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

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

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 www.cancer.org

Most sarcomas have a recurrent translocation

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

SPECTRAL KARYOTYPING (SKY)  Allows Simultaneous visualization

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

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

Centrosome aberrations common in OS Saos-2

OS characterized by ploidy change

Experimental Techniques DEAC FITC Texas Red Cy5 Spectrum Orange mBAND FISH

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)

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

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)

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.

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

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

Evidence of BFB events in OS Inverted duplications associated with BFB

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

Dicentrics in OS cell lines detected using pancentromeric PNA probe

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

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

Location of fragile sites

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