Ring Chromosome in Parosteal Osteosarcoma Clinical and Diagnostic Significance Jane F. Sinovic, Julia A. Bridge, and James R. Neff
ABSTRACT: In this study, two specimens of a parosteal osteosarcoma, a rare primary bone neoplasm comprising only 3-6% of all osteosarcomas, were cytogenetically analyzed utilizing standard techniques. In contrast to the complex karyotypes previously reported in osteosarcoma, this particular histologic subtype was characterized by a single chromosomal aberration, a ring chromosome. Ring chromosomes have been described as characteristic for two other low-grade malignant mesenchymal neoplasms, well-differentiated liposarcoma and dermatofibrosarcoma protuberans. We propose that the observation of a ring chromosome in osteosarcoma also correlates with a low-grade malignant potential.
INTRODUCTION
Parosteal osteosarcoma (POS), also referred to as juxtacortical osteosarcoma, is an unusual bone-producing sarcoma derived from the periosteum and/or parosteal tissue [1]. The neoplasm is most frequently located within the long bones of the limbs, in particular the distal femur. Histologically, POS most commonly presents in a low-grade pattern and prognostically, patients fare significantly better than patients with other forms of osteosarcoma [2]. Cytogenetic studies of osteosarcoma are few [3-6]. Of the reported cases, most appear to be characterized by complex karyotypes. The POS analyzed in this study was characterized by a single chromosomal abnormality, a ring chromosome. This finding may be of diagnostic and prognostic significance. CASE REPORT
A 17-year-old white male presented with a 6- to 8-week history of pain in the right popliteal fossa. A fixed hard mass was palpable on the posterior aspect of the distal femur. Radiographic studies including plain films, CT, and MRI revealed an extra-osseous mass adjacent to the posterior aspect of the right distal femoral metaphysis separated from the shaft by a thin zone of soft non-mineralized tissue (Fig. 1). A biopsy was performed and histologically consisted of bone, fibrous tissue, and cartilage. Fibrous stroma containing focally prominent lymphocytes and plasma cells intermixed with islands of irregular osteoid formation and
From the Departments of Pathology and Microbiology, Pediatrics, and Orthopaedic Surgery, University of Nebraska Medical Center, Omaha, Nebraska. Address reprint requests to: Julia A. Bridge, M.D., Hattie B. Munroe Center for Human Genetics, University of Nebraska Medical Center, 600 So. 42nd St., Omaha, NE 68198-5540. Received March 12, 1992; accepted April 22, 1992. 50 Cancer Genet Gytogenet62:50-52 (1992) 0165-4608/92/$05.00
organized lamellar bone was observed (Fig. 2). A diagnosis of parosteal osteosarcoma was established. A local resection achieving wide margins was followed with reconstruction utilizing a full-thickness tricortical anterior iliac crest graft. Grossly, the resected specimen consisted of a firm white nodular mass measuring 2.5 x 2.5 x 3.0 cm. The histologic appearance was similar to that described for the biopsy specimen. Samples of the femoral biopsy and resection specimens were submitted for cytogenetic analysis. Follow-up evaluations indicate that the patient remains free from disease 1 year after surgery.
MATERIALS AND METHODS
Tissue samples measuring 2 cm 3 were obtained directly from surgery and placed into RPMI-1640 media supplemented with 20% fetal bovine serum and antibiotics. The specimens were minced with scissors and enzymatically disaggregated by incubating overnight in 200/x/ml collagenase. Following removal of the collagenase solution, the cells were resuspended in supplemented RPMI-1640 media and seeded in 25-cm 2 culture flasks and incubated for 5-9 days. Two hours prior to harvest, the cells were exposed to Colcemid (0.02/x/ml). Following incubation in hypotonic solution (0.074 M KC1) for 30 minutes, the preparations were fixed three times with methanol and glacial acetic acid (3:1). Cells were dropped onto cold wet slides and Gbanded with Wright stain. Biotin-labeled probes for chromosomes X, 2, 6, 7, ,8, 10, 15, 17, and 18 centromeric c~-satellite sequences (ONCOR Scientific Chromosome "In-situ" Kit, Gaithersburg, MD 20877) were hybridized under stringent conditions to metaphase cells. The probes were detected by fluorescein-tagged avidin. The signal was amplified by addition of a layer of biotinylated anti-avidin followed by a second layer of fluorescein-tagged avidin. Signals were analyzed with fluorescent microscopy. :G 1992 Elsevier SciencePublishing Co.. inc. 655 Avenueof the Americas, New York. NY 10010
Ring C h r o m o s o m e in Osteosarcoma
51
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2
3
4
5
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9
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10
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18
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.| XY
Figure 3 Representative karyotype demonstrating the ring chromosome: 47,XY, + r. Figure 1 This lateral x-ray demonstrates an ossifying lesion adjacent to the posterior aspect of the distal femur of a 17-year-old male. Note the lucent region between the peripherally more dense and mature lesion from the underlying posterior cortex.
