Chromosome rearrangements at 12q13 in two cases of chondrosarcomas

Chromosome Rearrangements at 12q13 in Two Cases of Chondrosarcomas Yasuko Hirabayashi, Mitsuaki A. Yoshida, Tatsuro Ikeuchi, Tsuyoshi Ishida, Tatsuyoshi Kojima, Shozo Higaki, Rikuo Machinami and Akira Tonomura

We analyzed the karyotypes of two moderately differentiated (grade 2) chondrosarcomas. Case 1 had a reciprocal translocation between chromosomes 6 and 12, t(6;12)(q25;q13) in most of the cells analyzed, as well as trisomies of chromosomes 7, 8, 11, 17, 19, and 21 and tetrasomy of chromosome 19. A reciprocal translocation involving chromosomes 12 and 19, t(12;19)(q13;q13), was noted as a highly clonal abnormality in the other case. Some cells had t(12;19) as the sole c h r o m o s o m e abnormality. Thus, c h r o m o s o m e rearrangements involving the long arm of chromosome 12 at the same region (q13) were c o m m o n l y identified in the two tumors. These findings suggest that the rearrangements at 12q13 are n o n r a n d o m acquired changes that characterize a subgroup of chondrosarcomas.

ABSTRACT:

INTRODUCTION

Recently, various types of sarcomas, as well as hematopoietic disorders and carcinomas have been studied cytogenetically [1]. N o n r a n d o m chromosome abnormalities have been identified in some sarcomas: t(12q;16p) in myxold liposarcomas, t(Xp;18q) in synovial sarcomas, t(2q;13q) in rhabdomyosarcomas, and t(11q;22q) in Ewing's sarcomas [2, 3]. Chromosome studies have been less frequently reported, however, in chondrosarcomas [4-8], which are the second most c o m m o n malignant neoplasm of bone in late adulthood. Chondrosarcoma is a cartilage-forming malignant tumor of bone, and approximately 60% of them show a lowgrade malignant histologic appearance [9, 10]. In general, the histologic diagnosis and grading of chondrosarcomas have been considered difficult because the biologic behavior of these tumors is not always reflected histologically. In particular, distinguishing low-grade chondrosarcomas from benign chondromas has been problematic [10-12]. Therefore, a more reliable means of defining the malignancy criteria of chondrosarcoma is required. In recent years, karyotype analyses have been performed to identify

From the Departments of Pathology, [Y. H., T. IS., R. M.] and Orthopedic Surgery [T. K.], Faculty of Medicine, University of Tokyo, Tokyo; the Department of Cytogenetics, Medical Research Institute, Tokyo Medical and Dental University [M. A. Y., T. IK., A. T.], Tokyo; and the Department of Orthopedic Surgery, School of Medicine, University of Teikyo [S. H.], Tokyo, Japan. Address reprint requests to: Dr. Yasuko Hirabayashi, Department of Pathology, Faculty of Medicine, University of Tokyo, 7-31 Hongo, Bunkyo-ku, Tokyo 113, Japan. Received September 23, 1991; accepted October 16, 1991. © 1992 Elsevier Science Publishing Co., Inc.

655 Avenue of the Americas, New York. NY 10010

nonrandom chromosome changes in relation to the genesis, classification, and prognosis of chondrosarcomas [4-8]. We report chromosome abnormalities in two consecutive patients with moderately differentiated chondrosarcoma, both of w h o m showed rearrangements of chromosome 12q. CASE REPORTS Case 1

