72 Background: Clonality testing is an integral part of the initial assessment of B and T cell malignancies. The further use of conventional clonality assays for monitoring of disease post treatment is limited by low assay sensitivity and inability to track clones based on their unique sequences. Clonality assessment by next generation sequencing (NGS) provides increased diagnostic capabilities, allowing the tracking of disease specific clones as well as the full spectrum of clonal sequences that arise in response to therapy. Here we describe our initial experience using an NGS based assay for monitoring and our comparison to conventional clonality assays and flow cytometry. Methods: DNA was extracted from hematologic samples received for routine clonality assessment including diagnostic (DS) and follow-up post therapy (PT) samples. Clonality testing was performed by conventional PCR-based assays using biomed II primers and by NGS utilizing Lymphotrack IGH FR1 and TRG kits (Invivoscribe). The amplified products were sequenced on Illumina MiSeq. For MRD assessment, we created dedicated laboratory protocols as well as in-house developed software, MSK-LymphoClone (LC), containing a data analysis pipeline, analytical tools for clonality assessment and a signout portal for easy search and retrieval of pertinent clones. Results: Samples from 48 patients were included (48 DS and 60 PT) encompassing 12 plasma cell neoplasms, 11 acute lymphoblastic leukemias, 16 mature B-cell and 9 T-cell lymphomas. All diagnostic samples showed clonal rearrangement with 100% concordance between conventional and NGS assays. Residual disease was detected in 27/60 (45%) PT samples using conventional fragment size based assays, 27/57 (47%) using flow and 38/60 (63%) using NGS. Diagnostic clonal sequences were detected in as low as 0.0019% of total reads in PT samples tested by NGS. 18/60 (30.0%) PT samples (17 patients) were disease negative by all assays; 16 patients remained disease-free (median follow-up - 2.4 months). Conclusions: NGS clonality testing is a valuable tool for monitoring patients with B and T cell neoplasms showing higher sensitivity and specificity than conventional assays.
Next generation sequencing reveals novel alterations in B-cell heavy chain receptor repertoires associated with acute-on-chronic liver failure