CytogeneUc Analysis of Two Sacral Chordomas Diane L. Persons, Julia A. Bridge, and James R. Neff
ABSTRACT: Cytogenetic analysis of two sacral chordomas revealed two distinct abnormal clones in one of the cases: 44,XY,t(1;3)(q42;q11),- 2,der(7)t(2;7)(q23;q32),- 21 and 46,X,t(Y;8)(q12;q22), t(1;14)(p34;q32),t(5;10)(q13;p11). All cells analyzed from the second case were cytogenetically normal. To the best of our knowledge, chordomas have not previously been subjected to cytogenetic analysis.
INTRODUCTION Chordomas represent 1-4% of primary malignant bone tumors and are theorized to arise from notochordal rests [1, 2]. Lesions are distributed throughout the axial skeleton and account for greater than 50% of primary malignancies of the sacrum. Chordomas most frequently occur in late middle age and have a 3 : 1 male to female ratio in tumors arising from the sacrum [3]. Although these slow-growing tumors metastasize late in their course, they are difficult to cure due to their axial location [4, 5]. In this report, we present the clinical and cytogenetic findings of two chordomas.
CASE REPORTS
Case 1
A 56-year-old white male was originally seen in January 1986 at an outside institution for bilateral buttock pain secondary to a biopsy-proven sacral chordoma. Treatment at this time i n c l u d e d surgically debulking the neoplasm and 66 Gy external beam radiation therapy. The patient remained free of symptoms for approximately 1 year, after which he developed persistent pain in the region of the sacrum. A complete re-evaluation at the University of Kansas Medical Center revealed local recurrence without evidence of distal disease. A radical sacrectomy was performed in October 1987 through the body of $1 i n c l u d i n g the rectum and adjacent soft tissue, followed by 17.5 Gy intraoperative radiation therapy. Analysis of the regional l y m p h nodes revealed u n s u s p e c t e d metastatic adenocarcinoma of the prostate. In view of persistent disease in the lumbar region, the patient received 59.4 Gy external beam radiation therapy in February of 1989. Later, in April 1990, an additional lesion was discovered in the right subtrochanter region where an open biopsy revealed metastatic chordoma. Material from this previously untreated and unirradiated lesion was submitted for cytogenetic analysis. From the Departmentof Pathologyand Oncology(D. P.. J. B., J. N.), Universityof KansasMedical Center. and the Departmentsof Pediatricsand Pathology/Microbiology(J. B.), Universityof Nebraska MedicalCenter. Address reprint requests to: Dr. Julia A. Bridge, Hattie B. Munroe Center for Human Genetics, University of Nebraska Medical Center, 42nd and Dewey Ave., Omaha, Nebraska 68198. Received January 17, 1991; accepted April 4, 1991.
197 © 1991 ElsevierSciencePublishingCo., Inc. 655 Avenue of the Americas,New York, NY 10010
Cancer Genet Cytogenet56:197-201 (1991) 0165-4608/91/$03.50
198
D.L. Persons et al.