A 54-year-old man was admitted to the University of Tokyo Hospital in June 1989 with complaints of insidious pain of 2 years' duration and swelling at the right shoulder. Roentgenography and cytologic examination of needle aspirates indicated chondrosarcoma of the right humerus. In addition, two other bone lesions were detected, one in the right scapula and one in the right third phalanx. The patient underwent right interscapulothoracic amputation. The tumor of the proximal humerus was a huge, grayish, lobulated translucent tumor, 12 x 14 x 7.7 cm, extending into the adjacent soft tissue. Histologic examination showed proliferation of atypical spindleshaped chondrocytes in a myxoid matrix. Hypercellularity and an increase in the number of cells with plump nuclei and of binucleate cells were also observed (Fig. la). Thus, the tumor was diagnosed as a grade 2 chondrosarcoma according to the classification of Evans et al. (1977) [9] and subjected to the present chromosome study. On the other hand, chondroid tumors of the scapula (2.5 x 4.5 cm), and of the third phalanx, (0.6 cm in diameter) were confirmed. Histologically, both of the tumors exhibited benign hyaline cartilage. The above pathologic findings led to the diagnosis of grade 2 chondrosarcoma of the right proximal 35 Cancer Genet Cytogenet60:35 40 (1992) 0165-4608/92/$05.00

36

Y. Hirabayashi et al

logic diagnosis of grade 2 chondrosarcoma of the right pubic bone was made.

MATERIALS AND METHODS Fresh tissues of chondrosarcomas were directly obtained from the surgical resection. The patients had had neither c h e m o t h e r a p y nor r a d i o t h e r a p y preoperatively. The chromosomes from mitotic cells obtained after short-term culture were analyzed as described previously [14]. The materials were finely m i n c e d with scissors after being washed with Hanks' balanced salt solution containing antibiotics. The m i n c e d tissues were digested with 0.8% collagenase (type II) solution for 1 hour at 37°C, and the separated cells were cultured in RPMI 1640 m e d i u m s u p p l e m e n t e d with 18% fetal bovine serum in 5% C Q at 37°C. After sufficient growth of tumor cells, Colcemid was a d d e d to the cultures at a final concentration of 0.01 ~g! ml. After 17 hours, the cells were detached from the culture flasks with 0.25% trypsin containing 0.02% EDTA and incubated with 0.075 M KC1 for 30 minutes at 37°C. The cells were then fixed with methanol:acetic acid (3 : 1) and spread on clean slides by conventional air-drying method. Q-staining was used for karyotype analysis.

Cytogenetic Findings

Figure 1 Representative histopathologic sections from two chondrosarcomas. (a) Case 1, grade 2 chondrosarcoma showing hypercellularity with hyperchromatic nuclei and myxoid matrix (H&E x 340). (b) Case 2, grade 2 chondrosarcoma showing atypical chondrocytes with plump nuclei and binucleation (H&E x 340).

humerus associated w i t h m u l t i p l e e n c h o n d r o m a t o s i s (O1lier's disease) [13] involving the right scapula and right phalanx. Case 2 A 34-year-old w o m a n c o m p l a i n e d of difficulty in walking for the last 10 months and was admitted to the University of Tokyo Hospital in September 1989. Roentgenography and a c o m p u t e d t o m o g r a p h y (CT) scan s h o w e d an osteolytic lesion of the right pubic bone. An atypical cartilaginous tumor of the right pubic bone was suggested by open biopsy before operation. Local extensive resection, including the right pubic bone and partial hip joint, was performed. The surgically resected t u m o r was 5.5 x 3 x 2.5 cm and a p p e a r e d grayish and translucent. Histologically, there was proliferation of atypical chondrocytes with enlarged h y p e r c h r o m a t i c nuclei, with high cellularity and a m y x o i d appearance. Binucleate cells were frequently found (Fig. lb). The n e o p l a s m destroyed the cortex of the pubic bone and infiltrated the soft tissue. A histopatho-

