- Email: [email protected]
Deletion of chromosome 13 in osteosarcoma secondary to irradiation
Deletion of Chromosome 13 in Osteosarcoma Secondary to Irradiation Yavuz Y. Ozisik, Aurelia M. Meloni, Mark M. Zalupski, James R. Ryan, Faisal Qureshi, and Avery A. Sandberg
ABSTRACT: The cytogenetic analysis of a radiation-induced osteosarcoma in a 31-year-old male is presented. Complex karyotypic changes with numerical and structural abnormalities, including a del(13)(q12.3q21.1), were observed. This deletion may indicate that loss of RB1 gene (locus in 13q14) m a y be involved in the development of radiation-induced osteosarcoma.
INTRODUCTION Osteosarcoma has been reported as a secondary malignancy in patients previously treated with radiotherapy [1]. The interval between radiation therapy and the development of osteosarcoma ranges from 3.5-33 years, with a median interval of lO years [2]. Children who survive treatment for retinoblastoma, and to a lesser extent Ewing's sarcoma, appear to be at especially high risk of developing osteosarcoma [1, 3]. The addition of alkylating agent chemotherapy to radiation therapy increases the risk of developing a secondary neoplasm [1]. We report here the cytogenetic analysis of an osteosarcoma arising secondary to radiation therapy for the treatment of Ewing's sarcoma.
proximal tibia. Biopsy confirmed the clinical suspicion of osteosarcoma and an above-knee amputation was performed in December 1992. Grossly, on sectioning the tibia, the tumor measured 15 x 4 cm. The tumor distended the tibial cortex with focal encroachment of the cortical bone, but without extension into the soft tissues. On histologic examination, the tumor was high grade, with foci of poor differentiation. In other loci, osteoid was prominent (Fig. 1), Nuclear pleomorphism and mitotic figures were frequent. Osteoclastlike giant cells were present. The patient is currently receiving postoperative adjuvant chemotherapy.
MATERIALS AND METHODS CASE REPORT The patient is a 31-year-old white male diagnosed with localized Ewing's sarcoma of the right fibula in September, 1976. He was treated according to a protocol of the Ewing's Sarcoma Intergroup Study [4] beginning in October 1976. Treatment consisted of radiation therapy, 50 Gy delivered in 200cGy fractions to the entire right fibula and tibia, with a boost dose of 10 Gy to the tumor. Additional therapy included prophylactic lung irradiation and chemotherapy consisting of cyclophosphamide, vincristine, and dactinomycin for 18 months. The patient was well until the summer of 1992 when he experienced progressive discomfort and pain in his right leg. Radiographs demonstrated extensive lytic changes in the
From the Cancer Center of Southwest Biomedical Research Institute and Genetrix, Inc. (Y. Y. 0., A. M. M., A. A. S.), Scottsdale, Arizona, and Departments of Medicine (M. M. Z.), Orthopedic Surgery (J. R. R.), and Pathology (F. Q.), Wayne State Univerity, Detroit, Michigan. Address reprint requests to: AveryA. Sandberg, M.D., D.Sc., The Cancer Center of Southwest Biomedical Research Institute and Genetrix, Inc., 6401 E. Thomas Road, Scottsdale, Arizona 85251. Received February 17, 1993; accepted April 16, •993.
The tumor sample was collected aseptically during surgery and shipped for cytogenetic analysis in sterile medium. The sample was processed according to previously described methods [5]. The tissue was disaggregated in collagenase (200 U/ml) overnight and the resulting suspension seeded in flasks and coverslips using RPMI medium supplemented with 17% fetal bovine serum, 1% L-glutamine, and 2% antibiotics (penicillin 100 U/ml, streptomycin 100 ttg/ml) and incubated at 37°C and 5% CO2. The harvest procedure was performed with Colcemid exposure, hypotonic shock, and fixation in methanol:acetic acid (3:1]. Coverslips were prepared using trypsin-Giemsa and karyotyped according to the guidelines of the International System for Human Cytogenetic Nomenclature [6].
