Comparing Data Generated Using Multiparametric Flow Cytometry, Capillary Electrophoresis, and Next-Generation Sequencing in a Cohort of B-Cell Lymphoproliferative Disorder Samples

Abstract

Background: Determining whether suspected lymphoid proliferation disorder (LPD) samples are benign or malignant remains a significant challenge for the hematopathologist. For the vast majority of LPDs, the current gold standard includes assessment of clinical characteristics, cytomorphology, and immunophenotyping by flow cytometry; however, 10-15% of LPDs require further resolution by molecular methods, such as PCR-based capillary electrophoresis or next-generation sequencing, to rule out clonality based on V-D-J rearrangements in the immunoglobulin heavy-chain (IGH) gene. Invivoscribe (San Diego, CA) has developed multiple complementary methodologies to evaluate LPDs; including multiparametric flow cytometry (MFC), capillary electrophoresis (CE), and next-generation sequencing (NGS). To compare performance between these three methods, a cohort of 21 clinical samples were tested in a pilot study. Method: The CAP/CLIA Hematolymphoid Screening assay, a 10-color flow panel, was used to identify all major types of hematopoietic neoplasia according to the 2006 Bethesda Consensus and 2017 WHO Classification. For molecular testing, DNA was extracted from peripheral blood (PB), then PCR-based assays were used to determine presence or absence of clonality. The Research Use Only (RUO) molecular assays were designed for CE (ABI 3500xL) and NGS (MiSeqTM) detection, utilizing framework multiplex PCR master mixes targeting the conserved framework regions FR1, FR2, or FR3 within the VH and the JH regions of the IGH gene. Twenty-one (21) de-identified PB samples were collected from informed consensus donors (LabPMM, San Diego, CA) or from residual samples (Lahey Hospital, Burlington, MA) and tested under an IRB approved protocol. An aliquot of each PB sample was used for MFC evaluation, then the remaining DNA was extracted and tested using the CE and NGS methods. Test results were reported as "present abnormal" or "normal" for MFC and "clonal" or "non-clonal" for molecular assays based on pre-defined criteria. Results: The MFC assay identified 12 out of 21 (57%) samples as "present abnormal" and 9 out of 21 (43%) samples as "normal." Both molecular assays (CE and NGS) reported 12 out of 21 (57%) samples as "clonal" and 9 out of 21 (43%) samples as "non-clonal." The results between CE and NGS assays were 100% in agreement for clonality detection. Comparisons between the CE and MFC assays again resulted in 100% agreement; all 12 "present abnormal" samples by MFC were reported as "clonal" by CE, while all 9 "normal" samples by MFC were "non-clonal" by CE. In addition to presence of clonality, the NGS method was able to detect the prevalence of a specific clonal V-D-J rearrangement sequence (expressed as % reads), which ranged from 10% to 99% in the 12 clonal samples. This pilot study demonstrated excellent concordance (21/21) between these 3 methods (MFC, CE and NGS). Accordingly, MFC, CE and NGS are all suitable methods for the evaluation and stratification of B-cell LPD samples. The combined use of all 3 methods provides a comprehensive assessment of clonality: including surface markers identified by MFC, DNA rearrangement lengths from CE, and the specific DNA rearrangement sequences from NGS. In addition, the NGS method allows for tracking of these clonal sequences in relation to disease status. Conclusion: This pilot study demonstrated 100% concordance between 3 different methods (MFC, CE, and NGS) when evaluating samples from LPD subjects. Depending on laboratory capabilities and the type of information desired, any one of these technologies can provide appropriate results when studying clonality. The alignment between MFC and NGS allows the combined use of both methods to provide rapid diagnosis via the MFC service and identification of specific biomarkers for subsequent tracking of these clonal populations in clinical studies.