GA, et al. Detection of male DNA in the liver of female patients with primary biliary cirrhosis. J Hepatol 2000;33:690–5.[Medline]
14. Endo Y, Negishi I, Ishikawa O. Possible contribution of microchimerism to the pathogenesis of Sjogren’s syndrome. Rheumatology 2002;41:490–5.
15. Johnson KL, Samura O, Nelson JL, McDonnell WM, Bianchi DW. Significant fetal cell microchimerism in a nontransfused woman with hepatitis C: Evidence of long-term survival and expansion. Hepatology 2002;36: 1295–7.[Medline]
16. Saslow D, Runowicz CD, Solomon D, Moscicki A, Smith RA, Eyre HJ, et al. American Cancer Society guidelines for the early detection of cancer. CA Cancer J Clin 2002;52: 342–62.
17. La Vecchia C, Franceschi S, Decarli A, Fasoli M, Gentile A, Parazzini F, et al. Sexual factors, venereal diseases, and the risk of intraepithelial and invasive cervical neoplasia. Cancer 1986;58:935–41.[Medline]
18. Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV, et al. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 1999;189:12–9.[Medline]
19. Holowaty P, Miller AB, Rohan T, To T, Holowaty P, Miller AB, et al. Natural history of dysplasia of the uterine cervix. J Natl Cancer Inst 1999;91:252–8.
20. Moscicki AB, Shiboski S, Broering J, Powell K, Clayton L, Jay N, et al. The natural history of human papillomavirus infection as measured by repeated DNA testing in adolescent and young women. J Pediatr 1998;132:277–84.[Medline]
21. Barrasso R, De Brux J, Croissant O, Orth G. High prevalence of papillomavirus-associated penile intraepithelial neoplasia in sexual partners of women with cervical intraepithelial neoplasia. N Engl J Med 1987;317:916–23.[Abstract]
22. Ozsaran AA, Ates T, Dikmen Y, Zeytinoglu A, Terek C, Erhan Y, et al. Evaluation of the risk of cervical intraepithelial neoplasia and human papilloma virus infection in renal transplant patients receiving immunosuppressive therapy. Eur J Gynaecol Oncol 1999;20:127–30.[Medline]
23. Castellsague X, Bosch FX, Munoz N. Environmental cofactors in HPV carcinogenesis. Virus Res 2002;89:191–9.[Medline]
24. Khosrotehrani K, Stroh H, Bianchi DW, Johnson KL. Combined FISH and immunolabeling on paraffin-embedded tissue sections for the study of microchimerism. Biotechniques 2003;34:2–4.
25. Johnson KL, Nath J. Fluorescence in situ hybridization (FISH): DNA probe production and hybridization criteria. Biotech Histochem 1998;73:6–22.[Medline]
26. Johnson KL, Zhen DK, Bianchi DW. The use of fluorescence in situ hybridization (FISH) on paraffin-embedded tissue sections for the study of microchimerism. Biotechniques 2000;29:1220–4.[Medline]
27. Holmegren L, Szeles A, Rajnavolgyi E, Folkman J, Klein G, Ernberg I, et al. Horizontal transfer of DNA by the uptake of apoptotic bodies. Blood 1999;93:3956–63.
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