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Cytogenetic findings in 14 benign cartilaginous neoplasms
Cancer Genetics 204 (2011) 180e186
Cytogenetic findings in 14 benign cartilaginous neoplasms Nilo Sakai Junior*, Kikue Terada Abe, Lia Menezes Formigli, Marcio Fernandes Pereira, Maria Dulce Valverde de Oliveira, Deborah Afonso Cornelio, Alessandra de La Roque Ferreira, Ricardo Karam Kalil Molecular Pathology Division, Sarah Network of Rehabilitation Hospitals, Brasilia, DF, Brazil Benign cartilaginous tumors represent a spectrum of neoplastic processes with variable clinical and pathologic presentations. These tumors are histologically characterized by the presence of chondrocytes surrounded by a cartilaginous matrix. Few studies describe karyotypic abnormalities in these benign lesions. We report a series of 14 chondromas from a single institution. Conventional cytogenetics was performed on short term cultures from all cases. Clonal chromosome aberrations were found in nine tumors. One soft tissue chondroma contained three clones with t(6;12)(q12;p11.2), t(3;7)(q13;p12), and der(2)t(2;18)(p11.2;q11.2). Three periosteal chondromas displayed random structural aberrations of chromosomes 2, 3, 6, 7, and 11 and loss of chromosome 13. Among the enchondromas, three tumors displayed chromosome losses, one contained a complex translocation involving chromosomes 12, 15, and 21 as well as an inv(2)(p21q31),t(12;15;21)(q13;q14;q22) and a separate enchondroma showed a translocation involving chromosomes 12 and 22. Our data suggest that considerable cytogenetic heterogeneity exists among benign chondromatous tumors. Keywords Chondromas, cytogenetics ª 2011 Elsevier Inc. All rights reserved.
Benign cartilage tumors, including enchondromas, soft tissue chondromas, and periosteal chondromas, represent approximately 10e25% of all primary bone tumors. Enchondromas typically occur in the small tubular bones of the hands and feet, whereas the rare periosteal chondromas predominantly appear on the surface of long tubular bones (1). These tumors are not usually clinically apparent and therefore often diagnosed as incidental findings. The differential diagnosis between chondroma and low-grade chondrosarcoma is one of the most difficult in bone tumor pathology. The histological differences between benign lesions and well-differentiated malignant tumors are frequently tenuous, and welldifferentiated chondrosarcomas may display borderline or intermediate features of malignancy. Histologic grade has been shown to be of some prognostic importance; however, discrepancies between histologic appearance and biologic behavior are not infrequent. Chromosomal studies have proven to be of value in the diagnostic assessment of many
Received February 17, 2010; received in revised form February 9, 2011; accepted February 14, 2011. * Corresponding author. E-mail address: [email protected] 2210-7762/$ - see front matter ª 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergen.2011.02.004
mesenchymal neoplasms (2). Cytogenetic analysis can be used as a complementary histological assessment tool to yield increased biologic information on cartilaginous tumors. Few cytogenetic reports of enchondroma, periosteal chondroma, and soft tissue chondroma exist (3e12). To date, the Catalog of Chromosome Aberrations in Cancer has published only 27 cytogenetically abnormal bone and soft tissue chondromas (13). In this study, we present a review of the literature as well as the karyotypic findings of eight enchondromas, five periosteal chondromas, and one soft tissue chondroma.
Materials and methods Clinical and histopathologic data Between 2001 and 2008, a total of 35 chondromas were collected in our pathology department, of which 28 were enchondromas, 6 were periosteal chondromas, and 1 was a soft tissue chondroma. Tumor tissue samples from 14 patients were received directly from surgery for cytogenetic analysis. Six patients were male and 8 were female. Their ages ranged from 1 to 51 years old. All of the lesions studied
Cytogenetic findings in chondromas
181
were untreated primary tumors. The tumors were histologically classified into subtypes, examined to confirm the absence of features suggestive of malignant transformation, and the results correlated with radiological findings. Eight tumors were identified as enchondromas, 5 were periosteal chondromas, and 1 was a soft tissue chondroma. None of the patients had Maffucci syndrome or Ollier disease. Our study was conducted in accordance with the guidelines of the Medical Ethics Commission of the SARAH Network of Rehabilitation Hospitals, Brazil.
manufacturer’s instructions. This total RNA was reverse transcribed with random primers with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). The resulting cDNA was amplified by PCR using EWSR1 501F and DDIT3 194R primers (15). In parallel, the viability of the extracted RNA was verified by assaying the cDNA product with primers for the housekeeping gene PGK. Positive and negative controls were tested simultaneously with the tumor sample. Amplified products were analyzed on a 1.8% agarose gel.
Cell culture and cytogenetic analysis
Results
The tumor biopsy samples obtained for cytogenetic analysis were mechanically disaggregated with a caliper and a scalpel and incubated for 2 hours in a collagenase II solution (8 mg/ mL). The resulting cell suspension was washed and cultured at 37 C, 5% CO2 in 25 cm2 T flasks with McCoy 5A medium supplemented with L-glutamine, antibiotics, and 10e20% fetal bovine serum. After 5e18 days of primary culture, chromosomes were prepared according to standard protocols. Wright stain was used for trypsin G-banding. At least 20 metaphases were analyzed, and chromosome aberrations were classified according to the International System for Human Cytogenetic Nomenclature (ISCN 2005) (14).
