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Cytogenetics of trophoblasts from complete hydatidiform moles
Cytogenetics of Trophoblasts from Complete Hydatidiform Moles Rezvan Habibian and Urvashi Surti
ABSTRACT: The risk of developing chariocarcinoma following a complete hydatidiform mole (CHM) is 2000-4000 times greater than the risk following a normal pregnancy. To understand more fully the increased susceptibility of the molar trophoblast to malignant transformation, we separated the trophoblastic cells from the stromal cells in 14 complete moles and cultured them for cytogenetic analysis. The numerical and structural abnormalities found were compared with those found in the trophoblasts from normal pregnancy and malignant choriacarcinoma cell lines. The percentage of polyploid cells was 2.8 times greater in molar trophablasts than in normal trophoblasts. Although we found no consistent chromosomal abnormality in the molar trophoblasts, these cells were significantly more vulnerable to chromosomal breakage than the molar fibroblasts, normal trophoblasts, normal fibroblasts, and maternal decidual cells. Out of a total of 103 breakpoints observed in 338 cells, 42 coincided with known fragile sites, 18 with the location of protooncogenes, 27 with breakpoints reported in other neoplasia, and 18 with breakpoints found in four choriocarcinoma cell lines. The chromosomes in choriocarcinoma cell lines have hypotetraplaid mode and many structural rearrangements. Our results suggest that the genetic instability found in the molar trophoblosts may be responsible for progressive karyotypic changes and greater susceptibility to malignant transformation.
INTRODUCTION
A complete hydatidiform mole (CHM) is a conceptus without an embryo displaying generalized swelling of all placental villi, gross trophoblastic hyperplasia, and is associated with a high risk (5%-10%) of choriocarcinoma and persistent trophoblastic disease. [1] The complete moles have a 46,XX or, rarely, 46,XY karyotype, exclusively of paternal origin, resulting from duplication of a haploid sperm and dispermy respectively [2-5]. The m e c h a n i s m of increased susceptibility to malignant transformation of the trophoblast from the complete moles is not well understood. When molar fragments are grown in tissue culture, p r e d o m i n a n t l y mesenchymal cells (fibroblasts) proliferate. These cells are derived from the i n n e r cell mass, separate from an independent of the trophoblasts. Chromosomal analysis of mesenchymal cells reveals normal-looking karyotypes [2-6]. Any information pertaining to the trophoblast, accordingly, must be derived from a culture of its cells. In contrast to the molar mesenchyme, choriocarcinoma cells show extensive chromosomal rearrangements with a modal n u m b e r in hypotetraploid range [7-9]. Cytogenetic analysis of trophoblasts from complete moles has not been reported. From the Departmentof Pathology, Universityof Pittsburgh. Pittsburgh, PA Address requests for reprints to Dr. Urvashi Surti, Department of Pathology, Magee-Womens Hospital, Forbes Ave. and Halket St., Pittsburgh, PA 15213. Received February 20, 1987; accepted June 8, 1987.
271 © 1987 ElsevierSciencePublishingCo., Inc. 52 Vanderbi|tAve., New York, NY 10017
Cancer Genet Cytogenet29:271 287(1987) 0165-4608/87/$03.50
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R. Habibian and U. Surti
At present there is no way of predicting malignancy at the time of delivery of a hydatidiform mole, and a lengthy hCG follow-up stretching over 1 year or more is necessary [1]. Chemotherapy is initiated if hCG levels rise or plateau, as determined by Beta subunit r a d i o i m m u n o a s s a y , but early diagnosis is important to insure thoroughness and r a p i d i t y of cure by chemotherapy. We report in this paper, the results of the isolation, tissue culture, and chromosomal analysis of trophoblastic cells from CHM. The objectives of our study were to: a) determine what differences exist, if any, between the chromosomes of separated molar trophoblasts and those of fibroblasts from the same complete moles; b) compare the cytogenetic findings from molar trophoblasts with the findings from non-molar control cells in terms of numerical and structural abnormalities; c) determine whether structural and/or numerical chromosomal abnormalities present in choriocarcinoma cells are also present in any of the molar trophoblastic cells from the patients who are at high risk of developing persistent trophoblastic disease or frank choriocarcinoma; and d) determine if cytogenetic analysis of trophoblasts from CHM can be used as a prognostic tool in predicting d e v e l o p m e n t of postmolar choriocarcinoma.
MATERIALS AND METHODS Source of Tissue Molar tissues were obtained from 14 patients at Magee-Womens Hospital as part of an ongoing study on molar pregnancies. A portion of molar tissue was preserved in formalin and used for histologic studies. All the specimens were diagnosed as CHM on the basis of gross m o r p h o l o g y and histopathology. First-trimester placental tissues from therapeutic abortions were similarly examined and their trophoblasts (three cases) and fibroblasts (five cases) were used as controls. Other controls used were d e c i d u a l cells from eight first-trimester placentas (maternal decidual cells were thought to be appropriate because trophoblast and decidua share the same environment in vivo), and the cells from each mole without separation of trophoblast for w h i c h the cells were dissociated with 1% trypsin (GIBCO) in calcium and magnesium-free saline and 0.064% (85 p.g/ml) collagenase respectively. The cultured dissociated cells were mainly fibroblasts sometimes mixed with some trophoblasts. (These cells were used as controls because this or very similar procedures have been used in p u b l i s h e d reports of chromosomes in moles).
Separation of Human Trophoblasts The placental villi were separated from the rest of the tissue and w a s h e d in Waymouth's m e d i u m . The villi were m i n c e d into fine fragments (0.5-1.0 mm), and washed in 10 ml of plain W a y m o u t h ' s culture m e d i u m to remove erythrocytes and debris. The fragments were incubated for 15 minutes following the a d d i t i o n of 20 ml 0.125% trypsin (Worthington, Cooper Biomedical) in W a y m o u t h ' s m e d i u m and 2.5% Hepes buffer and 0.5% antibiotic solution of p e n i c i l l i n and streptomycin (pH 7.5). During incubation, the cells were gently agitated every 5 minutes. The trypsinization was repeated five times and supernatants from each trypsinization were collected into separate 15-ml plastic tubes containing 1 ml of fetal calf serum. The cells were centrifugated, r e s u s p e n d e d in 1 ml of Chang's m e d i u m , plated in Primaria tissue culture flasks (Falcon), and incubated at 37°C in an atmosphere of 95%
Trophoblasts from CHM
273
air, 5% CO2, and high humidity. After 24-48 hours, 4 ml of Chang's m e d i u m was a d d e d to each flask. The caps were screwed tightly, as r e c o m m e n d e d by Lueck and A l a d j e m [10], sealed with parafilm, and incubated u n d i s t u r b e d for 1 week. After 1 week, if the trophoblastic proliferation had begun the culture was then refed with 4 ml of fresh m e d i a and incubated with loose caps. The next day, cultured cells were harvested by standard technique. Air-dried slides were Q-banded with (CMA)2S stain [11].
Cytogenetic Studies A n attempt was made to find at least 20 suitable Q-banded metaphases, or to scan until all the slides had been examined. Each metaphase was scored for chromosome number, and metaphases with morphologically identifiable chromosomes were analyzed for the occurrence of breaks and gaps. Photographs were taken of wellspread metaphases for further cytogenetic analysis. Microscopic analysis of heterom o r p h i s m s located on chromosomes #3 #4, #13, #14, #15, #21, and #22 of molar cells and parental cells was a c c o m p l i s h e d and then confirmed by subsequent use of photographs. The h e t e r o m o r p h i s m s in the moles and parental blood were then c o m p a r e d for the purpose of determining the paternal origin of molar chromosomes and the presence of maternal cell contamination in the trophoblasts culture. All the cases were androgenetic in origin.
