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Chromosomes of a malignant ovarian teratoma
Chromosomes of a malignant ovarian teratoma LOUIS ARIAS·BERNAL, M.D. HOWARD W. JONES, JR., M.D. Baltimore, Maryland A malignant ovarian t,ratoma was studi,d with special "/lTence to th, sex chromatin and the chromosomes 0/ th, tumor. Many SIX chromatin-positive "l/s wlTe found. Karyotyp, studies "v.al,d 47 chromosom,s with th, IXtra chomosom, closely r,s,mbling a norma' A3 chromosom,.
THE C H ROM 0 S 0 M E S of malignant teratomas are of interest on three scores: first, because of their bearing on the sex of these so-called totipotential tumors; second, because of their possible bearing on the histogenesis of these tumors; and third, because of their possible bearing on the process of malignancy. Since the understanding of the relation between the sex chromatin and the sex chromosomes, several workers have studied these sex structures in many benign cystic teratomas, and a few malignant teratomas. This subject has been thoroughly reviewed and brought up to date by Rashad, Fathalla, and Kerr.1 Although there are a few reported exceptions, it seems generally agreed that teratomas arising in the ovary are sex chromatin positive. On the other hand, about half of teratomas arising in the male have been reported to be sex chromatin positive and half sex chromatin negative. However, in a study of the chromosomes of 7 malignant teratomas arising in males, some of which seemed to be sex chromatin positive, Galton and associates' were unable to identify any XX sex chromosome complements. The status of the sex characteristics of teratomas in males must, therefore, be considered to be, at the moment, in some doubt.
Teratomas are generally considered to be of germ cell origin, but at what stage of mitosis or meiosis of that cell? If the germ cell has completed the first meiotic (reduction) division, the "cytes" have but 23 chromosomes, i.e., half the normal diploid number. A study of the chromosomes of teratomas is, therefore, relevant to this particular question. However, it should be commented that there has been some misconception about this problem in the literature. With respect to the female, no germ cell in the ovary is normally beyond the prophase of meiosis I, except possibly an ovocyte about to be ovulated. Thus, normally, no germ cells should have gone through their reduction division. In view of this, it would be a great surprise to ~liscover 23 chromosomes in an ovarian teratoma, and it seems scarcely necessary to think in terms of parthenogenesis, autofertilization, or other suggestions to be found in the literature to explain a finding of diploid chromosomes in a teratoma. Therefore, the finding of normal diploid chromosomes in a modest number of studies of benign teratomas in the female is scarcely surprising,t. a although the use of tilSue culture in such studies possibly introduces errors in the cell selection and identification which must be kept in mind in interpreting these data. The relation of aneuploidy to malignancy is a subject of current interest, and many more data are needed to study this problem. For these three reasons it has been con-
From th, D,pa,'mlrU of G)lfllCol0f' afld Obstltrics, Johns Hopl"fls UfI;v,rsll, School of M,dic;fI,. Aid,d ~)' ,h, Am,rican Canc" Soci", G,an, No. T426.
785
786
Arias-Bernal and Jones
sidered suitable to report a study of the chromosomes of a malignant teratoma. Case report
B. J. L. (J. H. H. No. 1188953). A l4-year-old white female presented with an abdominal mass. Her menses started at the age of l3Y2 years, had an interval of 4 to 6 weeks, and a duration of 3 to 12 days. There was no intermenstrual pain or bleeding. Physical examination revealed a protruding abdomen with a large cystic mass arising from the pelvis reaching up to 3 cm. above the umbilicus. Chest roentgenogram was normal. She underwent laparotomy and was found to have a large cystic teratoma of the right ovary containing 2,500 ml. of clear fluid, along with various adult tissues containing embryonal tissue. It was felt from the frozen section that the tumor was malignant and for this reason the patient underwent bilateral salpingo-oophorectomy and total hysterectomy. The final pathology report was: teratoma with low-grade malignancy. Material and methods
Representative pieces of tissue were taken from the entire tumor and divided into two
March 15, 1968
Am.
J. Ohot. I: Gynec.
parts, one for histologic study and the other for tissue culture. The histologic sections were stained with hematoxylin and eosin. The tissue was cultured according to Lejeune's method with a slight modification.' Satisfactory metaphase spreads were obtained successfully from primary cultures of a monolayer of fibroblast-like cells at 34 days, and the cell line was kept growing for another fifteen subcultures in falcon flasks. Those were terminated by the addition of Coleemide, 0.002 p,g per milliliter, for 3 hours, then removed with a weak solution of trypsin. Hypotonic treatment was carried out for 30 minutes and then the cells were fixed with acetic acid and methanol, 1: 3. The cell suspension was spread by the air-drying method on chilled, moist slides and stained with Giemsa's stain in distilled water, 1: 15. After counting one hundred suitable nuclei, ten were chosen for photography with highcontrast copy film and critical karyotype analysis performed. The genetic sex of the patient was assessed by the sex chromatin
Fir. 1. Histopathologic section showing chromatin bodies. (Hematoxylin and eosin stain. )(1,000.)
