The activated B-cell (ABC) type of diffuse large B-cell lymphoma (DLBCL) is clinically more aggressive than the germinal center (GCB) type. The gene expression profile of ABC DLBCL resembles that of mature B cells upon stimulation via their B-cell receptor (BCR). In up to 30% of ABC DLBCL cases, this signature can be explained by gain-of-function mutations in CD79A, CD79B, or CARD11. Antigen-independent, constitutively active (BCR) signaling is a general oncogenic mechanism of CLL (Dühren-von Minden, Nature 2012). We hypothesized that autonomous BCR signaling akin to CLL may operate in ABC DLBCL and could explain the characteristic ABC gene expression signature.
BCR transcripts were identified from fresh-frozen biopsies from 12 histologically confirmed, IgM-expressing DLBCL by ARTISAN PCR, a novel anchored RT-PCR for unbiased amplification of BCR transcripts facilitated by template switching. Clonal full-length BCR sequences were identified by PacBio next-generation sequencing. Triple KO (TKO) cells were transduced with functional DLBCL BCR. TKO cells are arrested at the pre-B-cell stage due to lack of the rag2 and lambda5 genes. In addition, TKO cells have their wild-type SLP65 adaptor replaced with a tamoxifen-dependent SLP65. When transduced with expression constructs encoding a functional BCR, autonomous or antigen-induced BCR signalling can be measured as calcium flux upon induction with tamoxifen.
TKO cells transduced with seven of the twelve DLBCL BCR (58%) showed robust calcium flux and proliferation upon activation of SLP65 by tamoxifen without additional BCR crosslinking. By histomorphology and immunohistochemistry for CD10, PAX5, MUM1, and Bcl-6, these cases were classified as non-GCB-type DLBCL, including one primary CNS DLBCL and two primary mediastinal (PM-)DLBCL. In accordance with our hypothesis, BCR from three DLBCL classified as GCB lacked an autonomous BCR signal. The remaining two negative cases were non-GCB-type and are currently being analyzed for activating CD79/CARD11 mutations.
To further explore the detection of autonomous BCR signaling in DLBCL, PM-DLBCL and ABC DLBCL cell lines were tested in vitro by comparing calcium flux with and without blockade of Syk signaling by the tyrosine kinase inhibitor R406. All four PM-DLBCL and two of five ABC DLBCL cell lines had higher calcium flux in the absence of R406, indicating autonomous signaling activity without antigenic stimulation or artificial BCR crosslinking.
In summary, we demonstrate autonomous BCR activity in a substantial fraction (72%) of non-GCB DLBCL (including PM-DLBCL). These results point to an oncogenic role of structurally normal BCR in non-GCB DLBCL akin to CLL. Autonomous BCR signaling is a candidate alternative mechanism to the previously described activating mutations of components of the BCR signaling cascade to induce the characteristic gene expression signature of ABC DLBCL. Formal delineation of autonomous BCR signalling to activating mutations in the BCR signalling cascade is ongoing.
Citation Format: Marvyn T. Koning, Rudolf Übelhart, Arjen H.G. Cleven, Willem H. Zoutman, Sander A.J. van der Zeeuw, Philip Kluin, Marieke Griffioen, Hans G. Drexler, Cornelis P. Tensen, Maarten H. Vermeer, Rein Willemze, Szymon M. Kielbasa, Marcelo A. Navarrete, Hassan Jumaa, Hendrik Veelken. Autonomous, antigen-independent B-cell receptor signalling as a novel pathogenetic mechanism in non-GCB DLBCL. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-012.