Constitutional trisomy 8 mosaicism and gestational trophoblastic disease

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Constitutional Trisomy 8 Mosaicism and Gestational Trophoblastic Disease Hon Fong L. Mark, Jennifer Ahearn, and John C. Lathrop ABSTRACT: Concurrence of congenital trisomy 8 mosaicism and gestational trophoblastic disease in a 42-year-old gravida IV, para IV female is described in the present report. In contrast to other cases described in the literature, our patient had no known additional confounding chromosomal abnormalities other than trisomy 8. The finding of trisomy 8 masaicism in yet another type of cancer provides further s u p p o r t for the hypothesis of an increased predisposition to cancer in tissues with constitutional genomic imbalance, which can manifest itself as numerical chromosomal abnormalities (e.g., trisomies) or structural chromosomal abnormalities (e.g., translocations). To the best of our knowledge, this is the only report in the English literature of constitutional trisomy 8 mosaicism associated with gestational trophoblastic disease, a rare gynecologic disease entity in itself.

INTRODUCTION Mosaic trisomy 8 has been demonstrated quite frequently in m a l i g n a n t cells, either alone or in conjunction w i t h other chromosomal abnormalities. From studies of malignant cells of chronic myelogenous leukemia and other hematologic malignancies, Hsu et al. [1] as well as other investigators have reported a total of 12 cases of acquired trisomy 8. Progogina and F l e i s c h m a n [2] studied 15 patients with Ph 1 chromosome, i(17q), and trisomy 8. Rowley [3] studied bone marrow cells from patients w i t h hematologic disorders and found that an a d d i t i o n a l c h r o m o s o m e 8 was the most c o m m o n abnormality. Because PHA-stimulated lymphocytes from these patients showed a n o r m a l karyotype, it was c o n c l u d e d that the c h r o m o s o m e changes in the bone marrow cells probab l y represented somatic mutations s u p e r i m p o s e d on the individual's n o r m a l c h r o m o s o m e pattern. No c h r o m o s o m e studies were performed prior to the clinical diagnosis of leukemia. In 1975, Riccardi et al. [4] reported trisomy 8 m o s a i c i s m in the bone marrow blasts and skin cells of a 42-year-old male patient w i t h acute granulocytic leukemia and t(Tp;20p). Alt h o u g h trisomy 8 m o s a i c i s m has been demonstrated quite frequently in m a l i g n a n t cells, Riccardi's finding of trisomy 8 in skin fibroblasts was the first report suggesting that con-

From the Departments of Pathology, Rhode Island Hospital (H. F L. M.) and Roger Williams Medical Center (J. A.); and Department of Gynecology (J. C. L.), Rhode Island Hospital and Brown University School of Medicine (H. F. L. M., J. A., J. C. L.), Providence, Rhode Island. Address reprint requests to: Dr. Hon Fong Louie Mark, Director, Laboratory of Cytogenetics, FISH and Genotoxicology, Rhode Island Hospital, 593 Eddy Street, Providence, RI 02903. Received May 26, 1994; accepted September 1, 1994. Cancer Genet Cytogenet 80:150-154 (1995) © Elsevier Science Inc., 1995 655 Avenue of the Americas, New York, NY 10010

genital trisomy 8 may have a causative role in the developm e n t of cancer. However, Riccardi's patient carried another translocation in a d d i t i o n to the trisomy 8, therefore obscuring the relationship. Gafter et al. [5] reported the occurrence of aplastic anemia followed by preleukemia and acute leukemia in a 40-year-old female with congenital trisomy 8 mosaicism. However, Gafter's patient also exhibited sex chromosome m o s a i c i s m and therefore, the association between the congenital trisomy 8 and leukemia was also less definitive t h a n it might have been had trisomy 8 been found alone. Lastly, Niss and Passarge [6], in the course of re-examining c u l t u r e d fibroblasts stored in liquid nitrogen from a patient with developmental retardation, a solitary left kidney, and Wilms tumor, found a cell line trisomic for chromosome 8 in a d d i t i o n to pericentric inversion of a chromosome 2, the latter of w h i c h had been found to be segregating in the family for the past three generations. However, because the trisomy was restricted to fibroblasts in the first 22 subcultures and was not present in lymphocytes, and because multiple spontaneous chromosomal rearrangements were also seen in several late subcultures, the possibilities of spontaneous in vitro n o n d i s j u n c t i o n and mycoplasma infection in culture could not be ruled out. Therefore, although the possibility of an association between congenital mosaic trisomy 8 and Wilms t u m o r was interesting to consider, there were too many confounding factors involved to permit any definitive inferences. We wish to report here the c o i n c i d e n c e of congenital trisomy 8 and gestational trophoblastic disease in the case of a 42-year-old woman, w h i c h first came to our attention many years ago. Unlike other cases, such as the three described above, our patient had no chromosomal abnormalities other than the trisomy. Our interest in this old and almost forgotten case was rekindled by a recent report in which

