Molecular analysis of a myxoid chondrosarcoma with rearrangements of chromosomes 10 and 22.

Myxoid chondrosarcoma is a rare tumor of adulthood. An associated nonrandom reciprocal translocation between chromosome 9 and 22 was previously reported in this tumor. We performed cytogenetic and molecular genetic analysis of a myxoid chondrosarcoma derived from the sphenoid bone. Using restriction fragment length polymorphism (RFLP) analysis, we demonstrated rearrangement and a possible allele loss close to the chromosome 22 breakpoint. In addition, structural rearrangement in the chromosome 10q21.1 region and an allele loss in the chromosome 10q21-q23 region were also detected. In tumor DNA an additional hybridization fragment was detected by pAS-1 probe, which recognizes multiple pseudogenes of argininosuccinate synthetase dispersed in various chromosomes. We were unable to detect chromosomal abnormalities with two additional chromosome 9q probes. This study suggests that multiple gene rearrangements occurred in the myxoid chondrosarcoma and the significance of this is discussed.


INTRODUCTION
Identification of the specific cytogenetic abnormalities in cancers is important in disease diagnosis, defining tumor subtypes as well as providing the clue to the location of the genes involved in formation of the tumors. Nonrandom structural chromosome abnormalities have been identified in various soft tissue sarcomas [1]. Reciprocal translocation between chromosome 9 and 22 has been reported in two rare myxoid chondrosarcomas [2,3]. Recently, molecular analysis was used to study the genotype of solid tumors. The molecular genetic study can be performed directly on the tumor tissue and avoids the possible secondary change occurring in in vitro propagation. The method can detect small chromosome deletions and rearrangements beyond the resolution of cytogenetic study. We performed molecular analysis of a sphenoid myxoid chrondrosarcoma. In this tumor, rearrangement and a possible allele loss were detected in the chromosome 22q11-q12 region with the DNA probes and were consistent with the previous cytogenetic observation. We also demonstrated rearrangement and allele loss on chromosome 10. kV-t' V. 5h,~J~ ~,~t al

CASE REPORT
A 27-year-old white woman was admitted in Mar(:h 1987 with tire ~,hiel (.Oml)laint ot right-sided vision and hearing loss and right facial mmd)uess. C(mq)uterize,(t hmlogrdphy and magnetic resonance imaging scans of the head sbowed tire presen(:e ot a large mass at the right frontotemporal region. A large encat)sulate(I tumor mass, ~e,r} mucoid and with focal calcification, was surgically removed. The major bistologic constituent was a mildly to moderately cellular neol)lasm (:omposed ot small h) medium-sized polyhedral cells with bright pink (:ytoplasm and small, romlde(t, single or multiple nucleolated nuclei (Fig. 1 ). The patbologi(: diagnosis was (:bondrosar(:oma of the right sphenoid ring. Two years after operation the patient is alive without recurrence.

Isolation of DNA and RFLP Analysis
Tumor that had been frozen on dry ice at the time of removal and stored at 70°(] was used for DNA isolation. The frozen tumor was pulverized in liquid nitrogen and then suspended in a lysis buffer containing 20 mM Tris-HCl, 1 mM EDTA, 1% sodium dodecylsulfate (SDS) and 100 /~g/ml proteinase K and incubated at 37°C up to 18 hours. DNA was then extracted with phenol-chloroform-isoamyl alcohol (CHISM) and precipitated with 0.4 M ammonium-acetate and cold ethanol. The DNA pellets were dissolved in Tris-EDTA buffer. The patient's lymphocyte DNA was used as a control. Mononuclear cells isolated from the peripheral blood were resuspended in 10 mM Tris and then lysed with lysis buffer and extracted as above.
Ten micrograms of the control and the tumor DNA were digested to completion with the appropriate restriction enzymes, fractionated by 0.8% agarose gel electrophoresis, and transferred to Gene Screen plus nylon membrane according to the manufacturer's protocol. Prehybridization and hybridization were performed in a solution containing 1% SDS, 1 M NaCI, 10% dextran sulfate, 0.05 M Tris pH 7.5, and 0,5 mg/ml denatured herring sperm DNA at 65°C with shaking. The probe was radiolabeled with 32p-ATP by random oligonucleotide primer procedure [4]. Filters were washed twice in 2 x SSC/0.1% SDS at room temperature, once in 1 x SSC/0.1% SDS for 15 minutes, and once in 0.1 × SSC/0.1% SDS at 60°C for 30 minutes. Autoradiography was performed using Kodak XAR-5 film with intensifying screen at -70°C overnight or for several days depending on the particular probe used.

