Recurrent chromosomal copy number alterations in sporadic chordomas

Abstract

The molecular events in chordoma pathogenesis have not been fully delineated, particularly with respect to copy number changes. Understanding copy number alterations in chordoma may reveal critical disease mechanisms that could be exploited for tumor classification and therapy. We report the copy number analysis of 21 sporadic chordomas using array comparative genomic hybridization (CGH). Recurrent copy changes were further evaluated with immunohistochemistry, methylation specific PCR, and quantitative real-time PCR. Similar to previous findings, large copy number losses, involving chromosomes 1p, 3, 4, 9, 10, 13, 14, and 18, were more common than copy number gains. Loss of CDKN2A with or without loss of CDKN2B on 9p21.3 was observed in 16/20 (80%) unique cases of which six (30%) showed homozygous deletions ranging from 76 kilobases to 4.7 megabases. One copy loss of the 10q23.31 region which encodes PTEN was found in 16/20 (80%) cases. Loss of CDKN2A and PTEN expression in the majority of cases was not attributed to promoter methylation. Our sporadic chordoma cases did not show hotspot point mutations in some common cancer gene targets. Moreover, most of these sporadic tumors are not associated with T (brachyury) duplication or amplification. Deficiency of CDKN2A and PTEN expression, although shared across many other different types of tumors, likely represents a key aspect of chordoma pathogenesis. Sporadic chordomas may rely on mechanisms other than copy number gain if they indeed exploit T/brachyury for proliferation.

Figure 1. Heat map of array CGH results.

Copy number gains (red) and losses (green) are displayed for each individual chordoma case (rows) with chromosomes organized in columns (separated by white vertical lines) and indicated by labels at the bottom. Note that cases CH34 and CH37 are recurrent tumors from the same patient.

Figure 2. Frequency plot by genomic position.

Array CGH data from all chordoma cases from 20 unique patients were combined and presented as copy number gain/loss frequencies relative to chromosome and genomic position. Note that chromosome Y is not depicted. The second recurrent CH37 tumor was excluded from analysis.

Figure 3. Chromosome 9 array CGH results.

Array CGH results for chromosome 9 are shown for select chordoma cases showing homozygous CDKN2A deletion. Plots were generated with the Agilent Genomic Workbench Standard Edition 5.0 software. The sizes of the homozygous deletions for the five respective cases are as follows: 512 kb, 4.7 Mb, 76 kb, 76 kb, and 158 kb. Note that cases CH34 and CH37 are recurrent tumors from the same patient. Cases CH7 (158 kb) and CH36 (1.9 Mb) also harbor homozygous CDKN2A deletions and are not depicted above. kb = kilobases, Mb = megabases.

Figure 4. Top Panels: CDKN2A immunohistochemistry.

Representative chordoma cases showing lack of expression (left, CH39) and strong expression (right, CH35). Bottom Panels: PTEN Immunofluorescence. Representative chordoma cases were immunostained with anti-cytokeratin to highlight tumor cells (green) and anti-PTEN (red). Tumor cells show lack of PTEN expression in the left panel and expression of PTEN in the right panel. Note in both left panels that stromal tissue or normal cells show expression of CDKN2A and PTEN, but tumor areas indicated by arrows show lack of expression.

Figure 5. CDKN2A and PTEN methylation specific PCR.

Bisulfite-treated chordoma DNA samples were tested with methylation specific PCR to evaluate for hypermethylation of the CDKN2A and PTEN promoter regions. Two sets of unmethylated (U) and methylated (M) PCR primers were used for each target gene (bottom labels, MSP1 and MSP2). Unmethylated and methylated controls are shown along with results for case CH33. Results for other tested cases are summarized in Tables 3 and 4 under the CDKN2A and PTEN MSP1 and MSP2 columns. Tick marks on the left and right of each panel indicate 100, 200, 300, and 400 base pair sizes (bottom to top).

Figure 6. T quantitative real-time PCR.

Two primer/probe sets were used to quantitate T (6q27) and MCM7 (7q21.3-q22.1) (n = 3). Relative T∶MCM7 ratios were normalized against an average ratio established from ten non-chordoma DNA samples (normal). The normalized ratios were corrected for MCM7 copy number from array CGH data and approximate tumor percentage based on histological review. Corrected normalized ratios were multiplied by 2 to obtain the absolute T copy number. The dashed line represents a normal copy number of 2.