High surface IgM levels associate with shorter response to ibrutinib and BTK bypass in patients with CLL.

Chronic lymphocytic leukemia (CLL) cells have variably low surface IgM (sIgM) levels/signaling capacity, influenced by chronic antigen engagement at tissue sites. Within these low levels, CLL with relatively high sIgM (CLLhigh) progresses more rapidly than CLL with low sIgM (CLLlow). During ibrutinib therapy, surviving CLL cells redistribute into the peripheral blood and can recover sIgM expression. Return of CLL cells to tissue may eventually recur, where cells with high sIgM could promote tumor growth. We analyzed time to new treatment (TTNT) following ibrutinib in 70 patients with CLL (median follow-up of 66 months) and correlated it with pretreatment sIgM levels and signaling characteristics. Pretreatment sIgM levels correlated with signaling capacity, as measured by intracellular Ca2+ mobilization (iCa2+), in vitro (r = 0.70; P < .0001). High sIgM levels/signaling strongly correlated with short TTNT (P < .05), and 36% of patients with CLLhigh vs 8% of patients with CLLlow progressed to require a new treatment. In vitro, capacity of ibrutinib to inhibit sIgM-mediated signaling inversely correlated with pretherapy sIgM levels (r = -0.68; P = .01) or iCa2+ (r = -0.71; P = .009). In patients, sIgM-mediated iCa2+ and ERK phosphorylation levels were reduced by ibrutinib therapy but not abolished. The residual signaling capacity downstream of BTK was associated with high expression of sIgM, whereas it was minimal when sIgM expression was low (P < .05). These results suggested that high sIgM levels facilitated CLL cell resistance to ibrutinib in patients. The CLL cells, surviving in the periphery with high sIgM expression, include a dangerous fraction that is able to migrate to tissue and receive proliferative stimuli, which may require targeting by combined approaches.

Biological and Clinical Insight from Analysis of the Tumor B-Cell Receptor Structure and Function in Chronic Lymphocytic Leukemia.

The B-cell receptor (BCR) is essential to the behavior of the majority of normal and neoplastic mature B cells. The identification in 1999 of the two major CLL subsets expressing unmutated immunoglobulin (Ig) variable region genes (U-IGHV, U-CLL) of pre-germinal center origin and poor prognosis, and mutated IGHV (M-CLL) of post-germinal center origin and good prognosis, ignited intensive investigations on structure and function of the tumor BCR. These investigations have provided fundamental insight into CLL biology and eventually the mechanistic rationale for the development of successful therapies targeting BCR signaling. U-CLL and M-CLL are characterized by variable low surface IgM (sIgM) expression and signaling capacity. Variability of sIgM can in part be explained by chronic engagement with (auto)antigen at tissue sites. However, other environmental elements, genetic changes, and epigenetic signatures also contribute to the sIgM variability. The variable levels have consequences on the behavior of CLL, which is in a state of anergy with an indolent clinical course when sIgM expression is low, or pushed towards proliferation and a more aggressive clinical course when sIgM expression is high. Efficacy of therapies that target BTK may also be affected by the variable sIgM levels and signaling and, in part, explain the development of resistance.

Insertion of atypical glycans into the tumor antigen-binding site identifies DLBCLs with distinct origin and behavior.

Glycosylation of the surface immunoglobulin (Ig) variable region is a remarkable follicular lymphoma-associated feature rarely seen in normal B cells. Here, we define a subset of diffuse large B-cell lymphomas (DLBCLs) that acquire N-glycosylation sites selectively in the Ig complementarity-determining regions (CDRs) of the antigen-binding sites. Mass spectrometry and X-ray crystallography demonstrate how the inserted glycans are stalled at oligomannose-type structures because they are buried in the CDR loops. Acquisition of sites occurs in ∼50% of germinal-center B-cell-like DLBCL (GCB-DLBCL), mainly of the genetic EZB subtype, irrespective of IGHV-D-J use. This markedly contrasts with the activated B-cell-like DLBCL Ig, which rarely has sites in the CDR and does not seem to acquire oligomannose-type structures. Acquisition of CDR-located acceptor sites associates with mutations of epigenetic regulators and BCL2 translocations, indicating an origin shared with follicular lymphoma. Within the EZB subtype, these sites are associated with more rapid disease progression and with significant gene set enrichment of the B-cell receptor, PI3K/AKT/MTORC1 pathway, glucose metabolism, and MYC signaling pathways, particularly in the fraction devoid of MYC translocations. The oligomannose-type glycans on the lymphoma cells interact with the candidate lectin dendritic cell-specific intercellular adhesion molecule 3 grabbing non-integrin (DC-SIGN), mediating low-level signals, and lectin-expressing cells form clusters with lymphoma cells. Both clustering and signaling are inhibited by antibodies specifically targeting the DC-SIGN carbohydrate recognition domain. Oligomannosylation of the tumor Ig is a posttranslational modification that readily identifies a distinct GCB-DLBCL category with more aggressive clinical behavior, and it could be a potential precise therapeutic target via antibody-mediated inhibition of the tumor Ig interaction with DC-SIGN-expressing M2-polarized macrophages.