RESULTS T w e n t y m e t a p h a s e cells were analyzed from the biopsy specimen. Six of the t w e n t y cells were k a r y o t y p i c a l l y normal. Two abnormal clones were detected in the remaining cells and consisted of the following: twelve cells with
Figure 2 Parosteal osteosarcoma with well-formed bone and intervening fibrous stroma (H & E, × 100),
47,XY, + r (Fig. 3) and two ceils with 47,XY, + mar (Fig. 4). The origin of the ring c h r o m o s o m e and the marker chromosome was felt to be likely the same. Twenty-one metaphase cells from the definitive surgical s p e c i m e n were analyzed and again a ring c h r o m o s o m e was identified in ten of the m e t a p h a s e cells: 47,XY, + r. Fluorescence " i n - s i t u " h y b r i d i z a t i o n analysis of approximately 5 - 1 5 m e t a p h a s e cells and 5 0 - 2 0 0 interphase cells utilizing centromeric probes for c h r o m o s o m e s X, 2, 6, 7, 8, 10, 15, 17, and 18 were negative for staining of the ring chromosome in m e t a p h a s e cells. The majority of i n t e r p h a s e cells showed only one signal for the X c h r o m o s o m e probe and two signals for the r e m a i n i n g c h r o m o s o m a l probes tested. Therefore, the exact nature of the ring c h r o m o s o m e remained undefined. DISCUSSION Parosteal osteosarcoma (POS), an u n c o m m o n histologic subtype of osteogenic sarcoma, generally occurs equally in both sexes between the age of 15 and 40 years [7]. POS may recur, but metastases are infrequent. W h e n distant metastasis occurs, they most c o m m o n l y involve the lung and usually present after several local recurrences [8]. Histologically, POS is c o m p o s e d of a s p i n d l e cell and collagen stroma, containing osteoid and osseous trabecnlae. Occasionally, a malignant cartilage is also present. In this study, a single ring c h r o m o s o m e was detected in the analysis of a POS b i o p s y and definitive surgical s p e c i m e n from the distal femur of a 17-year-old male. The cytogenetic literature regarding classical osteosarcoma is
52
/. F. Sinovic el al.
I!
x
3
2
4
Jl...... 7
13
19
8
5
.", 9
10
14
20 XY
11
12
17
18
21
22
mar
Figure 4 Representative karyotype demonstrating the marker chromosome: 46,tas(Xq;1q/,- Y, ~-mar. The telomeric association and the loss of the Y chromosome were random.
scarce; however, the majority of cases studied have exhibited complex karyotypes with modal chromosome numbers in the hypodiploid, triploid, and hypertetraploid ranges. In three of the four reported cytogenetic studies, loss of chromosome 13 (location of retinoblastoma gene) was frequently observed [3-6]. Notably, however, clonal losses of chromosomes other than 13 were also frequently detected. Interestingly, a few cases of POS have been studied with flow cytometry. The majority of parosteal osteosarcomas studied with this technique revealed a normal diploid content [9]. H i d d e n m a n et al. [10] have shown that measurem e n t of DNA in patients with osteosarcoma can distinguish between highly malignant osteosarcomas (hyperdiploid tumors) and parosteal variants (diploid tumors) with welldifferentiated histologic features, and a relatively favorable prognosis. A flow cytometric study of histologically highgrade osteosarcomas revealed improved relapse-free and overall survival times in the near-diploid tumors [10]. The authors suggested that tumor-cell ploidy could be used to predict the sensitivity of histologically high-grade osteosarcoma to chemotherapeutic agents. In general, ring chromosomes as a neoplasm-specific a n o m a l y represent an u n u s u a l cytogenetic p h e n o m e n o n . Nevertheless, ring chromosomes have been shown to be characteristic for a few m e s e n c h y m a l neoplasma. Specifically, ring chromosomes are characteristically observed in i n t r a m u s c u l a r lipoma, well-differentiated liposarcoma [12], and dermatofibrosarcoma protuberans [13, 14]. Analogous to POS, well-differentiated liposarcoma and dermatofibrosarcoma protuberans are of low-grade malignant potential.
