1. CHROMOSOMAL MECHANISMS OF TUMOUR PROGRESSION IN OSTEOSARCOMA
Selvarajah S, Lim G, Beheshti B, Watson S, Bayani J, Yoshimoto M, Vukovic B, Braude I, Marrano P, Al-Romaih K, Thorner P, Lam W, Zielenska M and Squire J
Department of Laboratory Medicine & Pathobiology, University of Toronto
Department of Paediatric Laboratory Medicine, The Hospital for Sick Children

2. OSTEOSARCOMA
Osteosarcoma (OS) – most common (20%) of primary bone tumors
Bimodal distribution – 75% in young patients <20 yrs
Defined according to –
matrix production, location, histology, grade
Conventional OS
mostly high-grade, aneuploid

3. BACKGROUND: Occurrence Age Comments Conventional OS >90% of OS
2nd decade of life (80%) (childhood OS)
Mostly high-grade & aneuploid
Small Cell OS
Rare
1st & 2nd decade of life (70%)
90% diploid
High-grade surface OS
Childhood & Adult
Mostly high-grade
Telangiectactic OS
Multifocal OS
Intracortical OS
10-30 yrs
Mostly high grade
Periosteal OS
<2% of OS
~21 yrs
Good prognosis
Parosteal OS
Up to 5% of OS
More common in Adult
Well-differentiated OS
1-2% of OS
Adult
Adapted from Mills and Fechner, 1993, and

4. Most sarcomas have a recurrent translocation

5. Hypertetraploid – proximal tibia (9 year old female)
CYTOGENETICS OF OS:
Hypertetraploid – proximal tibia (9 year old female)
Complex karyotypes –
different for each case

6. SPECTRAL KARYOTYPING
(SKY)
 Allows Simultaneous visualization

7. Examples of SKY analysis of OS
A-C: inverted DAPI and RGB images of chromosomes are illustrated
D: only SKY-classified colours are illustrated
A: aberrations involving chr. 8
B: evolution of new rearrangements
C: aberrations involving chr. 20
D: SKY analysis of OS9

8. Summary of SKY and met.CGH in OS
SKY ANALYSIS:
Analysis and positional mapping of recurrent breakpoint regions reveal ‘hotspots’ for rearrangement (10 OS tumours & 4 OS cell lines)
Bayani et al., 2003
Summary of SKY and met.CGH in OS

9. Centrosome aberrations common in OS
Saos-2

10. OS characterized by ploidy change

11. Experimental Techniques
DEAC
FITC
Texas Red
Cy5
Spectrum Orange
mBAND FISH

12. OBSERVATIONS: COMPLICONS in OS
mBAND analysis shows pattern of interspersal gain of 6p21, 8q24 and 9p22.
Ladder amplification pattern.
Indicates co-amplification & co-translocation of non-syntenic regions (6p21.3-p22.1, 8q24 and 9p21-p22)

13. BAC Array CGH – MG-63
6p21.3
~6 Mb
8q24
~32 Mb
Chr 9
9p21~22
~17 Mb

14. Complicon organization in MG-63 marker chromosome
Amplification of drug resistance genes has been shown to generate
markers bearing ladder-like amplicons
Breakage-fusion-bridge mechanism is employed to acquire resistance via
oncogene amplification
Complicon organization in MG-63 marker chromosome
Coquelle et al., 1997 Bridge-Breakage-Fusion (BFB)

15. The distinct ladder pattern observed in the MG-63 marker chromosome suggests that the BFB cycle may be responsible for regions of focal amplifications. Such aberrant areas, which are found to over-express important oncogenes, may contribute to tumorigenesis and/or tumor progression in OS.

16. BREAKAGE-FUSION-BRIDGE
Fragile sites set amplicon boundaries
Inverted duplications
Amplified gene forms the ‘rungs’ of a
ladder in the marker chromosome

17. In MG63 BFB took place after rearrangements between chromosomes 6, 8 and 9

18. Evidence of BFB events in OS
Inverted duplications associated with BFB

19. amplification of alpha-satellite sequences (FISH probes)
ANAPHASE BRIDGE CONFIGURATIONS
amplification of alpha-satellite sequences (FISH probes)

20. Dicentrics in OS cell lines detected using pancentromeric PNA probe

21. SMRT array (Vancouver tiling set) MG-63
Resolution: 79kb
can map chromosome arm breakpoints to a single BAC locus
allows genome-wide mapping of precise microamplification/ deletions containing oncogenes, tumor-suppressor genes and new genes that may be associated with multiple tumor type
Resolution provided by clones in SMRT array

22. Tiling array CGH mapping of deletions and amplifications in OS MG63 cell line

23. Location of
fragile sites

25. Somatic chromosomal mechanisms generating genetic diversity in tumors
Segregation anomalies
Cell cycle checkpoint defects
Ploidy alterations
DNA repair defects
Translocation frequency
Telomere crisis
Genomic architecture - fragile sites, microhomologies
Epigenetic alterations