RESULTS The tumor cells were harvested after 7 days of culture; fifteen metaphases were analyzed and showed the following karyotype: 43,X, -Y, - 1, - 2,der(2)t(2 ;?)(p23;?], - 3,add(4) (p16), - 5,delC6)(q11),der(7)t(7;?)(q32;?), - 8, - 9,del(11)(p14), der(12)t(9;12)(q12;q24),der(12)t(5;12)(q23;p13),del(13)(q12.3 q21.1),der(17)t(17;?)(q25;?), - 19,add(21)(q22),del(22)(q13), + der(?)t(?;5) (?;p11), + 4mar (Fig. 2). 35
© 1993 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010
Cancer Genet Cytogenet 69:35-37 (1993) 0165-4608/93/$06.00
36
Y. Y. Ozisik et al.
Figure 1 Photomicrograph of the lesion demonstrates a cellular tumor with nuclear pleomorphism. In this betterdifferentiated area, prominent osteoid seams along with a more delicate lace-work of osteoid can be seen, some of it surrounding single cells. Scattered osteoclast-like giant cells are also present (hematoxylin and eosin x 100).
DISCUSSION Cytogenetic analysis of spontaneously occurring osteosarcoma has demonstrated very complex karyotypic changes with numerical and structural abnormalities involving chromosomes 1, 6, 8, 11, 13, 15, 17, 19, and 22 [7-121. Specific chromosome abnormalities have not yet been identified; however, structural abnormalities frequently consist of nonreciprocal translocations (derivatives) and deletions [9-111. The presence of monosomy of chromosome 13 has been observed in several cases [9, lo]. Interestingly, del(l3q) has not been observed in any of these previous cases. Cytogenetic analysis of cell lines from three reported radiation-induced osteosarcomas showed a variety of karyotypic changes, but without involvement of chromosome 13 [13]. Although the present case shows several abnormalities, the deletion chromosome 13 is thought to play a crucial role, indicated by reports of loss of heterozygosity in the deleted region, as discussed below. The retinoblastoma (RBl) gene, a well-known tumor suppressor gene, has been mapped to 13q14. Deletion of this region affects the RBl gene (loss of heterozygosity) and is associated with retinoblastoma development [l&15]. A clinical relationship between retinoblastoma and the subsequent development of osteosarcoma has been recognized. More recently, using molecular analysis, loss of heterozygosity of chromosome 13 has been found in osteosarcoma and retinoblastoma, suggesting that the RBl gene may be involved in the development of these tumors [16, 171.
Other chromosomes that may influence the development and/or progression of osteosarcoma include losses or deletions of chromosomes 1, 3, 5, 6, 9, 11, and 22, primarily because it is presumed that all of these chromosomes contain putative tumor suppressor genes. As in retinoblastoma, a similar tumor development mechanism has also been proposed for neuroblastoma (tumor suppressor gene locus in lp), breast carcinoma (locus in lq), renal cell and lung cancers (locus in 3p), colorectal carcinoma [locus in 5q), endometrial carcinoma (locus in chromosome 6), bladder carcinoma (locus in chromosome 9), Wilms’ tumor and liver cancer (locus in llp), meningioma, and acoustic neuroma (locus in 22q) [18,19]. All these genes may represent growth regulatory factors which could, alternatively or supplementary to the RBl gene, contribute to the osteosarcoma phenotype. In summary, we have observed the development of a highgrade osteosarcoma in the radiation port of a patient treated for Ewing’s sarcoma. The chromosomal changes observed in this case, particularly of chromosome 13, suggest a similar relationship between the RBl suppressor gene and radiation-induced osteosarcoma, as is seen in retinoblastomaassociated and spontaneous osteosarcoma. The authors wish to thank Shirley Frazzini for secretarial assistance, Mary Powellfor technical help, and James R. Kmvitzfor photographic assistance. This work was supported in part by Grant CA 41185 from the National Cancer Institute.
del(13q) in O s t e o s a r c o m a
37
-t t" 1
2
5
3 .,d
6 l - - , ~ t ) ~. . . . . 13
7 ~!~
8
9
-I
10
11
12
16
1T
18
........
14
15
I 19
20
i_ . . . . . .
