Project 3: NGS Control round

An international control round for deep sequencing analysis of BCR-ABL kinase domain mutations in 12 laboratories from 7 European countries

Thomas Ernst1, Simona Soverini2, Katerina Machova Polakova3, Nicholas C. P. Cross4, Andreas Hochhaus1

1.) Abteilung Hämatologie/Onkologie, Klinik für Innere Medizin II, Universitätsklinikum Jena, Jena, Germany

2.) Department of Hematology-Oncology 'L. and A. Seràgnoli', University of Bologna, Bologna, Italy

3.) Institute of Hematology and Blood Transfusion, Prague, Czech Republic

4.) Wessex Regional Genetics Laboratory, Salisbury, UK

Background: Point mutations in the BCR-ABL kinase domain are the most frequently identified mechanisms of acquired resistance towards tyrosine kinase inhibitors in chronic myeloid leukemia (CML). Although Sanger sequencing is still regarded as the gold standard technique for routine BCR-ABL mutation screening, next-generation sequencing (NGS) has evolved rapidly and is accessible to an increasing number of diagnostic laboratories. Thus far, data is limited on the technical performance of NGS for BCR-ABL mutation testing in a clinical diagnostic setting.

Methods: The European Treatment and Outcome Study (EUTOS) has established a network of CML reference laboratories across Europe. We sought to investigate the utilization, robustness, precision, and reproducibility of NGS for BCR-ABL mutation analysis. An online survey was conducted to obtain information about NGS performance in 48 EUTOS laboratories from 27 countries. To evaluate NGS performance, 20 blinded control samples were sent out recently to 12 laboratories (a total of 240 samples). Control samples contained cDNA of Ba/F3BCR-ABL cell lines harboring different BCR-ABL kinase domain mutations that were mixed with non-mutated Ba/F3BCR-ABL to produce dilutions ranging from 1% to 100% of mutant alleles. Control samples containing cDNA of non-mutated Ba/F3BCR-ABL were also included as wild-type controls. All samples were diluted into HL60 cells to simulate a BCR-ABL level of 10% on the International Scale.

Results: Twenty-nine of 48 labs (60%) have finalized the online survey so far. Fifteen labs reported to have an established NGS method for BCR-ABL mutation analysis of which 10 labs use Illumina and five labs Ion Torrent technology. Only 7 labs indicated to use NGS for routine BCR-ABL mutation analysis of diagnostic samples. The control samples were sent out recently and will be analyzed within the next 1-2 months by the 12 participating laboratories. NGS data will be analyzed according to quality (number of high quality sequencing reads and read length), robustness (number of mixed and dots filtered reads), performance (correct identification of mutations and wild-type samples), sensitivity (identification of low-level mutations) and concordance of mutation quantification. All results will be reported at the meeting.

Conclusion: This multicenter analysis will give important information about utilization, robustness, precision, and reproducibility of NGS for BCR-ABL mutation analysis in CML patients.