IGH-FR1 and Leader Analysis By Next Generation Sequencing for the Detection of Clonality and Somatic Hypermutation in Lymphoid Malignancies

The standard method to analyze clonality and somatic hypermutation of immunoglobulin genes in B-cell non Hodgkin lymphomas is a multiplex PCR followed by capillary electrophoresis and/or Sanger sequencing. Recently, Next Generation Sequencing (NGS) of immune receptor genes was demonstrated to be highly effective, with increased sensitivity for detecting sequences of interest. Based on that, it was proposed for the diagnosis and monitoring of lymphoid neoplasms. A phase 3 diagnostic accuracy study was designed to compare the value of the first commercially available kit for NGS of IGH/FR1 with the gold standard analysis. One hundred twenty samples were evaluated for IGH rearrangements with both traditional capillary electrophoresis (IdentiClone™ IGH Gene Clonality Assay by Invivoscribe Technologies) and NGS (LymphoTrack^TM IGH Assay – MiSeq and LymphoTrack® IGH Somatic Hypermutation Assay - MiSeq by Invivoscribe Technologies on an Illumina MiSeq).

The NGS clonality assay compared to conventional analysis had an overall diagnostic accuracy of 96% (63/66 cases), sensitivity (ST), specificity (SP), positive predictive value (PPV), and negative predictive value (NPV) of 95%, 100%, 100%, and 75%, respectively.

Regarding mutational analysis, conventional detection and NGS gave comparable results. Notably, NGS was capable of analyzing all cases, including those failed by Sanger sequencing. In discrepant cases NGS results were confirmed by a different set of primers that provided coverage of the IGH Leader sequence.

In conclusion NGS was effective for IGH analysis in lymphoid disease (even on FFPE samples). In order to apply them for routine diagnostics, NGS based approaches should be evaluated prospectively and cost-effectiveness assessments should be performed.