Designations of Chromosomoal Abnormalities Karyotypic analysis was performed according to the International System for Human Cytogenetic Nomenclature (ISCN) [12]. The p l o i d y designation used is as follows: h y p o d i p l o i d y , less than 46 chromosomes per cell; h y p e r d i p l o i d y , 4 7 - 5 0 chromosomes; p o l y p l o i d y , as more than 51 chromosomes. The triplet (mar) was used to indicate an unidentified structurally abnormal chromosome (i.e., marker). RESULTS
Clinical Findings Clinical data and follow-up information, including preevacuation hCG level and the time intervals required for return of hCG levels to normal (<5 mIU/ml of serum), are s u m m a r i z e d in Table 1. F o l l o w - u p information was available for 13 cases (cases 1-12 and 14). Of these, ten r e s p o n d e d well to evacuation of the mole, and the urinary hCG fell to normal levels w i t h i n 4.5-10 weeks (mean, 5.6). Postmolar trophoblastic disease d e v e l o p e d in two of 13 patients (15.4%). One patient (case 6) with high initial hCG value, was diagnosed as having choriocarcinoma 6 months after evacuation of the mole. The other (case 8) developed persistent trophoblastic disease with nodules in the lungs after four months. Chemotherapy resulted in complete remission in both patients. The urinary hCG level in case 6 d r o p p e d to a normal level w i t h i n 29.5 weeks, whereas, in case 8 it took m u c h longer (48 weeks).
Cytogenetic Results From 14 cases of CHMs with cultured trophoblasts, 338 cells were scanned for their chromosome number, 304 were analyzed for occurrence of breaks and/or gaps, and 142 k a r y o t y p e d for further structural abnormalities (Table 2). In control, 441 cells were scanned for their c h r o m o s o m e number, 393 cells, analyzed for occurrence of
10.5 7.5 8.5 13.5 15.5 13.5 13.0 16.0 7.5 8.5 NA 12.5 14.0 10.0
1 2 3 4 5 6 ~' 7 8 ~' 9 10 11 12 13 14
17 27 26 22 21 NA 28 22 18 18 24 28 15 16
Mother's age (yr) a
f r o m 14 p a t i e n t s w i t h C H M
55,000 >40,000 NA NA 35,800 4,048,000 896,000 639,200 NA 60,000 78,000 22,920 NA NA
Preevacuation hCG l e v e l (mlU/ml)
NA-Not available.
~Developed post-molar trophoblastic disease.
"Geslational age ranges from 7.5 to 16 wk and maternal age extends from 15 to 28 yr. Case 13 has 11o follow-up information.
G es tat io nal age (wk) °
Clinical data and follow-up informatian
Case number
Table 1
4.5 6.0 6.5 6.0 7.5 29.5 10.0 48.O 6.0 6.5 8.5 5.5 NA 6.0
HCG < 5 (wk after evacuation)
bo ",,3
34 17 38 30 31 42 26 26 13 27 13 6 16 19
338
Case number
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Total
142
18 6 12 14 8 11 17 8 8 8 2 5 7 18
N u m b e r of cells karyotyped
151
4 10 11
0 4 4 86
15 7 20 17 8 24 16 8 2 9 0
46
10 6 7 8 5 8 8 13 3 10 0
<46
Chromosome counts
Cytogenetic results of cultured trophoblasts from 14 CHM
N u m b e r of cells examined
Table 2
101
2 4
2
9 4 11 5 18 10 2 5 8 8 13
>46
94 (28.0)
2 (12.5) 3 [16.0)
0 (0)
(23.5) (23.5) (29.0) (13.3) (58.0) (24.0) (7.7) (19.2) (46.0) (30.0) (100.0)
55/304 (18.0)
9/32 (28.0) 0/17 (0) 3/27 (11.0) 6/30 (20.0) 1/27 (3.7) 2/35 (5.7) 3/26 (11.5) 6/26 (23.0) 5/11 (45.5) 7/24 (29.0) 2/10 (20.0) 2/6 (33.3) 1/14 (7.0) 8/19 (42.0)
cells (%)
cells (%} 8 4 11 4 18 10 2 5 6 8 13
N u m b e r of cells w i t h breaks or gaps/analyzed
N u m b e r of polyploid
107
13 0 5 10 2 3 3 10 18 14 2 8 4 13
Total n u m b e r of breaks and gaps
t',O ",,1
160
91
56
104
9 0 2 5 11
5 10 6 11 8 8 8
8
46
23
0 8 2 0 6
3 0 0 2 0 0 0
0
>46
(O) (87.5) b (25) (0) (25) 21 (13)
0 7 2 0 6
3 (18.7) 0 (0) 0 (0) 1 (4.8) 0 (0) O (0) 0 (0)
0 (0)
N u m b e r of polyploid cells (%) (16.6) (0} (4O) (0)
11/113 (9.7)
1/4 (25) 2/2 (100) 0/5 (0) 1/5 (20) 2/24 (8.3)
1/5 (20) O/5 (0) 1/6 (16.6)
0/17 (0)
1/6 0/9 2/5 O/5
N u m b e r of cells with breaks or gaps/ analyzed cells (%)
hThe morphology of the cells in culture resembled trophoblastic cell morr3hology. This may account for the high incidence of polyploidy.
~No growth of molar fibroblasts. All (:ells examined revealed maternal heteromorphisms.
Total
2 0 4 5 7
2
4 2 5 5 13
16 10 10 21 10 10 12 0a 11 8 8 10 24
8 0 4 8 2 2 4
6
10
1
2 3 4 5 6 7 8 9 10 11 12 13 14
<46
C h r o m o s o m e counts
9 5 5 6 5 5 6
N u m b e r of cells karyotyped
C y t o g e n e t i c a n a l y s i s of c u l t u r e d f i b r o b l a s t s f r o m 14 C H M
N u m b e r of cells examined
Case number
Table 3
12
Total n u m b e r of breaks or gaps
[',0
277
Trophob]asts from CHM
151 -
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20
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1 16
24
32
46
56
64
72
80
92
100 +
CHROMOSOME NO.
Figure 1
Pooled data of modal chromosome number in trophoblasts of 14 CHM.
breaks and/or gaps, and 270 cells karyotyped for further structural abnormalities. The results are shown in Tables 2 and 3.
Analysis of the polymorphic markers for exclusion of maternal cell contamination in trophoblast culture. In this series of 14 specimens, case 1 is heterozygous (46,XY) and the rest are homozygons CHM (46,XX). By using Q-banding and comparing the polymorphic markers on chromosomes #3, #4, #13, #14, #15, #21, and #22 of molar and maternal blood chromosomes, it was found that, of 14 cases, five had some maternal c o n t a m i n a t i o n in trophoblast culture (cases 9-13), and one
Figure 2 Pooled modal chromosome number in control ceils (molar fibroblasts, normal fibroblasts, normal trophoblasts, and decidual cells). 297
50
4O
30
d z
20
10
OL.
16
24
32
46
56
64
72
C H R O M O S O M E NO.
80
92
100+
90 160 101 90
441
NT MF D NF
Total
270
9O 91 59 30
N u m b e r of cells karyotyped
114
21 56 27 10
~46
287
6O 104 61 62
46
63
9 23 13 18
~46
Chromosome counts
NT, normal trophoblasts; MF, molar fibroblasts; D, decidual cells; NF, normal fibroblasts.