Histologically, all cases of chondromas in our series showed the usual pattern described in the literature. Thirteen of the 14 patients experienced local pain as the main symptom, and enchondroma was an occasional finding in only one patient with carpal tunnel syndrome. All of the soft tissue and surface lesions were excised, and the enchondromas were also subjected to curettage. Follow-up of all cases included in the current study are listed in Table 1. The cytogenetic analysis of each of the 14 chondroma specimens was successful. Normal karyotypes were detected in samples from five patients (patients 1, 2, 3, 7, and 8): four were enchondromas, and one was a periosteal chondroma. Non-clonal alterations were found in tissue from four patients: losses of chromosomes 4, 6, 9, 12, 14, and 16 were found in patients 1, 3, and 7; patient 2 showed the alteration del(7)(p12p15); and patient 3 contained the alterations tas(14;15)(q32;q26), ?del(X)(p?), t(1;?5). Nine patients (patients 4, 5, 6, 9, 10, 11, 12, 13, and 14) displayed clonal chromosome aberrations (Table 2), including diploid or near-diploid clones. One soft tissue chondroma contained four clones: 44w46,XY,t(6;12) (q12;p11.2)[4]/40w46,XY,t(3;7)(q13;p12)[3]/46,XY,der(2) t(2;18)(p11.2;q11.2)[2]/46,XY[13]. Three periosteal chondromas had random structural aberrations of chromosomes 2, 3, 6, 7, and 11, loss of chromosome 13, and a normal clone. Three enchondromas contained both a clone with chromosome losses and a normal clone, and one enchondroma had a clone with 46,XY,inv(2)(p21q31),t(12;15;21) (q13;q14;q22)[18]. Finally, one enchondroma contained a clone with the 46,XY,t(12;22)(q13;q13)[20] alteration only
Fluorescence in situ hybridization (FISH) analysis The FISH procedure was performed on G-banded slides according to the manufacturer’s instructions. The LSI EWSR1 (22q12) Dual Color, Break Apart Rearrangement Probe (Abbott, Downers Grove, IL) was used to detect EWSR1 gene rearrangements. Hybridization signals were observed by FISHView software (Applied Spectral Imaging, Migdal Haemek, Israel).
Reverse transcriptaseepolymerase chain reaction (RT-PCR) analysis Total RNA was isolated from frozen tumor tissue with the RNeasy Mini Kit (Qiagen, Valencia, CA) according to the Table 1
Histologic and clinical data for soft tissue chondroma, enchondroma and periosteal chondroma cases
Case
Diagnosis
Sex/age (y)
Location/size (cm)
Follow-up (mo)
1/N 2/N 3/N 4/A 5/A 6/A 7/N 8/N 9/A 10/A 11/A 12/A 13/A 14/A
Enchondroma Enchondroma Periosteal chondroma Periosteal chondroma Enchondroma Enchondroma Enchondroma Enchondroma Enchondroma Soft tissue chondroma Periosteal chondroma Periosteal chondroma Periosteal chondroma Enchondroma
F/35 F/35 F/8 M/22 F/40 F/35 M/4 F/26 F/45 M/51 M/24 F/1 M/23 M/33
Metacarpal/2.5 Hand phalanx/3.0 Metatarsal/3.2 Proximal femur/3.5 Hand phalanx/1.0 Halux/2.0 Distal femur/1.5 Hand phalanx/1.3 Hand phalanx/1.2 Foot phalanx/4.5 Tibia/2.0 Tibia/3.0 Radius/1.5 Proximal femur/2.0
40/NED 40/NED 6/NED 8/NED 28/NED 30/NED 22/NED 12/NED 30/NED 10/NED 7/NED 36/NED 30/NED 17/NED
Abbreviations: F, female; M, male; N, normal karyotype; A, abnormal karyotype; NED, no evidence of disease.
182 Table 2 Case
N. Sakai Junior et al. Clinical and cytogenetic data for soft tissue chondroma, enchondroma, and periosteal chondroma Sex/Age (Yrs)
Site
Karyotype
Reference
Soft tissue chondroma 10 M/51
Foot phalanx
Current study
1 4
F/56 M/62
Knee Elbow
11 9
M/66 M/61
Subpatellar Arm
8 1
M/30 F/3
Foot Sublingual
4b 3
F/48 F/16
Ankle Knee
44w46,XY,t(6;12)(q12;p11.2)[4]/40w46,XY,t(3;7) (q13;p12)[3]/46,XY,der(2)t(2;18)(p11.2;q11.2)[2]/ 46,XY[13] 46,XX,ins(4;12)(q3?4;q1?5q2?3)[17] 46,XY,t(3;12)(q27;q15)[2]/45,idem,t(1;4)(q12;q23), add(6)(q11),t(7;16)(p15;p13),13[20] 46,XY,inv(12)(p13.3q15)[10] 46,XY,t(11;19)(p15;q13)c/47,idem,þ5/47,idem,add (5)(q31),del(6)(q13),del(7)(p11p15),8,þder(?) t(?;8)(?;q11)x2 46,XY,6,11,þr,þmar 48w51,XX,þder(?)r(?;12)(?;q14q24)x2w5[18]/ 49w50,XX,þ17,þder(?)r(?;12)x2w3[4] 47,XX,þ5 46,X,t(X;12;8;13)(q24;q15;q11;q14),del(8)(p22)[22]
Enchondroma 4 M/23 5 F/40
Proximal femur Hand phalanx
6 9 14 1 2 1
F/35 F/45 M/33 F/65 F/57 M/50
Halux Hand phalanx Proximal femur Distal femur Proximal humerus Proximal fibula
3
F/9
Humerus
3 5 10 11
F/23 M/13 M/27 F/55
Finger Finger Finger Finger
12
M/38
Finger
13 14
M/13 M/50
Radius Toe