Determination of Allelic Dosage
BgllI blot hybridized with the D22S1 probe was stripped in 0.4N NaOH at 65°C for 30 minutes followed by 0.1 x SSC, 0.1% SDS, and 0.2 M Tris-HCl pH 7.5 at 65°C for 30 minutes and was rehybridized with NGF-B. Taql blot was hybridized with D22S9, stripped in the same fashion and rehybridized with the D10S4 and D10S5 probes. HindlII blot was used for both pAS-1 and D10S5 probes. The stripped blot was checked with an autoradiogram before reuse. The intensity of the hybridization signals were determined by laser densitometry (LKB ultrascan XL}, and peak areas corresponding to each hybridization signal were calculated by electronic integration.

Rearrangement Detected at Locus on Chromosome 22
The tumor and control DNA were compared with three RFLP probes on chromosome 22. D22S1 is an anonymous genomic DNA segment mapped to 22q11-22q13 that gives rise to two RFLP alleles with the BgllI restriction enzyme. D22S9 is an anonymous RFLP probe mapped to 22q11. c-sis is a protooncogene (PDGF-B chain) that detects two RFLP alleles with a HindIII digestion (Fig. 2). The results are shown in Fig. 3 and summarized in Table 2. The control DNA was homozygous for D22S9 and had a 5.8kitobase (kb) hybridization signal with TaqI digestion; the tumor DNA displayed an extra hybridization signal at 5.2 kb, suggesting that the tumor DNA had a possible small internal deletion or rearrangement of one allele in the region of chromosome 22q11. Analysis of control DNA with the D22S1 probe revealed homozygous hybridization sigLlal at (,).5 kb. A marked decrease in allele signal intensity was observed in the tumor sample, indicating a possible hemizygous deletion ot chromosome 22 sequences in the tumor (:ells. This was supported by rebybridizing the same DNA blot with NGF-b probe (mappe(t to chromosome 1); both tumor and (:onstitutional DNA had hybridization signals that were (1t equal intensity. Densitometry analysis is shown in Table 1

Loss of Heterozygosity and Rearrangement at Loci on Chromosome 10
The tumor and control DNA were compared with three chromosome 10 RFLP probes: D10S4, D10S5, and D10S1. The chromosomal localization of these probes is shown in Fig. 2  -TaqI, HindlII, and Hint2 (Fig. 3). Because both tumor,DNA and control DNA were homozygous at D10S1 locus they were not informative for allele loss. These findings suggest that the tumor has rearrangement occurring at chromosome 1{) q21.1 region and an allele loss at chromosome 10q21-10q23 region.

RFLP Analysis of Loci on Other Chromosomes
A breakpoint in the 9q31 and 22q12.2 regions was previously reported in two myxoid chondrosarcomas [11. We studied tumor and coutrol DNA with chromosome 9q RFLP probes including pAS-1 (eDNA for argininosuccinate synthetase enzyme; sequence showed that RFLP mapped to 9q11-q22) and Aldo B (mapped to 9q21.3 to q32). The tumor and the control DNA were homozygous for Aldo B probe as well as pAS-1 probe.
Tumor DNA had all extra 2.4-kb hybridization signal with pAS-1 probe, however, displayed decreased signal intensity with a 2.1-kb band {Fig. 4). Multiple pseudogenes for human argininosuccinate synthetase are dispersed in multiple autosomes, and the X chromosome pAS-1 detects one argininosuccinate synthetase pseudogene mapped in the 9q11-q22 region and the argininosuccinate synthetase structural gene mapped in the 9q34 region [5,6]. Pseudogenes on chromosome 9q11-q22 give HindlII polymorphism 3.9 kb and 2.7 kb. The 2.]-kb band is derived form the chromosome 12 argininosuccinate synthetase pseudogene (mapped to 12p13-qter). Hence the extra 2.4-kb band may be derived from the chromosome 12. The abl oncogene probe (located at 9q34) did not show a difference between tumor and control DNA. Both tumor and control DNA were homozygous for HoxII (chromosome 17) probe and heterozygous for NGF-B probe (chromosome 1).