N u m b e r of cells e x a m i n e d
C y t o g e n e t i c r e s u l t s of c u l t u r e d c o n t r o l c e l l s
Cell type
Table 4
(10.0) (13.0) (10.9) (11.0)
51 (11.5)
9 21 11 10
N u m b e r of polyploid cells (%)
30/393 (7.6)
7/90 (7.77) 11/113 (9.7) 8/100 (8.0) 4/90 (4.4)
N u m b e r of cells w i t h breaks a n d gaps/ a n a l y z e d cells (%)
36
7 12 10 7
Total n u m b e r of breaks a n d gaps
Co
ix3
Trophoblasts from CHM
279
y i e l d e d all (100%) maternal cells in fibroblast culture (case 9). However, the cultured cells form eight of 14 cases (53.3%) were thought to be trophoblasts without maternal cell contamination (cases 1-8) on the basis of heteromorphisms. Numerical abnormalities. With the exception of case 11, all of 14 CHM examined had variable counts with a modal n u m b e r of 46. Case 11 was characterized by a mode of 92. In case 11, the cultured cells without the separation of trophoblast (trypsin and collagenase) had trophoblast-like m o r p h o l o g y and did not resemble fibroblast morphology. This finding might account for the high percentage of polyp o i d y in the fibroblast control of this case. Figure 1 shows the distribution of chromosome numbers of trophoblastic cells and Figure 2 shows the distribution of chromosome numbers in the controls. As shown, controls are also characterized by variable counts and a mode of 46. The most c o m m o n numerical abnormality found was p o l y p l o i d y . Thirteen of 14 cases (92.8%) had p o l y p l o i d trophoblasts, while molar fibroblasts showed polyp l o i d y in 38% of the cases. As shown in Table 4, 94 of 338 (28.%) trophoblasts examined were polyploid, whereas the percentage of p o l y p l o i d cells in normal trophoblasts, molar fibroblasts, decidual cells and normal fibroblasts, was 10%, 13%, 10.9%, and 11%, respectively. In conclusion, the percentage of p o l y p l o i d y in molar trophoblasts (28%) was 2.4 times greater than that of the controls (11.5%), as indicated in Table 5. Loss of i n d i v i d u a l chromosomes in cells with d i p l o i d and tetraploid range in molar trophoblasts and controls was not significantly different except for the loss of X chromosome. From a total of 123 d i p l o i d and 19 p o l y p l o i d k a r y o t y p e d molar trophoblasts, eight (6.5%) and five (26.3%) cells, respectively, were missing an X chromosome. In the karyotyped cells from the controls, two of 265 (0.75%) d i p l o i d cells and none of five p o l y p l o i d cells were missing an X chromosome. Gain of various chromosomes was found in five cases of trophoblast cultures. All occurred in single cells and are as follows: 4 8 , X Y , + ? X , + 3 (case 1); 92,XXX, - X, + 5, - 8, + 18,?del?(4)(q12q22) (case 3); 93,XXXX,- 4, + 21, + mar, ctb(9)(q22) (case 4); 4 5 , X X , - 4 , - 1 9 , + 2 0 (case 7); and 47,XX,+ 3 (case 14). Extra chromosomes were also found in single cells of molar fibroblasts but not in the other control cells. Those include: 47,XX, + 2 (case 5); 46,XX, + 2 , - 17 (case 8); 93,XXXX, + 2, - 9 21,12p - , + 2mar; and 93,XXXX, + C (case 14). Structural Abnormalities. No c o m m o n chromosome rearrangements were obvious in the 14 cases, but arrangements of c h r o m o s o m e #8 were found more frequently than the others. The chromosome constitution of 16 molar trophoblastic cells with recognizable rearrangements is listed below. All of the rearranged chromosomes were found in single cells only, Case 1 4 4 , X Y , - 1 2 , - 22,t(3,22)(q27;ql 1),ctb(3)(p14),ctg(7)(q32) 46 ,XY, 13q + ,csb(18) (q21 ) 4 6 , X Y , 1 q - ,ctdel(2)(q33) Case 3 46,XX,del(18)(q11),ctg(11)(q21) 92,XXX,- X, + 5, - 8, + 18,?del(4)(q12q22) Case 4 46,XX,6q , 7 , - 10,ctg(3)(p13)+2mar Case 5 92,XXXX,- 17,del(8)(q22),t(8,?)(q22;?)
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R. Habibian and U. Surti
Table 5
Polyploidy in cultured trophoblasts and pooled controls
Cell type
Number of cells examined
Number of polyploid cells (%)
MT C
338 441
94 (28.0) 51 (11.5)
MT, molartrophoblasts:C, controls.
Case 9 4 7 , X X , - 2,4p - , + pvz(?),t(2;8)(?p11;p23),t(4;8)(p12;q24) + mar 89,XXX,- X , - 1 , - 16,dup(1)(p13p32),cxt(1;?),csg(5)(q34),csb(18)(q12), ctb(7)(q32),ctb(14) (q22) Case 11 92,XXXX,10q Case 12 47,X, - X , - 14,de1(1)(p34),del(2)(q21) ,csb(5)(q22) ,del(8)(q24),ctb(8) (p21) + 3mar 48,XX,5p ,10p ,det(X)(q22)+2mar Case 13 4 5 , X X , - 1 4 , - 16,t(14;16)(pl 1;p11),ctb(13)(q14),ctb(6)(p22) Case 14 45,XX,- 13,ctdel(3)(p25) 46,XX,dup(2)(q11--~qter),del(2)(p 16),ctb(9)(q22) 86,XX, - X, X, - 6, - 8, - 16, - 18, - 21, + 2f,del(7)(q22),9q- ,ctg(8)(q24) + mar Breaks and gaps were observed in all of the cases except case 2 and in most of the chromosomes. Figure 3 shows a complete karyotype of a trophoblast from case 10 with five breaks and gaps. Figure 4 illustrates partial karyotypes showing m a n y breaks, gaps, and other chromosome rearrangements from six different cases. Of the 304 analyzed metaphases from molar trophoblasts, 55 cells had breaks and/or gaps (18%), whereas, 30 of 393 (7.6%) analyzed cells in controls had these structural abnormalities (Table 6). A Chi-square test indicated that the cultured trophoblasts of CHM are significantly more vulnerable to chromosomal breakage than the control cells (X2(1] = 17.5; p = 0.01). The two cases that needed chemotherapy did not show increased breakage, compared with the benign cases. The average n u m b e r of breaks and/or gaps per molar trophoblast was four times greater than that in the control cell (Table 6). Figures 5 and 6 summarize the breakpoints of trophoblastic cells and control cells, and show the concordance of these sites with the generally accepted locations of h u m a n cellular oncogenes, k n o w n fragile sites, and the breakpoints involved in chromosomal rearrangements associated with various cancers including choriocarcinoma. Chromosomes #1 and # 8 had the highest frequency of breaks. Chromosomes #15, #20, and #21 were devoid of any structural abnormality. Of a total of 103 breakpoints determined in 304 analyzed metaphases, 42 (41%) coincided with k n o w n fragile sites, 45 (42%) with the breakpoints of chromosomal lesions as found in 20 neoplasia and four choriocarcinoma cell lines, and 18 (17.5%) with the location of protooncogenes [8,21] (Fig. 5). Only 18 (17.5%) of the observed breakpoints in molar trophoblasts coincided with breakpoints found in four choriocarcinoma cell lines. These breakpoints are: lp36, lp34, lq21, lq11, lcen, 4q12, 9q22, 12p12, 12q22, 14p11, and 22q11. Some of the breakpoints appear with relatively greater frequency. Of particular interest are breaks at 8q22, (a fragile site and also the location of the mos cellular oncogene) occurring in seven of 304
F i g u r e 3 Q-banded karyotype of the molar trophoblast from case 10. Arrows indicate chromosomes with breaks or gaps. Heteromorphisms on both homologues of chromosome #3, #13, and #15 are identical.
F i g u r e 4 Q-banded partial karyotypes of molar trophoblasts from six different cases. Abnormal chromosomes are marked with arrows and placed next to their normal homologs.
