Unexpected chronic lymphocytic leukemia B cell activation by bisphosphonates.

Chronic lymphocytic leukemia (CLL) is a disease of the elderly, often presenting comorbidities like osteoporosis and requiring, in a relevant proportion of cases, treatment with bisphosphonates (BPs). This class of drugs was shown in preclinical investigations to also possess anticancer properties. We started an in vitro study of the effects of BPs on CLL B cells activated by microenvironment-mimicking stimuli and observed that, depending on drug concentration, hormetic effects were induced on the leukemic cells. Higher doses induced cytotoxicity whereas at lower concentrations, more likely occurring in vivo, the drugs generated a protective effect from spontaneous and chemotherapy-induced apoptosis, and augmented CLL B cell activation/proliferation. This CLL-activation effect promoted by the BPs was associated with markers of poor CLL prognosis and required the presence of bystander stromal cells. Functional experiments suggested that this phenomenon involves the release of soluble factors and is increased by cellular contact between stroma and CLL B cells. Since CLL patients often present comorbidities such as osteoporosis and considering the diverse outcomes in both CLL disease progression and CLL response to treatment among patients, illustrating this phenomenon holds potential significance in driving additional investigations.

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.

LINC00152 expression in normal and Chronic Lymphocytic Leukemia B cells.

Long non-coding RNAs are emerging as essential regulators of gene expression, but their role in normal and neoplastic B cells is still largely uncharacterized. Here, we report on the expression pattern of the LINC00152 in normal B cells and Chronic Lymphocytic Leukemia B cell clones. Higher LINC00152 levels were consistently observed in memory B cell populations when compared to naïve B cells in the normal tissues analyzed [peripheral blood (PB), tonsils, and spleen]. In addition, independent stimulation via Immunoglobulins (IG), CD40, or Toll-like Receptor 9 (TLR9) upregulated LINC00152 in PB B cells. The expression of LINC00152 in a cohort of 107 early stage Binet A CLL patients was highly variable and did not correlate with known prognostic markers or clinical evolution. TLR9 stimulation, but not CD40 or IG challenge, was able to upregulate LINC00152 expression in CLL cells. In addition, LINC00152 silencing in CLL cell lines expressing LINC00152 failed to induce significant cell survival or apoptosis changes. These data suggest that, in normal B cells, the expression of LINC00152 is regulated by immunomodulatory signals, which are only partially effective in CLL cells. However, LINC00152 does not appear to contribute to CLL cell expansion and/or survival in a cohort of newly diagnosed CLL patients.

The spectrum of subclonal TP53 mutations in chronic lymphocytic leukemia: A next generation sequencing retrospective study.

Chronic lymphocytic leukemia (CLL) is a hematological disorder with complex clinical and biological behavior. TP53 mutational status and cytogenetic assessment of the deletion of the corresponding locus (17p13.1) are considered the most relevant biomarkers associated with pharmaco-predictive response, chemo-refractoriness, and worse prognosis in CLL patients. The implementation of Next Generation Sequencing (NGS) methodologies in the clinical laboratory allows for comprehensively analyzing the TP53 gene and detecting mutations with allele frequencies ≤10%, that is, "subclonal mutations". We retrospectively studied TP53 gene mutational status by NGS in 220 samples from 171 CLL patients. TP53 mutations were found in 60/220 (27.3%) samples and 47/171 (27.5%) patients. Interestingly, subclonal mutations could be detected in 31/60 samples (51.7%) corresponding to 25 patients (25/47, 53.2%). We identified 44 distinct subclonal TP53 mutations clustered in the central DNA-binding domain of p53 protein (exons 5-8, codons 133-286). Missense mutations were predominant (>80%), whereas indels, nonsense, and splice site variants were less represented. All subclonal TP53 variants but one [p.(Pro191fs)] were already described in NCI and/or Seshat databases as "damaging" and/or "probably damaging" mutations (38/44, 86% and 6/44, 14%, respectively). Longitudinal samples were available for 37 patients. Almost half of them displayed at least one TP53 mutant subclone, which could be alone (4/16, 25%) or concomitant with other TP53 mutant clonal ones (12/16, 75%); different patterns of mutational dynamics overtimes were documented. In conclusion, utilization of NGS in our "real-life" cohort of CLL patients demonstrated an elevated frequency of subclonal TP53 mutations. This finding indicates the need for precisely identifying these mutations during disease since the clones carrying them may become predominant and be responsible for therapy failures.

Old and New Facts and Speculations on the Role of the B Cell Receptor in the Origin of Chronic Lymphocytic Leukemia.