The results of c h r o m o s o m e analyses of the two tumors are s u m m a r i z e d in Table 1. In case 1, mitotic cells suitable for analysis were obtained after 4-day culture. The chromosome numbers ranged from 46 to 97, with a modal number of 52. Twenty-one metaphases were available for karyotype analysis. Two of them had the normal karyotype, 46XY. A reciprocal translocation between chromosomes 6 and 12, designated t(6;12)(q25;q13), was identified in 18 cells (86%), i n c l u d i n g a h y p e r t e t r a p l o i d cell with two copies of the translocation. All cells with the 6;12 translocation were s i m u l t a n e o u s l y a c c o m p a n i e d by trisomies of certain c h r o m o s o m e s (Fig. 2). More frequently involved were (in order of decreasing frequency) chromosomes 7 (100%), 11 (94%), 8 (89%), and 21 (72%). The others inc l u d e d trisomy and tetrasomy for c h r o m o s o m e 19, which were found in 44 and 22% of cells examined, respectively. The remaining cell showed m o n o s o m y 18 and an unidentiffed marker c h r o m o s o m e without showing the 6;12 translocation. In case 2, c h r o m o s o m e preparation was made 7 days after the initial cell culture. Fourteen metaphases were karyotyped. The m o d a l c h r o m o s o m e number in this case was 46, and Q-staining showed a reciprocal translocation between c h r o m o s o m e s 12 and 19, designated t(12;19)(q13;q13), w h i c h was identified in six cells (Fig. 3). Four of the six cells showed the translocation as the sole abnormality, and two cells with 45 chromosomes were monosomic for another autosome, possibly caused by a r a n d o m c h r o m o s o m e loss during preparation. Two metaphases showed the der(19) c h r o m o s o m e resulting from the, above translocation but lost the der(12) chromosome. Because these cells have only 42 and 43 chromosomes, the der(12) c h r o m o s o m e may also have been accidentally lost together with the others during preparation. Five mitotic

37

Table 1

R e s u l t s of c h r o m o s o m e a n a l y s e s i n t w o c a s e s of c h o n d r o s a r c o m a

Case no.

No. of cells analyzed

1

21

2

14

Karyotypes 46,XY 46,XY,- 18,+mar 50,XY,t(6;12)(q25;q13)+ 7,+8,+ 11,+21 a 50,XY,-3,t(6q;12q],+ 7,+ 1 1 , - 15,+ 19,+ 3mar 51,XY,t(6q;12q),+ 7,+ 8,+ 17,+ 19,+ 21 52,XY,t(6q;12q],+ 7,+8,+ 11,+ 3mar 52,XY,t(6q;12q),+ 7,+8,+ 11,+ 17,+ 19,+21 52,XY,t(6q;12q),+ 7,+8,+ 11,+ 19,+ 19,+21 52,XY,t(6q;12q),+ 7,+8,+ 11,+ 19,+21,+mar 52,XY,t(6q;12q),+ 7,+8,+ 11,+ 17,+ 19,+mar 52,XY,t(6q;12q),+ 7,+ 8,+ 11,-17,+21,+ 3mar 52,XY,t(6q;12q),+ 7,+ 8 , - 9 , + 11,- 16,+ 19,+4mar 53,XY,t(6q;12q),+ 7,+ 8,+ 11,+ 17,+ 19,÷ 21,+22 53,XY,t(6q;12q),+ 7,+8,+ 11,+ 17,+ 19,+ 19,+ 21 53,XY,t(6q;12q),+ 7,+8,+ 11,+ 17,+ 19,-20,+ 3mar 53,XY,t(6q;12q),+ 7 , + 8 , - 10,+ 11,+ 20,+ 21,+ 3mar 53,XY,t(6q;12q),+ 7,+8,+ 11,+ 17,+21,+ 2mar 54,XY,- 2,t(6q;12q),+ 7,+ 11,÷ 17,+ 19,+ 19,+21,+ 3mar 55,XY,t(6q;12q),+ 7,+ 8,+ 11,+ 17,+ 19,+ 19,+21,+2mar 97,XXYY,der(6]t(6q;12q)x 2,+8x 2 , - 10,+ 11x 2 , - 12 × 2,der(12)t(6q;12q),- 1 5 , - 1 6 , - 19,+ 21x 2 , - 22 42,XX,- 6 , - 1 2 , - 1 3 , - 16,der(19)t(12;19)(q13;q13) ° 44,XX,- 12,der(19)t(12q;19q),- 2 0 , - 22,+mar 45,XX,- 11,t(12q;19q) 45,XX,t(12q;19q],- 20 46,XX 46,XX,t(12q;19q) 46,XX,del(10p],- 13, +mar

The t(6;12)(q25;q13) and t(12;19)(q13;q13) are described in other cells as t(6q;12q) and t(12q;19q), respectively, abbreviating the breakpoints.

