Cytogenetics of Chordomas

Chordomas are a rare tumor, thought to arise from neoplastic transformation of embryonic notochordal rests. ¹ The genetic and molecular mechanisms underlying the development of chordoma are not well understood. Chromosomal analysis provides a valuable means for identifying the candidate genes and pathways important in tumor development. Early cytogenetic studies on chordoma using G-banding and spectral karyotyping (SKY) techniques identified abnormal karyotypes in fewer than 50% of chordoma cases. ²^,³^,⁴^,⁵^,⁶^,⁷^,⁸^,⁹^,¹⁰^,¹¹ Most abnormal karyotypes in chordoma are hypodiploid or near-diploid with numerous and various chromosomal aberrations. Importantly, no common tumor-specific alteration has been identified; however, a number of alterations occur with regularity.

More recent studies have utilized comparative genomic hybridization (CGH) or whole-genome single-nucleotide polymorphism (SNP) microarray analysis for the evaluation of copy number variants in chordomas. ¹²^,¹³^,¹⁴^,¹⁵^,¹⁶^,¹⁷^,¹⁸ CGH allows for an unbiased genome-wide analysis. CGH-based studies have identified chromosomal changes in 65 to 100% of cases studied and in 91 to 100% of cases utilizing the higher-resolution array CGH (aCGH) technique. ¹²^,¹³^,¹⁴^,¹⁵^,¹⁷ This finding, in addition to identifying aberrations by aCGH in cases with normal G-banding, has led authors to conclude that the previously noted high rate of normal karyotypes in chordomas were likely secondary to a growth advantage of normal cell populations in vitro. ¹²^,¹⁷

Chordoma is understood to be a genetically complex and heterogenous disease with no single deletion or gain common to all cases, as demonstrated by one series of 33 cases in which 166 unique aberrations were identified. ¹³ Changes are typically moderately complex and characterized primarily by losses of large chromosomal regions. ¹⁵^,¹⁷ Losses tend to occur more frequently than gains, with a frequency of about 9.2 losses per case and 4.9 gains per case. ¹²^,¹³^,¹⁶^,¹⁷ Frequently, implicated chromosomes include 1, 3, 7, 9, 10, 12, 17, 19, and 22, with partial losses of chromosomes 1, 3, 9, and 10 and gains of chromosome 7 occurring with the highest frequency. The cumulative frequencies of chromosomal involvement in the major aCGH studies are presented in ▶ Fig. 5.1. This chapter will provide a summary by loci of those chromosomal regions currently understood to be of interest in chordoma ( ▶ Table 5.1).

Fig. 5.1 Cumulative frequencies of copy number variation (CNV) in CGH studies. ¹³^,¹⁴^,¹⁵^,¹⁶^,¹⁷^,²⁵ In studies that included duplicated cases at times of recurrence, the latter case was excluded from analysis.

**Table 5.1** Chromosomes of interest in chordomas
Chromosome	Loss/Gain	Frequency^a	Candidate genes
1	Loss	47%	TNFRSF8, 9, 14
1	Loss	47%	RUNX3
3	Loss	53%	FHIT
			BCL6
			RASSF1A
6	Gain	15%	T (brachyury)
7	Gain	40%	EGFR
7	Gain	40%	MET
9	Loss	46%	CDKN2A
10	Loss	47%	PTEN
11	Loss	26%	ATM
22	Loss	28%	CHEK2
22	Loss	28%	SMARKB1
^aFrequencies calculated from available data from CGH and SNP studies (Le et al, ¹⁵ Scheil-Bertram et al, ¹³ Kitamura et al, ¹⁴ Rinner et al, ²⁵ Diaz et al, ¹⁸ Hallor et al, ¹⁷ Dewaele et al ¹⁶).

