Cytogenetic study of solid ovarian tumors

Cytogenetic Study of Solid Ovarian Tumors Cynthia G. Roberts and Martin H. N. Tattersall

ABSTRACT: With the use of a short-term tissue culture method, 27 solid ovarian tumor specimens (from

22 patients) were successfully karyotyped. The majority of the specimens were from serous carcinoma (18 specimens, 2 of which were not invasive). Adenocarcinomas (two specimens), two endometrioid carcinomas, and one each of clear cell, mucinous, sarcoma, squamous carcinoma, and an unclassified sex cord carcinoma were also analyzed. The specimens showed marked cytogenetic heterogeneity, ranging from a normal karyotype (46,XX) to very grossly aneuploid, with multiple rearrangements. All chromosomes, excepting 13, 15, 19, 20, and 21 were positively identified in at least one rearrangement. Chromosomes 1, 6, and 7 were most commonly involved. Identified rearrangements were not limited to one carcinoma type. The most common deletions of lp and 6q were identified in both serous carcinoma and adenocarcinoma. Deletion of 7q,(del(7)(q32)), was observed only in serous carcinoma but was limited to three patients. Correlations of modal chromosome count and number of marker chromosomes appeared to be associated with good prognosis for patients with serous carcinoma.

INTRODUCTION Approximately 90% of all cytogenetic information available in h u m a n cancer has been obtained from patients with leukemias and lymphoma, yet these represent only 3% of the total cancer incidence [1]. The major reason for this imbalance is the low mitotic yield and poor metaphase quality achieved in preparations from solid tumors. The bulk of cytogenetic information available for ovarian carcinoma is provided by the study of cell lines [2-6] (mostly established from cells in ascites fluid) or from direct preparations of ascites fluid cells [7-9]. However, ascites fluid cells may not be representative of the solid tumor from which they arise, and tissue culture selection may also result in a line that does not represent the original tumor. Clearly, studies of solid tumor tissue are preferable. The data available on solid ovarian tumor tissue to date are limited, both in terms of the n u m b e r of tumor specimens analyzed and in the n u m b e r and quality of metaphases karyotyped [10-17]. We recently reported a tissue culture method that improved the quality and quantity of metaphase preparations from solid ovarian tumors, while m i n i m i z i n g the risk of tissue culture selection [18]. The cytogenetic analyses of 27 tumor specimens using this method are now reported.

From CytogeneticsLaboratory(C. G. R.), RoyalNorth Shore Hospital,St. Leonards,Australia,and Department of Cancer Medicine(M. H. N. T.), Universityof Sydney,Sydney,Australia. Address reprint requests to: Cynthia G. Roberts, Cytogenetics Laboratory, 4th Floor WFP Block, Royal North Shore Hospital, St. Leonards, NSW 2065, Australia. Received February 10, 1988; accepted January 19, 1990.

243 © 1990 Elsevier SciencePublishingCo, Inc. 655 Avenueof the Americas,New York, NY 10010

Cancer GenetCytogenet48:243-253 (1999) 0165-4808/90/$03.50

Endometrioid (poorly differentiated) Endometrioid I Mucinous Sarcoma Serous (not invasive) Serous (not invasive) Serous I - I I Serous I - I I Serous II Serous II

4

5 6 7 [8] [9] 10 11 12 13

3

2

A d e n o c a r c i n o m a (poorly differentiated) A d e n o c a r c i n o m a (poorly differentiated Clear cell carcinoma

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47 66 84 56

58

41

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Age

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NK

C

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NK

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50

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N

43-47/92 69-153 46-122 46 46 43-47 47-49 46-136 53-82/ 138-150 +

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44-47

Range

46 134 b 65 ~' 46 46 46 48 69 71

75

46

68

46

Mode

Chromosome counts

Specimen source

Treatment prior to s p e c i m e n collection

Histological diagnosis and grade

successfully analyzed

P a t i e n t d a t a w i t h k a r y o t y p i c r e s u l t s f o r 27 s p e c i m e n s

1

No.

Table 1

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10

10

10

10

N

D(1.5) A(4.6) A(5.6) 46,XX(0) 46,XX(O) 46,XX(0) D(0) A(22.5) A(28.2)

A(5.9)

46,XX(0.4)

A(15.4)

46,XX(0.1)

Classification

Karyotype

2c 4 2c lb lb 3 lb 2c 4

2b

1-2

4

4

Stage

54 c 41 1 46 46 9 d 35 16

9

4

8

56':

Mo s u r v i v a l since s p e c i m e n collection

b~

Serous Serous Serous Serous Serous Serous Serous Serous Serous Serous Serous

Squamous Unclassified sex cord

[15] [16] 17 [18] [19] [20] 21 22 23 [24] [25]

26 27

54 50

42 40 57 71

50 55

63

43

LO LO

LO RO OM LO RO OM RO OM RO RO OM

LO

50 10

50 100 100 100 50 50 40 10 50

50

70

46-47/ 65-167 46-150 53-150 39-106 38-90 37-120 38-86 36-80 48-99 45-74 43-47 46-47/ 62-147 45-50 48-53

Months survival: % still alive, d lost to follow-up.

