Unmutated Chronic Lymphocytic Leukemia Patients Research Articles

MER Signaling Axis: A Novel Therapeutic Target in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of CD19+/CD5+/CD23+ monoclonal B-cells in peripheral blood, bone marrow and lymphoid tissues with a highly variable disease course. B-cell receptor (BCR)-signal inhibitors, specifically Ibrutinib which targets Bruton's tyrosine kinase (BTK) and Idelalisib which targets PI3Kδ, are effective in relapsed/refractory CLL patients. Despite BCR-signal-inhibitors are changing the management of CLL, these agents are not curative and resistance can develop, often leading to the transformation of CLL into more aggressive B-cell lymphomas limiting their therapeutics options. Thus, improved understanding of the nature and extent of other active receptor tyrosine kinase (RTK) signals in CLL cells is critical for development of additional therapies to treat these high-risk patients. Our recent discovery of MER, a RTK of the TAM (Tyro3, AXL, MER) family, in CLL cells may offer such an option. In order to analyze MER expression status in CLL cells from previously untreated CLL patients, we employed western blot, flow cytometry and qRT-PCR analyses. We detected low to high level expression of MER in CLL cells from ~50% of CLL patients as compared to normal, purified B cells. We also found that MER in CLL cells remained constitutively phosphorylated (active). Furthermore, our preliminary analysis suggested that MER expression in CLL cells may be positively associated with high-risk, unmutated IGHV CLL patients. In vitro ligation of Gas6 (growth arrest-specific gene 6), a specific ligand for all the TAM receptors, robustly activated MER as compared to AXL or Tyro3. Interestingly, activation of MER preferentially activated Erk1/2, NF-κB, and BTK of the BCR pathway in CLL cells. Partial depletion of MER in CLL cells using a specific-siRNA resulted in inhibition of P-Erk1/2, P-NF-κB and P-BTK, but not P-AKT, suggesting that MER may crosstalk with the BCR signaling pathway in CLL cells likely via interaction with CD79a, LYN or BTK. To address this hypothesis, we immunoprecipitated MER from CLL cell lysates and found a direct complex formation between MER and LYN. We also analyzed leukemic B cells from the Eµ-TCL1 transgenic mice which recapitulate highly aggressive human CLL for the expression of MER by western blot/flow cytometry. Results showed robust expression of MER in TCL1 leukemic B cells purified from mice with advanced CLL. Finally, treatment of CLL mice with a high-affinity MER inhibitor (UNC-2025) via oral gavage significantly reduced leukemic tumor burden. In total, our findings suggest that expression/activation of MER may potentiate CLL cell survival via a crosstalk with the BCR signal thus, could be an attractive therapeutic target for the progressive CLL patients.

NOTCH1 Aberrant Activation Is a Common Nonmutational Early Event in Chronic Lymphocytic Leukemia Hematopoietic Stem-Cells