The engagement of the B cell receptor (BcR) on the surface of leukemic cells represents a key event in chronic lymphocytic leukemia (CLL) since it can lead to the maintenance and expansion of the neoplastic clone. This notion was initially suggested by observations of the CLL BcR repertoire and of correlations existing between certain BcR features and the clinical outcomes of single patients. Based on these observations, tyrosine kinase inhibitors (TKIs), which block BcR signaling, have been introduced in therapy with the aim of inhibiting CLL cell clonal expansion and of controlling the disease. Indeed, the impressive results obtained with these compounds provided further proof of the role of BcR in CLL. In this article, the key steps that led to the determination of the role of BcR are reviewed, including the features of the CLL cell repertoire and the fine mechanisms causing BcR engagement and cell signaling. Furthermore, we discuss the biological effects of the engagement, which can lead to cell survival/proliferation or apoptosis depending on certain intrinsic cell characteristics and on signals that the micro-environment can deliver to the leukemic cells. In addition, consideration is given to alternative mechanisms promoting cell proliferation in the absence of BcR signaling, which can explain in part the incomplete effectiveness of TKI therapies. The role of the BcR in determining clonal evolution and disease progression is also described. Finally, we discuss possible models to explain the selection of a special BcR set during leukemogenesis. The BcR may deliver activation signals to the cells, which lead to their uncontrolled growth, with the possible collaboration of other still-undefined events which are capable of deregulating the normal physiological response of B cells to BcR-delivered stimuli.

Chronic lymphocytic leukemia cells impair osteoblastogenesis and promote osteoclastogenesis: role of TNFα, IL-6 and IL-11 cytokines.

Bone skeletal alterations are no longer considered a rare event in chronic lymphocytic leukemia (CLL), especially at more advanced stages of the disease. This study is aimed at elucidating the mechanisms underlying this phenomenon. Bone marrow stromal cells, induced to differentiate toward osteoblasts in osteogenic medium, appeared unable to complete their maturation upon co-culture with CLL cells, CLL-cell-derived conditioned media (CLL-cm) or CLL-sera (CLL-sr). Inhibition of osteoblast differentiation was documented by decreased levels of RUNX2 and osteocalcin mRNA expression, by increased osteopontin and DKK-1 mRNA levels, and by a marked reduction of mineralized matrix deposition. The addition of neutralizing TNFα, IL-11 or anti-IL-6R monoclonal antibodies to these cocultures resulted in restoration of bone mineralization, indicating the involvement of these cytokines. These findings were further supported by silencing TNFα, IL-11 and IL-6 in leukemic cells. We also demonstrated that the addition of CLL-cm to monocytes, previously stimulated with MCSF and RANKL, significantly amplified the formation of large, mature osteoclasts as well as their bone resorption activity. Moreover, enhanced osteoclastogenesis, induced by CLL-cm, was significantly reduced by treating cultures with the anti-TNFα monoclonal antibody infliximab. An analogous effect was observed with the use of the BTK inhibitor, ibrutinib. Interestingly, CLL cells co-cultured with mature osteoclasts were protected from apoptosis and upregulated Ki-67. These experimental results parallel the direct correlation between amounts of TNFα in CLL-sr and the degree of compact bone erosion that we previously described, further strengthening the indication of a reciprocal influence between leukemic cell expansion and bone structure derangement.

Validation of the Alternative International Prognostic Score-E (AIPS-E): Analysis of Binet stage A chronic lymphocytic leukemia patients enrolled into the O-CLL1-GISL protocol.

OBJECTIVES: To validate the predictive value on time to first treatment (TTFT) of AIPS-E and IPS-E evaluated in an independent cohort of newly diagnosed and non-referred Binet stage A CLL patients enrolled in the O-CLL1-GISL protocol (clinicaltrial.gov identifier: NCT00917540). METHODS: A cohort of 292 newly diagnosed Binet A CLL cases has been enrolled in the study. Patients from several Italian Institutions were prospectively enrolled within 12 months of diagnosis into the O-CLL1-GISL protocol. RESULTS: The majority of patients were male (62%); median age was 60.4 years, 102 cases (34.9%) showed unmutated IGHV genes, 8 cases (2.8) the presence of del(11q)/del(17p), 142 cases (48.6%) the presence of palpable lymph nodes and 146 cases (50%) and ALC > 15 × 109 /l. After a median follow-up of 7.2 years, 130 patients underwent treatment. According to the AIPS-E, 96 patients were classified as low-risk, 128 as intermediate-risk, and 68 as high-risk. These groups showed significant differences in terms of TTFT. The C-statistic was 0.71 (P < .0001) for predicting TTFT. According to IPS-E, 77 patients were classified as low-risk, 135 as intermediate-risk, and 80 as high-risk. These groups showed significant differences in terms of TTFT. The C-statistic was 0.705 (P < .0001) for predicting TTFT. CONCLUSIONS: Our data confirm an accurate prognostic utility of both AIPS-E and IPS-E at the individual patient level. These data may be useful for a precise stratification of early-stage patients.