In summary, we have described the cytogenetic analysis of a rare osteosarcoma variant, parosteal osteosarcoma. A single chromosomal aberration, n a m e l y a ring chromosome characteristic of other low-grade m e s e n c h y m a l neoplasma, was observed. This ring chromosome may be of diagnostic utility, because to date other osteosarcoma variants have been characterized by m u l t i p l e complex abnormalities. Moreover, our findings support the idea that the presence of a ring chromosome in c o n j u n c t i o n with ploidy determination can also serve as an important prognostic indicator for osteogenic sarcoma. The authors wish to thank Ms. Kathy Sarratt for her expert secretarial assistance. This work was supported in part by a grant through the Orthopaedic Research and Education Foundation (OREE).
REFERENCES 1. Huvos AG (1991): Bone Tumors: Diagnosis, Treatment and Prognosis. 2nd Ed. pp. 157-175. 2. Campanacci M (1986): Bone and soft tissue tumors. 195:433-453. 3. Biegel JA, Womer RB, Emanuel BS (1989): Complex karyotypes in a series of pediatric osteosarcomas. Cancer Genet Cytogenet 38:89-100. 4. Castedo SMMJ, Seruca R, Oosterhuis WI, de ]ong B, Koops HS, Leeuw JA (1988): Cytogenetics of a case of osteosarcoma. Cancer Genet Cytogenet 32:149-151. 5. Mandahl N, Helm S, Kristoffersson U, Mitehnan F, Rydholm A, R66ser B, Will6n H (1986): Multiple cytogenetic abnormalities in a case of osteosarcoma. Cancer Genet Cytogenet 23:257-260. 6. Mandahl N, Heim S. Brosj6 O, Bauer HCF, Tribukait B, Rydholm A, Mitelman F (1989): Cytogenetic and quantitative DNA analysis of primary and xenografted human osteosarcomas. Cancer Genet Cytogenet 42:27-34. 7. Unni KK, Dahlin DC, Beabout JW (1976): Parosteal osteogenic sarcoma. Cancer 2466-2475. 8. Campanacci M, Picci P, Gherlinzoni F, Bertoni F. Neff" JR (1984): Parosteal osteosarcoma: The experience of lnstituto Orthopesico Rizzali. J B J S 66B:313-321. 9. Bauer HCF (1988): DNA cytometry of osteosarcoma. Acta Orthop Scand Suppl No. 228. Vol. 59. 10. Hiddemann W, Rossner A, Wormann B, Mellin W, Klockenkemper B, B6sing T, BOchner T, Grundmann E (1987]: Tumor heterogeneity in osteosarcoma as identified by flow cytometry. Cancer 59:324-328. 11. Look AT, Douglass EC, Meyer WH (1988): Clinical importance of near-diploid tumor stem lines in patients with osteosarcoma of an extremity. N Eng J Med 24:1567-1572. 12. Heim S, Mandahl N, Kristoffersson U, Mitelman F, R60ser B, Rydholm A, Will6n (1981): Marker ring chromosome: A new cytogenetic abnormality characterizing lipogenic tumors. Cancer Genet Cytogenet 24:319-326. 13. Bridge JA, Neff JR, Sandberg AA (1990): Cytogenetic analysis of dermatofibrosarcoma protuberans. Cancer Genet Cytogenet 49:199-202. 14. Mandahl N, Heim S, Will6n H, Rydhotm A, Mitelman F (1990): Supernumerary ring chromosome as the sole cytogenetic abnormality in a dermatofibrosarcoma protuberans. Cancei Genet Cytogenet 49:273-275.