21
Y
22
X
-----I
F i g u r e 2 G-banded karyotype of the radiation-induced osteosarcoma. Arrowheads indicate the abnormal chromosomes besides the five shown as markers. See Results for karyotype description.
REFERENCES 1. Tucker MA, D'Angio GJ, Boice JD, Strong LC, Li FP, Stovall M, Stone BJ, Green DM, Lombardi F, Newton W, Hoover RN, Fraumeni JF, It., (1987): Bone sarcomas linked to radiotherapy and chemotherapy in children. N Engl J Med 317:588-593. 2. Huvos AG (1991): Bone tumors, diagnosis, treatment, and prognosis. 2nd ed. WB Saunders, Philadelphia, pp. 223-252. 3. Strong LC, Herson J, Osborne BM, Sutow WW (1973): Risk of radiation-related subsequent malignant tumors in survivors of Ewing's sarcoma. JNCI 62:1401-1406. 4. Nesbit ME, Perez CA, Tefft M, Burgert EO Jr., Vietti TJ, Kissane J, Pritchard DJ, Gehan EA (1981): Multi-modal therapy for the management of primary, non-metastataic Ewing's sarcoma of bone: An intergroup study. NCI Monograph 56:255262. 5. Limon J, Dal Cin P, Sandberg AA (1986): Application of longterm collagenase disaggregation for the cytogenetic analysis of human solid tumor. Cancer Genet Cytogenet 23:305-313. 6. ISCN (1991): Guidelines for Cancer Cytogenetics, Supplement to an International System for Human Cytogenetic Nomenclature, Mitelman F., ed. S. Karger, Basel. 7. Mandahl N, Helm S, Kristoffersson U, Mitelman F, Rydholm A, R66ser B, Will6n H (1986): Multiple cytogenetic abnormalities in a case of osteosarcoma. Cancer Genet Cytogenet 23:257-260. 8. Castedo SMMJ, Seruca R, Oosterhuis JW, de Jong B, Koops HS, Leeuw JA (1988): Cytogenetics of a case of osteosarcoma. Cancer Genet Cytogenet 32:149-151. 9. Biegel JA, Womer RB, Emanuel BS (1989): Complex karyotypes in a series of pediatric osteosarcoma. Cancer Genet Cytogenet 38:89-100.
10. Petkovic I, Cepulic M, Nakic M (1992): Chromosome abnormalities in a case of osteosarcoma. Cancer Genet Cytogenet 63:129, 11. L6pez-Gin~s C, Carda C, Diaz MP, Gil R, L6pez A, Pellin A, Llombart-Bosch A (1992): Cytogenetic analysis of four cases of xenografted human osteosarcomas. Cancer Genet Cytogenet 63:129. 12. Schwartz HS, Allen GA, Chudoba I, Butler MG (1992): Cytogenetic abnormalities in a rare case of giant cell osteogenic sarcoma. Cancer Genet Cytogenet 58:60-65. 13. Cowan JM, Beckett MA, Tarbell N, Weichselbaum RR (1990): Symmetrical chromosome rearrangements in cell lines established from human radiation-induced sarcomas. Cancer Genet Cytogenet 50:125-137. 14. Horsthemke B (1992): Genetics and cytogenetics of retinoblastoma. Cancer Genet Cytogenet 63:1-7. 15. Cowell JK, Hogg, A (1992): Genetics and cytogenetics of retinoblastoma. Cancer Genet Cytogenet 64:1-11. 16. Toguchida J, Ishizaki K, Sasaki MS, Ikenaga M, Sugimoto M, Kotoura Y, Yamamuro T (1988): Chromosomal reorganization for the expression of recessive mutation of retinoblastoma susceptibility gene in the development of osteosarcoma. Cancer Res 48:3939-3943. 17. Scheffer H, Kruize YCM, Osinga J, Kuiken G, Oosterhuis JW, Leeus JA, Koops HS, Buys CHCM (1991): Complete association of loss of heterozygosity of chromosomes 13 and 17 in osteosarcoma. Cancer Genet Cytogenet 53:45-55. 18. Sandberg AA (1990): The Chromosomes in Human Cancer and Leukemia. Elsevier, New York, pp. 968-984. 19. Weinberg RA (1991): Tumor suppressor genes. Sci 254:11381146.