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Constitutional Trisomy 8 Mosaicism a hypothesis concerning the meiotic origin of trisomic neoplasms was put forth by Haas and Seyger [7] (see also Mark [8]). The current view on the origin of trisomies commonly found in human neoplasms is that they result from a diseaserelated mitotic nondisjunction. The alternative hypothesis proposed is that at least a proportion of these trisomies results from meiotic events. The acceptance of this hypothesis will alter our current views on constitutional versus acquired abnormalities.

CASE REPORT F.V. was a 42-year-old gravida IV, para IV female, upon whom a bilateral partial salpingectomy had been performed in August, 1961. In October, 1975 she was admitted to the Rhode Island Hospital where a diagnostic curettage was performed for vaginal bleeding. The histopathologic diagnosis at that time was choriocarcinoma. This diagnosis was later revised to placental site trophoblastic tumor [9]. The latter diagnosis is an uncommon, recently defined tumor which shares some features with choriocarcinoma. Although the majority of these tumors pursue a relatively benign clinical course, highly aggressive cases have been reported. Choriocarcinoma and placental site trophoblastic tumor are considered to be manifestations of gestational trophoblastic disease [10].

151 The HCG titers at the time were greater than 100,000 IU. She underwent total hysterectomy and bilateral salpingooophorectomy. Hystereosalpingography done on the surgical specimen confirmed the nonpatency of both fallopian tubes. Chemotherapy was instituted following surgery because of persistent elevation of HCG titers. In February 1976, pulmonary metastases developed. She was admitted for the evaluation of possible cerebral metastases. On the evening of admission she suddenly became comatose. She was placed on a respirator and died two days later. Because the patient had had a partial tubal resection, the possibility of de novo choriocarcinoma arising from trophoblastic ceils derived from the last pregnancy after a long period of latency was raised. Postmortem samples for tissue culture were therefore obtained from the metastatic lung tumor and from the skin for cytogenetic studies. It was reasoned at the time that should a Y chromosome be found in the tumor tissue, it would provide compelling evidence for a new, more recent pregnancy being the source of the tumor. MATERIALS AND METHODS

For pathologic examination, tissue specimens from the autopsy were fixed in 10% formalin, processed in the routine manner, and stained with hematoxylin and eosin.

Figure la Gestational trophoblastic disease showing syncytiotrophoblast and cytotrophoblast. Hematoxylin and eosin, 100 x.

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H . F . L . Mark et al.

For cytogenetic studies, disaggregated lung and skin tissues were grown in BME (Basel M e d i u m Eagles) supp l e m e n t e d with 15% fetal calf serum, vitamins, and antibiotics until a monolayer of fibroblast-like cells was achieved. T h e r e u p o n each s a m p l e was d i v i d e d into two subcultures (passage 1), one culture being retained and the other harvested for karyotyping. Harvesting was done according to a modification of the method of M o o r h e a d et al. [11]. Briefly, on the second or third day after the s a m p l e was split, the flasks were e x a m i n e d and harvesting p r o c e d u r e s were initiated w h e n there was e n o u g h mitotic activity. One and a half hours before harvesting, Colcemid at a concentration of 0.3 ~g/mL was a d d e d to the cell culture to arrest mitosis. The flask was t h e n t r y p s i n i z e d and incubated at 37°C until the majority of the cells were lifted. After the cells had detached, two to three drops of fetal calf serum were a d d e d to the flask to arrest any further action of the trypsin. T h e r e u p o n hypotonic treatment was initiated by a d d i n g 5 mL of 0.075 M KC1 to each flask for a duration of 5-15 minutes at room temperature. At the end of the treatment, 5 mL of Carnoy's fixative (3:1, methanol:glacial acetic acid) was a d d e d a n d the cell suspension was poured into centrifuge tubes and spun at 800 r p m for 5 minutes. The supernatant was removed from the tubes a n d the pellet r e s u s p e n d e d . Fresh fixative was a d d e d and the cells were rinsed for an additional three or four times before slides were made. Slides were air-dried for better results in banding. Usually at least 30 cells were counted

