Inhibition of B Cell Receptor Signaling by Ibrutinib in Primary CNS Lymphoma

Abstract

Primary CNS lymphoma (PCNSL) harbors mutations that reinforce B cell receptor (BCR) signaling. Ibrutinib, a Bruton's tyrosine kinase (BTK) inhibitor, targets BCR signaling and is particularly active in lymphomas with mutations altering the BCR subunit CD79B and MYD88. We performed a proof-of-concept phase Ib study of ibrutinib monotherapy followed by ibrutinib plus chemotherapy (DA-TEDDi-R). In 18 PCNSL patients, 94% showed tumor reductions with ibrutinib alone, including patients having PCNSL with CD79B and/or MYD88 mutations, and 86% of evaluable patients achieved complete remission with DA-TEDDi-R. Increased aspergillosis was observed with ibrutinib monotherapy and DA-TEDDi-R. Aspergillosis was linked to BTK-dependent fungal immunity in a murine model. PCNSL is highly dependent on BCR signaling, and ibrutinib appears to enhance the efficacy of chemotherapy.

Keywords: ABC DLBCL; Aspergillus fumigatus; B cell receptor signaling; BTK; ibrutinib; macrophage; primary CNS lymphoma.

Figure 1. Ibrutinib-chemotherapy cytotoxicity models and DA-TEDDi-R Schema

(A) 10 x10 dose titration experiments were performed by combining a range of ibrutinib concentrations with a range of concentrations of the indicated agents in the TMD8 and OCI-Ly10 ABC DLBCL lines. The DBSumPos metric was used to summarize antagonism across the entire 10 × 10 block whereas the DBSumNeg metric was used to summarize drug synergy. For each selected ibrutinib combination pair, the average, plus or minus the SEM, is shown (2 biological replicates). Representative 10 × 10 dose-titration blocks are shown at the right with dose concentration combinations showing antagonism (blue) or synergy (red) highlighted. (B) DA-TEDDi-R treatment schema. Patients received ibrutinib alone in a 14-day ‘window’ (day -14 to -1) prior to cycle 1 of DA-TEDDi-R (temozolomide, etoposide, liposomal doxorubicin, dexamethasone, ibrutinib and rituximab) with intraventricular cytarabine. Abbreviations: IV- intravenous; PO-by mouth; IT-intrathecal; ICV-intracerebro-ventricular; ANC-absolute neutrophil count.

Figure 2. Pharmacokinetics of ibrutinib and metabolite (PCI-45227) and liposomal doxorubicin

A. Pharmacokinetics of ibrutinib and it metabolite PCI-45227 at the ibrutinib 840 mg dose level. Data represents the mean and standard deviation of patients 11–18 (8 patients). Ventricular CSF via ommaya reservoir and plasma were drawn during the ibrutinib window prior to day -14; post-dose on day -14 at 1, 2, 4, 6, 8, between 10 to 18, and at 24 hours and assayed for ibrutinib and PCI-45227 as indicated. B. Pharmacokinetics of liposomal doxorubicin in 4 patients. Data represent mean and standard deviation of total doxorubicin concentration (liposome bound + protein bound + free) in plasma and CSF. The mean Cmax in plasma and CSF were 41.4 (range: 23-2–61.5) and 0.61 (range: 0.15–2.83) ng/mL, respectively.

Figure 3. Clinical outcome of ibrutinib and DA-TEDDi-R treatment

A. Waterfall plot of maximum change from baseline of the product of the lesions for subjects with evaluable tumors on ibrutinib alone administered during the ‘window’ from day -14 to day -1 before initiation of DA-TEDDi-R (n=18). Two patients (*) with CSF and intraocular disease were assessed using quantitative estimates of tumor reductions. B. Representative brain MRI image before and after ibrutinib monotherapy in PCNSL patient 1. C. Best overall response rate achieved during DA-TEDDi-R therapy (n=16) and proportion of CR/CRu responses based on the International PCNSL workshop response criteria. The proportion of patients with negative research FDG-PET is shown. D. Swim lane plot of treatment duration and response for all patients on study (n=18). Prior treatment history is indicated by untreated, relapsed or refractory. Relapsed patients recurred following a complete response to prior treatment and refractory patients did not respond to prior chemotherapy. Abbreviations: CR-complete response; CRu-complete response unconfirmed; PR-partial response; PD-progressive disease; SD-stable disease; NE-not evaluable; FDG-PET-fluorodeoxy-glucose-positron emission tomography.