~o
283
Trophoblasts from CHM
Table 6
Breaks and gaps in cultured trophoblasts and pooled controls
Cell type
Number of cells with breaks or gaps/total analyzed cells (%)
Total number of breaks or gaps/total analyzed cells
Average number of breaks or gaps/cell
MT C
55/304 (18.0) 30/393 (7.6)
107/304 36/393
0.352 0.090
MT, molar trophoblasts; C, controls,
cells. Other vulnerable points are: the centromeric regions of chromosomes #1 and #10 (with four and three breaks, respectively), and 18q12 (fragile site; with three breaks). Figure 5 shows the location of the r e m a i n d e r of the breakpoints and their relative frequencies. Among the total number of karyotypes studied, 25 different markers of u n k n o w n origin were observed. No specific abnormality for trophoblasts of the CHM was found. DISCUSSION The technique described for culturing trophoblasts from CHM has proved satisfactory for cytogenetic analysis. P o l y p l o i d y was the most c o m m o n numerical abnormality. The percentage of p o l y p l o i d y in molar trophoblasts is 2.4 times greater than that of the controls (Table 5). Increased tetraploidy in cultures established from heritable colorectal cancer syndromes has been reported to occur solely in cells with the potential to undergo malignant transformation in vivo [13]. Sugimori et al. [14] s h o w e d that the DNA distribution of normal trophoblasts had a sharp peak at the d i p l o i d region, whereas, choriocarcinoma exhibited an extremely wide distribution of nuclear DNA content with no peak. Two patterns of DNA distribution were found in CHM. One type with DNA values similar to the first trimester normal trophoblast (type I), and the other showed a wide distribution of nuclear DNA (type II). The occurrence of subsequent trophoblastic disease was seen in 17% of type II and none of type I. Type II pattern resembled that of invasive mole. These findings suggest that the gradual increase in w i d t h of the DNA distribution pattern from normal trophoblast to CHM, invasive mole, choriocarcinoma may be related to the disease process. In the present study, only two cases developed postmolar trophoblastic disease and they had 19.2% and 24% p o l y p l o i d cells, respectively. A study analyzing more cases and using flow cytometry to measure the nuclear DNA content of the large n u m b e r of uncultured dissociated cells might help to further elucidate w h e t h e r or not moles with a higher percentage of polyploid cells are more prone to develop postmolar choriocarcinoma than moles with less p o l y p l o i d cells. The m o d a l chromosome numbers of four choriocarcinoma cell l i n e s - - B e W o , Jar, E1Fa, and D o S m i - - w e r e 75, 74/76, 80, and 81, respectively [8]. In the first three cell lines, m a n y chromosomes, especially the A and B group chromosomes, were each present in four copies suggesting that the lines were derived from cells that were in the tetraploid range but that had lost chromosomes during tumor progression or during m a n y passages in culture. For example, BeWo had a m o d e l chromosome n u m b e r of 86 w h e n first established in tissue culture in 1968 [7]. The role of n o n r a n d o m loss of X c h r o m o s o m e in 6.5% of d i p l o i d and 26.3% of p o l y p l o i d k a r y o t y p e d molar trophoblasts is not clear. It is interesting to note that a missing X c h r o m o s o m e alongside other karyotypic anomalies has been seen in acute myeloblastic leukemia (AML), meningioma, and l y m p h o m a [15]. A missing X
284
R. Habibian and U. Surti
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Figure 5 Diagrammatic representation of human chromosomes showing a total of 107 breakpoints (78 breaks and 29 gaps) observed in 304 trophoblastic cells from 13 CHM. Pertinent bands are marked on the left of each chromosome, and locations of protooncogenes are marked on the right. Note chromosomes #8, and #1 have the highest frequency of breaks. A total of four breaks are not shown because the exact breakpoints could not be determined. * breakpoints; • fragile sites; ~ location of observed breakpoints in four choriocarcinoma cell lines; • location of observed breakpoints in 20 neoplasias.
c h r o m o s o m e is also reported in older w o m e n and in w o m e n w i t h recurrent pregnancy loss. The o n l y other chromosomal abnormality reported in CHM involves clones (most probably fibroblasts) w i t h additional c h r o m o s o m e 20 in longitudinal cytogenetic studies [16]. In our laboratory long-term cytogenetics of fibroblasts from two benign cases of CHM also s h o w e d clones w i t h extra c h r o m o s o m e # 2 0 and # 2 . Because extra c h r o m o s o m e s # 2 0 and # 2 frequently are f o u n d in amniotic fluid cells, it is u n l i k e l y that these play a role in malignant transformation of trophoblast.
285
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22
* bcl-2 •
18
y
X
Figure 6 Schematic presentation of human chromosomes showing 36 breakpoints (nine gaps and 23 breaks} in a total of 393 control cells. * breakpoints in decidual cells (one breakpoint is not shown); • breakpoints in normal trophoblasts; [3 breakpoints in molar fibroblasts; 0 breakpoints in normal fibroblasts (one breakpoint is not shown}; ~> location of observed breakpoints in four choriocarcinoma cell lines; I~ location of observed breakpoints in 20 neoplasias; • fragile sites.
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The cytogenetic evaluation of the choriocarcinomas together with that of the antecedent molar pregnancy would be of interest. Use of improved techniques for the culture of cells and high-resolution b a n d i n g of the chromosomes may reveal a specific chromosomal rearrangement for choriocarcinoma. A comparison of the karyotypes for structural abnormalities i n c l u d i n g the occurrence of breaks and gaps, revealed the preferential i n v o l v e m e n t of chromosome # 8 in 50% of 14 cases, followed by chromosomes #1 and # 2 in 43%, and chromosomes #3, #6, #7, and #14 each, in 36% of the cases. Probably the most frequently involved chromosome in h u m a n neoplasia is # 8 [17]. The t(8;21) is a consistent abnormality in AML and acute n o n l y m p h o c y t i c leukemia (ANLL), and t(8;14) is characteristic for Burkitt's lymphoma. Various rearrangements of chromosome #1 have been reported in four choriocarcinoma cell lines [8, 18], in over 80% of ovarian cancer, and in m a n y other tumors [19, 20]. Because choriocarcinoma cells have m a n y different chromosomal rearrangements, it is reasonable to suggest that the significantly high frequency of breaks in molar trophoblast cells may predispose them to certain chromosomal rearrangements, which play an important role in the pathway leading to the development of trophoblastic malignancy. The extent of chromosomal breakage needs further confirmation. Studies using molecular techniques and prophase b a n d i n g are necessary to evaluate the concordance of the breakpoints found. Supported by NIH Grants CA43331-01 and HD17572 and Magee Womens Hospital Research Fund.
REFERENCES
1. Park WW (1971): Choriocarcinoma: A Study of Its Pathology. Philadelphia, F. A. Davis Co., pp. 55-138. 2. Kajii T, Ohama K (1977): Androgenetic origin of hydatidiform mole, Nature 268: 633-634. 3. Surti U, Szulman AE, O'Brien S (1979): Complete (classic) hydatidiform mole with 46,XY karyotype of paternal origin. Hum Genet 51:153-155. 4. Jacobs PA, Wilson CM, Sprenkle JA, Rosenshein NB, Migeon BR (1980): Mechanism of origin of complete hydatidiform moles. Nature 286:714-716. 5. Surti U, Szulman AE, O'Brien S (1982): Dispermic origin and clinical outcome of three complete hydatidiform moles with 46,XY karyotype. Am J Obstet Gynecol 144:84-87. 6. Szulman AE, Surti U (1978): The syndromes of hydatidiform moles: I. Cytogenetic and morphologic correlations. Am J Obstet Gynecol 131: 665-671. 7. Pattilo RA, Gey GO (1969): The establishment of a cell line of human hormone-synthesizing trophoblastic cells in vitro. Cancer Res 28:1231-1236. 8. Sheppard DM, Fisher RA, Lawler SD (1985): Karyotypic analysis and chromosome polymorphisms in four choriocarcinoma cell lines. Cancer Genet Cytogenet 16: 251-258, 9. Sasaki S, Katayama PK, Roesler M, Pattillo RA, Mattingly RF, Ohkawa K (1982): Cytogenetic analysis of choriocarcinoma cell lines. Acta Obstet Gynaec Jpn 34: 2253-2254. 10. Lueck J, Aladjam (1980): Time-lapse study of normal human trophoblast in vitro. Am J Obstet Gynecol 138:288-292. 11. Deugau KV, Vande Sande JH, Lin CC (1978): (CMA)2S: A double intercalating analogue of quinacrine for chromosome banding. Am J Hum Gen 30: 78A. 12. ISCN (1985): An International System for Human Cytogenetic Nomenclature, Harnden DG and Klinger HP (eds.); published in collaboration with Cytogenet Cell Genet (Karger, Basel, 1985); also in Birth Defects: Original Article Series, Vol. 21, No. 1 (March of Dimes Birth Defects Foundation, New York, 1985). 13. Danes BS (1978): Increased in vitro tetraploidy: Tissue specific within the heritable colorectal cancer syndromes with polyposis coli. Cancer 41:2330-2334.