46,XY,inv(2)(p21q31),t(12;15;21)(q13;q14;q22)[18] 38w45,XX,X,4,5,6,8,9,12[cp11]/46, XX[5] 43w45,XX,20[cp3]/46,XX[28] 38w45,XX,7,13[cp4]/46,XX[30] 46,XY,t(12;22)(q13;q12)[20] 47,XX,þmar[4]/46,XX[16] 45,XX,12,der(17)t(12;17)(q12;q11.2)[3]/46,XX[16] 40w46,XY,t(8;17)(q23;p13)[4],9[3],19[3],22[3] [cp11]/46,XY[9] 45w46,XX, der(6)t(6;15)(q13;q11)t(15;22) (q22;q13),t(7;3;5;7)(p22;p13;q22;q31)þ17, der(22)t(15;22)(q22;q13)[30],17,i(17)(q10)[2], 13,17,i(17)(q10),þ20[3],der(16)t(16;17) (q24;q21),17[9],der(16)t(16;17)(p13.3;q21), 17[9],der(16)t(16;17)(q24;q21),t(16;17) (p13.3;q21),17[4][cp30] 46,XX,t(12;15)(q13;q26)[4]/46,XX[14] 46,XY,i(6p)[13]/47,XY,idem,þ20[7] 46,XY,ins(3;?12)(q1?;q1?q2?)[20] 45,XX,1,?t(3;12)(q12;p11),der(6)t(1;6) (q12;q12),add(9)(q22),der(13)t(1;13) (p32;p11),der(19)t(9;19)(q22;q13),add(21)(p11), del(22)(q11)[7] 78w81,XXYY,2,2,der(6)t(6;17)(q21;q21) x2,þ7,þadd(7)(q11),8,der(8)t(4;8)(q21;q24), 9,10,10,11,12,add(14)(p1?),15, ?inv(15)(q12q15),16,16,17,19,20,21, 21,22,22,þ3w5mar[cp6] 46,XY,t(2;11)(q37;q13)[23] 45,XY,6[13]
n et al., 2003 (27) Dahle n et al., 2003 (27) Dahle Buddingh et al., 2003 (2) Tallini et al., 2002 (7)
Tallini et al., 2002 (7) Shadan et al., 2000 (11) Bridge et al., 1993 (8) Mandahl et al., 1990 (5)
Current study Current study Current study Current study Current study Buddingh et al., 2003 (2) Buddingh et al., 2003 (2) Gunawan et al., 2000 (16) Sawyer et al., 1998 (3)
Bridge et al., 1992 (4) Bridge et al., 1992 (4) n et al., 2003 (27) Dahle n et al., 2003 (27) Dahle
n et al., 2003 (27) Dahle
n et al., 2003 (27) Dahle n et al., 2003 (27) Dahle (Continued on the next page)
Cytogenetic findings in chondromas Table 2 Case
183
(Continued ) Sex/Age (Yrs)
Site
Karyotype
Reference
Periosteal chondroma 11 M/23 12 F/1 13 M/22 7 M/53
Tibia Tibia Radius Proximal phalanx
Current study Current study Current study Buddingh et al., 2003 (2)
8 10
M/14 M/49
Distal femur Bone, metatarsal
1
M/35
Distal femur
46,XY,t(7;11)(q22;q15)[2]/46,XY[18] 46,XX,t(2;11)(q37;q13)[19]/46,XX[2] 44,XY,del(3)(q?21),del(6)(q11),13[cp3]/46,XY[17] 46,XY,t(4;13)(q21;q34)[2]/81w91,XXYY,del(1) (p32)x2,2,add(2)(q37),6,7,þ8,þ19, þ1w4mar,inc[3]/46,XY[15] 46,XY,t(4;?14;7)(q25;?q21;q11.2)[3]/46,XY[14] 46,XY,t(14;14)(q11;q24),46,idem,t(11;15) (p15;q22w23)/46,XY 45,XY,der(7)t(7;12)(q32;q13),dic(12;12) (q13;q13),der(16)t(12;16)(q13;q22)[3]/ 45,idem,add(5)(q13)[4]/46,XY[5]
Buddingh et al., 2003 (2) Buddingh et al., 2003 (2) Mandahl et al., 1993 (10)
Abbreviations: M, male; F, female.
(Figure 1). In this case, metaphases were re-evaluated and a rearrangement in chromosome 22 was modified to 22q12 according to FISH studies with a 22q12 (EWSR1) breakapart probe (Figure 2). The results from RT-PCR did not reveal a EWSR1eDDIT3 fusion gene that has previously been reported in myxoid liposarcoma, a soft-tissue sarcoma that is morphologically and clinically distinct from enchondroma (Figure 3).
Discussion Cartilage tumors are a heterogeneous group with respect to clinical presentation, histomorphology, and biologic behavior.
Figure 1
This diversity is reflected in the cytogenetic literature on cartilage forming tumors, which demonstrates the presence of many varied numerical and structural chromosome aberrations (16). Cartilage tumors are probably derived from chondrocytes of the actively proliferating cartilaginous tissue of growth plates. For unknown reasons, the cells that give rise to these tumors likely do not proceed as normal to undergo hypertrophy and death. These cells instead remain as an isolated group of viable chondrocytes that may eventually proliferate in a neoplastic fashion (17). Soft tissue chondromas, on the other hand, may have a synovial origin according to Dahlin and Salvador (18). Alternatively, others have considered these chondromas to be developmental faults or metaplasia (19,20). No primary cytogenetic
G-banded karyotype of a metaphase of patient 14 showing the t(12;22)(q13;q12) and a non-clonal loss of chromosome 18.
184
N. Sakai Junior et al.
Figure 2 Metaphase FISH of case 14 with rearrangement of EWSR1 on 22q12. Split red/green signals are on the der(22)t(12;22) (green signal) and der(12)t(12;22) (red signal). Fused red/green signals mark one intact 22q12 region. (A color figure can be found in the online version of this article.)
abnormality has been identified in chondromas. Enchondromas are successfully treated by intra-lesional curettage in most cases, and local recurrences are uncommon. Higher expression of JunB protein was found in low-grade chondrosarcoma when compared to enchondromas. Therefore,
expression of this protein may be used as a potential diagnostic tool in differential diagnosis (21). Comparative genomic hybridization studies (22) have reported frequent losses of chromosomes in benign cartilaginous tumors, particularly losses of whole chromosomes
Figure 3 RT-PCR analysis of EWSR1eDDIT3 (A) and PGK (B) in an ethidium bromideestained 1.8% agarose gel. Size markers: 50- and 100-bp ladders. C1 and C2 indicate positive tumor cDNA controls for EWSR1eDDIT3 type 1 transcript. NTC, no template control (negative control). No specific EWSR1eDDIT3 translocation was found in a patient with the t(12;22)(q13;q12).