Volume 100 Numb« i
Chromosomes of malignant ovarian teratoma
A
c
0
_
J}) I { I
1
H~ d
F
'I
I)
19 10
I~
8 __
-- J _
nI
In
.~
K
I
l _
6 I'
6 I~
}i E
G
$
al
17 _ _ 11 _
16
J
) AA
'1-"
It
•
..,~ b Fir. 2. Example of one of 47 celli with an extra chromosome ,Unitar to AS. (Giemu'l.tam.
x3,OOO.)
787
788
Arias-Bernal and Jones
Am.
pattern of the buccal smear and by peripheral blood culture.6 Chromosomal analysis. Sex chromatin was identified in the mesodermal stromal and epithelial cells of sections of the tumor (Fig. 1). One hundred metaphase spreads of the primary culture after 29 days were counted. Twenty cells were of hypodiploid range; 22 were normal diploid cells; 47 cells contained 47 chromosomes with an extra chromosome similar to an A3 (Fig. 2) ; and 5 cells were hyperdiploid ranging from 47 to 94 (Table I). The sex chromosome complement was
XX. Chromosomal analysis was also carried out on subculture No. 10 after 169 days. The fibroblast-like cell monolayer growth had the modal number of chromosomes shown in Table II. Endoreduplication became more frequent after subculture No. 10 with an incidence of 7 per cent. The majority of the cells from primary cultures and subculture No. 10 yielded 47 chromosomes. The karyotype of these cells showed an extra A3-like chromosome in all the preparations. The
Table I r--....,....-=-....----,--~--I
Teratoma
47
20
.5
Total cdls counted 100
Table II Total No. 01 chromosomes cells <451 45 1 46 1 47 1<47 counted Teratoma ( subculture No. 10)
23
5
25
33
14
100
Table III No. 01 chromosomes
< 451 Blood
45 1 46 1 47 12 32 2
I > 47 o
Total cells counted .50
J.
March 15, 1968 ~bot. a: Gynec:.
cells with 46 chromosomes had a normal female karyotype. Chromosomal analysis of the patient's lymphocytes as control yielded the results shown in Table III. Of the spreads with 45 chromosomes, 6 cells were karyotyped and found to have a random loss of one chromosome and the 2 cells with 47 chromosomes had a random gain. It was concluded that the peripheral blood had a normal female chromosomal pattern. Comment
The finding of sex chromatin in cells in the tumor of this patient is consistent with previous studies. The finding of chromosomes in the near diploid range again fails to support the idea that teratomas are possibly of germ cell origin in the ovocyte stage after chromosome reduction. As noted above, other studies of chromosomes in benign teratomas· have had similar findings. If, as seemed likely, teratomas arise from germ cells, it is likely that the origin in the ovary, at least, is from a cell in the oogonium stage, or even earlier. It does not seem necessary to invoke the concept of parthenogenesis or autofertilization to explain the finding of 46 chromosomes. It is inappropriate in this case report to comment upon the vast literature bearing on the relation of aneuploidy to malignancy. Suffice it to say that there seems to be more and more evidence to associate these two phenomena. Only one other malignant ovarian teratoma has been studied, that of Galton and Benirschke. 8 In this instance a normal female karyotype was obtained from explants of tissue from a recurrent solid well-differentiated ovarian teratoma. As was mentioned above, use of tissue culture makes the exact nature of the cell whose chromosomes are studied in some doubt. While the same objection applies to our study, the consistent finding of an abnormal karyotype supports the probability that these chromosomes are from the neoplastic cells. In the only study of malignant teratomas·
Volume 100 Number 6
in the male, 2 aneuploidy was a consistent finding in all 7 tumors studied. We are pleased to acknowledge the coopera-
REFERENCES
1. Raahad, M. N., Fathalla, M. F., and Kerr, M. G.: AM. J. DSST. & GVNEC. 96: 461, 1966. 2. Galton, M., Benirschke, K., Baker, M. C., and Atkin, N. B.: Cytogenetica 5: 261, 1966. 3. Codman, P. A., and Richart, R. M.: New England J. Med. 271: 1241, 1964.
Chromosomes of malignant ovarian teratoma
tion of Dr.
J.
789
H. Dorsey, who provided the sur-
gical specimen, and Dr. K. Rafferty, Jr., for his help and kind permission to use the facilities of his laboratory.