Karyotype classification: see Results.

Chromosome Analysis: b median value (no clear modal value).

a Technical loss of majority of cells.

Treatmenl: C--cytotoxic chemotherapy, S--surgery, R--abdominal radiotherapy, NK--not known.

left ovary, RO--right ovary, OV

None None

None None None None

C None

None

None

Bracketed specimens are from the same patient. Specimen source:LO

II II II-III II-III II-Ill II-[II II-IV III IIl III III

Serous II

14

10 5

5 10 5 10 10 10 10 10 5 5 10

20

46,XX(O]/ A(32.7) A(9.4) A(9.4) D(7.0) D(17.1) D(19.O) D(17.2) D(9.5) A(12.0) A(7.6) 46,XX(O) 46,XX(O)/ A(12.9) D(0) A(1.6) 4 1

2c 3 3 3

3 3

4

3

5 15 c'd

14 13 11 24

18 13

5

30

ovary (side not specified), OM--omentum, RF--right fallopian tube.

46/ 74 ~ 66 b 64 44 42 43 42 40 54 57 44 46/ 73 b 46 53

O1

246

c . G . Roberts and M. H. N. Tattersall

MATERIALS A N D METHODS

Solid tumor samples were received from patients undergoing surgery at King George V Hospital, Sydney, and were transported to the laboratory on ice. Initially, specimens were prepared by fine m i n c i n g with scalpels, with 72 hours culture at 37°C in air in 25-cm 2 plastic: culture flasks with RPMI-1640 m e d i m n and supplements [18]. However, this method had a very low rate of success (3/20 specimens karyotyped - 15%). Greater success (24/32 specimens ~ 75%) was achieved with the use of a collagenase digestion, followed by 48 hours culture in low oxygen on a collagen matrix (for full details, see [18]). Specimens n u m b e r e d 3, 10, and 12 (Table 1) were cultured with the use of the former method, while the latter conditions were used for the remainder. Air dried slides were prepared after the cultured cells were fixed in three changes of 3 : 1 methanol/acetic acid. Matured slides were G-banded prior to analyses [18[. In most cases, at least 50 metaphase spreads were counted for chromosome number, and 10 metaphases were fully karyotyped. A marker chromosome was identified as clonal if it was present in at least two cells.

RESULTS

Table I summarizes the results for the 27 specimens successfully analyzed. The range of chromosome count, over all the specimens was very large ( 3 6 - 1 5 0 + ) , although the majority were either in the diploid range or contained between 5 0 - 8 0 chromosomes. In three cases, because of the wide range, a modal chromosome n u m b e r could not be determined. For these three specimens, the median chromosome count is listed. Cytogenetic heterogeneity revealed by karyotypic analysis varied markedly between specimens. Each specimen was classified according to the chromosome count and the average n u m b e r of marker chromosomes (m). This was calculated for each specimen by averaging the n u m b e r of identified and nonidentified markers for each cell karyotyped. Thus: 46,XX(m)--normal karyotype (8 specimens) D ( m ) - - d i p l o i d / n e a r diploid (8 specimens) A(m)--grossly aneuploid (13 specimens) Two samples had both karyotypically normal and abnormal populations. Representative karyotypes are illustrated in Figures 1-5. The majority of clonal marker chromosomes were identified, and the break points are presented graphically in Figure 6. Deletions and translocations were the most c o m m o n rearrangement, although inversions and possible duplications were also seen. The complete characterization of the marker chromosomes was further complicated by the high degree of a n e u p l o i d y in many of the specimens. In many cases, only one of the derivative chrolnosomes from a translocation could be identified. The lack of a reciprocal translocation product in solid tumors has been noted in many solid tumors [19], in that some reciprocal translocation products cannot be identified or are lost. Moreover, some unidentified marker chromosomes appear to have been formed from multiple translocation. Homogeneous staining regions (HSR) and double minutes (DMs) were never seen. All chromosomes, except 13, 15, 19, 20, and 21 were identified in at least one structural rearrangement. Most changes were grouped to particular chromosome regions: 1, 6q, 7, 4, 3, 9, X, 5, 8 (in order of frequency). All chromosomes were involved in aneuploidy. Survival data was available for all but two patients who were lost to follow up.