F i g u r e 2 Q-banded karyotype of case 1 showing the reciprocal (6;12) translocation (arrowheads). Also apparent are extra copies of chromosomes 7, 8, 11, 19, and 21: 52,XY,t(6;12)(q25;q13),+7,+8,+11,+19,+19,+21.

No. of cells 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 4 1

38

Y. Hirabayashi et al

Figure 3 Q-banded karyotype of case 2 showing the reciprocal (12;19) translocation (arrowheads) as the sole chromosome abnormality: 46,XX,t(12;19)(q13;q13).

cells showed a normal karyotype, 46, XX. The remaining cell showed a del(lOp) and an unidentified marker chromosome. DISCUSSION

Two unselected cartilaginous tumors, which were histopathologically diagnosed as moderately differentiated (grade 2) chondrosarcoma, were analyzed cytogenetically. Banding analyses demonstrated highly clonal rearrangements involving the long arm of chromosome 12 in both specimens. In particular, the reciprocal 12;19 translocation observed in case 2 was the sole chromosome abnormality in six metaphases. In case 1, the reciprocal 6;12 translocation was identified in 86% of cells analyzed and

Figure 4 Partial karyotypes showing the rearranged chromosomes in the translocations noted in case 1 (a) and case 2 (b).

these metaphases simultaneously showed some clonal numerical changes. Of particular interest was the observation that the breakpoints on chromosome 12q in both translocations were c o m m o n l y localized to the q13 region (Fig. 4), indicating that the rearrangements at 12q13 may play a significant role(s} in the genesis of such chondrosarcomas. Although chondrosarcoma is the second most common malignant bone tumor, little information is available on nonrandom chromosomal abnormalities. The particular problem in characterizing this type of malignancy is distinguishing histopathologically between benign chondromas and low-grade chondrosarcomas. Thus, cytogenetic findings have been proposed to provide valuable information in relation to the classification and prognosis of chondrosarcomas, as well as to the understanding of the mechanism of tumor development. Mandahl et al. [7], analyzing chromosome rearrangements in 16 chondromatous tumors, observed various types of chromosome abnormalities in both structure and number. Complex structural abnormalities were noted in a case of well-differentiated chondrosarcoma [6] and rearrangements involving chromosomes 1, 4, 5, and 9 were identified in two cases of dedifferentiated type [8]. So far, the chromosome studies previously reported have suggested considerable cytogenetic heterogeneity in chondromatous tumors. On the other hand, cytogenetic data for two myxoid chondrosarcomas arising from extraskeletal soft tissues have been reported, and both showed consistently characteristic chromosome rearrangements at 9(q22-31) and 22(q11-12) [4, 51.