ABSTRACT: The cytogenetic analysis of a radiation-induced osteosarcoma in a 31-year-old male is presented. Complex karyotypic changes with numerical and structural abnormalities, including a del(13)(q12.3q21.1), were observed. This deletion may indicate that loss of RB1 gene (locus in 13q14) m a y be involved in the development of radiation-induced osteosarcoma.
INTRODUCTION Osteosarcoma has been reported as a secondary malignancy in patients previously treated with radiotherapy [1]. The interval between radiation therapy and the development of osteosarcoma ranges from 3.5-33 years, with a median interval of lO years [2]. Children who survive treatment for retinoblastoma, and to a lesser extent Ewing's sarcoma, appear to be at especially high risk of developing osteosarcoma [1, 3]. The addition of alkylating agent chemotherapy to radiation therapy increases the risk of developing a secondary neoplasm [1]. We report here the cytogenetic analysis of an osteosarcoma arising secondary to radiation therapy for the treatment of Ewing's sarcoma.
proximal tibia. Biopsy confirmed the clinical suspicion of osteosarcoma and an above-knee amputation was performed in December 1992. Grossly, on sectioning the tibia, the tumor measured 15 x 4 cm. The tumor distended the tibial cortex with focal encroachment of the cortical bone, but without extension into the soft tissues. On histologic examination, the tumor was high grade, with foci of poor differentiation. In other loci, osteoid was prominent (Fig. 1), Nuclear pleomorphism and mitotic figures were frequent. Osteoclastlike giant cells were present. The patient is currently receiving postoperative adjuvant chemotherapy.
MATERIALS AND METHODS CASE REPORT The patient is a 31-year-old white male diagnosed with localized Ewing's sarcoma of the right fibula in September, 1976. He was treated according to a protocol of the Ewing's Sarcoma Intergroup Study [4] beginning in October 1976. Treatment consisted of radiation therapy, 50 Gy delivered in 200cGy fractions to the entire right fibula and tibia, with a boost dose of 10 Gy to the tumor. Additional therapy included prophylactic lung irradiation and chemotherapy consisting of cyclophosphamide, vincristine, and dactinomycin for 18 months. The patient was well until the summer of 1992 when he experienced progressive discomfort and pain in his right leg. Radiographs demonstrated extensive lytic changes in the
From the Cancer Center of Southwest Biomedical Research Institute and Genetrix, Inc. (Y. Y. 0., A. M. M., A. A. S.), Scottsdale, Arizona, and Departments of Medicine (M. M. Z.), Orthopedic Surgery (J. R. R.), and Pathology (F. Q.), Wayne State Univerity, Detroit, Michigan. Address reprint requests to: AveryA. Sandberg, M.D., D.Sc., The Cancer Center of Southwest Biomedical Research Institute and Genetrix, Inc., 6401 E. Thomas Road, Scottsdale, Arizona 85251. Received February 17, 1993; accepted April 16, •993.
The tumor sample was collected aseptically during surgery and shipped for cytogenetic analysis in sterile medium. The sample was processed according to previously described methods [5]. The tissue was disaggregated in collagenase (200 U/ml) overnight and the resulting suspension seeded in flasks and coverslips using RPMI medium supplemented with 17% fetal bovine serum, 1% L-glutamine, and 2% antibiotics (penicillin 100 U/ml, streptomycin 100 ttg/ml) and incubated at 37°C and 5% CO2. The harvest procedure was performed with Colcemid exposure, hypotonic shock, and fixation in methanol:acetic acid (3:1]. Coverslips were prepared using trypsin-Giemsa and karyotyped according to the guidelines of the International System for Human Cytogenetic Nomenclature [6].
