Dissecting the functional pathways of B cell receptor immunoglobulin in antigen driven mature B cell lymphomas

Periodic Reporting for period 1 - IGpath (Dissecting the functional pathways of B cell receptor immunoglobulin in antigen driven mature B cell lymphomas)

Berichtszeitraum: 2018-05-01 bis 2020-04-30

Chronic lymphocytic leukemia (CLL) is a mature B cell lymphoma (B-NHL), the most common adult hematologic malignancy. Splenic marginal zone lymphoma (SMZL) is an indolent B-NHL characterized by massive splenomegaly and moderate lymphocytosis. Both CLL and SMZL affect the elderly population (median age at diagnosis >70 years). About two-thirds of CLL patients and one-half of SMZL patients will eventually require treatment at any stage of their disease. However, despite the therapeutic progress achieved in recent years, both entities remain currently incurable.
The cause and the exact pathogenesis mechanisms of the distinct types of B-NHLs remains unknown. However, accumulating evidence supports that the B cell receptor immunoglobulin (BcR IG) holds a pivotal role in B-NHL ontogeny mediating critical interactions with the microenvironment, including binding to antigen and B cell activation by initiating an intracellular signaling cascade. The clinical relevance of BcR IG-mediated signaling is highlighted by the efficiency of BcR signaling inhibitors in B-NHL management. Nevertheless, open issues abound regarding the precise role of the BcR IG in lymphomagenesis, particularly regarding the nature and the range of the antigenic interactions and the functional consequences on the (pre)malignant clones and the clinical outcome. To complicate things further, in CLL a novel type of BcR signaling has been reported, namely BcR autonomous signaling, that is independent of external antigen triggering and lays on the ability of CLL BcR IG to self-associate.
The overall objective of IGpath was to dissect the BcR IG-related processes implicated in the pathogenesis of CLL and SMZL. Furthermore, to refine our understanding of the early stages of lymphomagenesis we investigated these mechanisms also in BcR IG from individuals with monoclonal B lymphocytosis (MBL), a pre-malignant condition that is considered as a potential precursor state to CLL. The refinement of the IG BcR-related mechanisms of pathogenesis may have implications for the clinical management since it can assist in the ‘functional’ classification of patients (or people at high-risk of developing overt disease) based on the type of antigenic stimulation, a step towards personalized medicine.

A multidisciplinary three-pillar approach was employed.
The first referred to the extensive immunogenetic analysis using high throughput sequencing techniques (next generation sequencing, NGS) that unveiled the imprints of the interaction of BcR IGs with selecting antigens. The second concerned the investigation of the actual identity of the antigenic elements involved in the selection and activation of (pre)malignant B cell clones, by expressing the BcR IG as soluble molecules through recombinant DNA technology and performing binding assays against a broad range of antigenic targets, including self and foreign antigens. Collectively, these studies offered strong evidence for functional pressure by specific antigenic elements. In addition, our novel findings emphasized the importance of BcR IG isotype in modulating fine antigen specificity through the generation of a large library of soluble CLL IGs of the ‘authentic’ isotype (same as that expressed by the leukemic clone): by using these molecules in antigen binding assays we confirmed and further extended the existence of a tight link between self- and poly-reactive patterns of BcR IG and aggressive disease in CLL, in support of a scenario where abundancy of antigenic stimuli provide a selection advantage to B cell clones. Last but emphatically not least, within IGpath we examined by NGS the BcR IG repertoire of a large number of MBL cases and, for the first time, the MBL BcR IG in terms of antigenic reactivity properties, hence, contributing into the comprehension of the BcR IG-antigen interaction processes leading to monoclonal cell expansions that could eventually drive neoplastic transformation. In addition, we completed the largest to-date functional analysis of SMZL BcR IG and showed intriguing features of BcR IG-antigen patterns of interaction in the disease.
In the third pillar we explored the role of BcR homotypic interactions in shaping the clonal behavior in a large cohort of CLL cases. Using appropriate biochemical technologies, we assessed the magnitude of BcR self-association and significantly extended our previous observations unveiling links between the qualities of the BcR self-association and the disease outcome.
The main results of IGpath have been presented at the 60th and 61st Annual Meetings of the American Society of Hematology, the prime scientific event in hematology. At the national level, IGpath results have been presented at the 30th Annual Congress of Hellenic Society of Hematology. In addition, the progress of the project has been systematically communicated through IGpath’s website.

The aspects of BcR IG-antigen interaction in the pathogenesis of B-NHL are multiple and complicated and open scientific questions concern the nature and the duration of the interactions as well as their timing in disease trajectory. To-date mostly one-dimensional approaches have been followed that fail to provide a broader perspective of BcR IG-related routes to B-NHL pathogenesis. IGpath progressed beyond the state of the art in that a multidisciplinary and holistic approach was employed to overcome the complexity of the scientific questions. In specific, BcR IGs derived both from malignant B cell clones and pre-malignant monoclonal cell populations were analyzed thoroughly (i) at the DNA level by employing high throughput immunogenetics and (ii) in protein level by analyzing their functional and structural properties, followed by (iii) integration of the findings with clinicobiological information.
To the best of our knowledge, this is the first systematic characterization of the BcR IG in MBL and SMZL. Additionally, IGpath offered the first comprehensive profiling of conventional (auto)antigenic binding and BcR self-association respectively, in CLL BcR IGs expressed with the ‘original’, leukemic isotypes, providing solid links between BcR IG poly-reactivity and low affinity BcR self-interaction with aggressive disease.
Overall, IGpath results assist in improved understanding of the properties of the BcR IG in B-NHL and offer novel insight into the role of the BcR IG derived mechanisms in modulating the natural history and clinical outcome of B-NHLs. Refined stratification of patients based on the qualities of BcR IG interaction (i.e. self-antigens, BcR IG self-association, microbes; mono- or poly- antigen reactive pattern of BcR IG) could assist in advent of targeted therapies with obvious benefits for the patients and the health system.

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Periodic Reporting for period 1 - IGpath (Dissecting the functional pathways of B cell receptor immunoglobulin in antigen driven mature B cell lymphomas)

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