DC-SIGN binding to mannosylated B-cell receptors in follicular lymphoma down-modulates receptor signaling capacity.

In follicular lymphoma (FL), surface immunoglobulin (sIg) carries mandatory N-glycosylation sites in the variable regions, inserted during somatic hypermutation. These glycosylation sites are tumor-specific, indicating a critical function in FL. Added glycan unexpectedly terminates at high mannose (Mann) and confers capability for sIg-mediated interaction with local macrophage-expressed DC-SIGN lectin resulting in low-level activation of upstream B-cell receptor signaling responses. Here we show that despite being of low-level, DC-SIGN induces a similar downstream transcriptional response to anti-IgM in primary FL cells, characterized by activation of pathways associated with B-cell survival, proliferation and cell-cell communication. Lectin binding was also able to engage post-transcriptional receptor cross-talk pathways since, like anti-IgM, DC-SIGN down-modulated cell surface expression of CXCR4. Importantly, pre-exposure of a FL-derived cell line expressing sIgM-Mann or primary FL cells to DC-SIGN, which does not block anti-IgM binding, reversibly paralyzed the subsequent Ca2+ response to anti-IgM. These novel findings indicate that modulation of sIg function occurs in FL via lectin binding to acquired mannoses. The B-cell receptor alternative engagement described here provides two advantages to lymphoma cells: (i) activation of signaling, which, albeit of low-level, is sufficient to trigger canonical lymphoma-promoting responses, and (ii) protection from exogenous antigen by paralyzing anti-IgM-induced signaling. Blockade of this alternative engagement could offer a new therapeutic strategy.

Changes in Bcl-2 members after ibrutinib or venetoclax uncover functional hierarchy in determining resistance to venetoclax in CLL.

Chronic lymphocytic leukemia (CLL) cells cycle between lymph node (LN) and peripheral blood (PB) and display major shifts in Bcl-2 family members between those compartments. Specifically, Bcl-XL and Mcl-1, which are not targeted by the Bcl-2 inhibitor venetoclax, are increased in the LN. Because ibrutinib forces CLL cells out of the LN, we hypothesized that ibrutinib may thereby affect expression of Bcl-XL and Mcl-1 and sensitize CLL cells to venetoclax. We investigated expression of Bcl-2 family members in patients under ibrutinib or venetoclax treatment, combined with dissecting functional interactions of Bcl-2 family members, in an in vitro model of venetoclax resistance. In the PB, recent LN emigrants had higher Bcl-XL and Mcl-1 expression than did cells immigrating back to the LN. Under ibrutinib treatment, this distinction collapsed; significantly, the pretreatment profile reappeared in patients who relapsed on ibrutinib. However, in response to venetoclax, Bcl-2 members displayed an early increase, underlining the different modes of action of these 2 drugs. Profiling by BH3 mimetics was performed in CLL cells fully resistant to venetoclax due to CD40-mediated induction of Bcl-XL, Mcl-1, and Bfl-1. Several dual or triple combinations of BH3 mimetics were highly synergistic in restoring killing of CLL cells. Lastly, we demonstrated that proapoptotic Bim interacts with antiapoptotic Bcl-2 members in a sequential manner: Bcl-2 > Bcl-XL > Mcl-1 > Bfl-1. Combined, the data indicate that Bcl-XL is more important in venetoclax resistance than is Mcl-1 and provide biological rationale for potential synergy between ibrutinib and venetoclax.

International prognostic score for asymptomatic early-stage chronic lymphocytic leukemia.

