A balanced t(5;17) (p15;q22-23) in chondroblastoma: frequency of the re-arrangement and analysis of the candidate genes

COBRA-FISH based karyotyping of the index case (CB13) showed 46, XY, t(5;17)(p15;q22-23)[20]/idemx2[5]/46, XY[4] (Figure 1A). The presence of metaphase cells with both rearranged and normal karyograms excluded the possibility of a constitutional translocation. The balanced nature of the rearrangement was supported by array-CGH testing with no additional genomic copy number changes. By FISH mapping, the breakpoint in 5p15 was demonstrated to be proximal to the SRD5A1 gene region (Figure 1B). The breakpoint in 17q22-23 was proximal to the CA10 gene region with a possible involvement of the gene transcription regulatory element (19) (Figure 1B). RT-PCR using potential chimera primer combinations was negative (data not shown).

Interphase FISH on tissue sections of the index case demonstrated that the translocation only occurred in mononuclear cells, while it was absent in multinucleated giant cells (Figure 1B). Rearrangement of chromosome 5 and 17 was found only in the index case and not in the other investigated chondroblastomas (Figure 1B). One case, CB 10, showed three unsplit signals for both the probes on chromosome 5 as well as on chromosome 17, suggesting either a possible trisomy of the two chromosomes or a triploidy in 2% of the cells (data not shown).

The meta-analysis showed that only the gene BST2 (CDNA FLJ59809 complete cds, highly similar to Bone marrow stromal antigen 2) (accession number: NM_004335) had significantly higher expression in growth plates versus chondroblastomas (adjusted p = 0.008). Global testing did not show any significant differences in the group of genes present on the expression array that are involved in androgen and oestrogen metabolism.

We report a novel balanced translocation t(5;17) in a chondroblastoma case. No recurrent chromosomal changes have ever previously been described in chondroblastoma (Table 1) [11–15]. Even though involvement of either chromosome 5 or 17 has been described in 5 out of the 13 karyotypes reported thus far, no recurrent chromosome band involvement has been shown (Table 1) [11–15]. FISH mapping identified breakpoints that occurred close to the SRD5A1 and CA10 genes, which are located on chromosome 5 and 17, respectively. We found no chimera product by reverse transcriptase PCR. Since the FISH mapping identified that the breakpoints occurred outside the coding region, this translocation is hypothesised to interfere with regulatory regions of the nearby genes. In the region of the breakpoints, no other genes are reported.

CA-RP X (Carbonic anhydrase related protein X), the protein product of CA10, is a member of the CA family, and SRD5A1 is involved in sex steroid metabolism. The CAs (carbonic anhydrases) are monomeric zinc-metallo enzymes that catalyse the reversible hydration of CO₂. These enzymes participate in a variety of biologic processes, including calcification and bone resorption. The human CA gene family includes twelve active iso-enzymes that exhibit characteristic catalytic activity (CA I-IV, VA, VB, VI, VII, IX, XII, XIII and XIV) and three isoforms that lack the CA activity due to the absence of one or more of three zinc-binding His residues, which are critical for CA catalytic activity. Thus, the latter have also been designated CA-related proteins VIII, X, and XI. Recent studies have addressed the role of CAs in cancer; notably, CA IX and CA XII have been reported to be over-expressed in several carcinomas [23]. It has been hypothesised that these transmembrane CA iso-enzymes may contribute to the tumour micro-environment by maintaining the extracellular acidic pH [24, 25]. CA-RP VIII, which does not have the catalytic CA activity as explained above, has been shown to be predominantly expressed in colorectal cancer cells at the tumour-invasion front [23]. However, the mechanism driving this pattern of expression and its possible role in tumour invasion is largely unknown. CA-RP X expression was previously reported to occur only in neural tissue [25, 26]. The promoter region of the CA10 gene has been intensively investigated; however, no defined sequence has yet been identified [25]. Immunostaining revealed that CA-RP X protein expression was absent in the neoplastic cells of the index case and expressed at low levels in three other cases. The lack of expression may point to the disruption of a yet unknown control element by the translocation. Furthermore, it may indicate that CA10 gene has an allele-specific expression pattern. In the other three cases with low expression, another mechanism diminishing the level of expression is hypothesised to occur. In the remaining cases, a variable expression pattern of the protein was observed. However, a pathogenic role of other members of the CA family cannot be excluded.