Figure 4. CD79B and MYD88 mutations

A. Meta-analysis of the prevelance of CD79B ITAM and MYD88 L265P mutations in 6 published PCNSL studies, compared with systemic ABC DLBCL. B. CD79B and MYD88 sequence analysis of 4 patients on study based on accessions NP_000617 and NP_002459, respectively. The CD79B splice acceptor mutation in patient 5 involves exon 5, which encodes the Y196 residue of the ITAM motif and thus would be predicted to disrupt ITAM function.

Figure 5. Association of Aspergillus with ibrutinib

A. Aspergillus event time and dexamethasone exposure. Seven patients developed aspergillosis of which 2 occurred on ibrutinib alone and 5 occurred during DA-TEDDi-R treatment. Five patients were receiving pre-treatment dexamethasone for control of brain edema. The patient numbers correspond to the swim lane numbers in Figure 3D. B. Brain MRI scans of Aspergillus infection in patient 9. MRI T1 post-contrast brain image pre-ibrutinib shows enhancing tumor and 13-days post-ibrutinib institution shows new Aspergillus abscesses and marked reduction in tumor enhancement. FDG-PET scans pre-ibrutinib shows metabolically active tumor and 13-days post-ibrutinib institution shows new metabolically inactive Aspergillus abscesses and marked reduction in metabolically active tumor. C. Chest CT and brain MRI scans of Aspergillus infection in patient 13. Thirteen days post-ibrutinib, chest CT shows new cavitary Aspergillus abscess that was not present pre-ibrutinb. MRI T1 post-contrast brain images show new ring enhancing Aspergillus abscesses that were not present pre-ibrutinib. D. Survival in murine model of Aspergillus infection in Btk^−/− and Btk^+/+ mice. At 14-days of observation after pharyngeal aspiration of Aspergillus fumigatus, 7/26 Btk^−/− mice exhibited mortality compared to 0/20 Btk^+/+ mice (p = 0.013; log-rank test) in 3 independent experiments. E. Weight loss in murine model of Aspergillus fumigatus at day 3 post-infection shown with standard error of the mean bars (n = 9–11) (p< 0.0001; unpaired t-test). F. Histology of representative lung sections from murine model of Aspergillus fumigatus infection at day 4 post-infection (n = 7 Btk^−/− and wild-type mice). Hematoxylin and eosin stain mages in upper panels (scale bars, 500 μm). Note more extensive peribronchial mononuclear cell and neutrophil inflammation with focal interstitial pneumonia and large airway mucous plugs in Btk^−/− compared to Btk^+/+ mice lung as pointed out by arrows. Grocott-Gomori’s methenamine silver stain (GMS) images in lower panels demonstrate more extensive Aspergillus fumigatus conidia in Btk^−/− compared to Btk^+/+ mice as pointed out by arrows. G. Model of BTK and macrophage innate immunity to Aspergillus. Pulmonary macrophages are the first line of defense against inhaled Aspergillus. Toll-like receptors (TLRs) initiate signaling and the production of inflammatory cytokines on exposure to fungal conidia spores and hyphae. TLR2 and TLR4 are membrane bound and initiate signaling through BTK, which promotes the nuclear translocation of NF-κB (Horwood et al., 2006). Phagocytosis of Aspergillus fumigatus conidia induces TLR9 recruitment to the phagosome and through BTK, leads to activation of phospholipase Cγ and calcineurin-mediated NFAT nuclear translocation (Herbst et al., 2015). NF-κB acts in synergy with NFAT to produce TNFα that results in neutrophil recruitment. Neutrophils are chemotactically attracted to affected areas by TNFα and chemokines, and destroy fungal hyphae through oxidative and non-oxidative killing (Lionakis and Kontoyiannis, 2003). BTK can also affect the myeloid compartment, where it has a role in the differentiation and function of neutrophils (Fiedler et al., 2011; Stadler et al., 2017). Additionally, glucocorticoids impair the phagocytic, oxidative and chemotactic function of both macrophages and neutrophils, and are a risk factor for invasive aspergillosis infection (Lionakis and Kontoyiannis, 2003).