PF526 STRUCTURAL AND FUNCTIONAL VARIABILITY OF THE TUMOR B‐CELL RECEPTOR INDICATES A ROLE FOR ENVIRONMENTAL INFLUENCES ON BEHAVIOR OF MANTLE CELL LYMPHOMAS
Authors: Dutton D. , Chiodin G. , Agata W. , Drennan S. , Henderson I. , Gaidano G. , Johnson P. , Stevenson F. , Packham G. , Sander B. , Forconi F. 
Published: June 2019

Background:

Mantle cell lymphoma (MCL) is a mature CD5+ B‐cell tumor defined by common phenotypic and genetic features resulting in overexpression of cyclins (most commonly cyclin D1) and subsequent alterations of cell cycle progression. However, MCL has a heterogeneous course ranging from relatively indolent to very aggressive. In CLL, variability of B‐cell receptor (BCR) structure and function, consequent to antigen engagement, has provided key information on the variability of CLL behavior and clinical course. MCL typically express immunoglobulin M (IgM) and IgD. The association of IGHV status with different clinical outcome (Navarro, 2012; Walsh, 2003) and the sensitivity to BCR‐associated kinase inhibitors suggest the functional importance of BCR signaling in MCL.

Aims:

Our aim was to investigate the hypothesis that BCR surface IgM (sIgM) levels and function are variable and that its variability may be consequent to environmental influences acting on the BCR binding site.

Methods:

Peripheral blood mononuclear cells from 26 MCL patients were investigated. BCR structure was analyzed by IGHV‐D‐J transcript sequencing to characterize the antigen‐binding site, and by immunoblotting of the tumor sIgM, following EndoH or PNGase digestion, to investigate the functional status of the effector site. SIgM/D expression was determined on MCL cells by immunophenotype using F (ab’)2 anti‐IgM/D. SIgM/D levels were measured after culture of MCL cells at 0 (baseline), 24, 48 and 72 hours. MCL samples with sIgM levels increasing >20% over baseline in vitro were defined “recoverers”. SIgM signaling capacity was measured by calcium mobilization assay and by phosflow of SYK and BTK following anti‐IgM stimulation.

Results:

Analysis of the tumor IGHV revealed an enrichment of MCL cases with mutated IGHV (M‐IGHV, homology to germline<98%, 11/21), possibly reflecting selection of patients with a more indolent course in this cohort. SIgM levels were extremely variable (n = 26, MFI range 47–2597, median 810) while sIgD levels were low (MFI range 1–252, median 25). Interestingly, sIgM levels increased during ‘antigen‐free’ in vitro culture in 9/17 samples (53%). Analysis of the “recoverers” revealed that 6/9 expressed M‐IGHV (66%), while the remaining 3 cases expressed sIgM using unmutated IGHV4–39 (1/3) and IGHV3–21 (2/3), known to associate with a more indolent course in MCL (Walsh, 2003). Baseline sIgM levels in the “recoverers” were significantly lower (range 47–1534, median 325) than the non‐recovering group (range 119–2282, median 1272; p = 0.027). SIgM levels associated with signaling capacity, measured by calcium mobilization (r = 0.635, p = 0.009) and phosflow (pSYK[Y525]; r = 0.933, p = 0.007 and pBTK[Y223]; r = 0.952, p = 0.001). Immunoblotting of the tumor sIgM by EndoH and PNGase digestion revealed immature glycans in a recovering MCL with low sIgM baseline levels but not in a non‐recovering MCL with high sIgM. This provides further support of prior exposure of the “recovering” MCL sIgM to environmental influences in vivo (Krysov, S 2010, Drennan, 2018).

Summary/Conclusion:

Our data on this selected cohort indicate that sIgM levels are variable and correlate with their signaling capacity. The evidence that a group of MCL with M‐IGHV or IGHV3–21, that express relatively low levels of sIgM with ability to recover in vitro, suggests selective influences on the BCR of these MCL. This process may be prominent in indolent MCL, where genomic aberrations are less frequent. The evidence in these MCL with recovering sIgM claims analogies with CLL, in which variable antigen‐driven anergy plays a role in clinical behavior.