A subset of gestational trophoblastic disease characterized by abnormal chromosome 8 copy number detected by fluorescence in situ hybridization

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A Subset of Gestational Trophoblastic Disease Characterized by Abnormal Chromosome 8 Copy Number Detected by Fluorescence In Situ Hybridization H. F. L. Mark, Alaa Afify, William Taylor, Kathleen Santoro, and John C. Lathrop

ABSTRACT: The present p a p e r describes the results o f research conducted to ascertain whether the report by Mark et al. [1], describing the concurrence o f congenital trisomy 8 mosaicism and gestational trophoblastic disease (GTD) in a 42 year-old Gravida IV, Para I V patient was an isolated event. In contrast to other cases described in the literature, the patient described in Mark eta]. [1] had no additional confounding c h r o m o s o m a l abnormalities other than trisomy 8. To the best o f our knowledge, ours was the only reported case o f constitutional trisomy 8 mosaicism associated with gestational trophoblastic disease, a rare gynecological disease entity. The question arises whether there exists a subset o f patients with GTD characterized by an abnormal c h r o m o s o m e 8 c o p y number. The implicit hypothesis is that an abnormal n u m b e r o f c h r o m o s o m e 8 s o m e h o w predisposes to cancer. A pilot s t u d y o f 10 cases o f GTD was conducted using fluorescence in situ hybridization (FISH) and a commercial c h r o m o s o m e 8-specific a-satellite probe on formalin-fixed, paraffin-embedded patient tissues. A m o n g eight informative cases successfully completed, two cases (25%) were f o u n d to be trisomic, when a cut-off point o f 10% trisomic cells is adopted. A n o t h e r two cases (25%) were f o u n d to be triploid. The results o f our FISH s t u d y indicated that an abnormal c h r o m o s o m e 8 c o p y n u m b e r f o u n d in Mark et al. [1] is unlikely to be an isolated event. Our data are consistent with the hypothesis that a subset o f GTD indeed m a y exist which is characterized by more than two copies o f c h r o m o s o m e 8. The present findings corroborate those recently f o u n d in breast, prostate, and other cancers. © Elsevier Science Inc., 1997

INTRODUCTION Mark et al. [1] previously described the coincidence of congenital trisomy 8 mosaicism and gestational trophoblastic disease in a 42-year-old w o m a n [1-5]. The patient was a Gravida IV, Para IV female, who had a bilateral partial salpingectomy. Diagnostic curettage was performed for vaginal bleeding. The histopathological diagnosis at that time was choriocarcinoma. Because the patient had a partial tubal resection, the possibility of de novo choriocarcin o m a arising from trophoblastic cells derived from the last pregnancy after a long period of latency was raised. Cytogenetic studies were performed on skin fibroblasts and

From the Laboratory of Cflogenetics, FISH and Genotoxicology (14. F. L. M., K. S.), Department of Pathology (H. F. L. M., A.A., W. T.), and Department of Obstetrics and Gynecology (J. C. L.), Rhode Island Hospital, Providence, Rhode Island; and Brown University School of Medicine (14. F. L. M., J. C. L.), Providence, Rhode Island, U.S.A. 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 July 25, 1996; accepted December 7, 1996. Cancer GenetCytogenet99:24-29 (1997) © Elsevier ScienceInc., 1997 655 Avenueof the Americas,New York, NY 10010

lung tumor tissues to u n e q u i v o c a b l y establish the presence of mosaicism. Although the results of the conventional cytogenetic analysis revealed the presence of a cell line with chromosome 8 trisomy [1], the patient did not exhibit any phenotypic manifestations usually associated with this mosaicism, such as mild to severe mental deficiency, p r o m i n e n t forehead, deep set eyes, p r o m i n e n t ears, deep palmar creases, etc. With the advent of recombin a n t DNA technology and the ability to perform molecular cytogenetic analysis on a large n u m b e r of interphase cells derived from formalin-fixed, paraffin-embedded archival materials, we were able to further assess the extent of mosaicism in this patient. Results of that study were reported in Mark et al. [5]. Our case was considered u n i q u e because, unlike other cases in the literature, our patient had no other confounding chromosomal abnormalities besides the trisomy. The question arises whether this case of trisomy 8 in a GTD patient represents an isolated event. A n extensive search of the literature failed to reveal similar reports. In order to more systematically assess chromosome 8 copy n u m b e r in GTD, paraffin sections were prepared

0165-4608/97/$17.00 PII S0165-4608(96)00439-6

FISH Study of GTD

25

from archival materials of patients w i t h a diagnosis of GTD. To the best of our knowledge, this was the only such study ever p e r f o r m e d on this disease using the t e c h n i q u e of FISH.