Most patients with chronic lymphocytic leukemia (CLL) are diagnosed with early-stage disease and managed with active surveillance. The individual course of patients with early-stage CLL is heterogeneous, and their probability of needing treatment is hardly anticipated at diagnosis. We aimed at developing an international prognostic score to predict time to first treatment (TTFT) in patients with CLL with early, asymptomatic disease (International Prognostic Score for Early-stage CLL [IPS-E]). Individual patient data from 11 international cohorts of patients with early-stage CLL (n = 4933) were analyzed to build and validate the prognostic score. Three covariates were consistently and independently correlated with TTFT: unmutated immunoglobulin heavy variable gene (IGHV), absolute lymphocyte count higher than 15 × 109/L, and presence of palpable lymph nodes. The IPS-E was the sum of the covariates (1 point each), and separated low-risk (score 0), intermediate-risk (score 1), and high-risk (score 2-3) patients showing a distinct TTFT. The score accuracy was validated in 9 cohorts staged by the Binet system and 1 cohort staged by the Rai system. The C-index was 0.74 in the training series and 0.70 in the aggregate of validation series. By meta-analysis of the training and validation cohorts, the 5-year cumulative risk for treatment start was 8.4%, 28.4%, and 61.2% among low-risk, intermediate-risk, and high-risk patients, respectively. The IPS-E is a simple and robust prognostic model that predicts the likelihood of treatment requirement in patients with early-stage CLL. The IPS-E can be useful in clinical management and in the design of early intervention clinical trials.

Genome-wide promoter methylation of hairy cell leukemia.

Classic hairy cell leukemia (HCL) is a tumor of mature clonal B cells with unique genetic, morphologic, and phenotypic features. DNA methylation profiling has provided a new tier of investigation to gain insight into the origin and behavior of B-cell malignancies; however, the methylation profile of HCL has not been specifically investigated. DNA methylation profiling was analyzed with the Infinium HumanMethylation27 array in 41 mature B-cell tumors, including 11 HCL, 7 splenic marginal zone lymphomas (SMZLs), and chronic lymphocytic leukemia with an unmutated (n = 7) or mutated (n = 6) immunoglobulin gene heavy chain variable (IGHV) region or using IGHV3-21 (n = 10). Methylation profiles of nontumor B-cell subsets and gene expression profiling data were obtained from public databases. HCL had a methylation signature distinct from each B-cell tumor entity, including the closest entity, SMZL. Comparison with normal B-cell subsets revealed the strongest similarity with postgerminal center (GC) B cells and a clear separation from pre-GC and GC cellular programs. Comparison of the integrated analysis with post-GC B cells revealed significant hypomethylation and overexpression of BCR-TLR-NF-κB and BRAF-MAPK signaling pathways and cell adhesion, as well as hypermethylation and underexpression of cell-differentiation markers and methylated genes in cancer, suggesting regulation of the transformed hairy cells through specific components of the B-cell receptor and the BRAF signaling pathways. Our data identify a specific methylation profile of HCL, which may help to distinguish it from other mature B-cell tumors.

Ibrutinib Therapy Releases Leukemic Surface IgM from Antigen Drive in Chronic Lymphocytic Leukemia Patients.

PURPOSE: In chronic lymphocytic leukemia (CLL), disease progression associates with surface IgM (sIgM) levels and signaling capacity. These are variably downmodulated in vivo and recover in vitro, suggesting a reversible influence of tissue-located antigen. Therapeutic targeting of sIgM function via ibrutinib, an inhibitor of Bruton tyrosine kinase (BTK), causes inhibition and tumor cell redistribution into the blood, with significant clinical benefit. Circulating CLL cells persist in an inhibited state, offering a tool to investigate the effects of drug on BTK-inhibited sIgM. EXPERIMENTAL DESIGN: We investigated the consequences of ibrutinib therapy on levels and function of sIgM in circulating leukemic cells of patients with CLL. RESULTS: At week 1, there was a significant increase of sIgM expression (64% increase from pretherapy) on CLL cells either recently released from tissue or persisting in blood. In contrast, surface IgD (sIgD) and a range of other receptors did not change. SIgM levels remained higher than pretherapy in the following 3 months despite gradual cell size reduction and ongoing autophagy and apoptotic activity. Conversely, IgD and other receptors did not increase and gradually declined. Recovered sIgM was fully N-glycosylated, another feature of escape from antigen, and expression did not increase further during culture in vitro. The sIgM was fully capable of mediating phosphorylation of SYK, which lies upstream of BTK in the B-cell receptor pathway. CONCLUSIONS: This specific IgM increase in patients underpins the key role of tissue-based engagement with antigen in CLL, confirms the inhibitory action of ibrutinib, and reveals dynamic adaptability of CLL cells to precision monotherapy.See related commentary by Burger, p. 2372.

