DNA Fragmentation Simulation Method (FSM) and Fragment Size Matching Improve aCGH Performance of FFPE Tissues

Justin M Craig,D Ashley Hill,Ahmed Idbaih,Mark W Kieran,Mark W Kieran,Mark W Kieran,Sandro Santagata,Sandro Santagata,Sandro Santagata,Patrick Y Wen,Patrick Y Wen,Patrick Y Wen,Patrick Y Wen,Aydin Sav,Keith L Ligon,Keith L Ligon,Keith L Ligon,Keith L Ligon,Natalie Vena,Shaun D Fouse,Azra H Ligon,Azra H Ligon,Azra H Ligon,Massimo Loda,Massimo Loda,Massimo Loda,Charles G Eberhart,Charles G Eberhart,Andrew D Norden,Andrew D Norden,Andrew D Norden,Andrew D Norden,Memet Ozek,Shakti Ramkissoon,Shakti Ramkissoon,Linda R Margraf,Linda R Margraf,Brian Rubin

doi:10.1371/journal.pone.0038881

Abstract

Whole-genome copy number analysis platforms, such as array comparative genomic hybridization (aCGH) and single nucleotide polymorphism (SNP) arrays, are transformative research discovery tools. In cancer, the identification of genomic aberrations with these approaches has generated important diagnostic and prognostic markers, and critical therapeutic targets. While robust for basic research studies, reliable whole-genome copy number analysis has been unsuccessful in routine clinical practice due to a number of technical limitations. Most important, aCGH results have been suboptimal because of the poor integrity of DNA derived from formalin-fixed paraffin-embedded (FFPE) tissues. Using self-hybridizations of a single DNA sample we observed that aCGH performance is significantly improved by accurate DNA size determination and the matching of test and reference DNA samples so that both possess similar fragment sizes. Based on this observation, we developed a novel DNA fragmentation simulation method (FSM) that allows customized tailoring of the fragment sizes of test and reference samples, thereby lowering array failure rates. To validate our methods, we combined FSM with Universal Linkage System (ULS) labeling to study a cohort of 200 tumor samples using Agilent 1 M feature arrays. Results from FFPE samples were equivalent to results from fresh samples and those available through the glioblastoma Cancer Genome Atlas (TCGA). This study demonstrates that rigorous control of DNA fragment size improves aCGH performance. This methodological advance will permit the routine analysis of FFPE tumor samples for clinical trials and in daily clinical practice.

Highlights

Tumor-specific genomic aberrations are of great diagnostic and prognostic value
Each aliquot was labeled separately and paired in four combinations to create both size-matched and mismatched fragment pairs. These paired samples were hybridized to Agilent 180 K feature arrays to model the variation in DNA fragment size commonly present in test and reference samples competitively hybridized to arrays
Our results identify some of the major sources of array comparative genomic hybridization (aCGH) variability and provide new methods for improving the data generated from suboptimal DNA specimens

Summary

Introduction

Tumor-specific genomic aberrations are of great diagnostic and prognostic value. These aberrations are increasingly useful in selecting targeted therapies for individual patients [1]. Genome-wide technologies to determine copy number changes such as array comparative genomic hybridization (aCGH) and single nucleotide polymorphism (SNP) arrays were among the first whole-genome technologies developed [5]. These technologies have been able to query the genome at intra-exon resolution and, as demonstrated in recent large-scale projects such as The Cancer Genome Atlas [4], can offer high-throughput analysis and robust genome-wide copy number data

Methods

Results

Conclusion