Comparison of ibrutinib and idelalisib plus rituximab in real-life relapsed/resistant chronic lymphocytic leukemia cases.

OBJECTIVES: To compare the capacity of ibrutinib (IB) and idelalisib-rituximab (IDELA-R) of prolonging overall survival (OS) as in CLL patients, previously treated with chemotherapy only. METHODS: A real-life cohort of 675 cases has been identified and investigated in the database of the groups participating in the study. RESULTS: At an unadjusted univariate analysis, a significant death risk reduction was observed favoring IB (IDELA-R vs IB HR = 0.5, 95% CI = 0.36-0.71) although with some limitations due to the non-randomized and retrospective nature of the study and to the lower number of patients in the IDELA-R group (112 cases) related to the current prescribing practice. To overcome the potential problem of confounding by indication, we adjusted the association between the type of therapy and mortality for all variables significantly associated with OS at Cox univariate analysis. Furthermore, those variables, differently distributed between the two study groups, were introduced into the multivariate Cox model to improve the effectiveness of the analysis. By introducing all these variables into the multiple Cox regression model, we confirmed the protective effect of IB vs IDELA-R (HR = 0.67, 95% CI = 0.45-0.98, P = .04) independent of potential confounders. CONCLUSIONS: Although our analysis presents some constraints, that is, the unavailability of additional potential confounders, and the retrospective nature of the study, this observation may be of help for the daily clinical practice, particularly in the absence of randomized trials comparing the two schedules.

Tracing CLL-biased stereotyped immunoglobulin gene rearrangements in normal B cell subsets using a high-throughput immunogenetic approach.

BACKGROUND: B cell receptor Immunoglobulin (BcR IG) repertoire of Chronic Lymphocytic Leukemia (CLL) is characterized by the expression of quasi-identical BcR IG. These are observed in approximately 30% of patients, defined as stereotyped receptors and subdivided into subsets based on specific VH CDR3 aa motifs and phylogenetically related IGHV genes. Although relevant to CLL ontogeny, the distribution of CLL-biased stereotyped immunoglobulin rearrangements (CBS-IG) in normal B cells has not been so far specifically addressed using modern sequencing technologies. Here, we have investigated the presence of CBS-IG in splenic B cell subpopulations (s-BCS) and in CD5+ and CD5- B cells from the spleen and peripheral blood (PB). METHODS: Fractionation of splenic B cells into 9 different B cell subsets and that of spleen and PB into CD5+ and CD5- cells were carried out by FACS sorting. cDNA sequences of BcR IG gene rearrangements were obtained by NGS. Identification of amino acidic motifs typical of CLL stereotyped subsets was carried out on IGHV1-carrying gene sequences and statistical evaluation has been subsequently performed to assess stereotypes distribution. RESULTS: CBS-IG represented the 0.26% average of IGHV1 genes expressing sequences, were detected in all of the BCS investigated. CBS-IG were more abundant in splenic and circulating CD5+ B (0.57%) cells compared to CD5- B cells (0.17%). In all instances, most CBS IG did not exhibit somatic hypermutation similar to CLL stereotyped receptors. However, compared to CLL, they exhibited a different CLL subset distribution and a broader utilization of the genes of the IGHV1 family. CONCLUSIONS: CBS-IG receptors appear to represent a part of the "public" BcR repertoire in normal B cells. This repertoire is observed in all BCS excluding the hypothesis that CLL stereotyped BcR accumulate in a specific B cell subset, potentially capable of originating a leukemic clone. The different relative representation of CBS-IG in normal B cell subgroups suggests the requirement for additional selective processes before a full transformation into CLL is achieved.

Redefining the prognostic likelihood of chronic lymphocytic leukaemia patients with borderline percentage of immunoglobulin variable heavy chain region mutations.