5.2 Chromosome 1

Genetic lesions of chromosome 1 are among the most common described in chordoma. Gains in chromosome 1q have been reported in 16 to 67% of cases ¹⁴^,¹⁸^,¹⁹ as well as the frequent formation of isochromosome 1q. ¹¹ Losses of chromosome 1p are reported in between 21 and 100% of cases and 43.5% cumulatively of cases studied by CGH and SNP. ¹²^,¹³^,¹⁴^,¹⁵^,¹⁶^,¹⁷ Additionally, loss of heterozygosity (LOH) studies have identified a high incidence of 1p36 loss among sporadic chordomas. ²⁰^,²¹^,²² LOH studies performed by Riva et al and Longoni et al have identified 1p36 LOH in 85% and 75% of cases, respectively. ²⁰^,²¹

1p36 LOH has been suggested to play a role in the tumorigenesis of chordoma based on the findings that these changes were identified in primary tumors prior to radiation therapy and persisted at the time of recurrence, ²¹ and have also been described in familial chordomas. ²² Furthermore, the differing expression profiles of studied genes at this locus between chordoma cases and nucleus pulposus suggest a role in pathogenesis. However, the specific genes involved are yet to be determined.

Several genes have been investigated based on their localization to 1p36. Riva et al found absence of CASP9 expression in five of eight cases investigated by reverse transcriptase–polymerase chain reaction (RT-PCR). CASP9 is a protease that plays a central role in the execution phase of apoptosis. The group’s follow-up study identified additional cases of CASP9 inactivation but at a much lower rate, leading the authors to conclude that CASP9 inactivation occurs in a limited number of cases and may play a minor role in tumor development. TNFRSF8, TNFRSF9, and TNFRSF14, members of the tumor necrosis factor (TNF) superfamily that maps to 1p36, were demonstrated to be differently expressed from controls in 40 to 53% of chordoma cases. The TNF-receptor superfamily positively regulates apoptosis and also limits cell proliferation. ²⁰^,²¹

DVL1 is a key factor in WNT signaling pathways expressed during developmental processes, including segmentation. The gene is inactive in many tissues, but active in nucleus pulposus, and in one study it was absent in four of eight chordomas and truncated in the remaining four. This finding suggests a potential role for DVL1 silencing in the neoplastic transformation of notochordal cells to chordoma. ¹^,²⁰ RUNX3 is a transcription factor and tumor suppressor gene that has been demonstrated to be transcriptionally activated in various cancers and is involved in the maturation of chondrocytes. ¹⁵^,²³^,²⁴ RUNX3 maps to 1p36 and has been suggested as a candidate gene in the pathogenesis of chordoma based on this location, but its role in chordoma has not been further studied. ¹⁵

5.3 Chromosome 3

Chromosome 3 loss is a frequently described event in chordoma involving between 14 and 80% of cases. ¹⁰^,¹²^,¹³^,¹⁴^,¹⁵^,¹⁶^,¹⁷^,¹⁸^,¹⁹ Furthermore, SNP analysis identified losses of chromosome 3 in 10 out of 10 cases and whole chromosome 3 loss was identified in 71% of cases by Hallor et al. ¹⁷ The SNP analysis demonstrated 3p26.3–q29 as a commonly lost region in all cases, ²⁵ and aCGH analysis identified 3p24.1–p14.2 as the smallest overlapping region in 8 of 10 cases. ¹⁶ Candidate genes on chromosome 3 include FHIT, PIK3CA, BCL6, RASSF1A, RBM5, PTPRG, and VHL, although only FHIT has been investigated in chordoma. ¹⁶^,¹⁸^,²⁵^,²⁶

Fragile histidine triad (FHIT) is located on chromosome 3p14.2 and has been implicated as a tumor suppressor gene through transcription regulation, modulation of DNA damage checkpoint responses, and enhancement of apoptosis in various cancers. ¹⁸^,²⁶^,²⁷^,²⁸^,²⁹^,³⁰ Diaz et al observed a high rate of reduced or absent FHIT expression in chordoma by immunostaining; however, chromosomal loss of the FHIT locus occurred much less frequently, leading the authors to conclude that epigenetic mechanisms in addition to genomic instability are involved in FHIT expression in chordoma. ¹⁸ Rinner et al have demonstrated the presence of alteration in DNA methylation in common tumor suppressor genes in chordoma, providing credence to the epigenetic hypothesis, although FHIT methylation patterns were not specifically investigated in their study. ²⁵ Interestingly, Rinnner et al found that reduced FHIT expression occurred more frequently in sacral chordoma (98%) than skull base chordoma (67%) and only 21% of classic clival chordoma cases demonstrated loss or gain of the FHIT locus. ²⁵

