Validating The Prognostic Significance Of FCRL2 In Predicting IGHV Mutation Status, Clinical Disease Progression, and Survival In CLL
Authors: Li Fu Jun , Cantor Alan B , Shakhmatov Mikhail A. , Li Ran , Yan Xiao-Jie , Chiorazzi Nicholas , Davis Randall S. 
Published: October 2019

Abstract
B cell chronic lymphocytic leukemia (CLL) can be stratified into indolent and aggressive subtypes according to the degree of somatic hypermutation evident in the immunoglobulin heavy chain variable region gene (IGHV). Despite some discordance with IGHV mutation status, both CD38 and ZAP-70 are routinely analyzed in clinical practice and are considered useful prognostic factors for segregating patients with aggressive-unmutated CLL (U-CLL). Members of the Fc receptor-like (FCRL1-5) family have immunoregulatory function and are preferentially expressed by B cells. Their distinct distribution pattern among subsets in healthy donors suggests that the FCRLs may also have useful diagnostic and/or therapeutic potential in B lineage malignancies. Previous work employing a receptor-specific monoclonal antibody (mAb-7F2) and an indirect staining method demonstrated that FCRL2 is particularly upregulated on the surface of indolent-mutated CLL (M-CLL) cells. Among 107 CLL samples, 52 U-CLL and 55 M-CLL, FCRL2 overlapped with IGHV status with 94.4% concordance, relative to 76.6% for CD38 (cutoff of 30%), and 80.4% for ZAP-70 according to the T/B ratio (cutoff of 4.0) or 74.8% by the % method (cutoff of 20%). Because several years have passed since our initial analysis using this reagent, we updated the clinical information to reassess the relationship among these prognostic parameters with FCRL2 staining and to further determine the usefulness of this biomarker in CLL. Similar to IGHV status [Hazard ratio (HR)=5.03], FCRL2 could also, but more powerfully, predict survival (HR=8.58). Both these features were superior to CD38 (HR=1.29) and ZAP-70 by either the T/B ratio (HR=1.42) or % (HR=1.04) approaches. The median survival for FCRL2 positive cases was 26.2 years and for FCRL2 negative donors was 12.7 years. With regard to disease progression, FCRL2 positive cases had a median treatment free interval of 13.5 years, whereas for FCRL2 negative donors it was only 3.2 years. Given these promising results, we pursued optimizing FCRL2 detection and assayability by flow cytometry to validate its clinical utility for diagnostic and prognostic purposes in CLL. A screen of FCRL2-specific mAbs led to the identification of a novel subclone termed 3E11, which by epitope mapping reacted with a site discrete from that of 7F2. The binding kinetics of these two mAbs also differed. According to surface plasmon resonance studies using an FCRL2-Fc recombinant protein, 3E11 possessed ∼2.5X better nanomolar affinity and a slower dissociation rate than 7F2. The superiority of 3E11 was also evidenced by its relatively stronger surface staining reactivity in flow cytometry analyses. To achieve a one-step staining strategy, 3E11 was directly conjugated with PE. Use of this PE-labeled reagent demonstrated brighter and more selective FCRL2 reactivity on M-CLL versus U-CLL (N=30) cells using either the post-Ficoll or whole blood-lysis preparation methods. Importantly, its reactivity was also stable over time (Concordance Correlation Coefficient of 0.9). As a promising CLL biomarker capable of selectively detecting the MT-CLL subtype and predicting disease progression and survival, the current findings demonstrate the practicability and reliability of a newfound mAb for validating the prognostic value of FCRL2 in CLL.

Disclosures:
LI: University of Alabama at Birmingham: Filed Patent, Filed Patent Patents & Royalties. Davis:NIH an University of Alabama at Birmingham: Patent Filed, Patent Filed Patents & Royalties, Research Funding.