Ansell, Stephen M.

Abstract Purpose: Waldenström's macroglobulinemia is an incurable lymphoproliferative disorder driven by an L265P mutation in the myeloid differentiation primary response gene 88 (MYD88), which activates downstream NF-κB signaling through the Myddosome. As this pathway depends in part on activity of interleukin-1 receptor-associated kinases (IRAKs)-1 and -4, we sought to evaluate the potential of the IRAK1/4 inhibitor R191 in preclinical models. Experimental Design: Patient-derived cell lines and primary samples were used in both in vitro and in vivo experiments to model Waldenström's macroglobulinemia and its response to IRAK1/4 inhibitors. Results: R191 induced a dose- and time-dependent reduction in viability of BCWM.1 and MWCL-1 Waldenström's cell lines, and suppressed activation of IRAK1/4. This was associated with cell-cycle arrest at G0–G1, reduced levels of cyclin-dependent kinases 4 and 6, and induction of apoptosis in cell lines and primary patient samples. Further downstream, R191 exposure led to reduced activation of NF-κB, and of protein kinase B/Akt/mammalian target of rapamycin signaling, whereas expression of a constitutively active Akt mutant induced R191 resistance. Gene expression profiling and gene set enrichment analysis revealed a signature consistent with inhibition of c-Myc and activation of the endoplasmic reticulum stress response. In both subcutaneous and systemic murine models of Waldenström's, R191 showed antitumor activity. Finally, the activity of R191 was enhanced when it was combined with novel chemotherapeutics such as bortezomib, afuresertib, and ibrutinib. Conclusions: Taken together, these data support the translation of R191 as an approach to target IRAK1/4 to the clinic for patients with Waldenström's macroglobulinemia.

AbstractSymptomatic hyperviscosity is a well‐established phenomenon in Waldenström macroglobulinemia (WM). Monoclonal IgM can variably impact intrinsic serum viscosity, leading to widely disparate symptomatic thresholds for development of hyperviscosity‐related symptoms. Data regarding the predictors of symptomatic hyperviscosity and outcomes related to this complication remain scarce and a recent study proposed that IgM >6000 mg/dL be considered a new criterion for initiating therapy in otherwise asymptomatic (smoldering) WM to pre‐empt hyperviscosity‐related injury. Herein, we attempt to identify predictors of the development of symptomatic hyperviscosity and its impact in patients with WM. Of 997 WM patients evaluated from January, 1996 through June, 2017, symptomatic hyperviscosity was observed in 130 (13%) patients. Overall survival (OS) of these 130 patients was similar to that of patients without symptomatic hyperviscosity (median: 11.5 vs 11.6 years; P = 0.63). On multivariate‐analysis, only viscosity >1.8 cp (risk ratio: 4.0, P = 0.02) assessed at the time of WM diagnosis was an independent predictor for the development of subsequent symptomatic hyperviscosity. Among patients with smoldering WM and IgM >6000 mg/dL at diagnosis (n = 13) who were managed expectantly, the median time‐to‐initial therapy was 6.9 years and only 15% developed hyperviscosity‐related symptoms subsequently. In summary, the occurrence of symptomatic hyperviscosity does not impact OS. Serum viscosity at diagnosis of WM, and not IgM concentration, represents the single most important independent predictor for development of subsequent hyperviscosity‐related symptoms. Patients with smoldering WM and high serum IgM can be safely observed in the absence of any indications per the Consensus recommendations to initiate WM‐directed therapy.

