SHORT COMMUNICATIONS Cytogenetic Studies of an Adrenal Cortical Carcinoma James L. Marks, Herman E. Wyandt, Robert M. Beazley, Jeffrey M. Milunsky, Kieran Sheahan, and Aubrey Milunsky
ABSTRACT: Cytogenetic findings in the third reported case of adrenal cortical carcinoma are described. In contrast to the two previous cases, hypodiploidy characterized almost all cells, which had as m a n y as eight abnormal chromosomes in each cell analyzed.
INTRODUCTION
MATERIALS A N D METHODS
Cytogenetic findings have been reported in two cases of adrenal cortical carcinoma, one functional [1] and one nonfunctional [2]. The present case thus represents the third report of cytogenetic findings in an adrenal cortical carcinoma and differs from the previous reports in that most cells are h y p o d i p l o i d (modal number = 38), with as m a n y as eight abnormal chromosomes per cell.
Three methods of cell dissociation were attempted: mincing alone; m i n c i n g followed by trypsinization (Trypsinversene, 1:250, GIBCO, Grand Island, NY) for 2 hours; and mincing followed by treatment with collagenase (0.8% in RPMI, Sigma, St. Louis, MO) for 2 hours. Of these methods, mincing with and without trypsinization y i e l d e d sufficient numbers of single cells for cytogenetic preparations. Portions of the cells thus obtained were exposed to Colcemid (0.01 ~ g / m l final concentration) for times varying from 1 to 18 hours. After exposure to Colcemid, the cells were s u s p e n d e d in h y p o t o n i c (0.075 M) KC1 and then fixed in methanol : acetic acid (3 : 1) according to standard cytogenetic procedure. Slides were prepared and b a n d e d with trypsin and Giemsa. A t t e m p t e d culture of the remainder of the cells and small pieces of tissue in RPMI 1640, s u p p l e m e n t e d with either 10% fetal calf serum or an equivalent a m o u n t of low-protein serum replacement (LPSR-1, s.e.), was unsuccessful.
CASE REPORT
A n 82-year-old white w o m a n with a past medical history significant for hypertension, n o n i n s u l i n - d e p e n d e n t diabetes mellitus, and osteoarthritis, was noted to have a right adrenal mass while she was being evaluated for a urinary tract infection. A 6-cm right adrenal mass was noted on a c o m p u t e d tomography (CT) scan of the abdomen. The patient was a s y m p t o m a t i c except for right u p p e r quadrant discomfort. There was no family history of cancer. P h e o c h r o m o c y t o m a was ruled out by the finding of normal urinary levels of v a n i l l y l m a n d e l i c acid (VMA) and metanephrins. W h e n no metastases were noted, a right adrenalectomy was performed. Pathologic examination showed an intermediate- to high-grade carcinoma, 8 x 5 x 5 cm, with frequent mitoses and extensive necrosis (Fig. 1). The tumor invaded the vascular sinusoids of the residual adrenal gland but was separated from periadrenal fat by an intact fibrous capsule. Three months later, a chest roentgenogram showed m u l t i p l e diffuse lung nodules, more than 20 in each lung, involving all lobes. After 1 month of chemotherapy, liver metastases were noted. The patient received four rounds of c h e m o t h e r a p y until she died of her disease, just short of 6 months from the time the adrenal tumor was discovered.
From the Center for Human Genetics and the Departments of Pediatrics and Pathology (J. L. M., H. E. W., J. M. M., K. S., A. M.), and the Department of Surgery (R. M. B.), Boston University School of Medicine, Boston, Massachusetts. Address reprints requests to: Dr. Aubrey Milunsky, Center for Human Genetics, Boston University School of Medicine, 80 E. Concord Street, Boston, MA 02118. Received June 21, 1991 ; accepted October 11, 1991.