RESULTS The tumor cells were harvested after 7 days of culture; fifteen metaphases were analyzed and showed the following karyotype: 43,X, -Y, - 1, - 2,der(2)t(2 ;?)(p23;?], - 3,add(4) (p16), - 5,delC6)(q11),der(7)t(7;?)(q32;?), - 8, - 9,del(11)(p14), der(12)t(9;12)(q12;q24),der(12)t(5;12)(q23;p13),del(13)(q12.3 q21.1),der(17)t(17;?)(q25;?), - 19,add(21)(q22),del(22)(q13), + der(?)t(?;5) (?;p11), + 4mar (Fig. 2). 35
© 1993 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010
Cancer Genet Cytogenet 69:35-37 (1993) 0165-4608/93/$06.00
36
Y. Y. Ozisik et al.
Figure 1 Photomicrograph of the lesion demonstrates a cellular tumor with nuclear pleomorphism. In this betterdifferentiated area, prominent osteoid seams along with a more delicate lace-work of osteoid can be seen, some of it surrounding single cells. Scattered osteoclast-like giant cells are also present (hematoxylin and eosin x 100).
DISCUSSION Cytogenetic analysis of spontaneously occurring osteosarcoma has demonstrated very complex karyotypic changes with numerical and structural abnormalities involving chromosomes 1, 6, 8, 11, 13, 15, 17, 19, and 22 [7-121. Specific chromosome abnormalities have not yet been identified; however, structural abnormalities frequently consist of nonreciprocal translocations (derivatives) and deletions [9-111. The presence of monosomy of chromosome 13 has been observed in several cases [9, lo]. Interestingly, del(l3q) has not been observed in any of these previous cases. Cytogenetic analysis of cell lines from three reported radiation-induced osteosarcomas showed a variety of karyotypic changes, but without involvement of chromosome 13 [13]. Although the present case shows several abnormalities, the deletion chromosome 13 is thought to play a crucial role, indicated by reports of loss of heterozygosity in the deleted region, as discussed below. The retinoblastoma (RBl) gene, a well-known tumor suppressor gene, has been mapped to 13q14. Deletion of this region affects the RBl gene (loss of heterozygosity) and is associated with retinoblastoma development [l&15]. A clinical relationship between retinoblastoma and the subsequent development of osteosarcoma has been recognized. More recently, using molecular analysis, loss of heterozygosity of chromosome 13 has been found in osteosarcoma and retinoblastoma, suggesting that the RBl gene may be involved in the development of these tumors [16, 171.
Other chromosomes that may influence the development and/or progression of osteosarcoma include losses or deletions of chromosomes 1, 3, 5, 6, 9, 11, and 22, primarily because it is presumed that all of these chromosomes contain putative tumor suppressor genes. As in retinoblastoma, a similar tumor development mechanism has also been proposed for neuroblastoma (tumor suppressor gene locus in lp), breast carcinoma (locus in lq), renal cell and lung cancers (locus in 3p), colorectal carcinoma [locus in 5q), endometrial carcinoma (locus in chromosome 6), bladder carcinoma (locus in chromosome 9), Wilms’ tumor and liver cancer (locus in llp), meningioma, and acoustic neuroma (locus in 22q) [18,19]. All these genes may represent growth regulatory factors which could, alternatively or supplementary to the RBl gene, contribute to the osteosarcoma phenotype. In summary, we have observed the development of a highgrade osteosarcoma in the radiation port of a patient treated for Ewing’s sarcoma. The chromosomal changes observed in this case, particularly of chromosome 13, suggest a similar relationship between the RBl suppressor gene and radiation-induced osteosarcoma, as is seen in retinoblastomaassociated and spontaneous osteosarcoma. The authors wish to thank Shirley Frazzini for secretarial assistance, Mary Powellfor technical help, and James R. Kmvitzfor photographic assistance. This work was supported in part by Grant CA 41185 from the National Cancer Institute.
del(13q) in O s t e o s a r c o m a
37
-t t" 1
2
5
3 .,d
6 l - - , ~ t ) ~. . . . . 13
7 ~!~
8
9
-I
10
11
12
16
1T
18
........
14
15
I 19
20
i_ . . . . . .
21
Y
22
X
-----I
F i g u r e 2 G-banded karyotype of the radiation-induced osteosarcoma. Arrowheads indicate the abnormal chromosomes besides the five shown as markers. See Results for karyotype description.