T r o p h o b l a s t s f r o m CHM
28 7
14. Sugimori H, Kashimura Y, Kashimura M, Taki I (1978): Nuclear DNA content of trophoblastic tumors. ACTA Cytologica 22:542-545. 15. Zankle H, Seidel H, Zang KD (1975): Sex-chromosome loss in human tumours. Lancet i:221. 16. Hunt PA, Jacobs PA (1985): In vitro growth and chromosome constitution of placental cells. II. Hydatidiform moles. Cytogenet Cell Genet 39:7-13. 17. Sandberg AA (1980): The Chromosomes in Human Cancer and Leukemia. Elsevier, New York, pp. 579.. 18. Wake N, Tanaka K, Chapman V. Matsui S, Sandberg AA (1981): Chromosomes and cellular origin of choriocarcinoma. Cancer Res 41:3137-3143. 19. Whang-Peng J, Knutsen T, Douglass EC, Chu E, Ozolo RF, Hogen WM, Young RC (1984): Cytogenetic studies in ovarian cancer. Cancer Genet Cytogen 11:91-106. 20. Rowley JD (1978) Abnormalities of chromosome no. 1 in haematological malignancies. Lancet i:554 555. 21. Yunis JJ, Soreng AL (1984): Constitutive fragile sites and cancer. Science 226:1199-1204.
ABSTRACT: The risk of developing chariocarcinoma following a complete hydatidiform mole (CHM) is 2000-4000 times greater than the risk following a normal pregnancy. To understand more fully the increased susceptibility of the molar trophoblast to malignant transformation, we separated the trophoblastic cells from the stromal cells in 14 complete moles and cultured them for cytogenetic analysis. The numerical and structural abnormalities found were compared with those found in the trophoblasts from normal pregnancy and malignant choriacarcinoma cell lines. The percentage of polyploid cells was 2.8 times greater in molar trophablasts than in normal trophoblasts. Although we found no consistent chromosomal abnormality in the molar trophoblasts, these cells were significantly more vulnerable to chromosomal breakage than the molar fibroblasts, normal trophoblasts, normal fibroblasts, and maternal decidual cells. Out of a total of 103 breakpoints observed in 338 cells, 42 coincided with known fragile sites, 18 with the location of protooncogenes, 27 with breakpoints reported in other neoplasia, and 18 with breakpoints found in four choriocarcinoma cell lines. The chromosomes in choriocarcinoma cell lines have hypotetraplaid mode and many structural rearrangements. Our results suggest that the genetic instability found in the molar trophoblosts may be responsible for progressive karyotypic changes and greater susceptibility to malignant transformation.
INTRODUCTION
A complete hydatidiform mole (CHM) is a conceptus without an embryo displaying generalized swelling of all placental villi, gross trophoblastic hyperplasia, and is associated with a high risk (5%-10%) of choriocarcinoma and persistent trophoblastic disease. [1] The complete moles have a 46,XX or, rarely, 46,XY karyotype, exclusively of paternal origin, resulting from duplication of a haploid sperm and dispermy respectively [2-5]. The m e c h a n i s m of increased susceptibility to malignant transformation of the trophoblast from the complete moles is not well understood. When molar fragments are grown in tissue culture, p r e d o m i n a n t l y mesenchymal cells (fibroblasts) proliferate. These cells are derived from the i n n e r cell mass, separate from an independent of the trophoblasts. Chromosomal analysis of mesenchymal cells reveals normal-looking karyotypes [2-6]. Any information pertaining to the trophoblast, accordingly, must be derived from a culture of its cells. In contrast to the molar mesenchyme, choriocarcinoma cells show extensive chromosomal rearrangements with a modal n u m b e r in hypotetraploid range [7-9]. Cytogenetic analysis of trophoblasts from complete moles has not been reported. From the Departmentof Pathology, Universityof Pittsburgh. Pittsburgh, PA Address requests for reprints to Dr. Urvashi Surti, Department of Pathology, Magee-Womens Hospital, Forbes Ave. and Halket St., Pittsburgh, PA 15213. Received February 20, 1987; accepted June 8, 1987.
271 © 1987 ElsevierSciencePublishingCo., Inc. 52 Vanderbi|tAve., New York, NY 10017
Cancer Genet Cytogenet29:271 287(1987) 0165-4608/87/$03.50
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At present there is no way of predicting malignancy at the time of delivery of a hydatidiform mole, and a lengthy hCG follow-up stretching over 1 year or more is necessary [1]. Chemotherapy is initiated if hCG levels rise or plateau, as determined by Beta subunit r a d i o i m m u n o a s s a y , but early diagnosis is important to insure thoroughness and r a p i d i t y of cure by chemotherapy. We report in this paper, the results of the isolation, tissue culture, and chromosomal analysis of trophoblastic cells from CHM. The objectives of our study were to: a) determine what differences exist, if any, between the chromosomes of separated molar trophoblasts and those of fibroblasts from the same complete moles; b) compare the cytogenetic findings from molar trophoblasts with the findings from non-molar control cells in terms of numerical and structural abnormalities; c) determine whether structural and/or numerical chromosomal abnormalities present in choriocarcinoma cells are also present in any of the molar trophoblastic cells from the patients who are at high risk of developing persistent trophoblastic disease or frank choriocarcinoma; and d) determine if cytogenetic analysis of trophoblasts from CHM can be used as a prognostic tool in predicting d e v e l o p m e n t of postmolar choriocarcinoma.
MATERIALS AND METHODS Source of Tissue Molar tissues were obtained from 14 patients at Magee-Womens Hospital as part of an ongoing study on molar pregnancies. A portion of molar tissue was preserved in formalin and used for histologic studies. All the specimens were diagnosed as CHM on the basis of gross m o r p h o l o g y and histopathology. First-trimester placental tissues from therapeutic abortions were similarly examined and their trophoblasts (three cases) and fibroblasts (five cases) were used as controls. Other controls used were d e c i d u a l cells from eight first-trimester placentas (maternal decidual cells were thought to be appropriate because trophoblast and decidua share the same environment in vivo), and the cells from each mole without separation of trophoblast for w h i c h the cells were dissociated with 1% trypsin (GIBCO) in calcium and magnesium-free saline and 0.064% (85 p.g/ml) collagenase respectively. The cultured dissociated cells were mainly fibroblasts sometimes mixed with some trophoblasts. (These cells were used as controls because this or very similar procedures have been used in p u b l i s h e d reports of chromosomes in moles).
Separation of Human Trophoblasts The placental villi were separated from the rest of the tissue and w a s h e d in Waymouth's m e d i u m . The villi were m i n c e d into fine fragments (0.5-1.0 mm), and washed in 10 ml of plain W a y m o u t h ' s culture m e d i u m to remove erythrocytes and debris. The fragments were incubated for 15 minutes following the a d d i t i o n of 20 ml 0.125% trypsin (Worthington, Cooper Biomedical) in W a y m o u t h ' s m e d i u m and 2.5% Hepes buffer and 0.5% antibiotic solution of p e n i c i l l i n and streptomycin (pH 7.5). During incubation, the cells were gently agitated every 5 minutes. The trypsinization was repeated five times and supernatants from each trypsinization were collected into separate 15-ml plastic tubes containing 1 ml of fetal calf serum. The cells were centrifugated, r e s u s p e n d e d in 1 ml of Chang's m e d i u m , plated in Primaria tissue culture flasks (Falcon), and incubated at 37°C in an atmosphere of 95%
Trophoblasts from CHM
273
air, 5% CO2, and high humidity. After 24-48 hours, 4 ml of Chang's m e d i u m was a d d e d to each flask. The caps were screwed tightly, as r e c o m m e n d e d by Lueck and A l a d j e m [10], sealed with parafilm, and incubated u n d i s t u r b e d for 1 week. After 1 week, if the trophoblastic proliferation had begun the culture was then refed with 4 ml of fresh m e d i a and incubated with loose caps. The next day, cultured cells were harvested by standard technique. Air-dried slides were Q-banded with (CMA)2S stain [11].