RASSF1 is a tumor suppressor gene located at 3p21.3 and is involved in controlling cell cycle and DNA repair and has been inactivated in various cancers through DNA hypermethylation. ²⁵^,³¹ Rinner et al demonstrated hypermethylation of RASSF1 in chordoma. ²⁵ The VHL gene is a well-known tumor suppressor gene associated with von Hippel-Lindau syndrome and is located at 3p25.3; although the VHL gene has not been studied in chordoma, a case of chordoma occurring in a patient with VHL syndrome has been reported. ³²

5.4 Chromosome 6

Gains in chromosome 6 occur in 13 to 29% ¹⁴^,¹⁵^,¹⁷^,¹⁸ of chordomas and 20% cumulatively by CGH. Although infrequent in copy number variation (CNV) frequency, chromosome 6 warrants mention due to the high degree of interest in the brachyury (T) gene in chordoma pathogenesis. Brachyury is located on 6q27, and duplication of its locus is associated with familial chordoma. ³³ Brachyury is a member of the T-box family of transcription factors, which are required for the development of the notochord. ³⁴^,³⁵^,³⁶^,³⁷ Brachyury acts as a regulator in oncogenic transcription on a diversity of signaling pathways and has been demonstrated to affect the expression of numerous genes. ³⁸ Furthermore, in vitro suppression of brachyury halts cell proliferation in chordoma cell lines ³⁹^,⁴⁰ and overexpression of brachyury, in a cell line that did not express brachyury, caused increased proliferation, motility, and invasiveness. ⁴¹ However, multiple studies have failed to show significant copy number gains involving the T gene in the majority of sporadic cases, suggesting that the mechanism of T activation is likely from epigenetic phenomenon or upstream effects rather than copy number gains in many cases. ¹⁵^,¹⁷^,³⁹^,⁴²^,⁴³

5.5 Chromosome 7

Gains in chromosome 7 have been identified as one of the most common events in chordoma and have been suggested to play an important role in chordoma genesis. Chromosome 7 gains have been identified in 27 to 73% of cases by various techniques and have been demonstrated in both primary and recurrent cases. ¹²^,¹³^,¹⁴^,¹⁵^,¹⁶^,¹⁷^,¹⁸^,¹⁹^,²⁵^,⁴⁴^,⁴⁵^,⁴⁶^,⁴⁷ Early studies identified SHH and HLXB9 as candidate genes in chordoma genesis; however, further study has failed to demonstrate overexpression of these genes. ¹²^,¹³ Subsequently EGFR, MET, LMTK2, SSP, EPHA1, EPHB4, and EPHB6 have been suggested as regions of interest.

EGFR is located at 7p12 and encodes a transmembrane receptor protein that is part of the protein tyrosine kinase family. Receptor binding of epidermal growth factor leads to cell proliferation. Responses to EGFR antagonist in advanced chordoma cases suggests a role of EGFR in advanced disease. ⁴⁸^,⁴⁹^,⁵⁰ Copy number gains involving the EGFR locus were identified in 40 to 52% of cases evaluated by fluorescence in situ hybridization (FISH). ¹⁷^,⁴² Tamborini et al identified EFGR activation in 86% of cases by antibody arrays. ⁵¹ These findings demonstrate that EGFR is a frequently activated receptor tyrosine kinase in chordoma, suggesting the opportunity for therapeutic targeting. ¹⁷