96 Cancer Genet Cytogenet61:96-98 (1992) 0165-4608/92/$05.00
RESULTS
Twenty-one mitotic cells from direct preparations were examined. Seven of 21 cells (33%) had a m o d a l number of 38 chromosomes; the r e m a i n d e r (14 cells) had from 24 to 63 chromosomes per cell. I n c l u d e d in this latter group were six cells with c h r o m o s o m e numbers of either 36, 37, or 39. One cell with 45 chromosomes and another with 46 were also noted. The 45-count cell was missing a chromosome, but was otherwise normal. Poor chromosome morphology allowed detailed analysis of only a few cells. Three cells with the modal n u m b e r of 38 and one with 37 were karyotyped. These four cells contained up to nine abnormal c h r o m o s o m e s (Fig. 1), some of w h i c h were present in all four cells and others of w h i c h were missing from at least one cell (Table 1). Chromosome loss (excluding chromosomes involved in rearrangements) also was n o n r a n d o m in the four cells with loss of one homologue of chromosomes 5, 6, 12, 13, 17, 18, and 22, and of both homologues of c h r o m o s o m e 15. Structural abnormalities were d e t e r m i n e d as follows: a) del(1)(q21q42); b) der(2) t(2;?)(p13;?); c) del(3)(p13p24); d) der(11)t(1;11)(p13;q23); 69 1992 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010
Cytogenetics of an A d r e n a l Cortical Carcinoma
97
Figure I Representative area of tumor showing large polygonal cells with moderate bleomorphism. The diagnosis of adrenal carcinoma was based on this morphology in conjunction with the absence of frequent mitoses, extensive necrosis, and vascular invasion in an 8-cm tumor weighing 110 g.
and e) ?iso(19p). The origin of the r e m a i n i n g chromosome markers (M1-M3) could not be determined. One cell (Fig. 2) showed a der(1)t(1;?)(p11;?).
DISCUSSION
We believe that the cytogenetic findings are significant for several reasons. First, in the previous two reports, one tumor was diploid with a sole karyotypic anomaly, t(4;11)(q35;p13) and the other was hyperdiploid (modal n u m b e r = 54). The present case differs from each of these in being h y p o d i p l o i d with m u l t i p l e n u m e r i c a l and structural anomalies. One similarity is the c o m m o n breakpoint, 11p13, w h i c h is the site of the proposed Wilms' tumor
locus [3]. That this rearrangement, t(1;11), is present in all tumor cells analyzed suggests that if it is not the primary cytogenetic event it occurred in the early stages of tumor development. Other markers, which are present in some cells and absent i n others, probably represent evolutionary changes in the tumor. A second important consideration is that these findings are taken from direct preparations of the tumor and thus represent the in vivo condition. Therefore, the tumor is cytogenetically heterogeneous, but probably of m o n o c l o n a l origin. Finally, considering the very different cytogenetic findings in this tumor type, there is a clear need for more studies using not only cytogenetic analysis, but also molecular techniques to refine and clarify further our u n d e r s t a n d i n g of the genetic events underlying development of adrenal cortical carcinoma. REFERENCES
Table
1
Distribution of chromosome abnormalities in four cells karyotyped a
Cell
a
b
c
d
e
M1
M2
M3
1 2 3
+ +
+ +
+ +
+ + +
+ +
+ + +
+ + +
+ + +
4
+
+
+
+
+
+
+
+
a Described in text and legend to Figure 2.
1. Limon J, Dal Cin P, Kakati S, Huben RP, Rao U, Sandberg AA (1987): Cytogenetic findings in a primary adrenocortical carcinoma. Cancer Genet Cytogenet 2 6 : 2 7 1 - 2 7 7 . 2. Limon J, Dal Cin P, Gaeta J, Sandberg AA (1987): Translocation t(4;11)(q35;p13) in an adrenocortical carcinoma. Cancer Genet Cytogenet 28:343-348. 3. Rose EA, Glaser T, Jones C, Smith CL, Lewis WH, Call K, Minden M, Champagne E, Bonetta L, Yeger H, Housman DE (1990): Complete physical map of the WAGR region of 11p13 localizes a candidate Wilms' tumor gene. Cell 60:405-508.
6
19
13
I
al
14
8
15
3
21
9
10
22
16
i!
4
X
17
11
d
Y
i
5
"i
18
12
2 m3
F i g u r e 2 Karyotype (modal no = 38) showing representative structural rearrangements (a-e, described in text) and three marker chromosomes (M1-M3) of uncertain origin, der(1)t(1;?) was present in only one cell, (large arrow). Abnormal chromosome 2 (b, inset from two cells) was present in three cells (Table 1).