Cytogenetic Studies A n attempt was made to find at least 20 suitable Q-banded metaphases, or to scan until all the slides had been examined. Each metaphase was scored for chromosome number, and metaphases with morphologically identifiable chromosomes were analyzed for the occurrence of breaks and gaps. Photographs were taken of wellspread metaphases for further cytogenetic analysis. Microscopic analysis of heterom o r p h i s m s located on chromosomes #3 #4, #13, #14, #15, #21, and #22 of molar cells and parental cells was a c c o m p l i s h e d and then confirmed by subsequent use of photographs. The h e t e r o m o r p h i s m s in the moles and parental blood were then c o m p a r e d for the purpose of determining the paternal origin of molar chromosomes and the presence of maternal cell contamination in the trophoblasts culture. All the cases were androgenetic in origin.
Designations of Chromosomoal Abnormalities Karyotypic analysis was performed according to the International System for Human Cytogenetic Nomenclature (ISCN) [12]. The p l o i d y designation used is as follows: h y p o d i p l o i d y , less than 46 chromosomes per cell; h y p e r d i p l o i d y , 4 7 - 5 0 chromosomes; p o l y p l o i d y , as more than 51 chromosomes. The triplet (mar) was used to indicate an unidentified structurally abnormal chromosome (i.e., marker). RESULTS
Clinical Findings Clinical data and follow-up information, including preevacuation hCG level and the time intervals required for return of hCG levels to normal (<5 mIU/ml of serum), are s u m m a r i z e d in Table 1. F o l l o w - u p information was available for 13 cases (cases 1-12 and 14). Of these, ten r e s p o n d e d well to evacuation of the mole, and the urinary hCG fell to normal levels w i t h i n 4.5-10 weeks (mean, 5.6). Postmolar trophoblastic disease d e v e l o p e d in two of 13 patients (15.4%). One patient (case 6) with high initial hCG value, was diagnosed as having choriocarcinoma 6 months after evacuation of the mole. The other (case 8) developed persistent trophoblastic disease with nodules in the lungs after four months. Chemotherapy resulted in complete remission in both patients. The urinary hCG level in case 6 d r o p p e d to a normal level w i t h i n 29.5 weeks, whereas, in case 8 it took m u c h longer (48 weeks).
Cytogenetic Results From 14 cases of CHMs with cultured trophoblasts, 338 cells were scanned for their chromosome number, 304 were analyzed for occurrence of breaks and/or gaps, and 142 k a r y o t y p e d for further structural abnormalities (Table 2). In control, 441 cells were scanned for their c h r o m o s o m e number, 393 cells, analyzed for occurrence of
10.5 7.5 8.5 13.5 15.5 13.5 13.0 16.0 7.5 8.5 NA 12.5 14.0 10.0
1 2 3 4 5 6 ~' 7 8 ~' 9 10 11 12 13 14
17 27 26 22 21 NA 28 22 18 18 24 28 15 16
Mother's age (yr) a
f r o m 14 p a t i e n t s w i t h C H M
55,000 >40,000 NA NA 35,800 4,048,000 896,000 639,200 NA 60,000 78,000 22,920 NA NA
Preevacuation hCG l e v e l (mlU/ml)
NA-Not available.
~Developed post-molar trophoblastic disease.
"Geslational age ranges from 7.5 to 16 wk and maternal age extends from 15 to 28 yr. Case 13 has 11o follow-up information.
G es tat io nal age (wk) °
Clinical data and follow-up informatian
Case number
Table 1
4.5 6.0 6.5 6.0 7.5 29.5 10.0 48.O 6.0 6.5 8.5 5.5 NA 6.0
HCG < 5 (wk after evacuation)
bo ",,3
34 17 38 30 31 42 26 26 13 27 13 6 16 19
338
Case number
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Total
142
18 6 12 14 8 11 17 8 8 8 2 5 7 18
N u m b e r of cells karyotyped
151
4 10 11
0 4 4 86
15 7 20 17 8 24 16 8 2 9 0
46
10 6 7 8 5 8 8 13 3 10 0
<46
Chromosome counts
Cytogenetic results of cultured trophoblasts from 14 CHM
N u m b e r of cells examined
Table 2
101
2 4
2
9 4 11 5 18 10 2 5 8 8 13
>46
94 (28.0)
2 (12.5) 3 [16.0)
0 (0)
(23.5) (23.5) (29.0) (13.3) (58.0) (24.0) (7.7) (19.2) (46.0) (30.0) (100.0)
55/304 (18.0)
9/32 (28.0) 0/17 (0) 3/27 (11.0) 6/30 (20.0) 1/27 (3.7) 2/35 (5.7) 3/26 (11.5) 6/26 (23.0) 5/11 (45.5) 7/24 (29.0) 2/10 (20.0) 2/6 (33.3) 1/14 (7.0) 8/19 (42.0)
cells (%)
cells (%} 8 4 11 4 18 10 2 5 6 8 13
N u m b e r of cells w i t h breaks or gaps/analyzed
N u m b e r of polyploid
107
13 0 5 10 2 3 3 10 18 14 2 8 4 13
Total n u m b e r of breaks and gaps
t',O ",,1
160
91
56
104
9 0 2 5 11
5 10 6 11 8 8 8
8
46
23
0 8 2 0 6
3 0 0 2 0 0 0
0
>46
(O) (87.5) b (25) (0) (25) 21 (13)
0 7 2 0 6
3 (18.7) 0 (0) 0 (0) 1 (4.8) 0 (0) O (0) 0 (0)
0 (0)
N u m b e r of polyploid cells (%) (16.6) (0} (4O) (0)
11/113 (9.7)
1/4 (25) 2/2 (100) 0/5 (0) 1/5 (20) 2/24 (8.3)
1/5 (20) O/5 (0) 1/6 (16.6)
0/17 (0)
1/6 0/9 2/5 O/5
N u m b e r of cells with breaks or gaps/ analyzed cells (%)
hThe morphology of the cells in culture resembled trophoblastic cell morr3hology. This may account for the high incidence of polyploidy.
~No growth of molar fibroblasts. All (:ells examined revealed maternal heteromorphisms.
Total
2 0 4 5 7
2
4 2 5 5 13
16 10 10 21 10 10 12 0a 11 8 8 10 24
8 0 4 8 2 2 4
6
10
1
2 3 4 5 6 7 8 9 10 11 12 13 14
<46
C h r o m o s o m e counts
9 5 5 6 5 5 6
N u m b e r of cells karyotyped
C y t o g e n e t i c a n a l y s i s of c u l t u r e d f i b r o b l a s t s f r o m 14 C H M
N u m b e r of cells examined
Case number
Table 3
12
Total n u m b e r of breaks or gaps
[',0
277
Trophob]asts from CHM
151 -
m
40-
u.~ O~
30
5=
20
,,-I --I UJ
(~ II tUC
10
1 16
24
32
46
56
64
72
80
92
100 +
CHROMOSOME NO.
Figure 1
Pooled data of modal chromosome number in trophoblasts of 14 CHM.
breaks and/or gaps, and 270 cells karyotyped for further structural abnormalities. The results are shown in Tables 2 and 3.
