Molecular profiling of chordoma

Abstract

The molecular basis of chordoma is still poorly understood, particularly with respect to differentially expressed genes involved in the primary origin of chordoma. In this study, therefore, we compared the transcriptional expression profile of one sacral chordoma recurrence, two chordoma cell lines (U-CH1 and U-CH2) and one chondrosarcoma cell line (U-CS2) with vertebral disc using a high-density oligonucleotide array. The expression of 65 genes whose mRNA levels differed significantly (p<0.001; ≥6-fold change) between chordoma and control (vertebral disc) was identified. Genes with increased expression in chordoma compared to control and chondrosarcoma were most frequently located on chromosomes 2 (11%), 5 (8%), 1 and 7 (each 6%), whereas interphase cytogenetics of 33 chordomas demonstrated gains of chromosomal material most prevalent on 7q (42%), 12q (21%), 17q (21%), 20q (27%) and 22q (21%). The microarray data were confirmed for selected genes by quantitative polymerase chain reaction analysis. As in other studies, we showed the expression of brachyury. We demonstrate the expression of new potential candidates for chordoma tumorigenesis, such as CD24, ECRG4, RARRES2, IGFBP2, RAP1, HAI2, RAB38, osteopontin, GalNAc-T3, VAMP8 and others. Thus, we identified and validated a set of interesting candidate genes whose differential expression likely plays a role in chordoma.

Figure 1.

(A) Cytology of the U-CH2 chordoma cell line. (B) CGH analysis of U-CH2. The gains are given in green, the losses in red.

Figure 2.

Summary of chromosomal imbalances in our cohort of 33 chordomas.

Figure 3.

Summary of gene expression analysis of the chromosomal arms of the 65 candidate genes whose mRNA levels differed significantly (p<0.001; ≥6-fold change) between chordoma and control (vertebral disc).

Figure 4.

Expression of gene transcripts (RT-RCR) of: (A) T brachyury; (B) IGFBP2; (C) RARRES2; (D) ECRG4; (E) cytokeratin 18, KRT18; and (F) T1A1a–b. Each value is expressed as the ratio of three values of each tumor (6 chordomas of 5 patients; 6 chondrosarcoma of 6 patients)/control (vertebral disc).

Figure 5.

(A) Expression of CD24 transcript (RT-RCR). Each value was expressed as relative expression (n=3) of each tumor (6 chordomas of 5 patients; 10 chondrosarcoma of 10 patients)/control (vertebral disc). FACS analysis of: (B) CD24, CD20, EMA and negative control of the chordoma cell line U-CH2; (C) CD24 analysis of previous published chordoma cell line U-CH1 (7); and (D) the chondrosarcoma cell line U-CS2. Immunohistochemistry of CD24 (clone 24C02) frozen section of: (E) parental tumor of U-CH1 (case 1); (F) chordoma no. 2 (both bars, 200 μm; (G) CS1; and (H) CS6 focally weak immunoreactivity (both bars, 100 μm).