MATERIALS AND METHODS Subsequent to gross e x a m i n a t i o n and prosection, sections of the resected tissue s p e c i m e n s were processed in a routine manner. F i v e- m ic r o n - th ic k sections of the formalinfixed, p araff i n - em b e d d e d tissues were stained w i t h hematoxylin and eosin (H & E) for histopathological evaluation. Selected slides and blocks were chosen for further studies. For fluorescence in situ hybridization, modifications of the procedures of Pinkel et al. [6], Mark [7], Miranda et al. [8], and Afify et al. [9], as well as manufacturer's instructions (Oncor, Gaithersburg, MD) were followed. Briefly, formalin-fixed, paraffin-embedded, 4~ to 8-micron thick tissue sections were a p p li e d to s i la n i z e d slides. The slides were baked at 65°C for 2 to 4 hours. S p e c i m e n s were deparaffinized in x y l e n e and fixed in 100% ethanol for 10 m i n u t e s each. Pretreatment w i t h 30% bisulfide s o d i u m in 2X SSC, pH 7.0 for 30 m in u t e s was followed by 1-minute washes of 2XSSC pH 7.0 and 2 m i n u te s of d e h y d r at i o n in a series of 70%, 80%, 90%, and 100% ethanol solutions. Tr eat m en t with proteinase (400 p~l of a stock solution, 25 mg/ml, in 40 ml 2X SSC, pH 7.0) for 30 m i n u t e s was foll o w e d by three washes in 2XSSC (pH 7.0) for 1 m i n u t e each, and by dehydration with the same series of ethanol solutions mentioned above, plus 100% acetone for 2 minutes. Hybridization was p e r f o r m e d w i t h a m i x t u r e of 2 ~1 of c h r o m o s o m e 8 ~x-satellite digoxigenin-FITC probe and 30 ~1 of Hybrisol VI per specimen, w h i c h was then covered

Table I

with a glass coverslip and sealed. The specimens were denatured at 90°C for 12 m i n u t e s and left in a h u m i d chamber at 37°C overnight. The following day, the slides were post-washed two times in a 50% formamide/2XSSC solution (pH 7.0) at 43°C for 15 m i n u t es each, in 0.1X SSC (pH 7.0) for 30 m i n u t es at 43°C, and then a final wash in 1X PBD (phosphate buffered detergent). For detection, slides were treated with 50 ~1 of fluorescein-labeled anti-digoxigenin at 37°C for 20 minutes. The interphase nuclei were counterstained with 20 p~l of a 1:1 dilution of p r o p i d i u m i o d i d e/ an t i f ad e for a final concentration of 1.25 p~g/ml per slide. The slides were covered with glass coverslips and e x a m i n e d u n d e r a Zeiss epifluorescence p h o t o m i c r o s c o p e using an FITC exciter filter set and Ektachrome ISO 400 color slide film. Scoring was based on the n u m b e r of hybridizing signals per n u c l e u s identified in individual, n o n - o v e r l a ppi ng nuclei, as suggested by Kim et al. [10] and H o p m a n et al. [11] and according to established protocols of this laboratory. At least 100 interphase nuclei, as can be seen in Table 1, were scored. Prior to scoring, an H & E-stained, formalin-fixed, paraffin-embedded tissue section was also examined to localize the area of interest.

RESULTS AND DISCUSSION Gestational trophoblastic disease is a group of diseases that have a c o m m o n feature of trophoblastic proliferation. They i n cl u d e hydatidiform moles, placental site trophoblastic tumors, and choriocarcinoma. Choriocarcinoma is the most aggressive form of gestational trophoblastic disease. The natural history of untreated choriocarcinoma is characterized by the development

S u m m a r y of the distribution of c h r o m o s o m e 8 copy n u m b e r Number of chromosome 8

Case ID $85-9349 4273-CW71 (placenta) $86-5747 $81-11358 $93-10205 $85-3387 $80-871 (placenta) $75-11050-A

