Molecular analysis of the INK4A/INK4A‐ARF gene locus in conventional (central) chondrosarcomas and enchondromas: indication of an important gene for tumour progression

Abstract

Loss of heterozygosity (LOH) at chromosomal band 9p21 is one of the few consistent genetic aberrations found in conventional chondrosarcoma. This locus harbours two cell-cycle regulators, CDKN2A/p16/INK4A and INK4A-p14ARF, which are inactivated in various human malignancies. It was therefore hypothesized that this locus also plays a role in the development of chondrosarcoma and this locus was investigated at protein, genetic, and epigenetic levels. Loss of p16 protein expression was detected by immunohistochemistry in 12 of 73 central chondrosarcomas and it correlated with increasing histological grade (p = 0.001). Loss of p16 protein expression was not found in 51 enchondromas, which are presumed to be potential precursors of conventional central chondrosarcoma. LOH at 9p21 was found in 15 of 39 chondrosarcomas (38%) but it did not correlate with loss of p16 protein expression. SSCP analysis of p16 did not reveal any mutations in 47 cases. Also, p14 was not the target of LOH, since it gave no aberrant bands on SSCP. To investigate whether an epigenetic mechanism was operating, methylation-specific PCR was used to look at p16 promotor methylation, which was identified in 5 of 30 tumours. However, this did not correlate with protein expression, or with LOH at 9p21. Cytogenetic data were available in a subset of cases. All tumours that showed chromosome 9 alterations also showed LOH and loss of INK4A/p16 protein expression. It is concluded that although some alterations were found at the DNA level and at the promoter expression level, the lack of correlation between LOH, promotor methylation, and protein expression indicates that a locus other than CDKN2A/p16 must be the target of LOH at 9p21. The correlation between INK4A/p16 protein expression and tumour grade, and the retention of expression in enchondromas, indicates that loss of INK4A/p16 protein expression may be an important event during tumour progression from enchondroma to conventional central chondrosarcoma, and in the progression in grade after recurrence of chondrosarcoma.

Frequent deletion of the CDKN2A locus in chordoma: analysis of chromosomal imbalances using array comparative genomic hybridisation

“…Frequent loss of chromosome arm 9p has previously been described in chordomas (Scheil et al, 2001;Kuzniacka et al, 2004;Brandal et al, 2005), and particularly, the region covering the CDKN2A (p16 and p14) and CDKN2B (p15) loci in chromosomal band 9p21 has been shown to be deleted in many tumour types (Gil and Peters, 2006), also in chondrosarcoma (van Beerendonk et al, 2004). Here, we provide further evidence that loss of this region is an important event also in chordomas, with 70% of the tumours showing deletion of this region.…”

The role of noncartilage‐specific molecules in differentiation of cartilaginous tumors

“…7 Moreover, the lower expression of p16INK4A in HGCCS underscores the significance of its loss in chondrosarcoma progression, which was reported previously. 20 ALCAM/CD166 also had greater expression in CMF than in HGCCS. This is in agreement with the impres-sion that ALCAM up-regulation is specific for CMF, as discussed above.…”

“…in cell cycle progression (although with an inhibitory action), 7,20 was significantly higher in CMF versus HGCCS (P < .05). Specifically, 67% of CMF samples versus 27.5% of HGCCS samples were positive for p16INK4A.…”

“…Scores that were generated with the same evaluation system for p16INK4A and cyclin D1 (CCND1) in CMF and HGCCS were available from previous studies. 7,19,20 A cut-off value >2 was applied for the sum of intensity and the number of positive cells for p16INK4A and CCCND1, and a cut-off value >3 was applied for activated leukocyte adhesion molecule (ALCAM/CD166). Cut-off selection was set as the minimal value for the sum of intensity and percentage that best discriminated CMF versus HGCCS.…”

Cell‐cycle control and the cartilage growth plate

“…Several changes in the p16 gene have been described in human chondrosarcomas, including deletions and partial methylation (Jagasia et al, 1996a,b;Asp et al, 2000;Asp et al, 2001;Ropke et al, 2003). Loss of p16 protein expression is also detected in some, but not all human chondrosarcomas (Van Beerendonk et al, 2004). In addition, amplification of the CDK4 gene has been reported in a human chondrosarcoma (Kanoe et al, 1998), and overexpression of CDK4 in several cartilaginous tumors has been described (Si, 2000;Asp et al, 2001;Si and Liu, 2001).…”