Chronic lymphocytic leukemia (CLL) is a B cell disorder characterized by constitutive NOTCH1 activation. NOTCH1 mutations are recurrently associated with CLL providing a new clonal marker to track CLL origin. To investigate CLL-initiating cells, we assessed NOTCH1 mutational status and signaling in hematopoietic stem (HSCs) and progenitor cells from CLL bone marrow. We collected a total of 29 bone marrow (BM) samples including 22 CLL patients and 7 healthy donors (HDs). BM cells were sorted into CD34+CD38- HSCs (purity 94.23% ± 3.04% ) and CD34+CD38+ progenitor fraction (98.12% ± 1.34%). While Sanger sequencing analysis of the NOTCH1 failed to detect alterations in CD34+/CD38- HSCs, AS-PCR and Droplet digital PCR (ddPCR) indicated the presence of small HSCs mutated clones in 57% of cases. Altogether, these data confirm that NOTCH1 mutation is an early event in CLL hematopoiesis. The analysis of CD34+/CD38+ progenitors detected the NOTCH1 mutation in the majority of samples (66%). The NOTCH1 mutational burden progressively increased along specific stages of HSC differentiation (6.4%± 4.7 in CD34+CD38- to 14.9%±11.3 in CD34+CD38+CD10+CD19+ cells, 22.7%±6.5 in CD34-CD38+CD10+CD19+ cells and 40.5%±4.3 in neoplastic CD5+CD19+ cells). This suggests that the NOTCH1 lesion is selected and expands during HSC differentiation toward a B neoplastic cell, thus strengthening the hypothesis that the genetic alteration is an initial event associated with the stepwise malignant transformation of CLL. Conversely, CD34+ cells (1x106) from CLL patients carrying the mutation, when transplanted into NOD/SCID/IL2Rgnull (NSG) mice generated a mature CLL phenotype (the median percentage of CD5+CD19+ cells in hCD45+ cells was 38.5% and 43.8% in BM and spleen respectively) lacking NOTCH1 mutation (as showed by AS-PCR assay performed on enriched hCD45 cells). This data is more in line with the hypothesis that this genetic abnormality is acquired at the mature B cell stage as an additional leukemogenic event to transform into clinical CLL. Thus, we analyzed the NOTCH1 signaling status in HSCs and progenitor cells of NOTCH1-mutated and unmutated CLL samples. The flow-cytometric analysis of the active NOTCH1-ICN protein level showed unmutated and mutated CLL has a significantly higher NOTCH1-ICN level than HDs samples in both CD34+/CD38- HSCs and CD34+CD38+ (73.4%±22.9 and 83%±16.4 vs 33.3%±14.8; 94.4%±7.3 and 92.8%±4.3 vs 47.9%±13.8, p<0.01 and p<0.001, respectively). c-MYC expression was found significantly higher in HSCs cells from NOTCH1 mutated and unmutated CLL samples compared to HD (3.5±0.7 and 2.6±0.08 vs 1.3±0.1). Western blot analysis of the expression levels of the NOTCH1-TM subunit revealed that HSCs from mutated and unmutated CLL patients always expressed the NOTCH1-TM protein, compared to HDs where NOTCH1-TM was either absent or expressed at lower levels. The present study indicated that the pool of CD34+ cells, including HSC and progenitor compartments, have NOTCH1 aberrantly expressed and activated in CLL patients compared to HDs demonstrating a common nonmutational NOTCH1 activation occurring early in CLL hematopoiesis. This selective pressure might contribute to the onset of specific NOTCH1 mutations in a DNA context that is prone to spontaneous microdeletion. These data represent a rationale for the use of therapies targeting the NOTCH1 signaling in CLL aimed to inhibit the survival of CLL-initiating cells. DisclosuresNo relevant conflicts of interest to declare.

Different Time-Dependent Risk for Disease Evolution in Chronic Lymphocytic Leukemia with Distinct IGHV Gene Somatic Hypermutation Status