Case 2
A 77-year-old white female was originally evaluated at an outside hospital in 1985 for pain related to a biopsy-proven sacral chordoma. Subsequently, the neoplasm was surgically debulked. One year later the patient was re-evaluated at the University of Kansas Medical Center for local recurrence of disease. Treatment at this time consisted of a sacral amputation through the body of $1 sparing the $1 nerve roots. No external beam radiation was utilized. The patient did well for approximately 3 years until she developed another local recurrence. A portion of the tumor which was subsequently debulked was submitted for cytogenetic analysis. The patient shortly thereafter expired. Postmortem examination revealed multiple skin and liver metastases. MATERIALS AND METHODS
Tissue samples measuring 1-2 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 or by incubating with 0.8% collagenase for 1.5-3.0 hours at 37°C. Following removal of the collagenase solution, the cells were resuspended in supplemented RPMI°1640 media (as stated above) and seeded in 25 cm 2 culture flasks or plated onto coverslips and incubated for 9-15 days. Chromosome preparations were analyzed utilizing previously described standard GTG-banding procedures [6]. Briefly, 3-8 hours prior to harvest, the cells were exposed to Colcemid (0.02 /xg/mL). Following incubation in hypotonic solution /0.075 M KCL) for 30 minutes, the preparations were fixed three times with methanol and glacial acetic acid/3 : 1). Coverslips were GTG banded with Harleco Giemsa stain. Cells harvested from flasks were dropped onto cold wet slides and G-banded with Wright stain. Thirteen and 12 metaphase cells were analyzed for cases 1 and 2, respectfully. RESULTS Case 1
Thirteen metaphase cells were analyzed. Two distinct abnormal clones were detected. One clone consisted of four cells with structural rearrangements involving chromosomes 1, 2, 3, and 7 (Fig. 1). One of the four cells was polyploid, but the same structural abnormalities were observed. A second clone (three cells) consisted of structural rearrangements involving chromosomes 1, 5, 8, 10, 14, and the Y chromosome (Fig. 2). The chromosomal complements are as follows: 44,XY,t(1;3)(q42;q11),-2, der(7)t(2;7)(q23;q32),- 21 and 46,X,t(Y;8)(q12;q22),t(1;14)(p34;q32),t(5;10)(q13;p11).
Case 2
Eleven of 12 metaphase cells analyzed were karyotypically normal. One cell was monosomic for chromosome 21. DISCUSSION
Chordomas are primary bone tumors which arise throughout the axial skeleton and comprise greater than 50% of the primary malignancies of the sacrum. To the best of knowledge, this is the first cytogenetic report of chordomas. We
199
Cytogenetic Analysis of Two Sacral Chordomas
B
A
'e I
3
I
3
2
7
2
7
Figure 1 Partial karyotype of two metaphase cells (A and B) from one clone in case 1 demonstrating the t(1;3)(q42;q11) and the der(7)t(2;7)(q23;q32). Note*: The normal 3 homologue was randomly absent in the B metaphase cell. detected two apparently separate abnormal clones from one chordoma specimen. The presence of two separate clones suggests the possibility of multiclonal origin. Studies indicating that some tumors are multiclonal have been reported [7-13]. However, the possibility that the two clones are secondary to a primary event that was either lost or detectable only at the molecular level cannot be excluded. In addition, the findings are difficult to interpret because the tissue was from a metastasis, and the primary tumor had undergone radiation treatment. The i n v o l v e m e n t of the Y chromosome in a structural rearrangement, as seen in Figure 2 Partial karyotype from the second clone in case 1 demonstrating t(1;14)(p34;q32), t(5;10)(q13;p11), and t(Y;8)(q12;q22).
~,~
4
1
¢~
14
5
10
8
Y
200
D.L. Persons et al.
Figure 3 Small nests of viable chordoma cells (upper left to lower right), from case 2, consisting af classic physaliferous cells surrounded by extensive necrosis.
this study, is highly unusual. Only a few reports of Y chromosome rearrangements in solid tumors are present in the literature. Abnormalities have been observed in a salivary gland tumor [14], large intestinal tumors [15, 16], and k i d n e y neoplasms [17,
18]. In the second case, tissue was obtained from a recurrent sacral c h o r d o m a w h i c h had not previously undergone irradiation. No clonal abnormalities were observed in this case; however, one cell was m o n o s o m i c for c h r o m o s o m e 21. A l t h o u g h the most likely explanation for this m o n o s o m y is r a n d o m loss, it is of interest that a clonal m o n o s o m y 21 was present in case 1. The analysis of the second case was c o m p l i c a t e d by extensive tumor necrosis. Rare small nests of viable tumor cells were identified in only one histology section from the final debulking p r o c e d u r e from w h i c h tissue was submitted for cytogenetic analysis (Fig. 3). The marked tumor necrosis was most likely secondary to the large size of the neoplasm, as the patient had not been subjected to either radiation or chemotherapy. Therefore, only non-viable tumor m a y have been s a m p l e d resulting in overgrowth of normal s u p p o r t i v e stromal cells. In addition, the lack of cytogenetic abnormalities in this case does not exclude the possibility of grossly undetectable aberrations present at the m o l e c u l a r level. A d d i t i o n a l c h r o m o s o m a l studies are necessary to determine the significance of the observed abnormalities in this study.