ABSTRACT: In this study, two specimens of a parosteal osteosarcoma, a rare primary bone neoplasm comprising only 3-6% of all osteosarcomas, were cytogenetically analyzed utilizing standard techniques. In contrast to the complex karyotypes previously reported in osteosarcoma, this particular histologic subtype was characterized by a single chromosomal aberration, a ring chromosome. Ring chromosomes have been described as characteristic for two other low-grade malignant mesenchymal neoplasms, well-differentiated liposarcoma and dermatofibrosarcoma protuberans. We propose that the observation of a ring chromosome in osteosarcoma also correlates with a low-grade malignant potential.
INTRODUCTION
Parosteal osteosarcoma (POS), also referred to as juxtacortical osteosarcoma, is an unusual bone-producing sarcoma derived from the periosteum and/or parosteal tissue [1]. The neoplasm is most frequently located within the long bones of the limbs, in particular the distal femur. Histologically, POS most commonly presents in a low-grade pattern and prognostically, patients fare significantly better than patients with other forms of osteosarcoma [2]. Cytogenetic studies of osteosarcoma are few [3-6]. Of the reported cases, most appear to be characterized by complex karyotypes. The POS analyzed in this study was characterized by a single chromosomal abnormality, a ring chromosome. This finding may be of diagnostic and prognostic significance. CASE REPORT
A 17-year-old white male presented with a 6- to 8-week history of pain in the right popliteal fossa. A fixed hard mass was palpable on the posterior aspect of the distal femur. Radiographic studies including plain films, CT, and MRI revealed an extra-osseous mass adjacent to the posterior aspect of the right distal femoral metaphysis separated from the shaft by a thin zone of soft non-mineralized tissue (Fig. 1). A biopsy was performed and histologically consisted of bone, fibrous tissue, and cartilage. Fibrous stroma containing focally prominent lymphocytes and plasma cells intermixed with islands of irregular osteoid formation and
From the Departments of Pathology and Microbiology, Pediatrics, and Orthopaedic Surgery, University of Nebraska Medical Center, Omaha, Nebraska. Address reprint requests to: Julia A. Bridge, M.D., Hattie B. Munroe Center for Human Genetics, University of Nebraska Medical Center, 600 So. 42nd St., Omaha, NE 68198-5540. Received March 12, 1992; accepted April 22, 1992. 50 Cancer Genet Gytogenet62:50-52 (1992) 0165-4608/92/$05.00
organized lamellar bone was observed (Fig. 2). A diagnosis of parosteal osteosarcoma was established. A local resection achieving wide margins was followed with reconstruction utilizing a full-thickness tricortical anterior iliac crest graft. Grossly, the resected specimen consisted of a firm white nodular mass measuring 2.5 x 2.5 x 3.0 cm. The histologic appearance was similar to that described for the biopsy specimen. Samples of the femoral biopsy and resection specimens were submitted for cytogenetic analysis. Follow-up evaluations indicate that the patient remains free from disease 1 year after surgery.
MATERIALS AND METHODS
Tissue samples measuring 2 cm 3 were obtained directly from surgery and placed into RPMI-1640 media supplemented with 20% fetal bovine serum and antibiotics. The specimens were minced with scissors and enzymatically disaggregated by incubating overnight in 200/x/ml collagenase. Following removal of the collagenase solution, the cells were resuspended in supplemented RPMI-1640 media and seeded in 25-cm 2 culture flasks and incubated for 5-9 days. Two hours prior to harvest, the cells were exposed to Colcemid (0.02/x/ml). Following incubation in hypotonic solution (0.074 M KC1) for 30 minutes, the preparations were fixed three times with methanol and glacial acetic acid (3:1). Cells were dropped onto cold wet slides and Gbanded with Wright stain. Biotin-labeled probes for chromosomes X, 2, 6, 7, ,8, 10, 15, 17, and 18 centromeric c~-satellite sequences (ONCOR Scientific Chromosome "In-situ" Kit, Gaithersburg, MD 20877) were hybridized under stringent conditions to metaphase cells. The probes were detected by fluorescein-tagged avidin. The signal was amplified by addition of a layer of biotinylated anti-avidin followed by a second layer of fluorescein-tagged avidin. Signals were analyzed with fluorescent microscopy. :G 1992 Elsevier SciencePublishing Co.. inc. 655 Avenueof the Americas, New York. NY 10010
Ring C h r o m o s o m e in Osteosarcoma
51
jj
,it ¸
~W
1
2
3
4
5
ii 6
7
8
9
6 13 .
