Abstract
Whilst next generation sequencing can report point mutations in fixed tissue tumour samples reliably, the accurate determination of copy number is more challenging. The conventional Multiplex Ligation-dependent Probe Amplification (MLPA) assay is an effective tool for measurement of gene dosage, but is restricted to around 50 targets due to size resolution of the MLPA probes. By switching from a size-resolved format, to a sequence-resolved format we developed a scalable, high-throughput, quantitative assay. MLPA-seq is capable of detecting deletions, duplications, and amplifications in as little as 5ng of genomic DNA, including from formalin-fixed paraffin-embedded (FFPE) tumour samples. We show that this method can detect BRCA1, BRCA2, ERBB2 and CCNE1 copy number changes in DNA extracted from snap-frozen and FFPE tumour tissue, with 100% sensitivity and >99.5% specificity.
Highlights
Detection of copy number variations (CNVs) in cancer receives less attention than detection of mutations, despite CNVs being relatively common, and having an important role in tumour initiation, progression, and treatment response in multiple cancer types
DNA extracted from blood samples from 12 de-identified individuals with germline BRCA1 or BRCA2 deletions and duplications were obtained from PathWest, Western Australia, and the Australian Ovarian Cancer Study (AOCS) [9]
If the observed mean ratios for the targeted regions were greater than 1.3 in blood and the corresponding tumour sample, they were reported as a germline duplication, while if the increased mean ratios were only observed in the tumour sample, but not in blood, they were regarded as having a somatic amplification
Summary
Detection of copy number variations (CNVs) in cancer receives less attention than detection of mutations, despite CNVs being relatively common, and having an important role in tumour initiation, progression, and treatment response in multiple cancer types. Focal somatic gene amplifications are important targets for approved therapies, such as for trastuzumab, lapatinib, in ERBB2 (HER2) amplified breast cancer or gastroesophageal cancer [1], and for potential therapies in genes such as MET and CCNE1 in multiple cancers [2,3]. Loss of function due to germline or somatic deletions in tumour suppressor genes may confer drug.
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