This work was supported in part by a grant through the Orthopaedic Research Education Foundation (OREF).
C y t o g e n e t i c A n a l y s i s of T w o S a c r a l C h o r d o m a s
201
REFERENCES 1. Healey JH, Lane JM (1989): Chordoma: A critical review of diagnosis and treatment. Ortho Clinics of North Am 20:417-426. 2. Mirra JM (1989): Bone tumors: Clinical, radiologic, and pathologic correlations. Lea and Febiger, Philadelphia, pp. 648-672. 3. Rich TA, Schiller A, Suit HD, Mankin HJ (1985): Clinical and pathologic review of 48 cases of chordoma. Cancer 56:182-187. 4. Gray SW, Singhabhand WB, Smith RA, Skandalakis JE (1975): Sacrococcygeal chordoma: Report of a case and review of the literature. Surgery 78:573-582. 5. Chambers PW, Schwinn CP (1979): Chordoma: A clinicopathologic study of metastasis. Am J Clin Pathol 72:765-776. 6. Bridge JA, Sanger WG, Shaffer B, Neff JR (1987): Cytogenetic findings in malignant fibrous histiocytoma. Cancer Genet Cytogenet 29:97-102. 7. Jin YS, Helm S, Mandahl N, Biorklund A, Wennerberg J, Mite|man F (1990): Multiple clonal chromosome aberrations in squamous cell carcinoma of the larynx. Cancer Genet Cytogenet 44:209-216. 8. Heim S, Caron M, Jin Y, Mandahl N, Mitelman F (1989): Genetic convergence during serial in vitro passage of a polyclonal squamous cell carcinoma. Cancer Genet Cytogenet 52:133-135. 9. Heim S, Jin Y, Mandahl N, Biorklund A, Wennerberg J, Jonsson N, Mitelman F (1988): Multiple unrelated chromosome abnormalities in an in situ squamous cell carcinoma of the skin. Cancer Genet Cytogenet 36:149-153. 10. Jin YS, Helm S, Mandahl N, Biorklund A, Wennerberg J, Mitelman F (1988): Multiple apparently unrelated clonal chromosome abnormalities in a squamous cell carcinoma of the tongue. Cancer Genet Cytogenet 32:93-100. 11. Vogelstein B, Fearon ER, Hamilton SR, Preisinger AC, Willard HF, Michelson AM, Riggs AD, Orkin SH (1987): Clonal analysis using recombinant DNA probes from the X-chromosome. Cancer Res 47:4806-4813. 12. Hsu SH, Luk GD, Krush AJ, Hamilton SR, Hoover HH (1983): Multiclonal origin of polyps in Gardner syndrome. Science 221:951-953. 13. Failkow PJ, Jackson CE, Block MA, Greenwald KA (1977): Multicellular origin of parathyroid "adenomas". N Engl J Med 297:696-698. 14. Mark J, Dahlenfors R, Ekedahl C (1983): Cytogenetics of the h u m a n benign mixed salivary gland tumour. Hereditas 99:115-129. 15. Martin P, Levin B, Golomb HM, Riddell RH (1979): Chromosome analysis of primary large bowel tumors. A new method for improving the yield of analyzable metaphases. Cancer 44:1656-1664. 16. Reichmann A, Martin P, Levin B (1981): Chromosomal banding patterns in h u m a n large bowel cancer. Int J Cancer 28:431-440. 17. Kovacs G, Szucs S, de Riese W, Baumgartel H (1987): Specific chromosome aberration in h u m a n renal cell carcinoma. Int J Cancer 40:171-178. 18. Szucs S, Muller-Brechlin R, DeRiese W, Kovacs G (1987): Deletion 3p: The only chromosome loss in a primary renal cell carcinoma. Cancer Genet Cytogenet 26:369-373.