10
!1
t 14
15 4- ~
20
O
t6
12
j 17
18
21
22
ring
.| XY
Figure 3 Representative karyotype demonstrating the ring chromosome: 47,XY, + r. Figure 1 This lateral x-ray demonstrates an ossifying lesion adjacent to the posterior aspect of the distal femur of a 17-year-old male. Note the lucent region between the peripherally more dense and mature lesion from the underlying posterior cortex.
RESULTS T w e n t y m e t a p h a s e cells were analyzed from the biopsy specimen. Six of the t w e n t y cells were k a r y o t y p i c a l l y normal. Two abnormal clones were detected in the remaining cells and consisted of the following: twelve cells with
Figure 2 Parosteal osteosarcoma with well-formed bone and intervening fibrous stroma (H & E, × 100),
47,XY, + r (Fig. 3) and two ceils with 47,XY, + mar (Fig. 4). The origin of the ring c h r o m o s o m e and the marker chromosome was felt to be likely the same. Twenty-one metaphase cells from the definitive surgical s p e c i m e n were analyzed and again a ring c h r o m o s o m e was identified in ten of the m e t a p h a s e cells: 47,XY, + r. Fluorescence " i n - s i t u " h y b r i d i z a t i o n analysis of approximately 5 - 1 5 m e t a p h a s e cells and 5 0 - 2 0 0 interphase cells utilizing centromeric probes for c h r o m o s o m e s X, 2, 6, 7, 8, 10, 15, 17, and 18 were negative for staining of the ring chromosome in m e t a p h a s e cells. The majority of i n t e r p h a s e cells showed only one signal for the X c h r o m o s o m e probe and two signals for the r e m a i n i n g c h r o m o s o m a l probes tested. Therefore, the exact nature of the ring c h r o m o s o m e remained undefined. DISCUSSION Parosteal osteosarcoma (POS), an u n c o m m o n histologic subtype of osteogenic sarcoma, generally occurs equally in both sexes between the age of 15 and 40 years [7]. POS may recur, but metastases are infrequent. W h e n distant metastasis occurs, they most c o m m o n l y involve the lung and usually present after several local recurrences [8]. Histologically, POS is c o m p o s e d of a s p i n d l e cell and collagen stroma, containing osteoid and osseous trabecnlae. Occasionally, a malignant cartilage is also present. In this study, a single ring c h r o m o s o m e was detected in the analysis of a POS b i o p s y and definitive surgical s p e c i m e n from the distal femur of a 17-year-old male. The cytogenetic literature regarding classical osteosarcoma is
52
/. F. Sinovic el al.
I!
x
3
2
4
Jl...... 7
13
19
8
5
.", 9
10
14
20 XY
11
12
17
18
21
22
mar
Figure 4 Representative karyotype demonstrating the marker chromosome: 46,tas(Xq;1q/,- Y, ~-mar. The telomeric association and the loss of the Y chromosome were random.
scarce; however, the majority of cases studied have exhibited complex karyotypes with modal chromosome numbers in the hypodiploid, triploid, and hypertetraploid ranges. In three of the four reported cytogenetic studies, loss of chromosome 13 (location of retinoblastoma gene) was frequently observed [3-6]. Notably, however, clonal losses of chromosomes other than 13 were also frequently detected. Interestingly, a few cases of POS have been studied with flow cytometry. The majority of parosteal osteosarcomas studied with this technique revealed a normal diploid content [9]. H i d d e n m a n et al. [10] have shown that measurem e n t of DNA in patients with osteosarcoma can distinguish between highly malignant osteosarcomas (hyperdiploid tumors) and parosteal variants (diploid tumors) with welldifferentiated histologic features, and a relatively favorable prognosis. A flow cytometric study of histologically highgrade osteosarcomas revealed improved relapse-free and overall survival times in the near-diploid tumors [10]. The authors suggested that tumor-cell ploidy could be used to predict the sensitivity of histologically high-grade osteosarcoma to chemotherapeutic agents. In general, ring chromosomes as a neoplasm-specific a n o m a l y represent an u n u s u a l cytogenetic p h e n o m e n o n . Nevertheless, ring chromosomes have been shown to be characteristic for a few m e s e n c h y m a l neoplasma. Specifically, ring chromosomes are characteristically observed in i n t r a m u s c u l a r lipoma, well-differentiated liposarcoma [12], and dermatofibrosarcoma protuberans [13, 14]. Analogous to POS, well-differentiated liposarcoma and dermatofibrosarcoma protuberans are of low-grade malignant potential.