Chromosome 12q A b n o r m a l i t i e s in Chondrosarcomas

In the present study, c o m m o n structural changes involving c h r o m o s o m e 12q13 were clonally identified in two cases, but w h e t h e r m o d e r a t e l y differentiated (grade 2) c h o n d r o s a r c o m a is generally characterized by the 12q abn o r m a l i t y is not known. The series of tumors studied by M a n d a h l et al. [7] involved three chondrosarcomas of grade 2, but none of t h e m s h o w e d the 12q abnormality, but rearrangements at the 12(q13-15) region were identified in one soft tissue c h o n d r o m a and one grade 1 chondrosarcoma [7]. Together, these findings and ours indicate that a subgroup of c h o n d r o m a t o u s tumors may exist w h i c h can be characterized by an a b n o r m a l i t y of 12(q13-15) region irrespective of the difference in the histopathologic types. Structural rearrangements a t c h r o m o s o m e 12ql 3 - 1 5 region have also been identified in tumors other than chondrosarcoma, e.g., m y x o i d l i p o s a r c o m a and lipoma [2, 15, 16], uterine l e i o m y o s a r c o m a [17, 18], m y o m a [19], and p l e o m o r p h i c a d e n o m a of the salivary gland [20]. Amplification of the cellular oncogene gli, w h i c h is localized to 12(q13-14.2), has been d e m o n s t r a t e d in rhabdomyosarcoma, osteosarcoma [21], and glioma [22], although the rearrangements of c h r o m o s o m e 12q have not been identified cytogenetically in these tumors. Molecular analyses in m y x o i d l i p o s a r c o m a s [231, l i p o m a s [24], and uterine l e i o m y o m a s [25], all of w h i c h were associated with chrom o s o m e changes at the 12(q13-15) region, showed no amplification or rearrangements in either of the oncogenes gli and int-1; the latter were located to 12q13 region [23]. Alteration in m e t h y l a t i o n patterns were detected with gli probes in m y x o i d lipsarcomas, however [26]. Whether these oncogenes are significantly associated with the 12q rearrangements in these and other reported chondromatous tumors m u s t still be elucidated. Loss or functional inactivation of tumor suppressor genes or both m a y p l a y a crucial role in the d e v e l o p m e n t of m a n y forms of m a l i g n a n c y [27, 28]. M a n d a h l et al. [7] reported frequent loss of c h r o m o s o m e s 6, 10, 11, 13, 18, and 22 in c h o n d r o s a r c o m a s and suggested that the accum u l a t e d loss of certain c h r o m o s o m e s m a y be associated with increasing proliferative or metastatic capacity. In the two cases we report, however, we did not detect loss of those chromosomes. W h e t h e r the translocation breakpoint 12q13 implicates the locus of a putative tumor suppressor gene associated w i t h genesis of c h o n d r o s a r c o m a is unknown. To u n d e r s t a n d the genetic role of the 12q13 rearrangement in d e v e l o p m e n t of chondrosarcomas and to clarify the possible r e l a t i o n s h i p between this c h r o m o s o m e a b n o r m a l i t y and histopathologic characteristics, further cytogenetic and m o l e c u l a r analyses are necessary. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture and by the Nippon Sharyo Research Fund.

REFERENCES 1. Sandberg AA (1990): The Chromosomes in Human Cancer and Leukemia. Elsevier Science Publishing Ca., New York. 2. Karakousis CP, Dal Cin P, Turc-Carel C, Limon J, Sandberg