1

2

3

4

2

85

36

36

5 273 86 41 146

137 775 540 528 829

32 8 8 3 37

22 2 3 0 20

25 3

118 85

25 28

4 87

Total scored

% trisomy 8

1

160

22.50%

5.48%

7

15.76% 0.76% 1.27% 0.52% 3.56%

15.91%

8

203 1058 631 572 1040 172 225

14.53% 12.44%

13.39%

22

>4

% trisomy 7

% trisomy 18

10.15%

$85-9349: Uterine curettage for a 23-year-old gravida 4, para 2 after she delivered a normal term female infant and was diagnosed with choriocarcinoma4 months after delivery. 4273-CW71: Placenta for a 19-year-old gravida 2, para 0 with a history of hydatidiform mole in 5/68. This placenta was from a normal living baby. $86-5747: Fallopian tube from a 23-year-old female with a history of hydatidiform mole. $81-11358: Uterine content for the same patient ($86-5747) with a 2O-weeksize uterus and diagnosis of hydatidiform mole. $93-10205: Uterine curettage for a 30-year-old female at 7 weeks pregnancy and a diagnosis negative for trophoblastic disease. Her termination of pregnancy was due to choriocarcinoma metastatic to the lung. $85-3387: Tissue for a question of a mole. $80-871: Placenta for the same patient (4273-CW71) from another normal living baby. $75-11050-A: Uterine curettage for a 41-year-old gravida 4, para 4 female with a diagnosis of choriocarcinoma.

26

H.F.L. Mark et al.

of early hematogenous metastases, for which the most common sites are the lung, brain, and liver. It has been estimated that about 50% of all choriocarcinomas occur in patients with past histories of complete hydatidiform moles [12]. Studies using locus-specific mini-satellite probes to identify restriction-fragment-length polymorphisms in DNA from these tissues can be useful in distinguishing gestational from non-gestational (germ cell) choriocarcinoma and in documenting the tissue derivation from antecedent complete moles [13]. Microscopically, the typical tumor we studied consisted of clusters of cytotrophoblasts separated by sheets of syncytiotrophoblasts, resulting in a characteristic dimorphic plexiform pattern (Fig. 1). The syncytiotrophoblastic cells were found to be multinucleated, highly developed, and lacking demonstrable mitotic activity, with deeply eosinophilic to basophilic dense cytoplasm. The cytotrophoblastic cells, in contrast, appeared primitive, with germinal components that showed mitotic activity. Typically, these cells were found to be small and mononucleated with a scant amount of pale cytoplasm. Both cell types showed nuclear pleomorphism, hyperchromasia, and nucleoli that were prominent. In Figure 1, portions of the tumor also showed a predominance of cytotrophoblastic or syncytiotrophoblastic cells. Areas of extensive hemorrhage, necrosis, and

vascular/lymphatic invasions were noted. No villi were identified in any of the cases. The presence of trisomic 8 cells in the tissues of a 42year-old woman with gestational trophoblastic disease was first reported by us [1-5]. Unlike other cases in the literature, our patient had no other confounding chromosomal abnormalities besides the trisomy. The existence of cells with trisomy for chromosome 8, site of the MYC (c-MYC) oncogene, was also observed in a number of other cancers. These include prostate carcinoma, fibrosarcoma, Wilms tumor, desmoid tumors, mesoblastic nephroma, leiomyosarcoma, mediastinal germ cell tumor, prostate carcinoma, ovarian carcinoma, salivary gland tmnors, squamous cell carcinoma of the head and neck, benign nevi, colon adenomas, solitary fibrous tumor, endometrial carcinoma, melanomas, sarcomas, the myelodysplastic syndromes, acute non-lymphocytic leukemia, chronic myeloproliferarive disorders, and lymphomas. Trisomy 8 mosaicism is also associated with the Rothmund-Thomson syndrome and Werner's syndrome, both of which are characterized by an increased predisposition towards cancer. Investigators at the Mayo Clinic have observed that trisomy 8 is an indicator of poor prognosis in prostate cancer (Jenkins, personal cominunications). Bullerdiek et ah [14] found trisomy 8 as the only or first aberration in two of 15 tumors

Figure 1 Choriocarcinoma (H &E) demonstrating the alternating arrangement of syncytiotxophoblasts(large arrow) and cytotrophoblasts (small arrow). Note vascular invasion.

