Dale C Wright1, Luke St Heaps1, Geoff McCowage2, Nicole Graf3.
1The Children’s Hospital at Westmead, Sydney Genome Diagnostics, Cytogenetics, Westmead
2The Children’s Hospital at Westmead, Oncology, Westmead
3The Children’s Hospital at Westmead, Anatomical Pathology, Westmead
BACKGOUND: Neuroblastomas show genetic and clinical heterogeneity. They can occur in infants with subsequent spontaneous regression, be localised with favourable outcome, or become refractory to treatment. Prognostic markers include age, histology and tumour stage, and genetic/genomic alterations. The latter includes DNA-ploidy, MYCN amplification (MYCNA), loss of heterozygosity (LOH) of 1p/11q and other segmental chromosome abnormalities (SCA); e.g. loss 17p, gain 1q/17q, among others. LOH/ SCA detection can be performed by FISH and/or microsatellite STR markers but compared to chromosome microarray (CMA) can be laborious. This study aimed to improve prognostic cytogenomic marker detection in neuroblastoma.
METHOD: Thirteen neuroblastoma patients [five retrospective, eight prospective] were investigated with median age at diagnosis 6 months [range: 2 – 72] and varying tumour stage: poorly differentiated, III, IV and IV-S. Fresh/frozen tissue was analysed using a customised 8x60K CGH+SNP cancer microarray [5Mb resolution] (Agilent Technologies). FISH was performed on touch-imprints using MYCN and chromosome 1p [TP73] probes on 12/13 and 2/13 patients, respectively. Karyotyping was attempted on three tumours.
RESULTS: CMA identified ploidy and MCYN status, and other SCAs in 11/13 tumours. One showed no abnormality and one failed. A single MYCNA tumour was diploid with LOH 1p and 12 SCAs. The remaining showed hyperdiploidy only (n=4), hyperdiploidy with SCA (n=5) [four with LOH 11q], hypodiploidy LOH 1p with three SCA (n=1), and diploidy with LOH 1p (n=1). These findings confirmed MYCN FISH, where one showed MYCNA and 9/11 tumours showed increased MYCN copy number (3-6) but NOT amplification. Two showed three 1p copies. Tumour karyotypes showed no abnormality (n=2) or failed (n=1).
CONCLUSION: CMA overcomes the challenges of karyotyping and targeted FISH, providing improved detection of ploidy and cytogenomic markers. However, rapid MYCN FISH remains important. Along with age, tumour stage and MYCN status, CMA can identify ploidy and LOH/SCA that refines patient risk-stratification.
Dale is a Principal Hospital Scientist and Head of Cytogenetics for Sydney Genome Diagnostics at The Children’s Hospital at Westmead, Sydney. He has wide experience in Clinical Cytogenetics, mostly involving infertility, prenatal and preimplantation genetic diagnosis, but more recently has been working with microarrays in multiple myeloma and paediatric solid tumours.