Development of a novel blood-based translational PCR screen for monoclonal B-cell lymphocytosis.

Abstract

e19033 Background: Over 10 million Americans have monoclonal B-cell lymphocytosis (MBL), a precursor to CLL, with age-associated increase in frequency, from ~5% at 45 years to >50% in nonagenarians. Despite significant associations with other malignancies and infectious disease hospitalizations, the natural history of MBL and CLL, and its causal link with allied conditions remains poorly defined. Our health system-wide screening based on routine CBC results identified 112 (10.5%) MBL cases among patients with persistent absolute lymphocytosis (PAL). However, flow cytometry diagnostic for monoclonal B cell expansion were only tested on 1070 out of 5747 PAL patients due to workflow constraints and lack of exposure to the clinical significance of MBL among non-oncology practitioners. Hence, we developed a cost-effective and high-throughput IGHV-D-J PCR screen to identify PAL cases with monoclonal B cells to streamline patients for definitive flow cytometry testing. Methods: Whole blood PAL samples were subjected to RBC lysis, RNA extraction, and reverse transcribed. Adapted from our published unique molecular identifier (UMI)-based methodology, linear amplification of the IGHV regions were performed with UMI-tagged primers to ensure amplification accuracy. Next, the NGS library was generated by semi-nested PCR using IGHV and IGHC primers and sequenced on the Illumina Miseq platform with depth of 40x based on estimated lymphocyte counts. Raw reads were processed and analyzed using a custom workflow built with MiXCR. Results: As research-based clonality testing is challenging for a system-wide screening test, we optimized the PCR strategies using varying cell counts expected for blood volume of CBC tests. No differences in the generation of IGVH-D-J libraries and average product sizes (~600bp) were observed between whole blood and FACS-sorted samples. To determine the correlation of monoclonal B cell percentage and UMI-based detection of unique IGVH-D-J rearrangements, healthy white blood cell were mixed with 0.5 - 12.5% of CLL B cells of known clonotype and clonality data by flow cytometry and our PCR screen were compared. This indicated a significant correlation between CLL-specific IGHV-D-J rearrangements and the percentage of spiked CD5 + CD19 + CLL cells determined by flow cytometry (R 2 = 1.0; P <0.001). Most importantly, we successfully identified the CLL-specific clones with sufficient sensitivity for all MBL counts. Conclusions: Despite reported associations of MBL with hematological malignancies and other conditions, large scale prospective studies remain challenging. To this end, we have developed an efficient semi-nested PCR test that identifies expanded monoclonal B cells using residual whole blood after routine CBC testing. With the catchment area of Northwell Health, this screening test will provide a crucial stepping stone to better understanding MBL as a clinical entity.