20
7
2
C
19
Ii
e
2
i|
b
2O
CO
ABSTRACT: Cytogenetic findings in the third reported case of adrenal cortical carcinoma are described. In contrast to the two previous cases, hypodiploidy characterized almost all cells, which had as m a n y as eight abnormal chromosomes in each cell analyzed.
INTRODUCTION
MATERIALS A N D METHODS
Cytogenetic findings have been reported in two cases of adrenal cortical carcinoma, one functional [1] and one nonfunctional [2]. The present case thus represents the third report of cytogenetic findings in an adrenal cortical carcinoma and differs from the previous reports in that most cells are h y p o d i p l o i d (modal number = 38), with as m a n y as eight abnormal chromosomes per cell.
Three methods of cell dissociation were attempted: mincing alone; m i n c i n g followed by trypsinization (Trypsinversene, 1:250, GIBCO, Grand Island, NY) for 2 hours; and mincing followed by treatment with collagenase (0.8% in RPMI, Sigma, St. Louis, MO) for 2 hours. Of these methods, mincing with and without trypsinization y i e l d e d sufficient numbers of single cells for cytogenetic preparations. Portions of the cells thus obtained were exposed to Colcemid (0.01 ~ g / m l final concentration) for times varying from 1 to 18 hours. After exposure to Colcemid, the cells were s u s p e n d e d in h y p o t o n i c (0.075 M) KC1 and then fixed in methanol : acetic acid (3 : 1) according to standard cytogenetic procedure. Slides were prepared and b a n d e d with trypsin and Giemsa. A t t e m p t e d culture of the remainder of the cells and small pieces of tissue in RPMI 1640, s u p p l e m e n t e d with either 10% fetal calf serum or an equivalent a m o u n t of low-protein serum replacement (LPSR-1, s.e.), was unsuccessful.
CASE REPORT
A n 82-year-old white w o m a n with a past medical history significant for hypertension, n o n i n s u l i n - d e p e n d e n t diabetes mellitus, and osteoarthritis, was noted to have a right adrenal mass while she was being evaluated for a urinary tract infection. A 6-cm right adrenal mass was noted on a c o m p u t e d tomography (CT) scan of the abdomen. The patient was a s y m p t o m a t i c except for right u p p e r quadrant discomfort. There was no family history of cancer. P h e o c h r o m o c y t o m a was ruled out by the finding of normal urinary levels of v a n i l l y l m a n d e l i c acid (VMA) and metanephrins. W h e n no metastases were noted, a right adrenalectomy was performed. Pathologic examination showed an intermediate- to high-grade carcinoma, 8 x 5 x 5 cm, with frequent mitoses and extensive necrosis (Fig. 1). The tumor invaded the vascular sinusoids of the residual adrenal gland but was separated from periadrenal fat by an intact fibrous capsule. Three months later, a chest roentgenogram showed m u l t i p l e diffuse lung nodules, more than 20 in each lung, involving all lobes. After 1 month of chemotherapy, liver metastases were noted. The patient received four rounds of c h e m o t h e r a p y until she died of her disease, just short of 6 months from the time the adrenal tumor was discovered.
From the Center for Human Genetics and the Departments of Pediatrics and Pathology (J. L. M., H. E. W., J. M. M., K. S., A. M.), and the Department of Surgery (R. M. B.), Boston University School of Medicine, Boston, Massachusetts. Address reprints requests to: Dr. Aubrey Milunsky, Center for Human Genetics, Boston University School of Medicine, 80 E. Concord Street, Boston, MA 02118. Received June 21, 1991 ; accepted October 11, 1991.