SummaryCD38 is expressed on Waldenström macroglobulinaemia (WM) cells, but its role as a therapeutic target remains undefined. With recent approval of the anti‐CD38 monoclonal antibody, daratumumab (Dara), we hypothesized that blocking CD38 would be lethal to WM cells.In vitroDara treatment of WM cells (including ibrutinib‐resistant lines) elicited antibody‐dependent cellular cytotoxicity (ADCC), complement‐dependent cytotoxicity (CDC), antibody‐dependent cell phagocytosis (ADCP) and direct apoptosis.In vivo, Dara treatment was well tolerated and delayed tumour growth in RPCI‐WM1‐xenografted mice. CD38 is reported to augment B‐cell receptor (BCR) signalling; we noted that Dara significantly attenuated phosphorylated SYK, LYN, BTK, PLCγ2, ERK1/2, AKT, mTOR, and S6 levels, and this effect was augmented by cotreatment with ibrutinib. Indeed, WM cells, including ibrutinib‐resistant WM cell lines treated with the ibrutinib + Dara combination, showed significantly more cell death through ADCC, CDC, ADCP and apoptosis relative to single‐agent Dara or ibrutinib. In summary, we are the first to report thein vitroandin vivoanti‐WM activity of Dara. Furthermore, we show a close connection between BCR and CD38 signalling, which can be co‐targeted with ibrutinib + Dara to induce marked WM cell death, irrespective of acquired resistance to ibrutinib.

SummaryWe evaluated the association of Human Pegivirus (HPgV) viraemia with risk of developing lymphoma, overall and by major subtypes. Because this virus has also been associated with better prognosis in the setting of co‐infection with human immunodeficiency virus, we further assessed the association of HPgV with prognosis. We used risk factor data and banked plasma samples from 2094 lymphoma cases newly diagnosed between 2002 and 2009 and 1572 frequency‐matched controls. Plasma samples were tested for HPgV RNA by reverse transcription polymerase chain reaction (RT‐PCR), and those with RNA concentrations <5000 genome equivalents/ml were confirmed using nested RT‐PCR methods. To assess the role of HPgV in lymphoma prognosis, we used 2948 cases from a cohort study of newly diagnosed lymphoma patients (included all cases from the case‐control study). There was a positive association of HPgV viraemia with risk of lymphoma overall (Odds ratio = 2·14; 95% confidence interval [CI] 1·63–2·80; P < 0·0001), and for all major subtypes except Hodgkin lymphoma and chronic lymphocytic leukaemia/small lymphocytic lymphoma, and this was not confounded by other lymphoma risk factors. In contrast, there was no association of HPgV viraemia with event‐free survival (Hazard ratio [HR] = 1·00; 95% CI 0·85–1·18) or overall survival (HR = 0·97; 95% CI 0·79–1·20) for lymphoma overall, or any of the subtypes. These data support the hypothesis for a role of HPgV in the aetiology of multiple lymphoma subtypes.

Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is a low-grade B-cell neoplasm and ∼2% to 9% patients develop an aggressive lymphoma, most commonly diffuse large B-cell lymphoma (Richter transformation, DLBCL-RT). Programmed death-1 (PD-1) pathway plays a crucial role in tumor host immunity evasion and its blockade has emerged as an effective anti-cancer immunotherapy. PD-L1 and PD-1 expression has shown predictive value in anti-PD cancer immunotherapy; however, it has not been well documented in CLL/SLL and DLBCL-RT. We evaluated PD-1 and PD-L1 expression by immunohistochemistry in 39 CLL/SLL, 15 DLBCL-RT, and 26 other DLBCL. In CLL/SLL, neoplastic B-cell PD-1 expression was weak and restricted to prolymphocytes/paraimmunoblasts within proliferation centers (PCs) and accentuated PCs of all sizes. Neoplastic B-cell PD-1 expression was highly prevalent and demonstrated increased intensity in DLBCL-RT, but in contrast was only rarely seen in other DLBCL (12/15 vs. 1/26; P<0.0001). An excellent correlation (90% concordance) was observed between neoplastic B-cell PD-1 immunohistochemistry positivity and molecularly defined CLL/SLL clonal relatedness in DLBCL-RT. PD-L1 expression was observed on the neoplastic B cells in rare DLBCL-RT and other DLBCL cases (1/15 vs. 1/26; P>0.05) as well as background histiocytes and dendritic cells. Overall survival of DLBCL-RT was significantly inferior to that of the other DLBCL (median, 16.9 vs. 106.1 mo; P=0.002). Our findings suggest a biological continuum from prolymphocytes/paraimmunoblasts in CLL/SLL PCs to the neoplastic B-cells in DLBCL-RT. The characteristic PD-1 expression in DLBCL-RT makes it a potential surrogate marker for determining clonal relatedness to CLL/SLL, which may have important prognostic and therapeutic implications.