In summary, we have described the cytogenetic analysis of a rare osteosarcoma variant, parosteal osteosarcoma. A single chromosomal aberration, n a m e l y a ring chromosome characteristic of other low-grade m e s e n c h y m a l neoplasma, was observed. This ring chromosome may be of diagnostic utility, because to date other osteosarcoma variants have been characterized by m u l t i p l e complex abnormalities. Moreover, our findings support the idea that the presence of a ring chromosome in c o n j u n c t i o n with ploidy determination can also serve as an important prognostic indicator for osteogenic sarcoma. The authors wish to thank Ms. Kathy Sarratt for her expert secretarial assistance. This work was supported in part by a grant through the Orthopaedic Research and Education Foundation (OREE).
REFERENCES 1. Huvos AG (1991): Bone Tumors: Diagnosis, Treatment and Prognosis. 2nd Ed. pp. 157-175. 2. Campanacci M (1986): Bone and soft tissue tumors. 195:433-453. 3. Biegel JA, Womer RB, Emanuel BS (1989): Complex karyotypes in a series of pediatric osteosarcomas. Cancer Genet Cytogenet 38:89-100. 4. Castedo SMMJ, Seruca R, Oosterhuis WI, de ]ong B, Koops HS, Leeuw JA (1988): Cytogenetics of a case of osteosarcoma. Cancer Genet Cytogenet 32:149-151. 5. Mandahl N, Helm S, Kristoffersson U, Mitehnan F, Rydholm A, R66ser B, Will6n H (1986): Multiple cytogenetic abnormalities in a case of osteosarcoma. Cancer Genet Cytogenet 23:257-260. 6. Mandahl N, Heim S. Brosj6 O, Bauer HCF, Tribukait B, Rydholm A, Mitelman F (1989): Cytogenetic and quantitative DNA analysis of primary and xenografted human osteosarcomas. Cancer Genet Cytogenet 42:27-34. 7. Unni KK, Dahlin DC, Beabout JW (1976): Parosteal osteogenic sarcoma. Cancer 2466-2475. 8. Campanacci M, Picci P, Gherlinzoni F, Bertoni F. Neff" JR (1984): Parosteal osteosarcoma: The experience of lnstituto Orthopesico Rizzali. J B J S 66B:313-321. 9. Bauer HCF (1988): DNA cytometry of osteosarcoma. Acta Orthop Scand Suppl No. 228. Vol. 59. 10. Hiddemann W, Rossner A, Wormann B, Mellin W, Klockenkemper B, B6sing T, BOchner T, Grundmann E (1987]: Tumor heterogeneity in osteosarcoma as identified by flow cytometry. Cancer 59:324-328. 11. Look AT, Douglass EC, Meyer WH (1988): Clinical importance of near-diploid tumor stem lines in patients with osteosarcoma of an extremity. N Eng J Med 24:1567-1572. 12. Heim S, Mandahl N, Kristoffersson U, Mitelman F, R60ser B, Rydholm A, Will6n (1981): Marker ring chromosome: A new cytogenetic abnormality characterizing lipogenic tumors. Cancer Genet Cytogenet 24:319-326. 13. Bridge JA, Neff JR, Sandberg AA (1990): Cytogenetic analysis of dermatofibrosarcoma protuberans. Cancer Genet Cytogenet 49:199-202. 14. Mandahl N, Heim S, Will6n H, Rydhotm A, Mitelman F (1990): Supernumerary ring chromosome as the sole cytogenetic abnormality in a dermatofibrosarcoma protuberans. Cancei Genet Cytogenet 49:273-275.
