Single-Step IGHV Next-Generation Sequencing Detects Clonality and Somatic Hypermutation in Lymphoid Malignancies: A Phase III Diagnostic Accuracy Study

SIMPLE SUMMARY: Clonality testing and somatic hypermutation analysis performed on B-cell receptor encoding genes are the most widely used molecular assays for lymphoma diagnostics. Currently, PCR-based methods standardized by the BIOMED2 consortium are regarded as the gold standard. In the last few years, new approaches based on next-generation sequencing (NGS) have been proposed and validated in phase I–II studies. Here, we present the first phase III diagnostic accuracy study, evaluating an NGS-based protocol (LymphoTrack(®) IGH assay, and LymphoTrack(®) IGH somatic hypermutation assay) compared to the gold standard. We formally documented a high diagnostic accuracy providing a clinical validation of the assays. ABSTRACT: Background: Multiplex PCR based on consensus primers followed by capillary electrophoresis and Sanger sequencing are considered as the gold standard method for the evaluation of clonality and somatic hypermutation in lymphoid malignancies. As an alternative, the next-generation sequencing (NGS) of immune receptor genes has recently been proposed as a solution, due to being highly effective and sensitive. Here, we designed a phase III diagnostic accuracy study intended to compare the current gold standard methods versus the first commercially available NGS approaches for testing immunoglobulin heavy chain gene rearrangements. Methods: We assessed IGH rearrangements in 68 samples by means of both the NGS approach (LymphoTrack(®) IGH assay, and LymphoTrack(®) IGH somatic hypermutation assay, run on Illumina MiSeq) and capillary electrophoresis/Sanger sequencing to assess clonality and somatic hypermutations (SHM). Results: In comparison to the routine capillary-based analysis, the NGS clonality assay had an overall diagnostic accuracy of 96% (63/66 cases). Other studied criteria included sensitivity (95%), specificity (100%), positive predictive value (100%) and negative predictive value (75%). In discrepant cases, the NGS results were confirmed by a different set of primers that provided coverage of the IGH leader sequence. Furthermore, there was excellent agreement of the SHM determination with both the LymphoTrack(®) FR1 and leader assays when compared to the Sanger sequencing analysis (84%), with NGS able to assess the SHM rate even in cases where the conventional approach failed. Conclusion: Overall, conventional Sanger sequencing and next-generation-sequencing-based clonality and somatic hypermutation analyses gave comparable results. For future use in a routine diagnostic workflow, NGS-based approaches should be evaluated prospectively and an analysis of cost-effectiveness should be performed.