The MET proto-oncogene is located at 7q31 and encodes the transmembrane receptor tyrosine kinase c-MET, which is in involved in the pathogenesis of various neoplasms. MET overexpression has been demonstrated in 70 to 95% of chordomas by immunohistochemistry. ⁴⁵^,⁴⁷^,⁵²^,⁵³^,⁵⁴ Two studies have demonstrated some correlation of MET overexpression and aneusomy of chromosome 7; however, this correlation is not universal to MET overexpression in chordoma. ⁴⁵^,⁴⁷ Furthermore, RT-PCR evaluation of common oncogenic fusions resulting in MET activation showed that these fusions were not present in chordoma, regardless of polysomy 7 status. These finding suggests that other unidentified mechanisms, aside from chromosome 7 copy number and oncogene fusions, exist for MET overexpression. ⁴⁵

5.6 Chromosome 9

Chromosome 9 losses occur with a relatively high frequency. 9p losses have been demonstrated in 16 to 76% of chordomas and 45% cumulatively in CGH and SNP studies and 9q loss in 16 to 81% of cases and 41% cumulatively. ¹⁴^,¹⁵^,¹⁶^,¹⁷^,¹⁸^,²⁵ CDKN2A, encoded on 9p21, blocks the function of CDK4 and CDK6–cyclin D complexes. CDK–cyclin complexes control the G₁/S-phase checkpoint of the cell cycle, through modifications of the retinoblastoma protein, and CDKN2A inactivation results in cell proliferation. ²⁴ Naka et al demonstrated frequent inactivation of CDKN2A protein by immunohistochemical staining. ⁷ Hallor et al ¹⁷ investigated the CDKN2A locus by aCGH and FISH and identified CDKN2A hemizygous loss in 58% of cases and homozygous loss in 12%. ¹⁷ Le et al demonstrated hemizygous loss in 50% and heterozygous loss in 30% with a higher-resolution array. In this study, immunohistochemistry staining for CDKN2A protein was negative in 83% of cases. ¹⁵ Furthermore, the authors studied promoter methylation status of CDKN2A and identified one case with a methylated promotor region, which had copy number maintenance but negative immunohistochemistry, demonstrating that methylation status is an infrequent mechanism of CDKN2A silencing.

A few reports of chordomas in patients with tuberous sclerosis complex exist. ⁵⁵^,⁵⁶^,⁵⁷^,⁵⁸^,⁵⁹ The TSC1 and TSC2 genes are located at 9q34 and 16p13, respectively. Lee-Jones et al demonstrated LOH in TSC1 in one case and TSC2 in a second case of chordomas associated with tuberous sclerosis. ⁵⁷ Although understudied and infrequently identified in sporadic cases, this correlation is of clinical value given the availability of mTOR inhibitors.

5.7 Chromosome 10

Chromosome 10 loss is reported in 19 to 80% of cases and 47% cumulatively by CGH. PTEN is a tumor suppressor gene located at 10q23 and has lipid phosphatase and protein phosphatase activity. ²⁴ Han et al demonstrated frequent loss of PTEN in chordoma, which was also associated with activation of the AKT/mTORC1 pathway. ⁶⁰ Those authors also demonstrated that rapamycin, an mTOR inhibitor, suppressed proliferation in a chordoma cell line. A later study, by the same group, demonstrated hemizygous deletion PTEN in 80% of chordoma cases by aCGH and negative immunofluorescence for PTEN in 68%. ¹⁵

5.8 Chromosome 11

Chromosome 11 deletions are reported in about 26% of cases in CGH studies. ¹²^,¹³^,¹⁵^,¹⁷ The ATM gene is located at 11q22 and is associated with p53 cell cycle checkpoints and cellular response to DNA damage. Hallor et al ¹⁷ found chromosome 11 deletions in 38% of studied cases and identified the minimally deleted region in these cases to contain the ATM loci. ¹⁷ The role of ATM in chordoma is otherwise not well studied.

