Immunoglobulin and T-cell receptor gene analysis by next generation sequencing

Introduction
Sanger sequencing of the monoclonal immunoglobulin (Ig) and T-cell receptor (TCR) rearrangements detected by PCR and Genescan analysis is needed for the design of a patient specific PCR for minimal residual disease (MRD) monitoring in patients with acute lymphoblastic leukemia (ALL) and for the assessment of the Ig hypermutation status - a prognostic factor - in patients with chronic lymphoblastic leukemia (CLL). PCR based next-gene-reaction sequencing (NGS) in an alternative technique for identification of all Ig/TCR rearrangend sequences present in a sample and is currently the focus of intense research in clonality assessment and MRD detection in lymphoid malignancies.
Aims
To assess the specificity of NGS compared to the Genescan/Sanger method for the identification of clonal Ig and TCR genes in ALL and CLL.
Methods
The IgH and TCR-gamma loci were analysed by the lymphotrack IGH+TRG assay from invivoscribe, te TCR beta locus through the immuno SEQ kit from Adaptive Biotechnologies. Sequencing was performed on the MISeq platform. For ALL, the IgH, the TCR-gamma and the TCR-beta were analysed in 29, 20 and 8 diagnostic samples respectively; for CLL the clonal IgH and the hypermutation status were analysed in 4 diagnostic samples.
Results and conclusion
All clonal Ig/TCR sequences identiefied with Sanger Sequencing were also detected by NGS. Additional clonal rearrangement(s) were detected in 17%, 44% and 43% of the samples for IgH, TCR-gamma negative for clonal IgH by Genescan, a clonal rearrangement was detected by NGS.
In conclusion, this PCR based NGS is a powerful alternative for the identification of clonal Ig and TCR sequences which extensively facilitates the design of patient specific PCR methods for MRD detection and the analysis of the somatic hypermutation status in CLL. The sensitivity of MRD quantification through NGS still has to be assessed exentsively.