A great challenge for early stage patients with chronic lymphocytic leukemia (CLL) is to determine if and, consequently, when they will require treatment, highlighting the need for robust prognostic assessment. Today, immunogenetic and genomic features are considered essential for accurate risk stratification in CLL. However, when utilizing such biomarkers, the prognosis is assessed assuming stable predictability over the disease course. Here, we assessed the risk for CLL evolution overtime by evaluating time-to-first-treatment (TTFT) for 2366 CLL patients not requiring treatment at the time of diagnosis (Binet A: n=1900, 80.3%/Binet B: n=287, 12.1%), hypothesizing that risk evolution might depend on both time and the biological characteristics of the clone. Based on the somatic hypermutation status (SHM) of the immunoglobulin heavy variable genes, 1364 (58%) and 1002 (42%) patients were classified as mutated (M-CLL) and unmutated CLL (U-CLL), respectively. Regarding genomic subgroups of CLL, we focused on high risk patients carrying aberrations within the TP53 gene (TP53 abn, deletion of chromosome 17p and/or TP53 mutations) and low risk patients exhibiting isolated deletion of chromosome 13q or normal FISH according to the Döhner hierarchical model (del(13q)/normal FISH).First, we estimated the risk overtime by the hazard curve. This enables to better visualize changes in risk overtime compared to a Kaplan-Meier curve by showing the estimated proportion of cases receiving treatment in a defined interval, given that a person is still treatment-free at the start of this interval. An interpolation method was then used to estimate the values of the hazard curve at each distinct year from the time of diagnosis, and the corresponding differences in hazard values between M-CLL and U-CLL. These differences constituted the basis for the overtime analysis. In particular, the overall time frame was divided in 5-year intervals, and the distributions of differences in hazard were statistically compared between consecutive 5-year intervals. P-values less than 0.05 would indicate different evolution for the hazard curve overtime for the M-CLL and U-CLL.First, we assessed all Binet A patients, regardless of their genomic aberrations background, and found significant differences between M-CLL vs U-CLL with p[0,5]Vs(5,10]=0.006, p(5,10]Vs(10,15]=0.009, p(10,15]Vs(15,20]=0.009, p(15,20]Vs(20,25]=0.014, and p(overall)<0.001, reflecting the constant decrease of the distance between the M-CLL and U-CLL hazard curves (Fig 1a), deemed as statistically significant between consecutive 5-year intervals. Next, we focused on different genomic risk subgroups and found that del(13q)/normal FISH patients followed the M-CLL/U-CLL rule with p[0,5]Vs(5,10]=0.006, p(5,10]Vs(10,15]=0.009, p(10,15]Vs(15,20]=0.009, and p(overall)<0.001, indicating little additional impact on risk evolution beyond that dictated by the SHM status (Fig 1b). In sharp contrast, within TP53 abn patients we found p[0,5]Vs(5,10]=0.006, which reflects the statistically significant increase of the distance between the two hazard curves for M-CLL and U-CLL after the 5th year (Fig 1c), hence indicating decreasing risk for TP53 abn M-CLL and, vice versa, constantly increasing risk for TP53 abn U-CLL after the 5th year. For Binet B patients, we did not observe any significant differences with p[0,5]Vs(5,10]=0.2, highlighting a similar hazard evolution for M-CLL and U-CLL (Fig 1d). Low numbers precluded assessing the impact of genomic aberrations within this subgroup of patients.In conclusion, within all early stage patients as well as in subgroup analysis of high or low genomic risk Binet A patients, M-CLL was found to display a rather stable overtime risk for TTFT, independently of the presence of TP53 abn. This sharply contrasted U-CLL patients, where the overtime risk for TTFT was clearly influenced by the genomic background since del(13q)/normal FISH patients showed a similar pattern with M-CLL whereas TP53 abn patients exhibited a continuously increasing risk for disease progression. From a different standpoint, these findings corroborate the concept that the precise prognostic value of TP53 abn depends on the biological background of the CLL clone that is altogether different in patients with distinct SHM status. [Display omitted] DisclosuresStrefford:Roche: Research Funding. Delgado:Abbvie, Jansen, Gilead, Roche: Consultancy, Speakers Bureau. Stavroyianni:Janssen Pharmaceuticals: Honoraria; Gilead Sciences: Honoraria; Takeda: Honoraria. Gaidano:Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Ghia:AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Pharmaceuticals: Honoraria, Research Funding; Roche: Honoraria; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Stamatopoulos:Abbvie: Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Novartis SA: Research Funding; Janssen Pharmaceuticals: Honoraria, Research Funding.