5.9 Chromosome 17

Loss of chromosome 17 occurs in 14 to 48% of chordoma cases by CGH and 21% cumulatively. The p53 gene (TP53), critical in the signal transduction pathway, mediating G1 arrest or apoptosis in response to DNA damage, ⁶¹ is located at 17p31. Alterations in the p53 pathway have been implicated in some chordoma cases based on immunohistochemistry studies. ²⁰^,⁶² However, FISH analysis of the 17p13 locus only rarely demonstrated rearrangements, suggesting that effects of the p53 pathway are infrequently due to copy number changes. ¹⁹

5.10 Chromosome 22

Chromosome 22 loss is reported in between 2 and 61% of CGH cases and 29% cumulatively, ¹³^,¹⁴^,¹⁵^,¹⁶^,¹⁷ whereas Scheil-Bertram et al reported chromosome 22 gains in 21% of cases. Candidate genes on chromosome 22 include the NF2 gene, CHEK2, and SMARKB1. CHEK2 is a tumor suppressor gene, which interacts with the p53 pathway to mediate the cell cycle. NF2 and CHEK2 have not been well studied in chordoma.

SMARCB1 is thought to function as a tumor suppressor gene and is a member of the adenosine triphosphate (ATP)-dependent SWI/SNF chromatin-remodeling complex encoded at 22q11. SMARCB1 was demonstrated to be absent in all cases of poorly differentiated chordoma by immunohistochemistry, whereas it was maintained in typical cases. FISH analyses for the SMARCB1 locus was negative in three of four these poorly differentiated cases. The findings of the study demonstrate a likely role for deletions of the SMARCB1 locus as a mechanism for aggressive behavior in chordoma.

5.11 Clinical Considerations

A few studies have evaluated the prognostic implications of cytogenetic changes in chordoma. The presence of an abnormal karyotype by G-banding has been associated with shorter recurrence-free and overall survival times, ⁶³ and is due to a higher frequency of an abnormal karyotype by G-banding in recurrent cases. Based on these findings, chromosomal aberrations were believed to be a late event in chordoma pathogenesis. ⁵^,¹¹ CGH studies have not identified a higher frequency of chromosomal aberrations in recurrent tumors compared with primary cases. ¹²^,¹⁷ However, DNA flow cytometry studies of both dedifferentiated and primarily malignant chordomas have suggested higher rates of aneuploidy in these cases, and Hruban et al demonstrated by cytogenetic techniques a high frequency of polyploidy in these cases. ⁶⁴^,⁶⁵^,⁶⁶

Aberrations in chromosomes 3, 4, 12, 13, and 14 have been associated with shorter recurrence and survival periods, with a particularly poor prognosis in cases involving chromosomes 3 and 13 ⁶³ ( ▶ Fig. 5.2). In a series of 37 patients with skull base chordomas, Kitamura et al demonstrated gain of 2p as poor prognostic indicator. ¹⁴ Horbinski et al found deletion of 9p21 to portend a worse prognosis. ⁶⁷ As previously discussed, Mobley et al demonstrated loss of the SMARCB1 locus to be associated with poorly differentiated cases. ⁶⁸ Naka et al have reported p53 overexpression to be associated with a poor prognosis. ⁷ Hallor et al ¹⁷ have reported that homozygous loss of CDKN2A and CDKN2B were present in all metastatic tumors studied. ¹⁷ Given that many of these loci are generally among the less frequently involved regions in chordoma, it is likely that they represent a state of genomic instability occurring in the later stages of chordoma as they progress to a more aggressive tumor. Alternatively, they may occur as the result of a chromothripsis event. Chromothripsis was recently described in chordoma and other tumors, particularly bone tumors, as an alternate mechanism of cancer development in which tens to hundreds of rearrangements occur in single catastrophic event. ⁶⁹

Fig. 5.2 Illustrative case demonstrating the effect of chromosome 3 and 13 involvement. Preoperative (a), postoperative (b), and follow-up (c) magnetic resonance images of a case that recurred just 7 months following resection. (d) SKY of the same case demonstrating involvement of both chromosomes 3 and 13. (e) Kaplan–Meier survival curves comparing cases that did (blue) and did not involve chromosomes 3 and 13.

Only gold members can continue reading. Log In or Register to continue