REFERENCES 1. Tucker MA, D'Angio GJ, Boice JD, Strong LC, Li FP, Stovall M, Stone BJ, Green DM, Lombardi F, Newton W, Hoover RN, Fraumeni JF, It., (1987): Bone sarcomas linked to radiotherapy and chemotherapy in children. N Engl J Med 317:588-593. 2. Huvos AG (1991): Bone tumors, diagnosis, treatment, and prognosis. 2nd ed. WB Saunders, Philadelphia, pp. 223-252. 3. Strong LC, Herson J, Osborne BM, Sutow WW (1973): Risk of radiation-related subsequent malignant tumors in survivors of Ewing's sarcoma. JNCI 62:1401-1406. 4. Nesbit ME, Perez CA, Tefft M, Burgert EO Jr., Vietti TJ, Kissane J, Pritchard DJ, Gehan EA (1981): Multi-modal therapy for the management of primary, non-metastataic Ewing's sarcoma of bone: An intergroup study. NCI Monograph 56:255262. 5. Limon J, Dal Cin P, Sandberg AA (1986): Application of longterm collagenase disaggregation for the cytogenetic analysis of human solid tumor. Cancer Genet Cytogenet 23:305-313. 6. ISCN (1991): Guidelines for Cancer Cytogenetics, Supplement to an International System for Human Cytogenetic Nomenclature, Mitelman F., ed. S. Karger, Basel. 7. Mandahl N, Helm S, Kristoffersson U, Mitelman F, Rydholm A, R66ser B, Will6n H (1986): Multiple cytogenetic abnormalities in a case of osteosarcoma. Cancer Genet Cytogenet 23:257-260. 8. Castedo SMMJ, Seruca R, Oosterhuis JW, de Jong B, Koops HS, Leeuw JA (1988): Cytogenetics of a case of osteosarcoma. Cancer Genet Cytogenet 32:149-151. 9. Biegel JA, Womer RB, Emanuel BS (1989): Complex karyotypes in a series of pediatric osteosarcoma. Cancer Genet Cytogenet 38:89-100.
10. Petkovic I, Cepulic M, Nakic M (1992): Chromosome abnormalities in a case of osteosarcoma. Cancer Genet Cytogenet 63:129, 11. L6pez-Gin~s C, Carda C, Diaz MP, Gil R, L6pez A, Pellin A, Llombart-Bosch A (1992): Cytogenetic analysis of four cases of xenografted human osteosarcomas. Cancer Genet Cytogenet 63:129. 12. Schwartz HS, Allen GA, Chudoba I, Butler MG (1992): Cytogenetic abnormalities in a rare case of giant cell osteogenic sarcoma. Cancer Genet Cytogenet 58:60-65. 13. Cowan JM, Beckett MA, Tarbell N, Weichselbaum RR (1990): Symmetrical chromosome rearrangements in cell lines established from human radiation-induced sarcomas. Cancer Genet Cytogenet 50:125-137. 14. Horsthemke B (1992): Genetics and cytogenetics of retinoblastoma. Cancer Genet Cytogenet 63:1-7. 15. Cowell JK, Hogg, A (1992): Genetics and cytogenetics of retinoblastoma. Cancer Genet Cytogenet 64:1-11. 16. Toguchida J, Ishizaki K, Sasaki MS, Ikenaga M, Sugimoto M, Kotoura Y, Yamamuro T (1988): Chromosomal reorganization for the expression of recessive mutation of retinoblastoma susceptibility gene in the development of osteosarcoma. Cancer Res 48:3939-3943. 17. Scheffer H, Kruize YCM, Osinga J, Kuiken G, Oosterhuis JW, Leeus JA, Koops HS, Buys CHCM (1991): Complete association of loss of heterozygosity of chromosomes 13 and 17 in osteosarcoma. Cancer Genet Cytogenet 53:45-55. 18. Sandberg AA (1990): The Chromosomes in Human Cancer and Leukemia. Elsevier, New York, pp. 968-984. 19. Weinberg RA (1991): Tumor suppressor genes. Sci 254:11381146.