Analysis of the polymorphic markers for exclusion of maternal cell contamination in trophoblast culture. In this series of 14 specimens, case 1 is heterozygous (46,XY) and the rest are homozygons CHM (46,XX). By using Q-banding and comparing the polymorphic markers on chromosomes #3, #4, #13, #14, #15, #21, and #22 of molar and maternal blood chromosomes, it was found that, of 14 cases, five had some maternal c o n t a m i n a t i o n in trophoblast culture (cases 9-13), and one
Figure 2 Pooled modal chromosome number in control ceils (molar fibroblasts, normal fibroblasts, normal trophoblasts, and decidual cells). 297
50
4O
30
d z
20
10
OL.
16
24
32
46
56
64
72
C H R O M O S O M E NO.
80
92
100+
90 160 101 90
441
NT MF D NF
Total
270
9O 91 59 30
N u m b e r of cells karyotyped
114
21 56 27 10
~46
287
6O 104 61 62
46
63
9 23 13 18
~46
Chromosome counts
NT, normal trophoblasts; MF, molar fibroblasts; D, decidual cells; NF, normal fibroblasts.
N u m b e r of cells e x a m i n e d
C y t o g e n e t i c r e s u l t s of c u l t u r e d c o n t r o l c e l l s
Cell type
Table 4
(10.0) (13.0) (10.9) (11.0)
51 (11.5)
9 21 11 10
N u m b e r of polyploid cells (%)
30/393 (7.6)
7/90 (7.77) 11/113 (9.7) 8/100 (8.0) 4/90 (4.4)
N u m b e r of cells w i t h breaks a n d gaps/ a n a l y z e d cells (%)
36
7 12 10 7
Total n u m b e r of breaks a n d gaps
Co
ix3
Trophoblasts from CHM
279
y i e l d e d all (100%) maternal cells in fibroblast culture (case 9). However, the cultured cells form eight of 14 cases (53.3%) were thought to be trophoblasts without maternal cell contamination (cases 1-8) on the basis of heteromorphisms. Numerical abnormalities. With the exception of case 11, all of 14 CHM examined had variable counts with a modal n u m b e r of 46. Case 11 was characterized by a mode of 92. In case 11, the cultured cells without the separation of trophoblast (trypsin and collagenase) had trophoblast-like m o r p h o l o g y and did not resemble fibroblast morphology. This finding might account for the high percentage of polyp o i d y in the fibroblast control of this case. Figure 1 shows the distribution of chromosome numbers of trophoblastic cells and Figure 2 shows the distribution of chromosome numbers in the controls. As shown, controls are also characterized by variable counts and a mode of 46. The most c o m m o n numerical abnormality found was p o l y p l o i d y . Thirteen of 14 cases (92.8%) had p o l y p l o i d trophoblasts, while molar fibroblasts showed polyp l o i d y in 38% of the cases. As shown in Table 4, 94 of 338 (28.%) trophoblasts examined were polyploid, whereas the percentage of p o l y p l o i d cells in normal trophoblasts, molar fibroblasts, decidual cells and normal fibroblasts, was 10%, 13%, 10.9%, and 11%, respectively. In conclusion, the percentage of p o l y p l o i d y in molar trophoblasts (28%) was 2.4 times greater than that of the controls (11.5%), as indicated in Table 5. Loss of i n d i v i d u a l chromosomes in cells with d i p l o i d and tetraploid range in molar trophoblasts and controls was not significantly different except for the loss of X chromosome. From a total of 123 d i p l o i d and 19 p o l y p l o i d k a r y o t y p e d molar trophoblasts, eight (6.5%) and five (26.3%) cells, respectively, were missing an X chromosome. In the karyotyped cells from the controls, two of 265 (0.75%) d i p l o i d cells and none of five p o l y p l o i d cells were missing an X chromosome. Gain of various chromosomes was found in five cases of trophoblast cultures. All occurred in single cells and are as follows: 4 8 , X Y , + ? X , + 3 (case 1); 92,XXX, - X, + 5, - 8, + 18,?del?(4)(q12q22) (case 3); 93,XXXX,- 4, + 21, + mar, ctb(9)(q22) (case 4); 4 5 , X X , - 4 , - 1 9 , + 2 0 (case 7); and 47,XX,+ 3 (case 14). Extra chromosomes were also found in single cells of molar fibroblasts but not in the other control cells. Those include: 47,XX, + 2 (case 5); 46,XX, + 2 , - 17 (case 8); 93,XXXX, + 2, - 9 21,12p - , + 2mar; and 93,XXXX, + C (case 14). Structural Abnormalities. No c o m m o n chromosome rearrangements were obvious in the 14 cases, but arrangements of c h r o m o s o m e #8 were found more frequently than the others. The chromosome constitution of 16 molar trophoblastic cells with recognizable rearrangements is listed below. All of the rearranged chromosomes were found in single cells only, Case 1 4 4 , X Y , - 1 2 , - 22,t(3,22)(q27;ql 1),ctb(3)(p14),ctg(7)(q32) 46 ,XY, 13q + ,csb(18) (q21 ) 4 6 , X Y , 1 q - ,ctdel(2)(q33) Case 3 46,XX,del(18)(q11),ctg(11)(q21) 92,XXX,- X, + 5, - 8, + 18,?del(4)(q12q22) Case 4 46,XX,6q , 7 , - 10,ctg(3)(p13)+2mar Case 5 92,XXXX,- 17,del(8)(q22),t(8,?)(q22;?)
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R. Habibian and U. Surti
Table 5
Polyploidy in cultured trophoblasts and pooled controls
Cell type
Number of cells examined
Number of polyploid cells (%)
MT C
338 441
94 (28.0) 51 (11.5)
MT, molartrophoblasts:C, controls.
Case 9 4 7 , X X , - 2,4p - , + pvz(?),t(2;8)(?p11;p23),t(4;8)(p12;q24) + mar 89,XXX,- X , - 1 , - 16,dup(1)(p13p32),cxt(1;?),csg(5)(q34),csb(18)(q12), ctb(7)(q32),ctb(14) (q22) Case 11 92,XXXX,10q Case 12 47,X, - X , - 14,de1(1)(p34),del(2)(q21) ,csb(5)(q22) ,del(8)(q24),ctb(8) (p21) + 3mar 48,XX,5p ,10p ,det(X)(q22)+2mar Case 13 4 5 , X X , - 1 4 , - 16,t(14;16)(pl 1;p11),ctb(13)(q14),ctb(6)(p22) Case 14 45,XX,- 13,ctdel(3)(p25) 46,XX,dup(2)(q11--~qter),del(2)(p 16),ctb(9)(q22) 86,XX, - X, X, - 6, - 8, - 16, - 18, - 21, + 2f,del(7)(q22),9q- ,ctg(8)(q24) + mar Breaks and gaps were observed in all of the cases except case 2 and in most of the chromosomes. Figure 3 shows a complete karyotype of a trophoblast from case 10 with five breaks and gaps. Figure 4 illustrates partial karyotypes showing m a n y breaks, gaps, and other chromosome rearrangements from six different cases. Of the 304 analyzed metaphases from molar trophoblasts, 55 cells had breaks and/or gaps (18%), whereas, 30 of 393 (7.6%) analyzed cells in controls had these structural abnormalities (Table 6). A Chi-square test indicated that the cultured trophoblasts of CHM are significantly more vulnerable to chromosomal breakage than the control cells (X2(1] = 17.5; p = 0.01). The two cases that needed chemotherapy did not show increased breakage, compared with the benign cases. The average n u m b e r of breaks and/or gaps per molar trophoblast was four times greater than that in the control cell (Table 6). Figures 5 and 6 summarize the breakpoints of trophoblastic cells and control cells, and show the concordance of these sites with the generally accepted locations of h u m a n cellular oncogenes, k n o w n fragile sites, and the breakpoints involved in chromosomal rearrangements associated with various cancers including choriocarcinoma. Chromosomes #1 and # 8 had the highest frequency of breaks. Chromosomes #15, #20, and #21 were devoid of any structural abnormality. Of a total of 103 breakpoints determined in 304 analyzed metaphases, 42 (41%) coincided with k n o w n fragile sites, 45 (42%) with the breakpoints of chromosomal lesions as found in 20 neoplasia and four choriocarcinoma cell lines, and 18 (17.5%) with the location of protooncogenes [8,21] (Fig. 5). Only 18 (17.5%) of the observed breakpoints in molar trophoblasts coincided with breakpoints found in four choriocarcinoma cell lines. These breakpoints are: lp36, lp34, lq21, lq11, lcen, 4q12, 9q22, 12p12, 12q22, 14p11, and 22q11. Some of the breakpoints appear with relatively greater frequency. Of particular interest are breaks at 8q22, (a fragile site and also the location of the mos cellular oncogene) occurring in seven of 304
F i g u r e 3 Q-banded karyotype of the molar trophoblast from case 10. Arrows indicate chromosomes with breaks or gaps. Heteromorphisms on both homologues of chromosome #3, #13, and #15 are identical.