Figure lb

and five good-quality b a n d e d metaphases were analyzed before further attempts at harvesting were discontinued. G-banding was done according to a modification of the t r y p s i n pretreatment m e t h o d of Seabright [12]. Briefly, airdried slides were incubated in 2 x SSC (0.3 M s o d i u m chloride, 0.03 M s o d i u m citrate) preheated to 60°C for 1-2 hours before trypsin treatment. After incubation, slides were airdried and passed through the following series: working trypsin solution (0.025 % solution in Hanks Balanced Salt Solution) at 4-10°C for 30-90 seconds, 1 x Hanks Balanced Salt Solution for a quick rinse, 70% ethyl alcohol for a quick rinse, 95% ethyl alcohol for a quick rinse, 4% Giemsa stain for approximately 5 minutes, and distilled water for final rinsing.

RESULTS

Pathologic Findings Autopsy revealed diffuse pulmonary metastases and implants of gestational trophoblastic t u m o r on the peritoneal surface of the urinary b l a d d e r (see Fig. 1). There was intracranial hemorrhage in the posterior portion of the right parietooccipital region, with a marked internal shift of the midline, tentorium, and brain stem. The cause of this hemorrhage and its relation to the t u m o r c o u l d not be ascertained. Small tumor emboli, if present, may have been difficult to identify. Angioinvasive metastasis seemed likely.

Multinucleated syncytiotrophoblast with marked anisonucleosis. Hematoxylin and eosin, 200 x.

Constitutional Trisomy 8 Mosaicism

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Figure 2 Representative karyotype derived from the lung tumor, showing trisomy for chromosome 8.

Cytogenetic Results Chromosomal analyses of metaphases derived from both the lung and skin tissues revealed the presence of two clones of cells: an apparently normal clone with a 46,XX karyotype and a clone of hyperdiploid cells with a 47,XX, + C karyotype. G-banding analyses showed the extra C-group chromosome in the abnormal clone to be a chromosome 8 [Fig. 2). Trisomy 8 was found to be present in 46% (16 of 35 cells} of the cells derived from the lung tumor. Sixteen cells derived from the lung tissue were normal. Three cells were hypodiploid, with a missing C, a missing G, and a missing F group chromosome each. This latter distribution of hypodiploidy was consistent with the null hypothesis of random loss due to technical factors (overspreading} and was not significantly different from that predicted by the Poisson {p > 0.05}. Forty-three percent {13 of 30 cells analyzed) of the cells from the skin tissue culture were trisomic. Seventeen of 30 cells from the skin were normal. No mitotic figures were seen in the direct preparation of the tumorous lung nodules.

DISCUSSION The cytogenetic results provided no definitive information regarding which pregnancy was the precursor of the placental

site trophoblastic tumor (PSTT}. The possibility of gestational tmphoblastic disease from a prior pregnancy (October, 1960} after a prolonged latent period is interesting due to its relatively rare occurrence [13-15]. The concordance of the karyotypes in the skin and lung tissues suggested that the chromosome 8 trisomy was a constitutional abnormality. The presence of this trisomy 8 cell line raises the provocative question of its etiologic relation to the tumor. There has been an ever-increasing number of reports on the association of constitutional chromosomal abnormalities and neoplasms. This topic was reviewed extensively and summarized years ago by Cervenka and Koulischer [16]. Similarly, increased incidences of neoplasms have been reported in the instability syndromes such as ataxia telangiectasia, Bloom syndrome, xeroderma pigmentosum, Fanconi anemia, and, to a lesser degree, Werner syndrome and Rothmund-Thomson syndrome. Interestingly, trisomy 8 has been mentioned recently in this list [17, 18]. The clinical features often cited to be associated with trisomy 8 mosaicism include mild to severe mental deficiency, prominent forehead, deep-set eyes, prominent ears, deep palmar creases, etc. [19]. Recently, the observation of phenotypic variability associated with trisomy 8, as described in the recent reports of Kapaun et al. [20] and of McDonaldMcGinn et al. [21], has attracted increased attention.