ABSTRACT: Cytogenetic analysis of two sacral chordomas revealed two distinct abnormal clones in one of the cases: 44,XY,t(1;3)(q42;q11),- 2,der(7)t(2;7)(q23;q32),- 21 and 46,X,t(Y;8)(q12;q22), t(1;14)(p34;q32),t(5;10)(q13;p11). All cells analyzed from the second case were cytogenetically normal. To the best of our knowledge, chordomas have not previously been subjected to cytogenetic analysis.
INTRODUCTION Chordomas represent 1-4% of primary malignant bone tumors and are theorized to arise from notochordal rests [1, 2]. Lesions are distributed throughout the axial skeleton and account for greater than 50% of primary malignancies of the sacrum. Chordomas most frequently occur in late middle age and have a 3 : 1 male to female ratio in tumors arising from the sacrum [3]. Although these slow-growing tumors metastasize late in their course, they are difficult to cure due to their axial location [4, 5]. In this report, we present the clinical and cytogenetic findings of two chordomas.
CASE REPORTS
Case 1
A 56-year-old white male was originally seen in January 1986 at an outside institution for bilateral buttock pain secondary to a biopsy-proven sacral chordoma. Treatment at this time i n c l u d e d surgically debulking the neoplasm and 66 Gy external beam radiation therapy. The patient remained free of symptoms for approximately 1 year, after which he developed persistent pain in the region of the sacrum. A complete re-evaluation at the University of Kansas Medical Center revealed local recurrence without evidence of distal disease. A radical sacrectomy was performed in October 1987 through the body of $1 i n c l u d i n g the rectum and adjacent soft tissue, followed by 17.5 Gy intraoperative radiation therapy. Analysis of the regional l y m p h nodes revealed u n s u s p e c t e d metastatic adenocarcinoma of the prostate. In view of persistent disease in the lumbar region, the patient received 59.4 Gy external beam radiation therapy in February of 1989. Later, in April 1990, an additional lesion was discovered in the right subtrochanter region where an open biopsy revealed metastatic chordoma. Material from this previously untreated and unirradiated lesion was submitted for cytogenetic analysis. From the Departmentof Pathologyand Oncology(D. P.. J. B., J. N.), Universityof KansasMedical Center. and the Departmentsof Pediatricsand Pathology/Microbiology(J. B.), Universityof Nebraska MedicalCenter. Address reprint requests to: Dr. Julia A. Bridge, Hattie B. Munroe Center for Human Genetics, University of Nebraska Medical Center, 42nd and Dewey Ave., Omaha, Nebraska 68198. Received January 17, 1991; accepted April 4, 1991.
197 © 1991 ElsevierSciencePublishingCo., Inc. 655 Avenue of the Americas,New York, NY 10010
Cancer Genet Cytogenet56:197-201 (1991) 0165-4608/91/$03.50
198
D.L. Persons et al.