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UNIDENTIFIED MARKERS Figure 3 Near diploid karyotype with multiple chromosome rearrangements (serous carcinoma II specimen 18), showing deletions (>) and derivative translocation products (~). Three of the patients (cases 1, 5, 8) were still alive and well at the time of preparation of this manuscript. For the 12 malignant serous carcinoma patients with survival data, correlations were calculated between months survival and stage (r = - 0 . 1 5 ) , histologic grade (r = - 0 . 1 6 ) , m o d a l c h r o m o s o m e n u m b e r (r = 0.42), and average n u m b e r of marker c h r o m o s o m e s (r = 0.47). DISCUSSION

The s p e c i m e n s analyzed reveal a perplexing degree of cytogenetic heterogeneity, both w i t h i n and between samples. Six specimens, i n c l u d i n g those classed as not invasive, had a normal karyotype (46,XXI, and a further two specimens had a k a r y o t y p i c a l l y normal population. Although it could be argued that normal, rather than tumor tissue had been karyotyped, this is unlikely. Tumors with a normal karyotype have been reported p r e v i o u s l y [8, 10, 12]. Thus, the neoplastic change need not be associated with a visible c h r o m o s o m a l rearrangement. Further, the very short culture period on the collagen matrix utilized in this study is u n l i k e l y to promote the r a p i d growth of normal tissue. Although normal karyotypes were identified, these samples s h o w e d a higher degree of a p p a r e n t l y r a n d o m error (involving both chromatid breaks and r a n d o m loss) than w o u l d be seen in, e.g., karyotypes prepared from fibroblast cultures.

249

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Figure 4

Grossly aneuploid karyotype with few abnormal chromosomes from a mucinous carcinoma (specimen 6). The copy number varies for each chromosome, from 3-10.

No chromosome anomalies were identified in all tumors or in all serous tumors. Thus, a c o m m o n cytogenetic rearrangement in the development of ovarian carcinoma was not found. The identified break points were distributed n o n r a n d o m l y and tended to be clustered in specific chromosome regions (Fig. 6). Chromosome 1 was most c o m m o n l y involved in structural rearrangements, particularly deletions (12 patients). Deletions of l p were identified in six patients with serous carcinoma and in one patient with adenocarcinoma. Rearrangements involving chromosome I are the most c o m m o n in ovarian cancer [2, 3, 5-7, 9, 11-16]. However, chromosome 1 abnormalities have been reported in all cancer types, and appear to be related to an event c o m m o n to neoplasia [12]. Deletions of the short arm of chromosome 3 were previously reported in solid ovarian tumors as well as in ascites fluid [9, 16] and cell lines [2, 3, 5, 6], while deletions of 3q have been identified in ascites fluid [6], cell lines [2, 4] and solid tumors [17]. A study of direct preparations of 14 ovarian carcinomas [17] indicated that n o n i n volvement of chromosomes 1 and 3 in rearrangments was important in patient survival. A comparison of the average survival (months) of patients in our study with (18.7 -+ 1.0) and without involvement (22.6 -+ 1.6) of chromosomes 1 and/or 3 was not significantly different (t = 0.47, p > 0.05). However, three patients in the latter group were still alive; thus, the average survival time for this group will increase. Chromosome 7 has also been reported to be involved in rearrangements, although not as c o m m o n l y as seen in this study. A 7p ÷ marker was also identified in ovarian

250

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UNIDENTIFIED HARKERS Figure 5 Aneuploid karyotype with multiple rearrangements (Serous carcinoma I-ll, specimen 12). Showing deletions (>), derivative translocation products (I~).

cancer by Panani and Ferti-Passantonopoulou [16]. Deletions of c h r o m o s o m e 7 have not previously been reported in solid ovarian tumors but have been identified in cell lines [2]. A deletion of 7q (q32--*qter) was found only in serous carcinoma, but identification of this marker was limited to only three patients. Marker chromosomes resulting from t(6;14)(q21;q24) have previously been reported in ovarian cancer [13,21]. In this study, neither translocation product was identified. However, deletions of the long arm of c h r o m o s o m e 6 was the second most c o m m o n c h r o m o s o m a l a n o m a l y (11 patients: 8 serous carcinoma, 1 adenocarcinoma, and 2 e n d o m e t r i o i d patients). Eight of the patients with a deleted 6q had the breakpoint at 6q21, the same point involved in the 6;14 translocation. Deletions of chromosome 6, however, are not restricted to ovarian cancer but rather are c o m m o n l y observed in solid tumors [22]. Ihara et al. [10] showed that the survival of malignant ovarian teratoma patients was d e p e n d e n t u p o n a n u m b e r of factors, i n c l u d i n g histologic grade, stage of disease, and the karyotype of the t u m o r cells. In this study, both m o d a l c h r o m o s o m e number and n u m b e r of marker c h r o m o s o m e s showed a greater correlation with survival than histologic grade and stage. The correlation between the m o d a l c h r o m o s o m e count and survival may a p p e a r to contradict the data of F r i e d t a n d e r et al. [23] that reported that a high DNA index in epithelial ovarian cancers correlated with a lower survival rate. However, a high DNA index does not necessarily indicate an increased chromo-