4. Lejeune, J., Turpin, R., and Gautier, M.: Rev. Fran\;. ttud. Clin. Bio\. 5: 406, 1960. 5. Moorhead, P. S., Nowell, P. C., Mellman, W. J., Batipps, D. M., and Hungerford, D. A.: Exper. Cell. Rei. 20: 613, 1960. 6. Galton, M., and Benirachke, K.: Lancet 2: 761, 1959.
THE C H ROM 0 S 0 M E S of malignant teratomas are of interest on three scores: first, because of their bearing on the sex of these so-called totipotential tumors; second, because of their possible bearing on the histogenesis of these tumors; and third, because of their possible bearing on the process of malignancy. Since the understanding of the relation between the sex chromatin and the sex chromosomes, several workers have studied these sex structures in many benign cystic teratomas, and a few malignant teratomas. This subject has been thoroughly reviewed and brought up to date by Rashad, Fathalla, and Kerr.1 Although there are a few reported exceptions, it seems generally agreed that teratomas arising in the ovary are sex chromatin positive. On the other hand, about half of teratomas arising in the male have been reported to be sex chromatin positive and half sex chromatin negative. However, in a study of the chromosomes of 7 malignant teratomas arising in males, some of which seemed to be sex chromatin positive, Galton and associates' were unable to identify any XX sex chromosome complements. The status of the sex characteristics of teratomas in males must, therefore, be considered to be, at the moment, in some doubt.
Teratomas are generally considered to be of germ cell origin, but at what stage of mitosis or meiosis of that cell? If the germ cell has completed the first meiotic (reduction) division, the "cytes" have but 23 chromosomes, i.e., half the normal diploid number. A study of the chromosomes of teratomas is, therefore, relevant to this particular question. However, it should be commented that there has been some misconception about this problem in the literature. With respect to the female, no germ cell in the ovary is normally beyond the prophase of meiosis I, except possibly an ovocyte about to be ovulated. Thus, normally, no germ cells should have gone through their reduction division. In view of this, it would be a great surprise to ~liscover 23 chromosomes in an ovarian teratoma, and it seems scarcely necessary to think in terms of parthenogenesis, autofertilization, or other suggestions to be found in the literature to explain a finding of diploid chromosomes in a teratoma. Therefore, the finding of normal diploid chromosomes in a modest number of studies of benign teratomas in the female is scarcely surprising,t. a although the use of tilSue culture in such studies possibly introduces errors in the cell selection and identification which must be kept in mind in interpreting these data. The relation of aneuploidy to malignancy is a subject of current interest, and many more data are needed to study this problem. For these three reasons it has been con-
From th, D,pa,'mlrU of G)lfllCol0f' afld Obstltrics, Johns Hopl"fls UfI;v,rsll, School of M,dic;fI,. Aid,d ~)' ,h, Am,rican Canc" Soci", G,an, No. T426.
785
786
Arias-Bernal and Jones
sidered suitable to report a study of the chromosomes of a malignant teratoma. Case report
B. J. L. (J. H. H. No. 1188953). A l4-year-old white female presented with an abdominal mass. Her menses started at the age of l3Y2 years, had an interval of 4 to 6 weeks, and a duration of 3 to 12 days. There was no intermenstrual pain or bleeding. Physical examination revealed a protruding abdomen with a large cystic mass arising from the pelvis reaching up to 3 cm. above the umbilicus. Chest roentgenogram was normal. She underwent laparotomy and was found to have a large cystic teratoma of the right ovary containing 2,500 ml. of clear fluid, along with various adult tissues containing embryonal tissue. It was felt from the frozen section that the tumor was malignant and for this reason the patient underwent bilateral salpingo-oophorectomy and total hysterectomy. The final pathology report was: teratoma with low-grade malignancy. Material and methods
Representative pieces of tissue were taken from the entire tumor and divided into two
March 15, 1968
Am.
J. Ohot. I: Gynec.
parts, one for histologic study and the other for tissue culture. The histologic sections were stained with hematoxylin and eosin. The tissue was cultured according to Lejeune's method with a slight modification.' Satisfactory metaphase spreads were obtained successfully from primary cultures of a monolayer of fibroblast-like cells at 34 days, and the cell line was kept growing for another fifteen subcultures in falcon flasks. Those were terminated by the addition of Coleemide, 0.002 p,g per milliliter, for 3 hours, then removed with a weak solution of trypsin. Hypotonic treatment was carried out for 30 minutes and then the cells were fixed with acetic acid and methanol, 1: 3. The cell suspension was spread by the air-drying method on chilled, moist slides and stained with Giemsa's stain in distilled water, 1: 15. After counting one hundred suitable nuclei, ten were chosen for photography with highcontrast copy film and critical karyotype analysis performed. The genetic sex of the patient was assessed by the sex chromatin
Fir. 1. Histopathologic section showing chromatin bodies. (Hematoxylin and eosin stain. )(1,000.)