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It was suggested by Mark [8] that the basis of this phenotypic variability may be due to the presence of different proportions of trisomy 8 cells in different tissues of the body. D e p e n d i n g on w h i c h tissue and on what proportion of the tissue contains the trisomic clone, different specific phenotypes can be observed. Mark further hypothesized that w h e n trisomy 8 is found in hematopoietic tissues such as the bone marrow, the risk of leukemia is drastically increased. An analogous situation will also be true for cancers occurring in other parts of the body. The possibility of the constitutional chrom o s o m e 8 trisomy playing a causative role in the development of gestational trophoblastic disease is intriguing. To the best of our knowledge, however, this is the only case of constitutional c h r o m o s o m a l abnormality in gestational trophoblastic disease found in the English literature thus far. Many more constitutional cytogenetic studies in hematopoietic and solid tumors will be n e e d e d before any conclusions can be drawn. Lastly, it should be noted that results reported here provide further evidence for the hypothesis of the meiotic origin of trisomic neoplasms [7, 8]. We would like to thank Robin Kieman for word processing, Drs. Roger Mark and Raed Sulaiman for reading the manuscript, and the following for their excellent technical assistance: Ms. E. Airall, Ms. K. Santoro, Ms. Kathleen Zolnierz, and Mr. D. Dunwoodie. The continued support of Drs. Roger Mark and Don B. Singer, and the remaining dedicated staff of the Laboratory of Cytogenetics, FISH and Genotoxicology are also acknowledged.

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5. Gafter U, Shabtai F, Kahn Y, Halbrecht I, Djaldetti M (1976): Aplastic anemia followed by leukemia in congenital trisomy 8 mosaicism. Clin Genet 9:134-142. 6. Niss R, Passarge E (1976): Trisomy 8 restricted to cultured fibroblasts. J Med Genet 13:229-234. 7. Haas OA, Seyger M (1993): Hypothesis: meiotic origin of trisomic neoplasms. Cancer Genet Cytogenet 70:112-116. 8. Mark HFL (1994): Phenotypic variability in trisomy 8 mosaicism is consistent with the hypothesis of meiotic origin of trisomic neoplasms. Cancer Genet Cytogenet 76:158. 9. Lathrop JC, Lauchlan S, Nayak R, Ambler M (1988): Clinical characteristics of placental site trophoblastic tumor (PSI'T). Gynecologic Oncol 31:32-42. 10. Mazur MT, KurmanRJ (1987): Gestational trophoblastic disease in Blaustein's Pathology of the Female Genital Tract, 3d Ed. Springer-Verlag, New York. 11. Moorhead PS, Nowell PC, Mellman WJ, Battips DM, Hungerford DA (1960): Chromosome preparation of leukocytes cultured from human peripheral blood. Exper Cell Res 20:613-616. 12. Seabright M (1971): A rapid banding technique for human chromosomes. Lancet 2:971-972. 13. Maizels G (1940): Probable chorioepithelioma in a virgin of seventy-one. Lancet 1:690-692. 14. Dorr W, Cutler O (1932): Chorion epithelioma in patient seventy years of age. California and Western Med 37:247-249. 15. White TGE (1955): A chorion-epithelioma of the uterus in a postmenopausal woman. J Obstet Gynaecol Br Emp 62:372-374. 16. Cervenka J, Koulischer L (1973): Chromosomes in human cancer. Charles C. Thomas, Publisher, Springfield, Illinois. 17. Der Kaloustian VM, McGill JJ, Vekemans M, Kopelman HR (1990): Clonal lines of aneuploid cells in Rothmund-Thomson syndrome. Am J Med Genet 37:336-339. 18. Ying KL, Oizumi, Curry CJR (1990): Rothmund-Thomson syndrome associated with trisomy 8 mosaicism. J Med Genet 27:258-260. 19. Jones KL (1988): Smith's Recognizable Patterns of Human Malformations, Fourth Edition. W.B. Saunders Company, Philadelphia. 20. Kapaun P, Kabisch H, Held KR, Walter TA, Hegewisch S, Zander AR (1993): Atypical chronic myelogenous leukemia in a patient with trisomy 8 mosaicism syndrome. Ann Hematol 66(1):57-58. 21. McDonald-McGinn DM, Grace K, Spinner NB, Emanuel BS, Zackai EH (1993): Clinical and cytogenetic variability in trisomy 8 mosaicism. Am J Hum Gen 53(Suppl)222.