Antigen-Independent, Autonomous B-Cell Receptor Signaling in Diffuse Large B-Cell Lymphoma

Gene expression profiling of diffuse large B-cell lymphoma (DLBCL) defines the Cell-of-origin (CoO) classification with two major subtypes, germinal center B-cell (GCB) and the more aggressive activated B-cell (ABC) type. Chronic activation of B-cell receptor (BCR) signaling with constitutive activation of the NF-κB pathway is an essential oncogenic mechanism in ABC DLBCL. BCR pathway activation with increased NF-κB activation can be caused by gain-of-function mutations of the BCR signaling cascade. We investigated whether antigen-independent, i.e. autonomous BCR signaling, akin to its essential pathogenetic role in chronic lymphocytic leukemia, acts as a functional driver mechanism in DLBCL without such gain-of-function mutations.

DNA and RNA were extracted from ABC DLBCL cell lines TMD8 and OCI-Ly3 and from fresh-frozen biopsies of 18 histologically confirmed DLBCL cases. Functional BCR transcripts were identified by ARTISAN PCR and transduced into murine triple knock out (TKO) cells. TKO cells lack the recombination activation gene 2 (Rag2) and lambda 5 gene (λ5) and are therefore incapable of expressing an endogenous BCR. In addition, the BCR signal transduction molecule SLP65 is replaced with a tamoxifen-inducible estrogen receptor (ERT2) - SLP65 fusion gene. Upon transduction of both heavy and light BCR chains and staining with Indo-1, background calcium flux can be measured as Indo-1 signal intensity ratio (SIR) by flow cytometry. After restoring functionality of the BCR signaling cascade by tamoxifen, antigen-independent BCR signaling is measured as SIR, followed by maximum BCR stimulation with an isotype-specific crosslinking antibody. Autonomous BCR signaling strength (SS^BCR) is quantified by an arithmetic product based on the relative Indo-1 SIR and the relative number of cells with SIR above background in the presence of tamoxifen and calibrated on crosslinking-induced maximum SIR.

According to the primary hypothesis, the BCR of CARD11 wild-type, IgM-expressing TMD8 cells demonstrated strong autonomous BCR signaling (SS^BCR=0.243), whereas the BCR of CARD11 mutated, IgG-expressing OCI-Ly3 cells completely lacked autonomous signaling (SS^BCR=0.022). BCR from 13 primary DLBCL signaled autonomously (SS^BCR=0.125-0.619), where 5 samples were negative (SS^BCR=0.004-0.023) Autonomous BCR signaling was absolutely dependent on IgM isotype (Fisher's exact test: p=0.0016). CoO classification by digital gene expression (Nanostring) analyses or immunohistochemistry demonstrated autonomous BCR signaling in 8/9 ABC (non-GCB) DLBCL, 2/6 GCB DLBCL, and 2 unclassifiable DLBCL, and a transformed follicular lymphoma DLBCL. Independent of CoO, DLBCL can be assigned to 6-7 clusters based on their genetic aberrations (Chapuy 2018, Wright 2020). Fifteen DLBCL were suited for WES and targeted panel sequencing. Four cases were assigned by a probabilistic calculation to Chapuy cluster C5, 2 cases to C2, and one case to C1, while 8 cases remained unclassifiable. Clustering by the LymphGen tool assigned 3 cases to cluster ST2, one case each into MCD, EZB, and N1, 4 cases into various clusters, and 5 cases unclassified. DLBCL with autonomous BCR signaling fell into clusters C1/unclassified, C2/unclassified, C5/unclassified (n=3), unclassified/EZB, unclassified/ST2, unclassified/N1 and unclassified/unclassified (n=2). Cases lacking autonomous BCR signaling were assigned to unclassified/ST2 (n=2), C5/MCD, and unclassified/unclassified. Eleven of 13 cases with an autonomous signaling BCR lacked activating CARD11 mutations.

In conclusion, autonomous BCR signaling is a potential immunological oncogenic driver in DLBCL that cannot be detected by sequencing studies, is independent of external antigen stimulation, and originates from a self-organizing conformation of the surface BCR (Minici 2017). Autonomous BCR signaling is present predominantly in ABC DLBCL and dependent on IgM isotype. Autonomous BCR signaling offers a convincing explanation for oncogenic NF-κB activation in DLBCL. Limited data suggest that the majority of DLBCL with autonomous BCR signaling fall into the ABC-dominated genetic clusters C1/C5, and MCD/N1, respectively. In summary, genetic aberrations, BCR function, and gene expression profile form 3 independent layers of DLBCL pathophysiology that need to be analyzed separately and offer independent targets for therapy.

Havranek:Bristol Myers Squibb: Research Funding.