Volume 100 Numb« i
Chromosomes of malignant ovarian teratoma
A
c
0
_
J}) I { I
1
H~ d
F
'I
I)
19 10
I~
8 __
-- J _
nI
In
.~
K
I
l _
6 I'
6 I~
}i E
G
$
al
17 _ _ 11 _
16
J
) AA
'1-"
It
•
..,~ b Fir. 2. Example of one of 47 celli with an extra chromosome ,Unitar to AS. (Giemu'l.tam.
x3,OOO.)
787
788
Arias-Bernal and Jones
Am.
pattern of the buccal smear and by peripheral blood culture.6 Chromosomal analysis. Sex chromatin was identified in the mesodermal stromal and epithelial cells of sections of the tumor (Fig. 1). One hundred metaphase spreads of the primary culture after 29 days were counted. Twenty cells were of hypodiploid range; 22 were normal diploid cells; 47 cells contained 47 chromosomes with an extra chromosome similar to an A3 (Fig. 2) ; and 5 cells were hyperdiploid ranging from 47 to 94 (Table I). The sex chromosome complement was
XX. Chromosomal analysis was also carried out on subculture No. 10 after 169 days. The fibroblast-like cell monolayer growth had the modal number of chromosomes shown in Table II. Endoreduplication became more frequent after subculture No. 10 with an incidence of 7 per cent. The majority of the cells from primary cultures and subculture No. 10 yielded 47 chromosomes. The karyotype of these cells showed an extra A3-like chromosome in all the preparations. The
Table I r--....,....-=-....----,--~--I
Teratoma
47
20
.5
Total cdls counted 100
Table II Total No. 01 chromosomes cells <451 45 1 46 1 47 1<47 counted Teratoma ( subculture No. 10)
23
5
25
33
14
100
Table III No. 01 chromosomes
< 451 Blood
45 1 46 1 47 12 32 2
I > 47 o
Total cells counted .50
J.
March 15, 1968 ~bot. a: Gynec:.
cells with 46 chromosomes had a normal female karyotype. Chromosomal analysis of the patient's lymphocytes as control yielded the results shown in Table III. Of the spreads with 45 chromosomes, 6 cells were karyotyped and found to have a random loss of one chromosome and the 2 cells with 47 chromosomes had a random gain. It was concluded that the peripheral blood had a normal female chromosomal pattern. Comment
The finding of sex chromatin in cells in the tumor of this patient is consistent with previous studies. The finding of chromosomes in the near diploid range again fails to support the idea that teratomas are possibly of germ cell origin in the ovocyte stage after chromosome reduction. As noted above, other studies of chromosomes in benign teratomas· have had similar findings. If, as seemed likely, teratomas arise from germ cells, it is likely that the origin in the ovary, at least, is from a cell in the oogonium stage, or even earlier. It does not seem necessary to invoke the concept of parthenogenesis or autofertilization to explain the finding of 46 chromosomes. It is inappropriate in this case report to comment upon the vast literature bearing on the relation of aneuploidy to malignancy. Suffice it to say that there seems to be more and more evidence to associate these two phenomena. Only one other malignant ovarian teratoma has been studied, that of Galton and Benirschke. 8 In this instance a normal female karyotype was obtained from explants of tissue from a recurrent solid well-differentiated ovarian teratoma. As was mentioned above, use of tissue culture makes the exact nature of the cell whose chromosomes are studied in some doubt. While the same objection applies to our study, the consistent finding of an abnormal karyotype supports the probability that these chromosomes are from the neoplastic cells. In the only study of malignant teratomas·
Volume 100 Number 6
in the male, 2 aneuploidy was a consistent finding in all 7 tumors studied. We are pleased to acknowledge the coopera-
REFERENCES
1. Raahad, M. N., Fathalla, M. F., and Kerr, M. G.: AM. J. DSST. & GVNEC. 96: 461, 1966. 2. Galton, M., Benirschke, K., Baker, M. C., and Atkin, N. B.: Cytogenetica 5: 261, 1966. 3. Codman, P. A., and Richart, R. M.: New England J. Med. 271: 1241, 1964.
Chromosomes of malignant ovarian teratoma
tion of Dr.
J.
789
H. Dorsey, who provided the sur-
gical specimen, and Dr. K. Rafferty, Jr., for his help and kind permission to use the facilities of his laboratory.
4. Lejeune, J., Turpin, R., and Gautier, M.: Rev. Fran\;. ttud. Clin. Bio\. 5: 406, 1960. 5. Moorhead, P. S., Nowell, P. C., Mellman, W. J., Batipps, D. M., and Hungerford, D. A.: Exper. Cell. Rei. 20: 613, 1960. 6. Galton, M., and Benirachke, K.: Lancet 2: 761, 1959.