Proteomic markers with prognostic impact on outcome of chronic lymphocytic leukemia patients under chemo-immunotherapy: results from the HOVON 109 study

Despite recent identification of several prognostic markers, there is still a need for new prognostic parameters able to predict clinical outcome in chronic lymphocytic leukemia (CLL) patients. Here, we aimed to validate the prognostic ability of known (proteomic) markers measured pretreatment and to search for new proteomic markers that might be related to treatment response in CLL. To this end, baseline serum samples of 51 CLL patients treated with chemo-immunotherapy were analyzed for 360 proteomic markers, using Olink technology. Median event-free survival (EFS) was 23 months (range: 1.25-60.9). Patients with high levels of sCD23 (>11.27, p = 0.026), sCD27 (>11.03, p = 0.04), SPINT1 (>1.6, p = 0.001), and LY9 (>8.22, p = 0.0003) had a shorter EFS than those with marker levels below the median. The effect of sCD23 on EFS differed between immunoglobulin heavy chain variable gene-mutated and unmutated patients, with the shortest EFS for unmutated CLL patients with sCD23 levels above the median. Taken together, our results validate the prognostic impact of sCD23 and highlight SPINT1 and LY9 as possible promising markers for treatment response in CLL patients.

Quantification of Differential Transcription Factor Activity and Multiomics-Based Classification into Activators and Repressors: diffTF

Transcription factors (TFs) regulate many cellular processes and can therefore serve as readouts of the signaling and regulatory state. Yet for many TFs, the mode of action-repressing or activating transcription of target genes-is unclear. Here, wepresent diffTF (https://git.embl.de/grp-zaugg/diffTF) to calculate differential TF activity (basic mode) and classify TFs into putative transcriptional activators or repressors (classification mode). In basic mode, it combines genome-wide chromatin accessibility/activity with putative TF binding sites that, in classification mode, are integrated with RNA-seq. We apply diffTF to compare (1) mutated and unmutated chronic lymphocytic leukemia patients and (2) two hematopoietic progenitor cell types. In both datasets, diffTF recovers most known biology and finds many previously unreported TFs. It classifies almost 40% of TFs based on their mode of action, which we validate experimentally. Overall, we demonstrate that diffTF recovers known biology, identifies less well-characterized TFs, and classifies TFs into transcriptional activators or repressors.

Study of gene expressions' correlation structures in subgroups of Chronic Lymphocytic Leukemia Patients.

In chronic lymphocytic leukemia (CLL) the interaction of leukemic cells with the microenvironment ultimately affects patient outcome. CLL cases can be divided in two subgroups with different clinical course based on the mutational status of the immunoglobulin heavy variable (IGHV) genes: mutated CLL (M-CLL) and unmutated CLL (U-CLL). Since in CLL, the differentiated relation of genes between the two subgroups is of greater importance than the individual gene behavior, this paper investigates the differences between the groups' gene interactions, by comparing their correlation structures. Fisher's test and Zou's confidence intervals are employed to detect differences of correlation coefficients. Afterwards, networks created by the genes participating in most differences are estimated with the use of structural equation models (SEM). The analysis is enhanced with graph modeling in order to visualize the between group differences in the gene structures of the two subgroups. The applied methodology revealed stronger correlations between genes in U-CLL patients, a finding in line with related biomedical literature. Using SEM for multigroup analysis, different gene structures between the two groups of patients were confirmed. The study of correlation structures can facilitate the detection of differential gene expression profiles in CLL subgroups, with potential applications in other diseases. Comparison of correlations can be very useful in understanding the complex internal structural differences which signify the variations of a disease.

Lipids and Their Effects in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is an incurable common B-cell malignancy with a spectrum of clinical outcomes. Over the past decade, our increasing understanding of the drivers of CLL progression has led to the development and use of novel therapeutics. For example, B-cell receptor (BCR) signaling was shown to be overactive in CLL, and subsequently the kinase inhibitors ibrutinib (BTK inhibitor) and idelalisib (PI3K delta inhibitor) were found to have clinical efficacy in this malignancy (Byrd et al., 2013, Furman et al., 2014). Despite these new treatments, CLL remains incurable and there remains a need to identify new therapeutic targets.