Cytogenetic findings in chondromas 19 and 22q. In our study, three enchondromas showed only nonspecific numerical chromosome aberrations (patients 5, 6, and 9). Structural chromosome aberrations were observed in six tumors (one soft tissue chondroma, two enchondromas, and three periosteal chondromas). Tumors with loss of chromosome 6, t(12;15;21)(q13;q14;q22), and del(6)(q11) were observed in patients 5, 4, and 13, respectively. Two enchondromas (patients 4 and 14) showed rearrangements in 12q13. Rearrangements of the long arm of chromosome 12 have been previously described in two enchondromas, two periosteal chondromas, and two soft tissue chondromas (4,5,10e12). Rearrangements of chromosome 6 and the long arm of chromosome 12 (particularly q13wq15) seem to be nonrandom (23). The long arm of chromosome 12 is a cytogenetic hot spot for abnormalities in many different mesenchymal neoplasms. Some biologic events associated with anomalies in 12q13w15 include amplification of MDM2, CDK4, and SAS genes in parosteal osteosarcoma (24,25). Cytogenetic studies of chondromas, enchondromas, and soft tissue chondromas are scarce. No consistent abnormality has been detected, although chromosomes 4, 5, 6, and 7 and the chromosomal region 12q13e15 seem to be nonrandomly involved in changes (26) (Table 2). We observed one case of a periosteal chondroma (patient 12) with a clone with a t(2;11)(q37;q13) as well as cells with a normal constitution. This alteration was n et al. previously described in one enchondroma by Dahle (27). We observed one enchondroma (patient 14) that presented with a t(12;22)(q13;q12) in all analyzed cells. This patient had a normal chromosome constitution in the peripheral blood. His femoral lesion was completely resected, examined, and histologically determined to be a typical enchondroma. To our knowledge, this chromosomal aberration has never been described in connection with enchondroma, although it has been previously described mainly in connection with myxoid liposarcoma, clear cell sarcoma of soft tissue, angiomatoid fibrous histiocytoma, and additional sporadic examples of other benign and malignant tumors (28e32). No specific EWSR1eDDIT3 translocation was found in this patient after RT-PCR studies. Sixteen studies describe a total of 19 patients with the t(12;22)(q13;q12) aberration; samples from these patients were classified in the Catalog of Chromosome Aberrations in Cancer as 15 clear cell sarcomas, two angiomatoid malignant fibrous histiocytoma, one multiple myeloma, and one peripheral neuroepithelioma (13). Malignant transformation of an enchondroma into a chondrosarcoma is a rare event (33e35). Enchondromas retain expression of the CDKN2A/ p16 protein, which has been correlated with tumor grade. This correlation and the retention of expression of this protein in enchondromas suggest that loss of CDKN2A/p16 protein expression may be an important event during tumor progression in enchondroma, as well as grade progression in recurrent chondrosarcoma (36). Chromosome banding analysis is an efficient technique when several loci need to be investigated. Abnormalities of chromosomes 5, 6, 7, and 12 and of chromosomal regions 6q13, 12q13, and 17p13 are shared by both malignant and benign cartilaginous tumors (5,8). At present, there are not sufficient data to allow a differential diagnosis between chondroma and low-grade chondrosarcoma based solely on cytogenetic studies. The simple presence of clonal abnormalities does not seem to
185 correlate with clinical behavior (4). However, we believe that cytogenetic and molecular studies may ultimately help define potential biological consequences of these recurrent abnormalities of cartilage tumors. These studies may eventually contribute to establishing an accurate differential diagnosis between benign and low-grade cartilaginous lesions.
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186 17. Milgram JW. The origins of osteochondromas and enchondromas: a histopathologic study. Clin Orthopaed Relat Res 1983;174:264e284. 18. Dahlin DC, Salvador AH. Cartilaginous tumors of the soft tissues of the hands and feet. Mayo Clin Proc 1974;49:721e726. 19. Uehara H, Becker FP. Extraskeletal cartilaginous tumours. Arch Surg 1960;80:319e326. 20. Smith MT, Farinacci CJ, Carpenter HA, et al. Extra-skeletal myxoid chondrosarcoma, a clinicopathological study. Cancer 1976;37:821e827. e JVMG. Bone: enchondroma. Atlas of 21. Pansuriya TC, Bove Genetics and Cytogenetics in Oncology and Haematology. July 2008. Available at: http://AtlasGeneticsOncology.org/Genes/ EnchondromaID5333.html. € fer KL, et al. Comparative genomic 22. Ozaki T, Wai D, Scha hybridization in cartilaginous tumors. Anticancer Res 2004;24: 1721e1725. 23. Sandberg AA, Bridge JA. Updates on the cytogenetics and molecular genetics of bone and soft tissue tumors: chondrosarcoma and other cartilaginous neoplasms. Cancer Genet Cytogenet 2003;143:1e31. 24. Gisselsson D, Palsson E, Hoglund M, et al. Differentially amplified chromosome 12 sequences in low- and high-grade osteosarcoma. Genes Chromosomes Cancer 2002;33:133e140. 25. Yokoyama R, Schneider-Stock R, Radig K, et al. Clinicopathologic implications of MDM2, p53 and K-ras gene alterations alterations in osteosarcomas: MDM2 amplification and p53 mutations found in progressive tumors. Pathol Res Pract 1998;194:615e621. 26. Vanni R. Bone: chondroma. Atlas of Genetics and Cytogenetics in Oncology and Haematology. May 2003. Available at: http:// AtlasGeneticsOncology.org/Genes/ChondromaID5147.html. n A, Mertens F, Rydholm A, et al. Fusion, disruption, and 27. Dahle expression of HMGA2 in bone and soft tissue chondromas. Mod Pathol 2003;16:1132e1140.