39 AA (1987): Chromosomal changes in soft-tissue sarcomas. Arch Surg 122:1257-1260. 3. Sandberg AA, Turc-Carel C, Gemmill RM (1988): Chromosomes in solid tumors and beyond. Cancer Res 48:1049-1059. 4. Hinrichs SH, Jaramillo MA, Gumerlock PH, Gardner MB, Lewis JP, Freeman AE (1985): Myxoid chondrosarcoma with a translocation involving chromosomes 9 and 22. Cancer Genet Cytogenet 14:219-226. 5. Turc-Carel C, Dal Cin P, Rao U, Karakousis C, Sandberg AA (1988): Recurrent breakpoints at 9q13 and 22q12.2 in extraskeletal myxoid chondrosarcoma. Cancer Genet Cytogenet 30:145-150. 6. Fletcher JA, Lipinski KK, Weidner N, Morton CC (1989): Complex cytogenetic aberrations in a well-differentiated chondrosarcoma. Cancer Genet Cytogenet 41:115-121. 7. Mandahl N, Heim S, Arheden K, Rydholm A, Will~n H, Mitelman F (1990): Chromosomal rearrangements in chondromatous tumors. Cancer 65:242-248. 8. Zalupski MM, Ensley JF, Ryan J, Selvaggi S, Baker LH and Wolman SR (1990): A common cytogenetic abnormality and DNA content alterations in dedifferentiated chondrosarcoma. Cancer 66:1176-1182. 9. Evans HL, Ayala AG, Romsdahl MM (1977): Prognostic factors in chondrosarcoma of bone. Cancer 40:818-831. 10. Sanerkin NG (1980): The diagnosis and grading of chondrosarcoma of bone. A combined cytologic and histologic approach. Cancer 45:582-594. 11. Campanacci M, Guernelli N, Leonessa C, Boni A (1975): Chondrosarcoma. A study of 133 cases, 80 with long term follow up. Ital J Orthop Traumatol 1:387-414. 12. Pritchard DJ, Lunke RJ, Taylor WF, Dahlin DC, Medley BE (1980): Chondrosarcoma: A clinicopathologic and statistical analysis. Cancer 45:149-157. 13. Ollier (1900): Dyschondroplasie. Lyon Med:93:23-24. 14. Yoshida MA, Ikeuchi T, Tachibana Y, Takagi K, Moriyama M, Tonomura A (1988): Rearrangements of chromosome 3 in nonfamilial renal cell carcinomas from Japanese patients. Jpn J Cancer Res 79:600-607. 15. Mandahl N, Heim S, Johansson B, Bennet K, Mertens F, Olsson G, R6/Sser B, Rydholm A, Will~n H, Mitelman F (1987): Lipomas have characteristic structural chromosomal rearrangements of 12q13-q14. Int J Cancer 39:685-688. 16. Turc-Carel C, Dal Cin P, Boghosian L, Leong SPL, Sandberg AA (1988): Breakpoints in benign lipoma may be at 12q13 or 12q14. Cancer Genet Cytogenet 36:131-135. 17. Heim S, Nilbert M, Vanni R, Floderus U-M, Mandahl N, Liedgren S, Lecca U, Mitelman F (1988): A specific translocation, t(12;14)(q14-15;q23-24), characterizes a subgroup of uterine leiomyomas. Cancer Genet Cytogenet 32:13-17. 18. Turc-Carel C, Dal Cin P, Boghosian L, Terk-Zakarian J, Sandberg AA (1988): Consistent breakpoints in region 14q22-24 in uterine leiomyoma. Cancer Genet Cytogenet 32:25-31. 19. Gibas Z, Griffin CA, Emanuel BS (1988): Clonal chromosome rearrangements in a uterine myoma. Cancer Genet Cytogenet 32:19-24. 20. Mark J, Sandros J, Wedell B, Stenman G, Ekedahl C (1988): Significance of the choice of tissue culture technique on the chromosomal patterns in human mixed salivary gland tumors. Cancer Genet Cytogenet 33:229-244. 21. Roberts WM, Douglass EC, Peiper SC, Houghton PJ, Look AT (1989): Amplification of the gli gene in childhood sarcomas. Cancer Res 49:5407-5413. 22. Kinzler KW, Bigner SH, Bigner DD, Trent JM, Law ML, O'Brien SJ, Wong AJ, Vogelstein B (1987): Identification of an amplified, highly expressed gene in a human glioma. Science 236:70-73.

40

23. Turc-Carel C, Pietrzak E, Kakati S, Kinniburgh AJ, Sandberg AA (1987): The h u m a n int-1 gene is located at chromosome region 12q12-q13 and is not rearranged in myxoid liposarcoma with t(12;16)(q13;p11), Oncogene Res 1:397-405. 24. Arheden K, Mandahl N, Heim S, Mitelman F (1989): The INT 1 oncogene is not rearranged or amplified in lipomas with structural chromosomal abnormalities of 12q13-15. Cancer Genet Cytogenet 42:143-146. 25. Arheden K, Nilbert M, Helm S, Mandahl N, Mitelman F (1989): No amplification or rearrangement of INT-1, GLI, or COL2A1 in uterine leiomyomas with t(12;14)(q14-15;q2324). Cancer Genet Cytogenet 39:195-210.

Y. H i r a b a y a s h i et al.

26. Paulien S, Turc-Carel C, Dal Cin P, Jani-Sait S, Sreekantaiah C, Leong SPL, Vogelstein B, Kinzler KW, Sandberg AA, Gemmill RM (1990): Myxoid liposarcoma with t(12;16)(q13;p11) contains site-specific differences in methylation patterns surrounding a zinc-finger gene mapped to the breakpoint region on chromosome 12. Cancer Res 5 0 : 7 9 0 2 - 7 9 0 7 . 27. Knudson AG Jr (1985): Hereditary cancer, oncogenes, and antioncogenes. Cancer Res 45:1437-1443. 28. Green AR (1988): Recessive mechanisms of malignancy. Br J Cancer 58:115-121.