Diffuse Large B-Cell Lymphoma Patient-Derived Xenograft Models Capture Molecular and Biologic Heterogeneity and Inform Therapy

Abstract Diffuse large B-cell lymphoma (DLBCL) is a clinically and biologically heterogeneous disease defined by different transcriptional classifications, associated signaling and survival pathways and additional recurrent genetic alterations. In the cell-of-origin (COO) scheme, DLBCLs subtypes share certain features with normal germinal center B-cells (GCB) and activated B-cells (ABC). In comparison to GCB DLBCLs, ABC tumors have increased baseline NFκB activity and more frequent genetic alterations of NFκB pathway components. DLBCLs with shared functional features are also defined by the consensus clustering classification (CCC) which delineates B-cell receptor (BCR), Oxidative Phosphorylation (OxPhos) and Host Response (HR) tumors. BCR DLBCLs have increased reliance on BCR-signaling and survival pathways and aerobic glycolysis. BCR-dependent DLBCLs with high or low baseline NFκB activity (which largely correspond to ABC or GCB tumors, respectively) have distinct SYK-PI3K-dependent survival pathways and shared sensitivity to proximal BCR pathway inhibitors. Although DLBCLs have infrequent inactivating somatic mutations of TP53, these tumors commonly have copy number alterations (CNAs) of TP53 and genes encoding cell cycle pathway components. Given the clinical and molecular heterogeneity of DLBCL, we sought to develop faithful subtype-specific model systems to assess targeted therapies. Fresh tumor biopsies from 27 primary LBCLs were implanted under the renal capsule of immune compromised NSG mice. Nine of 27 tumors were successfully expanded in vivo, serially propagated for > 5 generations and considered stable LBCL PDX models. All models were EBV- and had clonal IgH rearrangements. Morphological and immunohistochemical signatures defined 8 PDX models as DLBCL and 1 as EBV- plasmablastic lymphoma (PBL). All LBCL PDX models were subjected to RNA-Seq and classified with respect to COO and CCC subtypes. Models were also evaluated by whole exome sequencing with a modified bait set which captured coding mutations and selected chromosomal rearrangements. Six of 9 DLBCL PDX models were ABC type. These models exhibited mutations of MYD88 alone or in association with PIM1 or CD79B with other alterations, as reported in primary ABC DLBCLs. The remaining 2 DLBCL PDX models were GCB type, with characteristic alterations of GNA13 and EZH2, and chromosomal translocations involving IgH and either BCL2 or MYC. Of note, BCL2 and MYC translocations are known adverse prognostic features of primary GCB DLBCL. Certain PDX models had additional mutations including B2M, MLL2, TNFAIP3, MEF2B and TP53. Only 25% (2/8) of the DLBCL PDX models harbored inactivating TP53 mutations whereas 75% (6/8) of tumors exhibited CNAs of TP53 or its upstream modifier, CDKN2A. These data are consistent with the reported incidence and type of TP53 pathway alterations in primary DLBCLs and contrast sharply with the near-uniform presence of TP53 mutations in DLBCL cell lines. Using the CCC classification, 6/8 DLBCL PDX models (both GCBs and 4 of 6 ABCs) were defined as BCR-subtype and 2 models as non-BCR type. To assess the utility of the DLBCL PDX models for functional analysis of BCR signaling, we first assessed cell surface immunoglobulin (sIg) expression by flow cytometry. All 6 BCR-type DLBCLs expressed sIgM whereas the 2 non-BCR DLBCL models and the PBL model lacked sIg. Next, we treated viable tumor cell suspensions with a selective SYK inhibitor, entospletinib (GS-9973). SYK inhibition significantly decreased the proliferation of all 6 BCR-type DLBCLs, but had no effect on the non-BCR-type DLBCLs or the PBL PDX. Given the distinctive SYK/PI3K-dependent signaling and survival pathways in DLBCLs with low or high baseline NFκB, we also assessed selective apoptotic pathway readouts in entospletinib-treated PDX cell suspensions. SYK inhibition selectively upregulated the pro-apoptotic BH3 family member, HRK, in BCR-dependent GCB-type DLBCL PDX samples and significantly downregulated the anti-apoptotic BCL2 family member, BCL2A1, in BCR- dependent ABC-type DLBCL PDX tumors, effects consistent with those previously observed in primary DLBCL samples. In summary, we have established and molecularly characterized faithful PDX models of DLBCL and PBL and demonstrated their usefulness in evaluating novel BCR pathway inhibitors. Disclosures Rodig: Perkin Elmer: Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding. Shipp:Gilead: Consultancy; Sanofi: Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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