GLASGOW – Whole genome sequencing (WGS) appears capable of replacing cytogenetic testing and next generation sequencing (NGS) for the detection of clinically relevant molecular abnormalities in hematological malignancies, according to investigators.
A comparison of WGS with fluorescence in situ hybridization (FISH) showed that WGS caught all the same significant structural variants, plus some abnormalities that FISH had not detected, reported lead author Shirley Henderson, PhD, lead for cancer molecular diagnostics at Genomics England in Oxford.
Although further validation is needed, these findings, reported at the annual meeting of the British Society for Haematology, support an ongoing effort to validate the clinical reliability of WGS, which is currently reserved for research purposes.
“It’s vitally important that the clinical community engage with this and understand both the power and the limitations of this technique and how this work is going to be interpreted for the benefit of patients,” said, session chair from University College London’s Cancer Institute.
The investigators compared WGS with FISH for detection of clinically significant structural variants (SVs) and copy number variants (CNVs) in tumor samples from 34 patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL).
The 252 standard of care FISH tests – conducted at three separate clinical diagnostic centers in the United Kingdom – included 138 SVs and 114 CNVs. WGS relied on a combination of bioinformatics and visual inspection of Circos plots. WGS confirmed all of the SVs detected by FISH with high confidence; WGS detected four additional SVs, also with high confidence, including an ETV6-RUNX1 fusion not detected by FISH because of probe limitations.
Results for CNVs were similar, with WGS detecting 78 out of 85 positive CNVs. Six of the missed positives were associated with low quality samples or low level mutations in the FISH test, suggesting that at least some positives may have been detected with better samples. Only one negative CNV from FISH was missed by WGS.
Overall, WGS had a false positive rate of less than 5% and a positive percentage agreement with FISH that exceeded 90%.
“Further work is required to fully validate all aspects of the WGS analysis pipeline,” Dr. Henderson said. “But these results indicate that WGS has the potential to reliably detect SVs and CNVs in these conditions while offering the advantage of detecting all SVs and CNVs present without the need for additional interrogation of the sample by multiple tests or probes.”
Dr. Henderson noted that there is really no “perfect method” for identifying structural and copy number variants at the present time.
Small variants are relatively easy to detect with techniques such as karyotyping and gene banding, but these tests have shortcomings, namely, that they require live cells and have “fairly high failure rates for various reasons,” Dr. Henderson said.
“FISH is an incredibly useful test and it has higher resolution than gene banding, but the problem with FISH is that you only find what you’re looking at,” Dr. Henderson said. “It’s not genome wide; it’s very targeted.”
Similarly, polymerase chain reaction (PCR), including next generation sequencing (NGS), can detect molecular abnormalities, but only those that are targeted, which may necessitate multiple tests, she said.
“If you start looking for all of the structural variants [with existing techniques], then you’re going to be doing an awful lot of tests,” Dr. Henderson said.
Another potential benefit of WGS is that it is “future resistant,” Dr. Henderson said. “As new biomarkers are discovered, you don’t have to redesign a new targeted test. It will also detect emerging biomarkers, such as mutational signatures and burden.”
The study was sponsored by NHS England. The investigators reported having no conflicts of interest.
SOURCE: Henderson S et al. BSH 2019, .