N. Sakai Junior et al. 28. Lam KY, Lo CY, Shek TW, et al. Primitive small round cell tumour of the adrenal gland presenting with fever of unknown origin and t(12;22)(q13;q12) cytogenetic finding. J Clin Pathol 2001;54:966e969. 29. Hallor KH, Mertens F, Jin Y, et al. Fusion of the EWSR1 and ATF1 genes without expression of the MITF-M transcript in angiomatoid fibrous histiocytoma. Genes Chromosomes Cancer 2005;44:97e102. 30. Patel RM, Downs-Kelly E, Weiss SW, et al. Dual-color, breakapart fluorescence in situ hybridization for EWS gene rearrangement distinguishes clear cell sarcoma of soft tissue from malignant melanoma. Mod Pathol 2005;18:1585e1590. 31. Dunham C, Hussong J, Seiff M, et al. Primary intracerebral angiomatoid fibrous histiocytoma: report of a case with a t(12;22) (q13;q12) causing type 1 fusion of the EWS and ATF-1 genes. Am J Surg Pathol 2008;32:478e484. 32. Gabrea A, Martelli ML, Qi Y, et al. Secondary genomic rearrangements involving immunoglobulin or MYC loci show similar prevalences in hyperdiploid and nonhyperdiploid myeloma tumors. Genes Chromosomes Cancer 2008;47:573e590. 33. Middlemiss JH. Cartilage tumours. Br J Radiol 1964;37: 277e286. 34. Culver JE Jr, Sweet DE, McCue FC. Chondrosarcoma of the hand arising from a pre-existent benign solitary enchondroma. Clin Orthop Relat Res 1975;113:128e131. e JV, van der Heul RO, Taminiau AH, et al. Chon35. Bove drosarcoma of the phalanx: a locally aggressive lesion with minimal metastatic potential: a report of 35 cases and a review of the literature. Cancer 1999;86:1724e1732. 36. van Beerendonk HM, Rozeman LB, Taminiau AH, et al. Molecular analysis of the INK4A/INK4AeARF gene locus in conventional (central) chondrosarcomas and enchondromas: indication of an important gene for tumour progression. J Pathol 2004;202:359.
Cytogenetic findings in 14 benign cartilaginous neoplasms Nilo Sakai Junior*, Kikue Terada Abe, Lia Menezes Formigli, Marcio Fernandes Pereira, Maria Dulce Valverde de Oliveira, Deborah Afonso Cornelio, Alessandra de La Roque Ferreira, Ricardo Karam Kalil Molecular Pathology Division, Sarah Network of Rehabilitation Hospitals, Brasilia, DF, Brazil Benign cartilaginous tumors represent a spectrum of neoplastic processes with variable clinical and pathologic presentations. These tumors are histologically characterized by the presence of chondrocytes surrounded by a cartilaginous matrix. Few studies describe karyotypic abnormalities in these benign lesions. We report a series of 14 chondromas from a single institution. Conventional cytogenetics was performed on short term cultures from all cases. Clonal chromosome aberrations were found in nine tumors. One soft tissue chondroma contained three clones with t(6;12)(q12;p11.2), t(3;7)(q13;p12), and der(2)t(2;18)(p11.2;q11.2). Three periosteal chondromas displayed random structural aberrations of chromosomes 2, 3, 6, 7, and 11 and loss of chromosome 13. Among the enchondromas, three tumors displayed chromosome losses, one contained a complex translocation involving chromosomes 12, 15, and 21 as well as an inv(2)(p21q31),t(12;15;21)(q13;q14;q22) and a separate enchondroma showed a translocation involving chromosomes 12 and 22. Our data suggest that considerable cytogenetic heterogeneity exists among benign chondromatous tumors. Keywords Chondromas, cytogenetics ª 2011 Elsevier Inc. All rights reserved.
Benign cartilage tumors, including enchondromas, soft tissue chondromas, and periosteal chondromas, represent approximately 10e25% of all primary bone tumors. Enchondromas typically occur in the small tubular bones of the hands and feet, whereas the rare periosteal chondromas predominantly appear on the surface of long tubular bones (1). These tumors are not usually clinically apparent and therefore often diagnosed as incidental findings. The differential diagnosis between chondroma and low-grade chondrosarcoma is one of the most difficult in bone tumor pathology. The histological differences between benign lesions and well-differentiated malignant tumors are frequently tenuous, and welldifferentiated chondrosarcomas may display borderline or intermediate features of malignancy. Histologic grade has been shown to be of some prognostic importance; however, discrepancies between histologic appearance and biologic behavior are not infrequent. Chromosomal studies have proven to be of value in the diagnostic assessment of many
Received February 17, 2010; received in revised form February 9, 2011; accepted February 14, 2011. * Corresponding author. E-mail address: [email protected] 2210-7762/$ - see front matter ª 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergen.2011.02.004
mesenchymal neoplasms (2). Cytogenetic analysis can be used as a complementary histological assessment tool to yield increased biologic information on cartilaginous tumors. Few cytogenetic reports of enchondroma, periosteal chondroma, and soft tissue chondroma exist (3e12). To date, the Catalog of Chromosome Aberrations in Cancer has published only 27 cytogenetically abnormal bone and soft tissue chondromas (13). In this study, we present a review of the literature as well as the karyotypic findings of eight enchondromas, five periosteal chondromas, and one soft tissue chondroma.