Surface CD5 Protein Risk Stratifies Chronic Lymphocytic Leukemia

Introduction: Chronic Lymphocytic Leukemia (CLL) is a malignancy characterized by B-lymphocytes with aberrant expression of CD5. In normal T-lymphocytes, CD5 is an important regulator of T-cell receptor signaling. In CLL, CD5 acts as a repressor of B-cell receptor (BCR) signaling, whereby phosphorylated CD5 anchors SHP-1 at the cell membrane, resulting in inhibition of BCR-mediated second messenger signaling. The significance of CD5 on outcomes of CLL patients has not been evaluated. Because BCR signaling is an oncogenic stimulus in CLL, we hypothesized that higher levels of CD5 may be associated with superior clinical outcomes.Methods: Median fluorescence intensity (MFI) of CD5 on CLL cells was determined by flow cytometry. CLL samples and clinical data were obtained from patients enrolled in IRB-approved protocols at the Duke University and Durham VA Medical Centers. Molecular prognostic markers were measured as described previously. Descriptive and time to event (Cox proportional hazard and Kaplan-Meier) statistical analyses were performed in the statistical environment, R.Results: Between March 2011 and April 2015, 961 CLL samples were evaluated from 352 unique CLL patients. One to 10 samples were evaluated per CLL patient. The mean CD5 MFI was calculated for each unique patient, with a median of 206.59 (range 0.50 - 768.31) and median standard deviation of 33.02 (range 0.09 - 315.46) for CLL patients with multiple samples collected. 150 patients received therapy (44%), and there was no significant difference in CD5 MFI mean or standard deviation between patients who received and did not receive therapy. CD5 MFI values did not significantly differ among CLL demographic or prognostic groups. Cox proportional hazard ratio analyses showed that higher CD5 MFI is associated with longer time to therapy (TTT, p = 0.037), but not overall survival. Based on the distribution of CD5 MFI, we divided the CLL cohort into thirds, with low CD5 defined as MFI < 137, high CD5 defined as MFI > 283.5, and medium CD5 with MFI between these values. Kaplan-Meier analyses demonstrated a significant difference in TTT between these three groups (p = 0.004), with higher CD5 MFI associated with longer TTT. Moreover, CD5 MFI added to established molecular prognostic markers to risk stratify CLL patients (p < 0.0001 for CD38 and IGHV, p = 0.0003 for FISH, and p = 0.004 for ZAP70). Importantly, for those with good prognostic markers such as IGHV mutation or low CD38 expression, lower CD5 identified patients with shorter TTT (p = 0.01 for IGHV mutated; p = 0.0009 for CD38 negative). However, TTT was not significantly different in unmutated IGHV CLL patients or CD38 positive CLL patients with varying degrees of CD5 expression.Conclusions: Surface CD5 expression varies among CLL patients. For most individual patients, there appears to be a low level of variability in CD5 expression, regardless of CLL-directed therapy. Higher CD5 expression is associated with superior clinical outcomes in CLL, consistent with prior in vitro determination of CD5 as a negative regulator of BCR signaling. The relevance of CD5 in risk-stratifying IGHV mutated and CD38 negative CLL patients is of particular relevance, since these subgroups of CLL are particularly dependent on BCR-signaling as an oncogenic process. These findings are important both for prognostication and for development of therapies for this group of CLL patients. DisclosuresNo relevant conflicts of interest to declare.

SOX11 expression in chronic lymphocytic leukemia correlates with adverse prognostic markers.

The transcription factor SOX11 plays an important role in embryonic neurogenesis and tissue remodeling. Recent studies have shown aberrant expression of SOX11 in various types of aggressive B cell neoplasms. In this study, we have analyzed SOX11 transcription levels in 86 patients with diagnosis of chronic lymphocytic leukemia (CLL). Results were correlated with well-known prognostic factors such as immunoglobulin heavy chain variable (IGHV) gene mutational status, cytogenetics risk groups and clinicopathological characteristics of the disease. Overall, 35 % of cases showed SOX11 expression; meanwhile, the remaining 65 % lacked gene expression. The analysis taking into account the IGHV mutational status showed significant differences in SOX11 transcripts levels between mutated (0.004 ± 0.0001) and unmutated CLL patients (0.405 ± 0.011) (p < 0.0001), as well as a positive correlation between SOX11 mRNA expression and the percentage of IGHV homology (p = 0.0001). Furthermore, significantly lower SOX11 mRNA expression was detected in patients with deletion 13q14 as a single alteration (0.016 ± 0.008) than those observed in cases with deletions 11q/17p (0.35 ± 0.017) (p = 0.02). The correlation of gene expression with clinical evolution showed shorter treatment free survival (p = 0.043) and overall survival (p = 0.047) in SOX11 positive patients compared to SOX11 negative cases. Our findings show for the first time an association between SOX11 expression and some CLL poor prognostic factors. These results suggest SOX11 as a possible biomarker that adds new biological information that could contribute to a better understanding of this pathology.