Sex steroids accelerate longitudinal growth in puberty [8]. Specifically, upon sexual maturation the level of circulating hormones is markedly increased. This is associated with a dramatic increase in the length growth rate - the so-called pubertal spurt. In both boys and girls, oestrogen is a key determinant for the puberty-associated phenomena related to longitudinal growth and bone quality [8, 27]. Specifically in boys, most of the testosterone is converted to oestrogen by CYP19; this enzyme is expressed in and around the growth plate and regulates the local concentration of oestrogen [28]. However, a minor role might be also played by androgens. Specifically, androgen receptors are found in the growth plate [29]. SRD5A1 catalyses the conversion of testosterone into the more potent androgen, dihydrotestosterone. This latter hormone is able to bind androgen receptors, which will translocate to the nucleus thereby activating transcription of the downstream genes. Furthermore, dihydrotestosterone is a non-aromatisable androgen (i.e., it cannot be converted to oestrogen by aromatase). Despite the above-mentioned role of oestrogens in promoting the pubertal spurt, its action on growth plate chondrocyte proliferation rate is not completely clear [8]. A low dosage of circulating oestrogens may stimulate length growth through increased growth plate chondrocyte proliferation [8]. On the other hand, high circulating levels of oestrogens can induce growth plate closure through accelerated bone replacement of hypertrophic chondrocytes. Accordingly, ESR1 is mainly found in growth plate hypertrophic chondrocytes [30]. Remarkably, the diffuse ESR1 expression chondroblastoma closely resembles the expression in hypertrophic chondrocytes. We did not find a casual relationship between the presence of the translocation and changes in the levels of SRD5A1expression. However, diffuse expression of SRD5A1, ESR1 and CYP19 in all the chondroblastomas tested, including the index case, was found. AR showed a scattered expression in few cases, mainly in males.

Taken together, our findings show a great similarity of levels of expression, both at the protein level as well as at the mRNA level, in sex steroid signalling-related molecules between growth plates and chondroblastomas. This supports a possible role for this signalling in promoting chondroblastoma pathogenesis.

Finally, we observed intra-tumoural heterogeneity of the staining pattern of multinucleated giant cells. We previously reported that a similar pattern of expression occurs in multinucleated giant cells of chondroblastomas for other molecules [7]. Some multinucleated giant cells might be of syncytial origin or from impaired duplication of neoplastic cells; however, our present findings lead us to reject this latter hypothesis. In the index case, neoplastic mononuclear cells displayed rearrangement of chromosomes 5 and 17. In contrast, the nuclei from multinucleated giant cells did not display rearrangements in the specific chromosomal regions. Taken together, these results favour a clonal origin of the mononuclear cells of chondroblastoma and suggest that the multinucleated giant cells within the lesions might be reactive. Although we cannot exclude the multinucleated giant cells have a different clonal origin, we consider it more likely that the giant cells are osteoclast-like and recruited through the RANK-ligand, which is present on the surface of the chondroblast-like cells of chondroblastoma [31]. This is in parallel to the recruited nature of giant cells in giant cell tumours of bone, which appear to be blood born as shown by their immunophenotype [32].

In conclusion, we observed a case with a translocation at t(5;17) with potential involvement of two candidate genes. At the genome level, no disruption of the coding sequences was observed; however, involvement of regulatory elements may have occurred. Although direct correlation was observed only in the index case, a certain type of molecular deficiency caused by the translocation might be important for chondroblastoma pathogenesis. Diffuse expression of SRD5A1 and sex steroid signalling-related molecules confirms the role of this pathway in cartilaginous tumours. Additionally, we determined that multinucleated giant cells in chondroblastoma are most likely reactive.