Materials and methods Clinical and histopathologic data Between 2001 and 2008, a total of 35 chondromas were collected in our pathology department, of which 28 were enchondromas, 6 were periosteal chondromas, and 1 was a soft tissue chondroma. Tumor tissue samples from 14 patients were received directly from surgery for cytogenetic analysis. Six patients were male and 8 were female. Their ages ranged from 1 to 51 years old. All of the lesions studied
Cytogenetic findings in chondromas
181
were untreated primary tumors. The tumors were histologically classified into subtypes, examined to confirm the absence of features suggestive of malignant transformation, and the results correlated with radiological findings. Eight tumors were identified as enchondromas, 5 were periosteal chondromas, and 1 was a soft tissue chondroma. None of the patients had Maffucci syndrome or Ollier disease. Our study was conducted in accordance with the guidelines of the Medical Ethics Commission of the SARAH Network of Rehabilitation Hospitals, Brazil.
manufacturer’s instructions. This total RNA was reverse transcribed with random primers with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). The resulting cDNA was amplified by PCR using EWSR1 501F and DDIT3 194R primers (15). In parallel, the viability of the extracted RNA was verified by assaying the cDNA product with primers for the housekeeping gene PGK. Positive and negative controls were tested simultaneously with the tumor sample. Amplified products were analyzed on a 1.8% agarose gel.
Cell culture and cytogenetic analysis
Results
The tumor biopsy samples obtained for cytogenetic analysis were mechanically disaggregated with a caliper and a scalpel and incubated for 2 hours in a collagenase II solution (8 mg/ mL). The resulting cell suspension was washed and cultured at 37 C, 5% CO2 in 25 cm2 T flasks with McCoy 5A medium supplemented with L-glutamine, antibiotics, and 10e20% fetal bovine serum. After 5e18 days of primary culture, chromosomes were prepared according to standard protocols. Wright stain was used for trypsin G-banding. At least 20 metaphases were analyzed, and chromosome aberrations were classified according to the International System for Human Cytogenetic Nomenclature (ISCN 2005) (14).
Histologically, all cases of chondromas in our series showed the usual pattern described in the literature. Thirteen of the 14 patients experienced local pain as the main symptom, and enchondroma was an occasional finding in only one patient with carpal tunnel syndrome. All of the soft tissue and surface lesions were excised, and the enchondromas were also subjected to curettage. Follow-up of all cases included in the current study are listed in Table 1. The cytogenetic analysis of each of the 14 chondroma specimens was successful. Normal karyotypes were detected in samples from five patients (patients 1, 2, 3, 7, and 8): four were enchondromas, and one was a periosteal chondroma. Non-clonal alterations were found in tissue from four patients: losses of chromosomes 4, 6, 9, 12, 14, and 16 were found in patients 1, 3, and 7; patient 2 showed the alteration del(7)(p12p15); and patient 3 contained the alterations tas(14;15)(q32;q26), ?del(X)(p?), t(1;?5). Nine patients (patients 4, 5, 6, 9, 10, 11, 12, 13, and 14) displayed clonal chromosome aberrations (Table 2), including diploid or near-diploid clones. One soft tissue chondroma contained four clones: 44w46,XY,t(6;12) (q12;p11.2)[4]/40w46,XY,t(3;7)(q13;p12)[3]/46,XY,der(2) t(2;18)(p11.2;q11.2)[2]/46,XY[13]. Three periosteal chondromas had random structural aberrations of chromosomes 2, 3, 6, 7, and 11, loss of chromosome 13, and a normal clone. Three enchondromas contained both a clone with chromosome losses and a normal clone, and one enchondroma had a clone with 46,XY,inv(2)(p21q31),t(12;15;21) (q13;q14;q22)[18]. Finally, one enchondroma contained a clone with the 46,XY,t(12;22)(q13;q13)[20] alteration only
Fluorescence in situ hybridization (FISH) analysis The FISH procedure was performed on G-banded slides according to the manufacturer’s instructions. The LSI EWSR1 (22q12) Dual Color, Break Apart Rearrangement Probe (Abbott, Downers Grove, IL) was used to detect EWSR1 gene rearrangements. Hybridization signals were observed by FISHView software (Applied Spectral Imaging, Migdal Haemek, Israel).
Reverse transcriptaseepolymerase chain reaction (RT-PCR) analysis Total RNA was isolated from frozen tumor tissue with the RNeasy Mini Kit (Qiagen, Valencia, CA) according to the Table 1
Histologic and clinical data for soft tissue chondroma, enchondroma and periosteal chondroma cases
Case
Diagnosis
Sex/age (y)
Location/size (cm)
Follow-up (mo)
1/N 2/N 3/N 4/A 5/A 6/A 7/N 8/N 9/A 10/A 11/A 12/A 13/A 14/A
Enchondroma Enchondroma Periosteal chondroma Periosteal chondroma Enchondroma Enchondroma Enchondroma Enchondroma Enchondroma Soft tissue chondroma Periosteal chondroma Periosteal chondroma Periosteal chondroma Enchondroma
F/35 F/35 F/8 M/22 F/40 F/35 M/4 F/26 F/45 M/51 M/24 F/1 M/23 M/33
Metacarpal/2.5 Hand phalanx/3.0 Metatarsal/3.2 Proximal femur/3.5 Hand phalanx/1.0 Halux/2.0 Distal femur/1.5 Hand phalanx/1.3 Hand phalanx/1.2 Foot phalanx/4.5 Tibia/2.0 Tibia/3.0 Radius/1.5 Proximal femur/2.0
40/NED 40/NED 6/NED 8/NED 28/NED 30/NED 22/NED 12/NED 30/NED 10/NED 7/NED 36/NED 30/NED 17/NED
Abbreviations: F, female; M, male; N, normal karyotype; A, abnormal karyotype; NED, no evidence of disease.