Case 2
A 77-year-old white female was originally evaluated at an outside hospital in 1985 for pain related to a biopsy-proven sacral chordoma. Subsequently, the neoplasm was surgically debulked. One year later the patient was re-evaluated at the University of Kansas Medical Center for local recurrence of disease. Treatment at this time consisted of a sacral amputation through the body of $1 sparing the $1 nerve roots. No external beam radiation was utilized. The patient did well for approximately 3 years until she developed another local recurrence. A portion of the tumor which was subsequently debulked was submitted for cytogenetic analysis. The patient shortly thereafter expired. Postmortem examination revealed multiple skin and liver metastases. MATERIALS AND METHODS
Tissue samples measuring 1-2 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 or by incubating with 0.8% collagenase for 1.5-3.0 hours at 37°C. Following removal of the collagenase solution, the cells were resuspended in supplemented RPMI°1640 media (as stated above) and seeded in 25 cm 2 culture flasks or plated onto coverslips and incubated for 9-15 days. Chromosome preparations were analyzed utilizing previously described standard GTG-banding procedures [6]. Briefly, 3-8 hours prior to harvest, the cells were exposed to Colcemid (0.02 /xg/mL). Following incubation in hypotonic solution /0.075 M KCL) for 30 minutes, the preparations were fixed three times with methanol and glacial acetic acid/3 : 1). Coverslips were GTG banded with Harleco Giemsa stain. Cells harvested from flasks were dropped onto cold wet slides and G-banded with Wright stain. Thirteen and 12 metaphase cells were analyzed for cases 1 and 2, respectfully. RESULTS Case 1
Thirteen metaphase cells were analyzed. Two distinct abnormal clones were detected. One clone consisted of four cells with structural rearrangements involving chromosomes 1, 2, 3, and 7 (Fig. 1). One of the four cells was polyploid, but the same structural abnormalities were observed. A second clone (three cells) consisted of structural rearrangements involving chromosomes 1, 5, 8, 10, 14, and the Y chromosome (Fig. 2). The chromosomal complements are as follows: 44,XY,t(1;3)(q42;q11),-2, der(7)t(2;7)(q23;q32),- 21 and 46,X,t(Y;8)(q12;q22),t(1;14)(p34;q32),t(5;10)(q13;p11).
Case 2
Eleven of 12 metaphase cells analyzed were karyotypically normal. One cell was monosomic for chromosome 21. DISCUSSION
Chordomas are primary bone tumors which arise throughout the axial skeleton and comprise greater than 50% of the primary malignancies of the sacrum. To the best of knowledge, this is the first cytogenetic report of chordomas. We
199
Cytogenetic Analysis of Two Sacral Chordomas
B
A
'e I
3
I
3
2
7
2
7
Figure 1 Partial karyotype of two metaphase cells (A and B) from one clone in case 1 demonstrating the t(1;3)(q42;q11) and the der(7)t(2;7)(q23;q32). Note*: The normal 3 homologue was randomly absent in the B metaphase cell. detected two apparently separate abnormal clones from one chordoma specimen. The presence of two separate clones suggests the possibility of multiclonal origin. Studies indicating that some tumors are multiclonal have been reported [7-13]. However, the possibility that the two clones are secondary to a primary event that was either lost or detectable only at the molecular level cannot be excluded. In addition, the findings are difficult to interpret because the tissue was from a metastasis, and the primary tumor had undergone radiation treatment. The i n v o l v e m e n t of the Y chromosome in a structural rearrangement, as seen in Figure 2 Partial karyotype from the second clone in case 1 demonstrating t(1;14)(p34;q32), t(5;10)(q13;p11), and t(Y;8)(q12;q22).
~,~
4
1
¢~
14
5
10
8
Y
200
D.L. Persons et al.
Figure 3 Small nests of viable chordoma cells (upper left to lower right), from case 2, consisting af classic physaliferous cells surrounded by extensive necrosis.
this study, is highly unusual. Only a few reports of Y chromosome rearrangements in solid tumors are present in the literature. Abnormalities have been observed in a salivary gland tumor [14], large intestinal tumors [15, 16], and k i d n e y neoplasms [17,
18]. In the second case, tissue was obtained from a recurrent sacral c h o r d o m a w h i c h had not previously undergone irradiation. No clonal abnormalities were observed in this case; however, one cell was m o n o s o m i c for c h r o m o s o m e 21. A l t h o u g h the most likely explanation for this m o n o s o m y is r a n d o m loss, it is of interest that a clonal m o n o s o m y 21 was present in case 1. The analysis of the second case was c o m p l i c a t e d by extensive tumor necrosis. Rare small nests of viable tumor cells were identified in only one histology section from the final debulking p r o c e d u r e from w h i c h tissue was submitted for cytogenetic analysis (Fig. 3). The marked tumor necrosis was most likely secondary to the large size of the neoplasm, as the patient had not been subjected to either radiation or chemotherapy. Therefore, only non-viable tumor m a y have been s a m p l e d resulting in overgrowth of normal s u p p o r t i v e stromal cells. In addition, the lack of cytogenetic abnormalities in this case does not exclude the possibility of grossly undetectable aberrations present at the m o l e c u l a r level. A d d i t i o n a l c h r o m o s o m a l studies are necessary to determine the significance of the observed abnormalities in this study.
