Detection of clinically relevant copy number alterations in oral cancer progression using multiplexed droplet digital PCR

Curtis B Hughesman,X J David Lu,Charles Haynes,Yuqi Zhu,Kelly Y P Liu,Rebecca M Towle,Catherine F Poh

doi:10.1038/s41598-017-11201-4

Abstract

Copy number alterations (CNAs), a common genomic event during carcinogenesis, are known to affect a large fraction of the genome. Common recurrent gains or losses of specific chromosomal regions occur at frequencies that they may be considered distinctive features of tumoral cells. Here we introduce a novel multiplexed droplet digital PCR (ddPCR) assay capable of detecting recurrent CNAs that drive tumorigenesis of oral squamous cell carcinoma. Applied to DNA extracted from oral cell lines and clinical samples of various disease stages, we found good agreement between CNAs detected by our ddPCR assay with those previously reported using comparative genomic hybridization or single nucleotide polymorphism arrays. Furthermore, we demonstrate that the ability to target specific locations of the genome permits detection of clinically relevant oncogenic events such as small, submicroscopic homozygous deletions. Additional capabilities of the multiplexed ddPCR assay include the ability to infer ploidy level, quantify the change in copy number of target loci with high-level gains, and simultaneously assess the status and viral load for high-risk human papillomavirus types 16 and 18. This novel multiplexed ddPCR assay therefore may have clinical value in differentiating between benign oral lesions from those that are at risk of progressing to oral cancer.

Highlights

Copy number alterations (CNAs), somatic changes involving the gain or loss of genomic material, are common drivers for tumorigenesis[1,2]
We demonstrate that this droplet digital PCR (ddPCR)-based assay detects CNAs at the targeted loci in a manner that is consistent with data from benchmark methods (CGH arrays[31,32,33,34], single nucleotide polymorphism (SNP) arrays[35] and WG-NGS36) for both oral cell lines and clinical samples of different histopathological stages
To evaluate the performance of our multiplexed ddPCR assay to detect CNAs, we first analyzed a panel of genomic DNA samples taken from immortalized cell lines representing normal, dysplasia and oral squamous cell carcinoma (OSCC) phenotypes

Summary

Introduction

Copy number alterations (CNAs), somatic changes involving the gain or loss of genomic material, are common drivers for tumorigenesis[1,2]. Primers targeting the E6 regions of human papillomavirus (HPV) types 16 and 18 are included[27], allowing us to concurrently assess for status and viral load of high-risk HPV, an emerging risk factor associated with head and neck squamous cell carcinoma (HNSCC), especially at the oropharyngeal site[28,29,30] We demonstrate that this ddPCR-based assay detects CNAs at the targeted loci in a manner that is consistent with data from benchmark methods (CGH arrays[31,32,33,34], SNP arrays[35] and WG-NGS36) for both oral cell lines and clinical samples of different histopathological stages. The ability of the method to quantify different patterns of CNAs in various histopathological stages is demonstrated through application to cell lines having normal, dysplasia and OSCC phenotypes, and to clinical samples classified as non-progressing and progressing low-grade dysplasia (LGD), high-grade dysplasia (HGD), and OSCC

Methods

Results

Conclusion