Mutation status and immunoglobulin gene rearrangements in patients from northwest and central region of Spain with chronic lymphocytic leukemia.

The aim of this study was to investigate the frequency and mutation status of the immunoglobulin heavy variable chain (IGHV) in a cohort of 224 patients from northwest and central region of Spain diagnosed with chronic lymphocytic leukemia (CLL), and to correlate it with cytogenetic abnormalities, overall survival (OS) and time to first treatment (TTFT). 125 patients had mutated IGHV, while 99 had unmutated IGHV. The most frequently used IGHV family was IGHV3, followed by IGHV1 and IGHV4. The regions IGHV3-30, IGHV1-69, IGHV3-23, and IGHV4-34 were the most commonly used. Only 3.1% of the patients belonged to the subfamily IGHV3-21 and we failed to demonstrate a worse clinical outcome in this subgroup. The IGHV4 family appeared more frequently with mutated pattern, similar to IGHV3-23 and IGHV3-74. By contrast, IGHV1-69 was expressed at a higher frequency in unmutated CLL patients. All the cases from IGHV3-11 and almost all from IGHV5-51 subfamily belonged to the group of unmutated CLL.

Lipoprotein lipase expression in unmutated CLL patients is the consequence of a demethylation process induced by the microenvironment

Lipoprotein lipase expression in unmutated CLL patients is the consequence of a demethylation process induced by the microenvironment

Combining Expression of Hypervariably Expressed Genes with IGHV Mutation Status Is a More Robust Prognostic Indicator Than Mutation Status Alone in Chronic Lymphocytic Leukemia

Abstract 1774 Introduction:Chronic Lymphocytic Leukemia (CLL) follows a variable, difficult to predict course. Biomarkers (IGHV mutations, CD38 and ZAP-70) have improved prognostication but accuracy is only ∼80%, not sufficient to initiate preemptive therapy. To address this, we have incorporated another molecular biological parameter, hypervariably expressed genes (HVEGs) from global gene expression profiles (GEPs) [Dozmorov, 2011], along with IGHV mutation status to develop a more robust method of prognostication. Methods:RNA was purified from B cells of 65 CLL patients and 25 normal subjects. GEPs were defined with Illumina HumanHT12 beadchips and analyzed to identify HVEGs fluctuating coordinately in CLL patients but not expressed in normal circulating human B cells. A Cox proportional hazards regression model was used to compare expression levels with clinical outcome. Results:Unmutated CLL (U-CLL) and mutated CLL (M-CLL) samples were sorted based on the averaged normalized gene expression level in the largest HVEG cluster consisting of 45 genes. Samples were split into subgroups based on high and low expression of these genes. Four subgroups were identified: U-CLL with high (U-HVEhi), U-CLL with low (U-HVElo), M-CLL with high (M-HVEhi), and M-CLL with low (M-HVElo) expression of HVEGs. When these subgroups were analyzed for time-to-first treatment (TTFT), the findings were remarkably different. Using a Cox proportional hazards regression model we found that for M-CLL the presence of low levels of HVEGs denoted a subgroup with a prolonged median TTFT compared to M-CLL patients with high levels of HVEGs (23.46 yrs in M-HVElo versus 9.05 yrs in M-HVEhi; P=0.0075). This association was reversed in U-CLL, where low levels of HVEGs pinpointed a subgroup with a shortened median TTFT compared to U-CLL patients with high levels of HVEGs: median TTFT was 8.01 yrs in U-HVEhi and decreased by 50% to 3.12 yrs in U-HVElo (P=0.0151). Compared to the TTFT for M-HVElo, the TTFT for U-HVElo was dramatically shorter (3.12 vs. 23.46 years; P<0.0001). Discussion:Combining the HVEG approach with IGHV mutation status enabled us to define intra-group heterogeneity, and thereby improve prognostication for apparently homogeneous subgroups of U-CLL and M-CLL patients. This combination was superior to IGHV mutations alone in that it pinpointed a subset of M-CLL patients with shorter TTFTs and a subset of U-CLL patients with longer TTFTs, indicating that the combined approach selects those patients that IGHV mutation analysis misclassified. Therefore, combining IGHV mutation status and HVEG expression provides a more precise indicator with potentially valuable clinical implications. The data suggest that the HVEGs are responsible for the different outcomes in the two groups and could provide mechanistic insights into key aspects of CLL B-cell biology as well as therapeutic targets. Disclosures:No relevant conflicts of interest to declare.

