High Grade B-Cell Lymphoma with BCL2 and MYC Rearrangement (Double-Hit High-Grade Lymphoma; DH-HGL): Establishment of a Human Model System for Ex Vivo and In Vivo Studies

High-grade lymphomas with BCL2- and MYC-rearrangements (double-hit high-grade lymphomas, DH-HGL) pose a significant clinical challenge due to their aggressive course, high risk of CNS infiltration, and poor outcomes with R-CHOP-based therapies compared to diffuse large B-cell lymphoma. However, the molecular basis of this peculiar biology remains poorly understood, which hampers the development of improved therapeutic strategies. Addressing this gap necessitates representative model systems.

Here, we report the establishment of a genetically defined human DH-HGL model system for functional ex vivo and in vivo studies. Primary germinal center B-cells were isolated from human tonsillectomy specimens and immortalized by stable overexpression of BCL2 and MYC, modeling the effects of BCL2- and MYC-rearrangements and enabling long-term growth in co-culture with immortalized follicular dendritic cells (YK6) expressing CD40L and IL-21. As many DH-HGL carry additional recurrent gene mutations also found in primary CNS lymphoma, including genetic inactivation of CDKN2A and MYD88-mutations, we used the BCL2 and MYC overexpressing cells to generate DH-HGL-like cells by CRISPR-/Cas-9 mediated CDKN2A knockout and expression of (GFP-labeled) MYD88 with or without the L265P hotspot mutation. To enable ex vivo and in vivo bioluminescence imaging (BLI), we expressed (mCherry-labeled) enhanced firefly luciferase.

Western blot and ELISA revealed enhanced NF-κB activation in DH-HGL-like cells carrying MYD88^L265P compared to MYD88^wt. Additionally, MYD88^L265P cells showed sustained growth in co-culture with CD40L-negative YK6, unlike MYD88^wt cells, suggesting that the L265P mutation compensates for CD40L-mediated NF-κB signaling.

To confirm this growth advantage in vivo, NOD/SCID/gamma (NSG) mice were injected subcutaneously with DH-HGL-like cells carrying MYD88^wt into the right flank and DH-HGL-like cells carrying MYD88^L265P into the left flank (N=9, i.e., 3 donors and 3 replicates each). While MYD88^wt cells induced tumors in 4 out of 9 mice (44%), MYD88^L265P led to tumor development in all 9 mice (100%). Furthermore, MYD88^L265P-tumors were significantly larger, necessitating the sacrifice of the mice in every case (1.24 ± 0.24 cm vs. 0.80 ± 0.14 cm, P=0.006). Histopathological and immunohistochemical examination confirmed that all tumors resembled the morphological and immune phenotypical features of human DH-HGL, including high-grade GC B morphology, immunoblastic subtype with high expression of BCL2 and MYC and Ki67>90%.

Due to the robust engraftment of the MYD88^L265P DH-HGL-like cells upon subcutaneous injection, these cells were also injected intravenously/intrafemorally (N=2 each) for subsequent analysis of clinical course and CNS tropism. The cells engrafted in all 4 NSG mice (100%) as confirmed by BLI. Unexpectedly, the DH-HGL-like cells did not expand rapidly but displayed a persistent BLI signal predominantly in the bone marrow. Two mice developed intriguing neurological abnormalities at weeks 19 (tilted head positioning) and 29 (hind leg paralysis), respectively; however, they still had low BLI signals and were sacrificed without re-isolating human cells. At week 29, one of the two remaining mice lost the BLI signal, while the BLI signal of the other increased constantly in the bones. Both mice were sacrificed at week 39 without neurological abnormalities. Analyses of the brains of these mice are ongoing. DH-HGL-like cells were re-isolated from the spine of the mouse with increasing BLI signal. Serial re-transplantation (re-Tx) of these cells showed a high re-engraftment rate (1^st re-Tx: 2/2, 2^nd re-Tx 2/3) and highly aggressive growth behavior with rapidly increasing BLI signal and short survival of the mice (6-8 weeks, N=4). To investigate the evolution leading to this aggressive growth, DH-HGL-like cells were isolated from the 2^nd re-Tx and re-cultivated on YK6 for comparative analyses with their respective pre-injection samples, including whole exome and transcriptome sequencing. Our initial results indicate changes in cytokine and receptor signaling, including markedly increased CCL22 expression.

In summary, we introduce a novel human DH-HGL model which seems to exhibit the typical histopathological and clinical features of this challenging disease. This genetically well-defined model is suitable for ex vivo and in vivo studies.

Vick:Tubulis GmbH: Patents & Royalties: pending patent application FLT3-mAb 20D9. Strobl:iOmx Therapeutics AG: Current Employment. Hodson:Astra Zeneca, GSK: Research Funding. Jeremias:Tubulis GmbH: Patents & Royalties: pending patent application FLT3-mAb 20D9.