FISH Study of GTD

examined, a n d raised the possibility of trisomy 8 as a recurrent clonal abnormality in breast cancer. Cavalli et al. [15] s u b s e q u e n t l y confirmed trisomy 8 in the characterization of a subtype of ductal breast carcinomas. Rohen et al. [16] reported that trisomy 8 was not only found as a clonal aberration in 10 cases but was also the most frequent non-

27

clonal aberration in primary breast carcinoma. Preliminary data from this laboratory have also implicated a role of trisomy 8 in breast cancer [9]. In this paper, we address the question whether the findings of trisomy 8 cells in Mark et al. [1] was an isolated event. We wish to explore the possibility that a sub-

Figure 2 Fluorescence in situ hybridization using a chromosome 8-specific c~-satellite probe on formalin-fixed, paraffin-embedded tumor tissue, demonstrating the presence of abnormal chromosome 8 copy number.

28

set of GTD exists w h i c h is characterized by abnormal chromosome 8 copy n u m b e r of more than two. Towards this end, a pilot study of 10 cases of rare GTD specimens were selected by a pathologist, with the help of a gynecological oncologist, and analyzed blindly. The FISH data, based on eight informative cases successfully completed, together with brief clinical descriptions, are s u m m a r i z e d in Table 1. Two cases did not yield satisfactory results, due to technical difficulties (the sections lifted off the slides, despite the fact that the slides were coated and extra precautions were taken). Of the eight informative cases derived from slides given to us for molecular cytogenetic analysis, two demonstrated the presence of greater than 10% of cells with three chromosome 8 signals. The frequency of trisomic cells in the first case ($85-9349) was 22.5%, whereas the frequency of trisomic cells in the second case ($75-11050-A) was 12.4%, versus a m e a n for normal uterine tissues of 0.2% (with a standard deviation of 0.3%). This latter test specim e n ($75-11050-A), u p o n slide decoding, was found to be the same case studied by Mark et al. [5], thus validating the original study. As metaphase studies were performed on this patient, we know for certain that this case was trisomic and not triploid. However, w i t h o u t further analysis, we could not distinguish trisomy from triploidy in the first of these two cases. FISH using a chromosome 7-specific n-satellite probe was therefore undertaken. Results of this experiment using the control probe were consistent with trisomy and not triploidy. In contrast, two other cases ($80-871 and 4273-CW71) yielded three signals w h e n analyzed by the control chromosome 7 probe as well as the chromosome 8 test probe. The most likely conclusion is that these two cases were triploid, w h i c h is not a surprising finding in GTD (Roberts and Mutter, [17]). The findings of triploid cells versus trisomic cells, however, did not in any way d i m i n i s h the possible significance of high chromosome 8 copy number. On the contrary, it can be argued that the primary effect that an extra chromosome 8 exerts, whatever that turns out to be, persists regardless of the ploidy background. The presence of cells in another patient's tissues with an abnormal chromosome 8 copy n u m b e r even in this small sample raises the provocative question of the etiologic relation of chromosome 8 to the tumor. Although there have been reports of c-MYC RNA in choriocarcinoma (see for example, Sarkar et al., [18]) and c-MYC amplification in choriocarcinoma cell lines (Fujino, [19]; Roberts and Mutter, [17]), the role of chromosome 8, home of cMYC (8q24), in the genesis of GTD is not clear. Our previous findings in breast, however, lend support to the possibility that abnormal copies of chromosome 8 may play a significant role in the development of h u m a n cancer. The finding of yet another case besides the original case (which another observer i n d e p e n d e n t l y identified this time) with abnormal chromosome 8 copy n u m b e r in a total sample of only eight informative cases of these rare tumors is remarkable. Although additional studies from this and other laboratories need to be conducted in order to confirm and extend the results of the present study, the data do suggest that an abnormal chromosome 8 copy

