(P 03) CDK4 IS CO-AMPLIFIED WITH EITHER TP53 PROMOTER GENE FUSIONS OR MDM2 THROUGH DISTINCT MECHANISMS IN OSTEOSARCOMA

Objective: Osteosarcoma exhibits distinctive subgroups based on clinical, morphological and genetic characteristics. The most common is high-grade conventional osteosarcoma, whereas low-grade osteosarcomas (namely parosteal and low-grade central) form a smaller subgroup. Amplification of the MDM2 and CDK4 genes, along with other parts of chromosome arm 12q in osteosarcoma, is typically associated with low-grade tumours or tumours of a low-grade origin. Conventional osteosarcomas normally harbour genome-wide structural and numerical chromosome changes, coupled to loss of TP53 function. However, some of these high-grade tumours may simultaneously display 12q amplifications. We subjected 33 samples from 25 high- and low-grade osteosarcomas (conventional n=14, dedifferentiated parosteal n=3, parosteal n=5, and low-grade central n=3) with MDM2 and/or CDK4 amplification to high-resolution genomic copy number, structural variation and transcriptomic analyses and integrated the genetic information with clinical data with the aim of determining if additional genes among the chromosome 12 amplicons may contribute to tumour progression.

Methods: Whole genome DNA copy number alterations were investigated in two cohorts of conventional osteosarcomas (n=108 and n=36, respectively) from which cases with chromosome arm 12q amplifications including CDK4 and/or MDM2 were selected (Cytoscan HD arrays, Thermo Fischer Scientific). Additionally, cases diagnosed as low-grade osteosarcoma at the Skåne University and Karolinska University Hospitals (Sweden) were included. In total, 33 biopsies from 25 patients were selected for further investigation. Whole genome mate pair sequencing technology (Nextera Mate Pair, Illumina) was used to detect structural alterations which were verified in selected cases using longread whole-genome HiFi sequencing (PacBio). Low-pass single-cell whole genome sequencing was used to assess copy number alterations in individual cells from selected tumours. For 82 osteosarcomas and 13 control osteoblastomas, high-quality RNA was obtained, and global gene expression analysis and fusion gene detection was performed using RNA sequencing (TruSeq, Illumina).

Results: Four major subgroups were identified that varied in genetic complexity and harboured various instances of group-specific promoter swapping events and amplification mechanisms. The first harboured three low-grade osteosarcomas (low-grade central n=1, parosteal n=2) with chromosome 12 amplicons (including both CDK4 and MDM2) as the sole acquired genetic alteration. The second group contained a mixture of ten high- and low-grade tumours (low-grade central n=2, parosteal n=3, dedifferentiated parosteal n=3, and conventional n=2) with both CDK4 and MDM2 amplification in addition to some alterations outside of chromosome 12. Many cases in this group displayed rearrangement of the regulatory regions of the FRS2 or PLEKHA5 genes leading to upregulation of various 3′ partner genes. PLEKHA5-rearranged cases showed a poor clinical outcome (conventional n=1, dedifferentiated parosteal n=1). The third group, made up of seven conventional osteosarcomas with heavily rearranged genomes, harboured both CDK4 and MDM2 amplification intertwined with amplified regions from numerous other chromosomes. In these three groups, a chromothripsis event affecting chromosome arm 12q followed by genomic amplification was the likely cause of the observed amplifications. Finally, the fourth group, made up of five conventional osteosarcomas, harboured CDK4 – but not MDM2 – amplification and heavily rearranged genomes. CDK4 amplification was intertwined with an initial structural variant affecting TP53 that simultaneously lead to TP53 promoter swapping and the creation of a dicentric chromosome. No chromothripsis events were detected affecting chromosome arms 12q or 17p in this group.

Conclusion: Our results further characterise osteosarcomas with 12q amplification, showing that there are indeed differences between such tumours. We find evidence for the existence of two separate modes of genomic amplification affecting chromosome arm 12q and associated regions in high- and low-grade osteosarcomas. Additionally, previously unreported instances of promoter swapping might play a role in the development of low-grade osteosarcomas, although such findings would require further in vitro studies to be corroborated.