In chronic lymphocytic leukaemia (CLL), caution is warranted regarding the clinical implications of immunoglobulin variable heavy chain region (IGHV) rearrangements with a 'borderline' (BL) percentage of mutations (i.e. 97-97·9% IGHV identity). We analysed the IGHV mutational status in 759 untreated CLL patients (cohort 1). BL-CLL (n = 36, 5%) showed a time to first treatment (TFT) similar to that of M-CLL (n = 338) and significantly longer than that of UM-CLL (n = 385), despite the enrichment in subset #2 cases. In fact, CLLs belonging to subset #2 (n = 15/759, 2%) were significantly more frequent among BL-CLLs (n = 5/36, 14%), with a brief TFT. TFT of BL-CLL remained comparable to that of M-CLL also considering the 327 CLL patients evaluated at diagnosis. These findings were then validated in an independent cohort 2 of 759 newly diagnosed CLL patients (BL-CLL: n = 11, 1·4%) and in all newly diagnosed patients from cohorts 1 and 2 (n = 1 086, 84% stage A; BL-CLL: n = 47, 4·3%). BL-CLL at diagnosis showed a biological profile comparable to that of M-CLL with a low frequency of unfavourable prognostic markers, except for a significant enrichment in subset #2. Our data suggest that the prognosis of BL-CLL is good and similar to that of M-CLL, with the exception of subset #2 cases.

A laboratory-based scoring system predicts early treatment in Rai 0 chronic lymphocytic leukemia.

We present a laboratory-based prognostic calculator (designated CRO score) to risk stratify treatment-free survival in early stage (Rai 0) chronic lymphocytic leukemia (CLL) developed using a training-validation model in a series of 1,879 cases from Italy, the United Kingdom and the United States. By means of regression analysis, we identified five prognostic variables with weighting as follows: deletion of the short arm of chromosome 17 and unmutated immunoglobulin heavy chain gene status, 2 points; deletion of the long arm of chromosome 11, trisomy of chromosome 12, and white blood cell count >32.0x103/microliter, 1 point. Low-, intermediate- and high-risk categories were established by recursive partitioning in a training cohort of 478 cases, and then validated in four independent cohorts of 144 / 395 / 540 / 322 cases, as well as in the composite validation cohort. Concordance indices were 0.75 in the training cohort and ranged from 0.63 to 0.74 in the four validation cohorts (0.69 in the composite validation cohort). These findings advocate potential application of our novel prognostic calculator to better stratify early-stage CLL, and aid case selection in risk-adapted treatment for early disease. Furthermore, they support immunocytogenetic analysis in Rai 0 CLL being performed at the time of diagnosis to aid prognosis and treatment, particularly in today's chemofree era.

Hepatocyte Growth Factor: A Microenvironmental Resource for Leukemic Cell Growth.

Chronic lymphocytic leukemia (CLL) is characterized by the progressive expansion of B lymphocytes CD5+/CD23+ in peripheral blood, lymph-nodes, and bone marrow. The pivotal role played by the microenvironment in disease pathogenesis has become increasingly clear. We demonstrated that bone marrow stromal cells and trabecular bone cells sustain survival of leukemic B cells through the production of hepatocyte growth factor (HGF). Indeed the trans-membrane kinase receptor for HGF, c-MET, is expressed on CLL cells and STAT3 TYR705 or AKT phosphorylation is induced after HGF/c-MET interaction. We have further observed that c-MET is also highly expressed in a peculiar type of cells of the CLL-microenvironment showing nurturing features for the leukemic clone (nurse-like cells: NLCs). Since HGF treatment drives monocytes toward the M2 phenotype and NLCs exhibit features of tumor associated macrophages of type 2 we suggested that HGF, released either by cells of the microenvironment or leukemic cells, exerts a double effect: i) enhances CLL cells survival and ii) drives differentiation of monocytes-macrophages to an oriented immune suppressive phenotype. We here discuss how paracrine, but also autocrine production of HGF by malignant cells, may favor leukemic clone expansion and resistance to conventional drug treatments in CLL, as well as in other hematological malignancies. Novel therapeutic approaches aimed to block HGF/c-MET interactions are further proposed.

Toll-like receptor 9 stimulation can induce IκBζ expression and IgM secretion in chronic lymphocytic leukemia cells.

Chronic lymphocytic leukemia cells strongly depend on external stimuli for their survival. Both antigen receptor and co-stimulatory receptors, including Toll-like receptors, can modulate viability and proliferation of leukemic cells. Toll-like receptor ligands, and particularly the TLR9 ligand CpG, mediate heterogeneous responses in patients' samples reflecting the clinical course of the subjects. However, the molecular framework of the key signaling events underlying such heterogeneity is undefined. We focused our studies on a subset of chronic lymphocytic leukemia cases characterized by expression of CD38 and unmutated immunoglobulin genes, who respond to CpG with enhanced metabolic cell activity. We report that, while CpG induces NFKBIZ mRNA in all the samples analyzed, it induces the IκBζ protein in a selected group of cases, through an unanticipated post-transcriptional mechanism. Interestingly, IκBζ plays a causal role in sustaining CpG-induced cell viability and chemoresistance, and CpG stimulation can unleash immunoglobulin secretion by IκBζ-positive malignant cells. These results identify and characterize IκBζ as a marker and effector molecule of distinct key pathways in chronic lymphocytic leukemia.