DISCUSSION
Hinrichs et al. [2] reported a case of inyxoid chondrosarcama of the right shoulder with a nonrandom reciprocal translocation t(9;22)(q22;ql 1). Turc-Carel et al. [3] subsequently reported another case of extraskeletal myxoid chondrosarcoma from the left distal thigh with a clonal translocation t(9;15;22) (q31;q25;q12.2) and suggested that breakpoints 9q31 and 22q12.2 were associated with extraskeletal myxoid chondrosarcoma. Our case had abnormalities involving the 22qll-q12 region, as demonstrated Three chromosome 9q probes were used to detect molecular evidence of rearrangement; no differences were observed with the aldolase B probe (mapped to 9q21-32) and abl probe (9q34). pAS-1 probe revealed an aberrant band, however, owing to dispersed chromosome location of multiple argininosuccinate synthetase pseudogene, this aberrant band may not be derived from the chromosome 9. In addition a decreased hybridization signal of chromosome 12 argininosuccinate synthetase pseudogene derived sequence was observed. Because the rearrangement may not be detected by standard Southern analysis, the negative data on chromosome 9 do not exclude the possible involvement of chromosome 9 in myxoid chondrosarcoma. No abnormality was detected with probes from chromosome 1 and 17. Loss of heterozygosity or hemizygous deletion at a specific chromosome locus often indicates that chromosomal deletions have occurred; such deletions may also be accompanied by chromosomal rearrangement. In certain tumors, deletions in a particular gene region led to identification of a recessive cancer gene or tumor suppres-sor gene [7[ The hrst (:il,.a(,terJzed tumor SUl)pressor gene-retinobl,lstom,J ge.e (Rb) [81 is deleted or mutated not only m retlnoblaslomas but also m osleogenic sarcon],ls, small (;ell lung (:arcinom.s, brest (,enters. and bladder cancers [.(t-12 J. I)NA rearr.mg. lllell[ Call |]lOVe a (.eJlular l)rolooncogene a(lia(:enl to another ()ll(.()gl:Hle ()r llllo ~tll active transcription unit and result in increasc,(t pr()duction or pr(Mu(.tion ot an altered gene product revolved iIi (:ell growth. This has been I)es! illustrated in chrom( myelogenous leukmma (CML)and Burkitt's lymphoma [13. 141.
The present study demonstrates allele h)ss, rearrangement, ~)r a small deletion involving chromosomes 1() am[ 22 (~ccurring in a rare sporadic mvxoid chondrosar-COllie, a ttlnl(/r of mesenchvmal origin. Cytogeneti(: and molecular genetic abnornlalities involving chromosome 22q11-(t22 have been reported in various neuroectodermal tumors (eg, meningioma, Ewing's sarcoma, peripheral neuroepithelioma, acoustic neuroma, and astrocytoma [15][16][17][18][19]). Loss ot chromosome 10 is also commonly found in high-grade gliomas and meningiomas 120, 211 m~phcating a i)ossible (:ommon molecular ine(:hanism involved in the evolutmn ot these ll.eoplasms. The marker D10S5 showing rearraxlgement in this tumor is linked to multiple endocrine neoplasm (MEN) 2A lo(:us with a maximum lod score oi 3.58 tit a recoml)lnation frequency o| 0.19 1221. Patients with MEN 2 are predisposed to develop mechdlarv thyroid can(:er, pheochromocytoma, and parathyroid a(tenoma. These tindings suggest the presem:e of genes closed to D10S5 locus and chromosome 22qll-q22 region involved in the tumorigenesis. To determine whether chromosome 10 and 22 abnormalities are specific for myxoid chondrosarcoma, more tumors need to be studied with similar probes. Detailed mapping of the chromosome 22q 11-12 region and the chromosome 10q21.1 region may reveal genes involved in the formation of this rare tumor and possible other more common tumors.