182 Table 2 Case
N. Sakai Junior et al. Clinical and cytogenetic data for soft tissue chondroma, enchondroma, and periosteal chondroma Sex/Age (Yrs)
Site
Karyotype
Reference
Soft tissue chondroma 10 M/51
Foot phalanx
Current study
1 4
F/56 M/62
Knee Elbow
11 9
M/66 M/61
Subpatellar Arm
8 1
M/30 F/3
Foot Sublingual
4b 3
F/48 F/16
Ankle Knee
44w46,XY,t(6;12)(q12;p11.2)[4]/40w46,XY,t(3;7) (q13;p12)[3]/46,XY,der(2)t(2;18)(p11.2;q11.2)[2]/ 46,XY[13] 46,XX,ins(4;12)(q3?4;q1?5q2?3)[17] 46,XY,t(3;12)(q27;q15)[2]/45,idem,t(1;4)(q12;q23), add(6)(q11),t(7;16)(p15;p13),13[20] 46,XY,inv(12)(p13.3q15)[10] 46,XY,t(11;19)(p15;q13)c/47,idem,þ5/47,idem,add (5)(q31),del(6)(q13),del(7)(p11p15),8,þder(?) t(?;8)(?;q11)x2 46,XY,6,11,þr,þmar 48w51,XX,þder(?)r(?;12)(?;q14q24)x2w5[18]/ 49w50,XX,þ17,þder(?)r(?;12)x2w3[4] 47,XX,þ5 46,X,t(X;12;8;13)(q24;q15;q11;q14),del(8)(p22)[22]
Enchondroma 4 M/23 5 F/40
Proximal femur Hand phalanx
6 9 14 1 2 1
F/35 F/45 M/33 F/65 F/57 M/50
Halux Hand phalanx Proximal femur Distal femur Proximal humerus Proximal fibula
3
F/9
Humerus
3 5 10 11
F/23 M/13 M/27 F/55
Finger Finger Finger Finger
12
M/38
Finger
13 14
M/13 M/50
Radius Toe
46,XY,inv(2)(p21q31),t(12;15;21)(q13;q14;q22)[18] 38w45,XX,X,4,5,6,8,9,12[cp11]/46, XX[5] 43w45,XX,20[cp3]/46,XX[28] 38w45,XX,7,13[cp4]/46,XX[30] 46,XY,t(12;22)(q13;q12)[20] 47,XX,þmar[4]/46,XX[16] 45,XX,12,der(17)t(12;17)(q12;q11.2)[3]/46,XX[16] 40w46,XY,t(8;17)(q23;p13)[4],9[3],19[3],22[3] [cp11]/46,XY[9] 45w46,XX, der(6)t(6;15)(q13;q11)t(15;22) (q22;q13),t(7;3;5;7)(p22;p13;q22;q31)þ17, der(22)t(15;22)(q22;q13)[30],17,i(17)(q10)[2], 13,17,i(17)(q10),þ20[3],der(16)t(16;17) (q24;q21),17[9],der(16)t(16;17)(p13.3;q21), 17[9],der(16)t(16;17)(q24;q21),t(16;17) (p13.3;q21),17[4][cp30] 46,XX,t(12;15)(q13;q26)[4]/46,XX[14] 46,XY,i(6p)[13]/47,XY,idem,þ20[7] 46,XY,ins(3;?12)(q1?;q1?q2?)[20] 45,XX,1,?t(3;12)(q12;p11),der(6)t(1;6) (q12;q12),add(9)(q22),der(13)t(1;13) (p32;p11),der(19)t(9;19)(q22;q13),add(21)(p11), del(22)(q11)[7] 78w81,XXYY,2,2,der(6)t(6;17)(q21;q21) x2,þ7,þadd(7)(q11),8,der(8)t(4;8)(q21;q24), 9,10,10,11,12,add(14)(p1?),15, ?inv(15)(q12q15),16,16,17,19,20,21, 21,22,22,þ3w5mar[cp6] 46,XY,t(2;11)(q37;q13)[23] 45,XY,6[13]
n et al., 2003 (27) Dahle n et al., 2003 (27) Dahle Buddingh et al., 2003 (2) Tallini et al., 2002 (7)
Tallini et al., 2002 (7) Shadan et al., 2000 (11) Bridge et al., 1993 (8) Mandahl et al., 1990 (5)
Current study Current study Current study Current study Current study Buddingh et al., 2003 (2) Buddingh et al., 2003 (2) Gunawan et al., 2000 (16) Sawyer et al., 1998 (3)
Bridge et al., 1992 (4) Bridge et al., 1992 (4) n et al., 2003 (27) Dahle n et al., 2003 (27) Dahle
n et al., 2003 (27) Dahle
n et al., 2003 (27) Dahle n et al., 2003 (27) Dahle (Continued on the next page)
Cytogenetic findings in chondromas Table 2 Case
183
(Continued ) Sex/Age (Yrs)
Site
Karyotype
Reference
Periosteal chondroma 11 M/23 12 F/1 13 M/22 7 M/53
Tibia Tibia Radius Proximal phalanx
Current study Current study Current study Buddingh et al., 2003 (2)
8 10
M/14 M/49
Distal femur Bone, metatarsal
1
M/35
Distal femur
46,XY,t(7;11)(q22;q15)[2]/46,XY[18] 46,XX,t(2;11)(q37;q13)[19]/46,XX[2] 44,XY,del(3)(q?21),del(6)(q11),13[cp3]/46,XY[17] 46,XY,t(4;13)(q21;q34)[2]/81w91,XXYY,del(1) (p32)x2,2,add(2)(q37),6,7,þ8,þ19, þ1w4mar,inc[3]/46,XY[15] 46,XY,t(4;?14;7)(q25;?q21;q11.2)[3]/46,XY[14] 46,XY,t(14;14)(q11;q24),46,idem,t(11;15) (p15;q22w23)/46,XY 45,XY,der(7)t(7;12)(q32;q13),dic(12;12) (q13;q13),der(16)t(12;16)(q13;q22)[3]/ 45,idem,add(5)(q13)[4]/46,XY[5]
Buddingh et al., 2003 (2) Buddingh et al., 2003 (2) Mandahl et al., 1993 (10)
Abbreviations: M, male; F, female.
(Figure 1). In this case, metaphases were re-evaluated and a rearrangement in chromosome 22 was modified to 22q12 according to FISH studies with a 22q12 (EWSR1) breakapart probe (Figure 2). The results from RT-PCR did not reveal a EWSR1eDDIT3 fusion gene that has previously been reported in myxoid liposarcoma, a soft-tissue sarcoma that is morphologically and clinically distinct from enchondroma (Figure 3).