A non-invasive approach to monitor chronic lymphocytic leukemia engraftment in a xenograft mouse model using ultra-small superparamagnetic iron oxide-magnetic resonance imaging (USPIO-MRI).

Chronic lymphocytic leukemia (CLL) is the most prevalent leukemia among adults. Despite its indolent nature, CLL remains an incurable disease. Herein we aimed to monitor CLL disease engraftment and, progression/regression in a xenograft CLL mouse model using ultra-small superparamagnetic iron oxide-magnetic resonance imaging (USPIO-MRI). Spleen contrast enhancement, quantified as percentage change in signal intensity upon USPIO administration, demonstrated a difference due to a reduced USPIO uptake, in the spleens of mice injected with CLL cells (NSG-CLL, n=71) compared to controls (NSG-CTR, n=17). These differences were statistically significant both after 2 and 4weeks from CLL cells injection. In addition comparison of mice treated with rituximab with untreated controls for changes in spleen iron uptake confirmed that it is possible to monitor treatment efficacy in this mouse model of CLL using USPIO-enhanced MRI. Further applications could include the preclinical in vivo monitoring of new therapies and the clinical evaluation of CLL patients.

Prospective validation of predictive value of abdominal computed tomography scan on time to first treatment in Rai 0 chronic lymphocytic leukemia patients: results of the multicenter O-CLL1-GISL study.

OBJECTIVE: We performed an external and multicentric validation of the predictive value of abdominal computed tomography (aCT) on time to first treatment (TTFT) in early stage chronic lymphocytic leukemia (CLL) patients. METHODS: aCT was performed at diagnosis in 181 Rai 0 patients enrolled in the O-CLL1-GISL trial (clinicaltrial.gov ID:NCT00917549). RESULTS: Fifty-five patients showed an abnormal aCT. Patients with an abnormal aCT showed a significantly shorter TTFT than those with normal aCT (P < 0.0001). At multivariate analysis, aCT (P = 0.011), ß-2 microglobulin (P = 0.019), and CD38 expression (P = 0.047) correlated with TTFT. Following IWCLL 2008 criteria, 112 (61.9%) cases remained at Rai 0, while 69 (38.1%) satisfied the criteria of clinical monoclonal B-cell lymphocytosis (cMBL). Reclassified Rai 0 patients with an abnormal aCT showed a significantly shorter TTFT than those with a normal aCT (P < 0.0001). At multivariate analysis, only aCT (P = 0.011) correlated with TTFT. Eleven cMBL cases (15.9%) showed an abnormal aCT and were reclassified as small lymphocytic lymphomas (SLL); nonetheless, TTFT was similar for cMBLs and SLLs. CONCLUSION: Our results confirm the ability of the abnormal aCT to predict progression in early stage cases.

The chronic lymphocytic leukemia international prognostic index predicts time to first treatment in early CLL: Independent validation in a prospective cohort of early stage patients.

The chronic lymphocytic leukemia International Prognostic Index (CLL-IPI) combines 5 parameters (age, clinical stage, TP53 status [normal vs. del(17p) and/or TP53 mutation], IGHV mutational status, serum ß2-microglobulin) to predict survival and time-to-first-treatment (TTFT) in CLL patients. We performed an observational study in 337 prospectively collected, Binet stage A patients to validate the ability of the CLL-IPI to predict TTFT in an independent cohort of early stage CLL patients. The CLL-IPI score stratified Binet stage A patients into three subgroups with different outcome. Since the CLL-IPI was originally developed to predict survival, we next investigated the optimal cut-off score to predict TTFT in Binet stage A patients. Recursive partitioning analysis identified three subsets with scores of 0 (n = 139), 1 (n = 90), and ≥ 2(n = 108). The probability of remaining free from therapy 5 years after diagnosis was 85%, 67% and 46% in these three categories (P < 0.0001.; C-statistic:c = 0.72; 95% CI:0.58-0.81). This optimized CLL-IPI scoring for TTFT was subsequently validated in an independent cohort of Binet A patients from the Mayo Clinic (n = 525). The ability of either original or optimized CLL-IPI to predict TTFT was equivalent to other prognostic models specifically designed for this endpoint (2011 MDACC score and O-CLL1 score). Although originally developed to predict suvival, the CLL-IPI is useful for predicting TTFT in early stage CLL patients. Am. J. Hematol. 91:1090-1095, 2016. © 2016 Wiley Periodicals, Inc.