Clinical investigational studies for validation of a next-generation sequencing in vitro diagnostic device for cystic fibrosis testing

Abstract

Purpose: Clinical investigational studies were conducted to demonstrate the accuracy and reproducibility of the Illumina MiSeqDx CF System, a next-generation sequencing (NGS) in vitro diagnostic device for cystic fibrosis testing. Methods: Two NGS assays – a Clinical Sequencing Assay (Sequencing Assay) and a 139-Variant Assay (Variant Assay) – were evaluated in both an Accuracy Study and a Reproducibility Study, with comparison to bi-directional Sanger sequencing and PCR as reference methods. For each study, positive agreement (PA), negative agreement (NA), and overall agreement (OA) were evaluated. Results: In the Accuracy Study, the Sequencing Assay achieved PA of 99.7% including the polyTG/polyT region and PA of 100% excluding the region. The Variant Assay achieved PA of 100%. NA and OA were >99.99% for both Assays. In the Reproducibility Study, the Sequencing Assay achieved PA of 99.2%; NA and OA were both 99.7%. The Variant Assay achieved PA of 99.8%; NA and OA were both 99.9%. Sample pass rates were 99.7% in both studies for both assays. Conclusion: This is the first systematic evaluation of a NGS platform for broad clinical use as an in vitro diagnostic, including accuracy validation with multiple reference methods and reproducibility validation at multiple clinical sites. These NGS-based Assays had accurate and reproducible results which were comparable to or better than other methods currently in clinical use for clinical genetic testing of cystic fibrosis.

Keywords::

• clinical validation
• cystic fibrosis
• in vitro diagnostics
• next-generation sequencing
• sequencing-by-synthesis

Acknowledgement

The authors would like to thank the investigators for their participation in the clinical investigational study for reproducibility. They would like to thank G Cutting for his expert guidance. They also acknowledge L Hanson, N Marino and P Rupprecht of Mayo Clinic College of Medicine, as well as H Walline and T Goodman of Progenity, Inc.

Financial & competing interests disclosure

DS Grosu, L Hague, M Chelliserry, KM Kruglyak, R Lenta, B Klotzle, J San, WM Goldstein, S Moturi, P Devers, E Lin and FS Ong are employees of Illumina, Inc. E Peters was a former employee of Illumina Inc. and is now an employee of Genentech, Inc. J Woolworth, B Elashoff, J Stoerker, DJ Wolff, KJ Friedman and WE Highsmith were in receipt of project funding from Illumina, Inc. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties.

No writing assistance was utilized in the production of this manuscript.

Key issues

• This is the first published report of a systematic evaluation of a next-generation sequencing (NGS) platform for broad clinical use as an in vitro diagnostic, including accuracy validation with multiple reference methods and reproducibility validation at multiple clinical sites. The clinical investigational studies presented here demonstrate that the Illumina MiSeqDx Cystic Fibrosis (CF) System is able to identify a broad spectrum of CF transmembrane (CFTR) variants with high accuracy and reproducibility.

• The Illumina MiSeqDx CF System offers enhanced clinical utility for the management of CF due to the analytical accuracy of NGS and the inclusion of a comprehensive set of clinically and functionally validated CFTR variants. Furthermore, the simultaneous US FDA marketing authorization of the MiSeqDx as the first non-disease-specific NGS in vitro diagnostic platform is expected to allow any lab to test any sequence by NGS for any purpose, and represents a major step forward in the application of genomic information to improve patient care.

• The 139-Variant Assay includes all variants identified as pathogenic in the CFTR2 database as of September 2013 and is expected to provide enhanced clinical utility by detecting a higher proportion of CF carrier couples and a higher fraction of pathogenic variants in CF patients, thus allowing for more informed and more timely reproductive and treatment decisions.

• The Sequencing Assay, by interrogating with high accuracy the vast majority of CFTR genomic positions likely to harbor pathogenic variants, provides additional clinical utility in cases where CF is strongly suspected on clinical grounds but first-tier testing could not identify a genetic basis for the phenotype.

• The MiSeqDx CF Assays perform well even in regions that are known to induce Sanger errors, such as the polyTG/polyT region and genomic positions close to indels.

• The ability of NGS to simultaneously interrogate multiple types of CFTR pathogenic variants (including large deletions) may lead to the displacement of current multi-step sequential genetic testing paradigm for CF. As the cost of NGS continues to drop, and performance is shown to be comparable or superior to traditional methods, the application of NGS to many different clinical areas is expected to become ubiquitous.