F i g u r e 4 Q-banded partial karyotypes of molar trophoblasts from six different cases. Abnormal chromosomes are marked with arrows and placed next to their normal homologs.
~o
283
Trophoblasts from CHM
Table 6
Breaks and gaps in cultured trophoblasts and pooled controls
Cell type
Number of cells with breaks or gaps/total analyzed cells (%)
Total number of breaks or gaps/total analyzed cells
Average number of breaks or gaps/cell
MT C
55/304 (18.0) 30/393 (7.6)
107/304 36/393
0.352 0.090
MT, molar trophoblasts; C, controls,
cells. Other vulnerable points are: the centromeric regions of chromosomes #1 and #10 (with four and three breaks, respectively), and 18q12 (fragile site; with three breaks). Figure 5 shows the location of the r e m a i n d e r of the breakpoints and their relative frequencies. Among the total number of karyotypes studied, 25 different markers of u n k n o w n origin were observed. No specific abnormality for trophoblasts of the CHM was found. DISCUSSION The technique described for culturing trophoblasts from CHM has proved satisfactory for cytogenetic analysis. P o l y p l o i d y was the most c o m m o n numerical abnormality. The percentage of p o l y p l o i d y in molar trophoblasts is 2.4 times greater than that of the controls (Table 5). Increased tetraploidy in cultures established from heritable colorectal cancer syndromes has been reported to occur solely in cells with the potential to undergo malignant transformation in vivo [13]. Sugimori et al. [14] s h o w e d that the DNA distribution of normal trophoblasts had a sharp peak at the d i p l o i d region, whereas, choriocarcinoma exhibited an extremely wide distribution of nuclear DNA content with no peak. Two patterns of DNA distribution were found in CHM. One type with DNA values similar to the first trimester normal trophoblast (type I), and the other showed a wide distribution of nuclear DNA (type II). The occurrence of subsequent trophoblastic disease was seen in 17% of type II and none of type I. Type II pattern resembled that of invasive mole. These findings suggest that the gradual increase in w i d t h of the DNA distribution pattern from normal trophoblast to CHM, invasive mole, choriocarcinoma may be related to the disease process. In the present study, only two cases developed postmolar trophoblastic disease and they had 19.2% and 24% p o l y p l o i d cells, respectively. A study analyzing more cases and using flow cytometry to measure the nuclear DNA content of the large n u m b e r of uncultured dissociated cells might help to further elucidate w h e t h e r or not moles with a higher percentage of polyploid cells are more prone to develop postmolar choriocarcinoma than moles with less p o l y p l o i d cells. The m o d a l chromosome numbers of four choriocarcinoma cell l i n e s - - B e W o , Jar, E1Fa, and D o S m i - - w e r e 75, 74/76, 80, and 81, respectively [8]. In the first three cell lines, m a n y chromosomes, especially the A and B group chromosomes, were each present in four copies suggesting that the lines were derived from cells that were in the tetraploid range but that had lost chromosomes during tumor progression or during m a n y passages in culture. For example, BeWo had a m o d e l chromosome n u m b e r of 86 w h e n first established in tissue culture in 1968 [7]. The role of n o n r a n d o m loss of X c h r o m o s o m e in 6.5% of d i p l o i d and 26.3% of p o l y p l o i d k a r y o t y p e d molar trophoblasts is not clear. It is interesting to note that a missing X c h r o m o s o m e alongside other karyotypic anomalies has been seen in acute myeloblastic leukemia (AML), meningioma, and l y m p h o m a [15]. A missing X
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c h r o m o s o m e is also reported in older w o m e n and in w o m e n w i t h recurrent pregnancy loss. The o n l y other chromosomal abnormality reported in CHM involves clones (most probably fibroblasts) w i t h additional c h r o m o s o m e 20 in longitudinal cytogenetic studies [16]. In our laboratory long-term cytogenetics of fibroblasts from two benign cases of CHM also s h o w e d clones w i t h extra c h r o m o s o m e # 2 0 and # 2 . Because extra c h r o m o s o m e s # 2 0 and # 2 frequently are f o u n d in amniotic fluid cells, it is u n l i k e l y that these play a role in malignant transformation of trophoblast.
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The cytogenetic evaluation of the choriocarcinomas together with that of the antecedent molar pregnancy would be of interest. Use of improved techniques for the culture of cells and high-resolution b a n d i n g of the chromosomes may reveal a specific chromosomal rearrangement for choriocarcinoma. A comparison of the karyotypes for structural abnormalities i n c l u d i n g the occurrence of breaks and gaps, revealed the preferential i n v o l v e m e n t of chromosome # 8 in 50% of 14 cases, followed by chromosomes #1 and # 2 in 43%, and chromosomes #3, #6, #7, and #14 each, in 36% of the cases. Probably the most frequently involved chromosome in h u m a n neoplasia is # 8 [17]. The t(8;21) is a consistent abnormality in AML and acute n o n l y m p h o c y t i c leukemia (ANLL), and t(8;14) is characteristic for Burkitt's lymphoma. Various rearrangements of chromosome #1 have been reported in four choriocarcinoma cell lines [8, 18], in over 80% of ovarian cancer, and in m a n y other tumors [19, 20]. Because choriocarcinoma cells have m a n y different chromosomal rearrangements, it is reasonable to suggest that the significantly high frequency of breaks in molar trophoblast cells may predispose them to certain chromosomal rearrangements, which play an important role in the pathway leading to the development of trophoblastic malignancy. The extent of chromosomal breakage needs further confirmation. Studies using molecular techniques and prophase b a n d i n g are necessary to evaluate the concordance of the breakpoints found. Supported by NIH Grants CA43331-01 and HD17572 and Magee Womens Hospital Research Fund.
REFERENCES
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14. Sugimori H, Kashimura Y, Kashimura M, Taki I (1978): Nuclear DNA content of trophoblastic tumors. ACTA Cytologica 22:542-545. 15. Zankle H, Seidel H, Zang KD (1975): Sex-chromosome loss in human tumours. Lancet i:221. 16. Hunt PA, Jacobs PA (1985): In vitro growth and chromosome constitution of placental cells. II. Hydatidiform moles. Cytogenet Cell Genet 39:7-13. 17. Sandberg AA (1980): The Chromosomes in Human Cancer and Leukemia. Elsevier, New York, pp. 579.. 18. Wake N, Tanaka K, Chapman V. Matsui S, Sandberg AA (1981): Chromosomes and cellular origin of choriocarcinoma. Cancer Res 41:3137-3143. 19. Whang-Peng J, Knutsen T, Douglass EC, Chu E, Ozolo RF, Hogen WM, Young RC (1984): Cytogenetic studies in ovarian cancer. Cancer Genet Cytogen 11:91-106. 20. Rowley JD (1978) Abnormalities of chromosome no. 1 in haematological malignancies. Lancet i:554 555. 21. Yunis JJ, Soreng AL (1984): Constitutive fragile sites and cancer. Science 226:1199-1204.