Cytogenetic, telomere, and telomerase studies in five surgically managed lumbosacral chordomas

Abstract

Lumbosacral chordomas are rare skeletal sarcomas of the spine that originate from the remnant notochord. The understanding of this human cancer is limited to observations of its clinical behavior and its embryonic link. Thus, we performed chromosome and molecular analyses from five surgically harvested chordomas in an effort to document genetic and biochemical abnormalities which might aid in understanding the tumor biology of this understudied neoplasm. Cytogenetic analysis of the five chordomas revealed normal results in four patients and random abnormalities in only one tumor cell in the 100 cells studied from the fifth patient. A repeat telomeric probe (TTAGGG)50 was hybridized to genomic DNA isolated from chordoma cells (and HeLa cells) and digested with HinfI. The tumor DNA was paired with leukocyte DNA from age-matched controls and revealed telomere elongation in four of the four chordoma patients studied with molecular genetic techniques. Conversely, telomere length reduction has been reported during in vitro senescence of human fibroblasts, giant cell tumor of bone, colon cancer, intracranial tumors, childhood leukemia, Wilms tumor, and in HeLa cells. Telomerase activity (telomerase is required to maintain telomere integrity) was also determined by visualizing the extension of radioactive telomeric repeats on DNA sequencing gels. The telomeric fragments were assembled during incubation of the cytoplasmic extract containing telomerase. Telomerase activity was observed in HeLa (positive control and commercially available cell line), giant cell tumor of bone (positive control tumor cells from living patients), and in chordoma cells from one of the two chordoma patients (but to a lesser degree compared with HeLa). As expected, the chordoma patients' fibroblasts exhibited no telomerase activity.

Figure 1

Photomicrograph of chordoma from patient number 3 demonstrating physaliferous cells ( × 250, H&E stain).

Figure 2

GTG-banded karyotype, 46,X, − X,t(5;12)(q13;p13),t(6;7) (q25;q22), + 14, showing the alterations of 5q13, 7q22, and monosomy X, as reported previously and discussed in the text.

Figure 3

Southern hybridization analysis of the telomere region in chordoma paired with leukocyte DNA from the age-matched controls (5 μg per lane). DNA from smaller telomere regions migrate farther by electrophoresis and thus show a longer signal length. Tumor DNA shows significantly increased telomere length when compared to leukocyte (blood) DNA from the age-matched controls. Patient numbers correspond to the numbers in Table 1. Marker numbers (in kilobases) on vertical axis represent the approximate position of high-weight DNA fragments from a known DNA marker of known sizes (lambda DNA digested with HindIII).

Figure 4

DNA sequencing gel (8%) showing telomerase activity from cell extracts of tumor cells from HeLa, giant cell tumor of bone (GCT), and chordoma patients. Lane 1, primer (TTAGGG)₃; Lane 2, HeLa (12.5 μg); Lane 3, HeLa (25 μg); Lane 4, HeLa (50 μg); Lanes 5, 6, and Z GCT (250 μg); Lane 8, chordoma (250 μg) from patient number 4; Lane 9, chordoma (250 μg) from patient number 3; Lane 10, fibroblasts (250 μg) from patient number 3; and Lane 11, base pair fragments from the C-strand of a standard DNA sequencing reaction from intron III and exon III segments of the growth hormone gene (courtesy of J. Cogan). DNA fragment sizes can be determined by calculating the difference between the known base pair marker sizes represented by numbers on the vertical axis. Telomerase activity was observed for HeLa, GCT, and tumor cells from one of the two chordoma patients (patient number 3). No telomerase activity was observed for the fibroblasts from our chordoma patient number 3.