Discussion Cartilage tumors are a heterogeneous group with respect to clinical presentation, histomorphology, and biologic behavior.
Figure 1
This diversity is reflected in the cytogenetic literature on cartilage forming tumors, which demonstrates the presence of many varied numerical and structural chromosome aberrations (16). Cartilage tumors are probably derived from chondrocytes of the actively proliferating cartilaginous tissue of growth plates. For unknown reasons, the cells that give rise to these tumors likely do not proceed as normal to undergo hypertrophy and death. These cells instead remain as an isolated group of viable chondrocytes that may eventually proliferate in a neoplastic fashion (17). Soft tissue chondromas, on the other hand, may have a synovial origin according to Dahlin and Salvador (18). Alternatively, others have considered these chondromas to be developmental faults or metaplasia (19,20). No primary cytogenetic
G-banded karyotype of a metaphase of patient 14 showing the t(12;22)(q13;q12) and a non-clonal loss of chromosome 18.
184
N. Sakai Junior et al.
Figure 2 Metaphase FISH of case 14 with rearrangement of EWSR1 on 22q12. Split red/green signals are on the der(22)t(12;22) (green signal) and der(12)t(12;22) (red signal). Fused red/green signals mark one intact 22q12 region. (A color figure can be found in the online version of this article.)
abnormality has been identified in chondromas. Enchondromas are successfully treated by intra-lesional curettage in most cases, and local recurrences are uncommon. Higher expression of JunB protein was found in low-grade chondrosarcoma when compared to enchondromas. Therefore,
expression of this protein may be used as a potential diagnostic tool in differential diagnosis (21). Comparative genomic hybridization studies (22) have reported frequent losses of chromosomes in benign cartilaginous tumors, particularly losses of whole chromosomes
Figure 3 RT-PCR analysis of EWSR1eDDIT3 (A) and PGK (B) in an ethidium bromideestained 1.8% agarose gel. Size markers: 50- and 100-bp ladders. C1 and C2 indicate positive tumor cDNA controls for EWSR1eDDIT3 type 1 transcript. NTC, no template control (negative control). No specific EWSR1eDDIT3 translocation was found in a patient with the t(12;22)(q13;q12).
Cytogenetic findings in chondromas 19 and 22q. In our study, three enchondromas showed only nonspecific numerical chromosome aberrations (patients 5, 6, and 9). Structural chromosome aberrations were observed in six tumors (one soft tissue chondroma, two enchondromas, and three periosteal chondromas). Tumors with loss of chromosome 6, t(12;15;21)(q13;q14;q22), and del(6)(q11) were observed in patients 5, 4, and 13, respectively. Two enchondromas (patients 4 and 14) showed rearrangements in 12q13. Rearrangements of the long arm of chromosome 12 have been previously described in two enchondromas, two periosteal chondromas, and two soft tissue chondromas (4,5,10e12). Rearrangements of chromosome 6 and the long arm of chromosome 12 (particularly q13wq15) seem to be nonrandom (23). The long arm of chromosome 12 is a cytogenetic hot spot for abnormalities in many different mesenchymal neoplasms. Some biologic events associated with anomalies in 12q13w15 include amplification of MDM2, CDK4, and SAS genes in parosteal osteosarcoma (24,25). Cytogenetic studies of chondromas, enchondromas, and soft tissue chondromas are scarce. No consistent abnormality has been detected, although chromosomes 4, 5, 6, and 7 and the chromosomal region 12q13e15 seem to be nonrandomly involved in changes (26) (Table 2). We observed one case of a periosteal chondroma (patient 12) with a clone with a t(2;11)(q37;q13) as well as cells with a normal constitution. This alteration was n et al. previously described in one enchondroma by Dahle (27). We observed one enchondroma (patient 14) that presented with a t(12;22)(q13;q12) in all analyzed cells. This patient had a normal chromosome constitution in the peripheral blood. His femoral lesion was completely resected, examined, and histologically determined to be a typical enchondroma. To our knowledge, this chromosomal aberration has never been described in connection with enchondroma, although it has been previously described mainly in connection with myxoid liposarcoma, clear cell sarcoma of soft tissue, angiomatoid fibrous histiocytoma, and additional sporadic examples of other benign and malignant tumors (28e32). No specific EWSR1eDDIT3 translocation was found in this patient after RT-PCR studies. Sixteen studies describe a total of 19 patients with the t(12;22)(q13;q12) aberration; samples from these patients were classified in the Catalog of Chromosome Aberrations in Cancer as 15 clear cell sarcomas, two angiomatoid malignant fibrous histiocytoma, one multiple myeloma, and one peripheral neuroepithelioma (13). Malignant transformation of an enchondroma into a chondrosarcoma is a rare event (33e35). Enchondromas retain expression of the CDKN2A/ p16 protein, which has been correlated with tumor grade. This correlation and the retention of expression of this protein in enchondromas suggest that loss of CDKN2A/p16 protein expression may be an important event during tumor progression in enchondroma, as well as grade progression in recurrent chondrosarcoma (36). Chromosome banding analysis is an efficient technique when several loci need to be investigated. Abnormalities of chromosomes 5, 6, 7, and 12 and of chromosomal regions 6q13, 12q13, and 17p13 are shared by both malignant and benign cartilaginous tumors (5,8). At present, there are not sufficient data to allow a differential diagnosis between chondroma and low-grade chondrosarcoma based solely on cytogenetic studies. The simple presence of clonal abnormalities does not seem to
185 correlate with clinical behavior (4). However, we believe that cytogenetic and molecular studies may ultimately help define potential biological consequences of these recurrent abnormalities of cartilage tumors. These studies may eventually contribute to establishing an accurate differential diagnosis between benign and low-grade cartilaginous lesions.
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