This work was supported in part by a grant through the Orthopaedic Research Education Foundation (OREF).
C y t o g e n e t i c A n a l y s i s of T w o S a c r a l C h o r d o m a s
201
REFERENCES 1. Healey JH, Lane JM (1989): Chordoma: A critical review of diagnosis and treatment. Ortho Clinics of North Am 20:417-426. 2. Mirra JM (1989): Bone tumors: Clinical, radiologic, and pathologic correlations. Lea and Febiger, Philadelphia, pp. 648-672. 3. Rich TA, Schiller A, Suit HD, Mankin HJ (1985): Clinical and pathologic review of 48 cases of chordoma. Cancer 56:182-187. 4. Gray SW, Singhabhand WB, Smith RA, Skandalakis JE (1975): Sacrococcygeal chordoma: Report of a case and review of the literature. Surgery 78:573-582. 5. Chambers PW, Schwinn CP (1979): Chordoma: A clinicopathologic study of metastasis. Am J Clin Pathol 72:765-776. 6. Bridge JA, Sanger WG, Shaffer B, Neff JR (1987): Cytogenetic findings in malignant fibrous histiocytoma. Cancer Genet Cytogenet 29:97-102. 7. Jin YS, Helm S, Mandahl N, Biorklund A, Wennerberg J, Mite|man F (1990): Multiple clonal chromosome aberrations in squamous cell carcinoma of the larynx. Cancer Genet Cytogenet 44:209-216. 8. Heim S, Caron M, Jin Y, Mandahl N, Mitelman F (1989): Genetic convergence during serial in vitro passage of a polyclonal squamous cell carcinoma. Cancer Genet Cytogenet 52:133-135. 9. Heim S, Jin Y, Mandahl N, Biorklund A, Wennerberg J, Jonsson N, Mitelman F (1988): Multiple unrelated chromosome abnormalities in an in situ squamous cell carcinoma of the skin. Cancer Genet Cytogenet 36:149-153. 10. Jin YS, Helm S, Mandahl N, Biorklund A, Wennerberg J, Mitelman F (1988): Multiple apparently unrelated clonal chromosome abnormalities in a squamous cell carcinoma of the tongue. Cancer Genet Cytogenet 32:93-100. 11. Vogelstein B, Fearon ER, Hamilton SR, Preisinger AC, Willard HF, Michelson AM, Riggs AD, Orkin SH (1987): Clonal analysis using recombinant DNA probes from the X-chromosome. Cancer Res 47:4806-4813. 12. Hsu SH, Luk GD, Krush AJ, Hamilton SR, Hoover HH (1983): Multiclonal origin of polyps in Gardner syndrome. Science 221:951-953. 13. Failkow PJ, Jackson CE, Block MA, Greenwald KA (1977): Multicellular origin of parathyroid "adenomas". N Engl J Med 297:696-698. 14. Mark J, Dahlenfors R, Ekedahl C (1983): Cytogenetics of the h u m a n benign mixed salivary gland tumour. Hereditas 99:115-129. 15. Martin P, Levin B, Golomb HM, Riddell RH (1979): Chromosome analysis of primary large bowel tumors. A new method for improving the yield of analyzable metaphases. Cancer 44:1656-1664. 16. Reichmann A, Martin P, Levin B (1981): Chromosomal banding patterns in h u m a n large bowel cancer. Int J Cancer 28:431-440. 17. Kovacs G, Szucs S, de Riese W, Baumgartel H (1987): Specific chromosome aberration in h u m a n renal cell carcinoma. Int J Cancer 40:171-178. 18. Szucs S, Muller-Brechlin R, DeRiese W, Kovacs G (1987): Deletion 3p: The only chromosome loss in a primary renal cell carcinoma. Cancer Genet Cytogenet 26:369-373.