1.14 DNA Methylation Status is Associated to Lipoprotein Lipase (LPL) Expression and can be Modulated by FC Treatment in Unmutated CLL Patients

1.14 DNA Methylation Status is Associated to Lipoprotein Lipase (LPL) Expression and can be Modulated by FC Treatment in Unmutated CLL Patients

IGHV Unmutated Chronic Lymphocytic Leukemia (CLL) B Cells Are More Susceptible to Spontaneous Apoptosis Than Mutated CLL B Cells and Are Subject to the Anti-Apoptotic Effect of Environmental Signals

AbstractAbstract 2431Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease. A very reliable prognosticator is the mutational status of the tumor immunoglobulin heavy chain variable region (IGHV): patients with unmutated (UM) IGHV have a worse prognosis than patients with mutated (M) IGHV. Soluble factors (i.e. IL-4 and CD40L) and cellular components of the local microenvironment [i.e. bone marrow stromal cells (BMSC) and nurse-like cells (NLCs)] are important survival factors for CLL B cells. It is currently unknown to what extent UM and M CLL cells depend on the local microenvironment for their survival.We have evaluated the spontaneous apoptotic rate of tumor cells isolated by immunomagnetic selection from the peripheral blood (PB) of M and UM CLL patients. Both M and UM CLL B cells underwent spontaneous apoptosis throughout the culture period. However, the UM CLL B cells showed a significantly higher degree of apoptosis in 7-day cultures as compared to M CLL B cells. In both M and UM CLL B cells, high basal levels of Bcl-2 expression and NF-kB activity were detected. On day 7, the percentage of Bcl-2+ leukemic cells was significantly lower in UM than in M CLL B cells. EMSA test showed that NF-kB was totally inactivated in UM CLL B cells and only partially reduced in M CLL B cells. Quantitative analysis of RelA and RelB subunits showed that NF-kB inactivation in UM CLL B cells consisted in a strong reduction of both RelA and RelB nuclear expression. CD40L, IL-4 and stromal cells significantly improved UM CLL B cells viability and significantly recovered Bcl-2 expression. The protective effect exerted by these stimuli was totally independent from the recovery of NF-kB expression. Indeed, after 7 days of culture, the UM CLL B cells had completely lost the nuclear form of NF-kB, and none of the stimuli was capable of restoring it.We observed that UM CLL cells were less susceptible to spontaneous apoptosis when cultured as unfractionated peripheral blood mononuclear cells (M or UM PBMC) as compared to purified leukemic cells (M and UM CLL B cells). The reduced apoptosis detected in UM PBMC was accompanied by a retained expression of Bcl-2 and by a restored activity of NF-kB and suggested the presence of a pro-survival element in the peripheral blood of these patients. To investigate the role of NLC in rescuing UM CLL B cells from apoptosis we first evaluated whether M and UM PBMC generated NLC with the same efficiency. Unexpectedly, the former generated significantly higher numbers of NLC than UM PBMC. Despite the lack of generation of NLC, CLL B cells viability was very similar in the non-adherent fraction of M and UM PBMC on day 7 and 14 of culture. This observation ruled out a role for NLC in supporting UM CLL B cells survival.Conversely, a pro-survival effect on UM CLL B cells was exerted by autologous T cells. Indeed, a significant reduction in the apoptotic rate of leukemic cells was observed when purified UM CLL B cells were cultured in the presence of autologous peripheral blood T cells (UM CLL B cell/T cell co-cultures). NF-kB activity was completely lost in UM CLL B cells cultured for 7 days in medium alone whereas it was restored in UM CLL B cells / T cells co-cultures. The prosurvival effect of circulating T cells was exerted both in cell-to-cell contact and in trans-well condition and was associated to increased secretions of tumor necrosis factor-alpha (TNF-α), platelet-derived growth factor (PDGF)-BB and interleukin-8 (IL-8) as detected by analyses of supernatants through a Multiplex system.These data indicate that despite their more aggressive features, UM CLL B cells are more susceptible to spontaneous apoptosis and depend from environmental prosurvival signals. This vulnerability of UM CLL B cells can be exploited as a selective target of therapeutic interventions. Disclosures:Boccadoro: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Massaia: Novartis: Honoraria, Research Funding.