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some count, since a n e u p l o i d y and multiple c h r o m o s o m e rearrangements may combine to keep the modal c h r o m o s o m e n u m b e r near diploid, w h i l e increasing the DNA content of a cell. Further, because all specimens were not able to be karyotyped, the cytogenetic data may represent a biased sample compared with the flow cytometric study. From these data, it appeared that the average n u m b e r of marker c h r o m o s o m e s is a better prognostic indicator of survival in serous carcinoma patients. One possible explanation for the high correlation between numbers of abnormal c h r o m o s o m e s with i m p r o v e d ou t c o m e may be an inherent tendency to genetic instability and increased probability of response to chemotherapy, affording greater survival. The correlations of modal c h r o m o s o m e number and extent of c h r o m o s o m a l rearrangements with survival were not found in four cell lines by Sheer et al. [6], although tissue culture selection may have had a significant effect on their data. Although the overall picture obtained from cytogenetic studies of ovarian carcinoma is complex, because of the enormous heterogeneity, both within and between patients, these data indicate that karyotypic analysis can be achieved with a high degree of success and, in the case of malignant serous carcinoma, may be of prognostic value. Larger studies of other ovarian tumor types are indicated, to determine w h et h er the prognostic significance of marker c h r o m o so m es is applicable to survival prediction.

The authors are very grateful to Judy Hood for typing this manuscript and to Dr. Peter Russell (King George V Hospital, Sydney) for providing all specimens, with their histologic diagnosis, from patients operated on by staff of the Department of Gynecological Oncology. This work was performed while C. R. was employed by the Ludwig Institute for Cancer Research (Sydney Branch).

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Ovarian Cancer Cytogenetics

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12. Wake N, Hreshchyshyn MM, Piver SM, Matsui S, Sandberg AA (1980): Specific cytogenetic changes in ovarian cancer involving chromosomes 6 and 14. Cancer Res 40:4512-4518. 13. Wake N, Slocum HK, Rustum YM, Matsui S, Sandberg AA (1981) Chromosomes and causation of h u m a n cancer and leukemia. XLIV. A method for chromosome analysis of solid tumors. Cancer Genet Cytogenet 3:1-10. 14. Kusyk CJ, Seski JC, Medlin WV, Edwards CL (1981): Progressive chromosome changes associated with different sites of one ovarian carcinoma. J Natl Cancer Inst 66:1021-1025. 15. Trent JM, Salmon SE (1981): Karyotypic analysis of h u m a n ovarian carcinoma cells cloned in short term agar culture. Cancer Genet Cytogenet 3:279-291. 16. Panani A, Ferti-Passantonopoulou (1985): Common marker chromosomes in ovarian'cancer. Cancer Genet Cytogenet 16:65-71. 17. Atkin NB, Baker MC (1987) Abnormal chromosomes including small metacentrics in 14 ovarian cancers. Cancer Genet Cytogenet 26:355-361. 18. Roberts CG, Tattersall MHN (1987): High quality metaphases from solid ovarian tumors. Cancer Genet Cytogenet 27:9-13. 19. Atkin NB (1986): Lack of reciprocal translocations in carcinomas. Cancer Genet Cytogenet 21:275-278. 20. Atkin NB (1986): Chromosome I aberrations in cancer. Cancer Genet Cytogenet 21:279-285. 21. Atkin NB, Baker MC (1981): Specific chromosome change in ovarian cancer. Cancer Genet Cytogenet 3:275-276. 22. Bullerdieck J, Bartnitzke S (1985): The deleted long arm of chromosome 6: A secondary chromosome abnormality in solid tumors and lack of congenital aberrations monosomic for part of the critical segment? Cancer Genet Cytogenet 18:183-185. 23. Friedlander ML, Hedley DW, Taylor IW, Russell P, Coates AS, Tattersall MHN (1984): Influence of cellular DNA content on survival in advanced ovarian cancer. Cancer Res 44:397-400.