96 Cancer Genet Cytogenet61:96-98 (1992) 0165-4608/92/$05.00
RESULTS
Twenty-one mitotic cells from direct preparations were examined. Seven of 21 cells (33%) had a m o d a l number of 38 chromosomes; the r e m a i n d e r (14 cells) had from 24 to 63 chromosomes per cell. I n c l u d e d in this latter group were six cells with c h r o m o s o m e numbers of either 36, 37, or 39. One cell with 45 chromosomes and another with 46 were also noted. The 45-count cell was missing a chromosome, but was otherwise normal. Poor chromosome morphology allowed detailed analysis of only a few cells. Three cells with the modal n u m b e r of 38 and one with 37 were karyotyped. These four cells contained up to nine abnormal c h r o m o s o m e s (Fig. 1), some of w h i c h were present in all four cells and others of w h i c h were missing from at least one cell (Table 1). Chromosome loss (excluding chromosomes involved in rearrangements) also was n o n r a n d o m in the four cells with loss of one homologue of chromosomes 5, 6, 12, 13, 17, 18, and 22, and of both homologues of c h r o m o s o m e 15. Structural abnormalities were d e t e r m i n e d as follows: a) del(1)(q21q42); b) der(2) t(2;?)(p13;?); c) del(3)(p13p24); d) der(11)t(1;11)(p13;q23); 69 1992 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010
Cytogenetics of an A d r e n a l Cortical Carcinoma
97
Figure I Representative area of tumor showing large polygonal cells with moderate bleomorphism. The diagnosis of adrenal carcinoma was based on this morphology in conjunction with the absence of frequent mitoses, extensive necrosis, and vascular invasion in an 8-cm tumor weighing 110 g.
and e) ?iso(19p). The origin of the r e m a i n i n g chromosome markers (M1-M3) could not be determined. One cell (Fig. 2) showed a der(1)t(1;?)(p11;?).
DISCUSSION
We believe that the cytogenetic findings are significant for several reasons. First, in the previous two reports, one tumor was diploid with a sole karyotypic anomaly, t(4;11)(q35;p13) and the other was hyperdiploid (modal n u m b e r = 54). The present case differs from each of these in being h y p o d i p l o i d with m u l t i p l e n u m e r i c a l and structural anomalies. One similarity is the c o m m o n breakpoint, 11p13, w h i c h is the site of the proposed Wilms' tumor
locus [3]. That this rearrangement, t(1;11), is present in all tumor cells analyzed suggests that if it is not the primary cytogenetic event it occurred in the early stages of tumor development. Other markers, which are present in some cells and absent i n others, probably represent evolutionary changes in the tumor. A second important consideration is that these findings are taken from direct preparations of the tumor and thus represent the in vivo condition. Therefore, the tumor is cytogenetically heterogeneous, but probably of m o n o c l o n a l origin. Finally, considering the very different cytogenetic findings in this tumor type, there is a clear need for more studies using not only cytogenetic analysis, but also molecular techniques to refine and clarify further our u n d e r s t a n d i n g of the genetic events underlying development of adrenal cortical carcinoma. REFERENCES
Table
1
Distribution of chromosome abnormalities in four cells karyotyped a
Cell
a
b
c
d
e
M1
M2
M3
1 2 3
+ +
+ +
+ +
+ + +
+ +
+ + +
+ + +
+ + +
4
+
+
+
+
+
+
+
+
a Described in text and legend to Figure 2.
1. Limon J, Dal Cin P, Kakati S, Huben RP, Rao U, Sandberg AA (1987): Cytogenetic findings in a primary adrenocortical carcinoma. Cancer Genet Cytogenet 2 6 : 2 7 1 - 2 7 7 . 2. Limon J, Dal Cin P, Gaeta J, Sandberg AA (1987): Translocation t(4;11)(q35;p13) in an adrenocortical carcinoma. Cancer Genet Cytogenet 28:343-348. 3. Rose EA, Glaser T, Jones C, Smith CL, Lewis WH, Call K, Minden M, Champagne E, Bonetta L, Yeger H, Housman DE (1990): Complete physical map of the WAGR region of 11p13 localizes a candidate Wilms' tumor gene. Cell 60:405-508.
6
19
13
I
al
14
8
15
3
21
9
10
22
16
i!
4
X
17
11
d
Y
i
5
"i
18
12
2 m3
F i g u r e 2 Karyotype (modal no = 38) showing representative structural rearrangements (a-e, described in text) and three marker chromosomes (M1-M3) of uncertain origin, der(1)t(1;?) was present in only one cell, (large arrow). Abnormal chromosome 2 (b, inset from two cells) was present in three cells (Table 1).
20
7
2
C
19
Ii
e
2
i|
b
2O
CO