Cortactin, a Lyn substrate, is a checkpoint molecule at the intersection of BCR and CXCR4 signalling pathway in chronic lymphocytic leukaemia cells.

Cortactin (CTTN) is a substrate of the Src kinase Lyn that is known to play an actin cytoskeletal regulatory role involved in cell migration and cancer progression following its phosphorylation at Y421. We recently demonstrated that Cortactin is overexpressed in patients with chronic lymphocytic leukaemia (CLL). This work was aimed at defining the functional role of Cortactin in these patients. We found that Cortactin is variably expressed in CLL patients both in the peripheral blood and lymph nodes and that its expression correlates with the release of matrix metalloproteinase 9 (MMP-9) and the motility of neoplastic cells. Cortactin knockdown, by siRNA, induced a reduction in MMP-9 release as well as a decrease of migration capability of leukaemic B cells in vitro, also after chemotactic stimulus. Furthermore, Cortactin phosphorylation was lowered by the Src kinase-inhibitor PP2 with a consequent decrease of MMP-9 release in culture medium. An impaired migration, as compared to control experiments without Cortactin knockdown, was observed following CXCL12 triggering. Reduced Cortactin expression and phosphorylation were also detected both in vivo and in vitro after treatment with Ibrutinib, a Btk inhibitor. Our results highlight the role of Cortactin in CLL as a check-point molecule between the BCR and CXCR4 signalling pathways.

Cross-talk between chronic lymphocytic leukemia (CLL) tumor B cells and mesenchymal stromal cells (MSCs): implications for neoplastic cell survival.

Leukemic cells from Chronic Lymphocytic Leukemia (CLL) patients interact with stromal cells of the surrounding microenvironment. Mesenchymal Stromal Cells (MSCs) represent the main population in CLL marrow stroma, which may play a key role for disease support and progression. In this study we evaluated whether MSCs influence in vitro CLL cell survival. MSCs were isolated from the bone marrow of 46 CLL patients and were characterized by flow cytometry analysis. Following co-culture of MSCs and leukemic B cells, we demonstrated that MSCs were able to improve leukemic B cell viability, this latter being differently dependent from the signals coming from MSCs. In addition, we found that the co-culture of MSCs with leukemic B cells induced an increased production of IL-8, CCL4, CCL11, and CXCL10 chemokines.As far as drug resistance is concerned, MSCs counteract the cytotoxic effect of Fludarabine/Cyclophosphamide administration in vivo, whereas they do not protect CLL cells from the apoptosis induced by the kinase inhibitors Bafetinib and Ibrutinib. The evidence that leukemic clones are conditioned by environmental stimuli suggest new putative targets for therapy in CLL patients.

Integrated CLL Scoring System, a New and Simple Index to Predict Time to Treatment and Overall Survival in Patients With Chronic Lymphocytic Leukemia.

INTRODUCTION: Several prognostic factors have been identified to predict the outcome of patients with chronic lymphocytic leukemia (CLL), but only a few studies analyzed more markers together. PATIENTS AND METHODS: Taking advantage of a population of 608 patients, we identified the strongest prognostic markers of survival and, subsequently, in a cohort of 212 patients we integrated data of cytogenetic lesions, IGHV mutational status, and CD38 expression in a new and easy scoring system we called the integrated CLL scoring system (ICSS). ICSS defines 3 groups of risk: (1) low risk (patients with 13q(-) or normal fluorescence in-situ hybridization analysis results, mutated IGHV, and CD38) (2) high risk (all 11q(-) or 17p(-) patients and/or all unmutated IGHV and CD38(+) patients); and (3) intermediate risk (all remaining patients). RESULTS: Using only these 3 already available prognostic factors, we were able to properly redefine patients and better predict the clinical course of the disease. CONCLUSION: ICSS could become a useful tool for CLL patients' management.