H . F . L . Mark et al. n u m b e r (Fig. 2) as found in our previous study is u n l i k e l y to be an isolated event: At least a small subset of GTD, as in breast and other cancers, may be characterized by abnormal chromosome 8 copy number. To the best of our knowledge, this is the only systematic FISH study thus far of chromosome 8 copy n u m b e r in gestational trophoblastic disease found in the English literature. We would like to thank Dr. Roger Mark and Ms. Mary Rausch for reading the manuscript. The continued support of Dr. Mark and the dedicated staff of the Laboratory of Cytogenetics, FISH and Genotoxicology, which celebrates its third decade of service, is also acknowledged. REFERENCES 1. Mark HFL, Ahearn J, Lathrop JC (1994)'. Constitutional trisomy 8 mosaicism and gestational trophoblastic disease, Cancer Genet Cytogenet 80:150-154. 2. Lathrop JC, Wachtel TJ, Meissner GF (1978): Uterine choriocarcinoma fourteen years following bilateral tubal ligation. Obstet Gynaecol 51(4):477-482. 3. Lathrop JC, Lauchlan S, Nayak R, Ambler M (1988): Clinical characteristics of placental site trophoblastic tumor (PSTT). Gynecol Oncol 31:32-42. 4. Hecht F (1994): Trisomy and disomy in tumors, Cancer Genet Cytogenet 80:171. 5. Mark HFL, Grollino MG, Sulaiman RA, Lathrop JC (1995): Fluorescent in situ hybridization (FISH) assessment of chromosome copy number in gestational trophoblastic disease. Annal Clin Lab Science 25:291-296. 6. Pinkel D, Strawne R, Gray J (1986): Cytogenetic analysis using quantitative high sensitivity, fluorescence hybridization. Proc Nat Acad Sci USA 83:2934-2938. 7. Mark HFL (1994): Fluorescent in situ hybridization as an adjunct to conventional cytogenetics. Ann Clinic Lab Sci. 24(2):153-163. 8. Miranda RN, Mark HFL, Medeiros LJ (1994): Fluorescent in situ hybridization in routinely processed bone marrow aspirate clot and core biopsy sections. Am J Pathol 145:1-6. 9. Afify A, Bland KI, Mark HFL (1996): Fluorescent in situ hybridization assessment of chromosome 8 copy number in breast cancer. Breast Cancer Res Treatment 38:201-208. 10. Kim SY, Lee JS, Ro JY, Gay ML, Hong WK, Hittleman WN (1993): Interphase cytogenetics in paraffin sections of lung tumors by non-isotopic in situ hybridization: mapping/genotype heterogeneity. Am J Pathol 142:307-317. 11. Hopman AHN, Van Hooren E, Van de Kaa CA, Vooijs PG, Ramaekers FCS (1991): Detection of numerical chromosome aberrations using in situ hybridization in paraffin sections of routinely processed bladder cancer. Mod Pathol 4:503-513, 12. Lurain JR, Brewer JI, Torok EE, Halpern B (1982): Gestational trophoblastic disease: treatment results at the Brewer Trophoblastic Disease Center. Obstet Gynecol 60:354-360. 13. Fisher RA, Newlands ES, Jeffrey AJ, Boxer GM (1992): Gestational and nongestational trophoblastic tumors distinguished by DNA analysis. Cancer 69:839-849. 14. Bullerdiek J, Leuschner E, Taquia E, Bonk U, Bartnitzke S (1993): Trisomy 8 as a recurrent clonal abnormality in breast cancer. Cancer Genet Cytogenet 65:64-67. 15. Cavalli LR, Rogatto SR, Rainho CA, dos Santos MJ, Cavalli IJ, Grimaldi DM (1995): Cytogenetic report of a male breast cancer. Cancer Genet Cytogenet 81: 66-71.

F I S H S t u d y of GTD

16. Rohen C, Meyer-Bolte K, Bonk U, Ebel T, Staats B, Leuschner E, Gohla G, Caselitz J, Bartnitzke S, Bullerdiek J (1995): Trisomy 8 and 18 as frequent clonal and single-cell aberrations in 185 primary breast carcinomas. Cancer Genet Cytogenet 80:33-39. 17. Roberts DJ, Mutter GL (1994): Advances in the molecular biology of gestational trophoblastic disease. J Reprod Med 39:201-208.

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18. Sarkar S, Kacinski BM, Kohorn EI, Mekino MJ, Carter D, Blakemore KJ (1986): Demonstration of myc and ras oncogene expression by hybridization in situ in hydatidiform mole and in the BeWo choriocarcinoma cell line. Am J Obstet Gynecol 154:390-393. 19. Fujino T (1987): Analysis of c-onc genes in choriocarcinoma cells. Hokkaido Igaku Zasshi 62:798-807.