High expression of AID and active class switch recombination might account for a more aggressive disease in unmutated CLL patients: link with an activated microenvironment in CLL disease

AbstractInteraction of chronic lymphocytic leukemia (CLL) B cells with tissue microenvironment has been suggested to favor disease progression by promoting malignant B-cell growth. Previous work has shown expression in peripheral blood (PB) of CLL B cells of activation-induced cytidine deaminase (AID) among CLL patients with an unmutated (UM) profile of immunoglobulin genes and with ongoing class switch recombination (CSR) process. Because AID expression results from interaction with activated tissue microenvironment, we speculated whether the small subset with ongoing CSR is responsible for high levels of AID expression and could be derived from this particular microenvironment. In this work, we quantified AID expression and ongoing CSR in PB of 50 CLL patients and characterized the expression of different molecules related to microenvironment interaction. Our results show that among UM patients (1) high AID expression is restricted to the subpopulation of tumoral cells ongoing CSR; (2) this small subset expresses high levels of proliferation, antiapoptotic and progression markers (Ki-67, c-myc, Bcl-2, CD49d, and CCL3/4 chemokines). Overall, this work outlines the importance of a cellular subset in PB of UM CLL patients with a poor clinical outcome, high AID levels, and ongoing CSR, whose presence might be a hallmark of a recent contact with the microenvironment.

Differential Effects of Microenvironmental Elements On Tumor Cells Survival in Chronic Lymphocytic Leukemia Patient Subsets with Good or Poor Prognosis.

Abstract 2333Poster Board II-310Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease. A very reliable prognosticator is the mutational status of the tumor immunoglobulin heavy chain variable region (IgVH): patients with unmutated (UM) IgVH have a worse prognosis than patients with mutated (M) IgVH. Soluble factors (i.e. IL-4 and CD40L) and cellular components of the local microenvironment [i.e. bone marrow stromal cells (BMSC) and nurse-like cells (NLCs)] are important survival factors for CLL B cells. It is currently unknown to what extent UM and M CLL cells depend on the local microenvironment for their survival.We have evaluated the spontaneous apoptotic rate of tumor cells isolated by immunomagnetic selection from the peripheral blood (PB) of M and UM CLL patients. Leukemic cells purified by negative selection from the PB of UM CLL patients showed significantly higher rates of spontaneous apoptosis after long-term in vitro culture as compared to CLL cells isolated from M patients. Both M and UM CLL cells showed high basal level of Bcl-2 expression and NF-kB activity soon after purification. In vitro spontaneous apoptosis of purified UM CLL cells was associated with a progressive downregulation of the intracellular expression of Bcl-2 and with a complete loss of the active nuclear form of NF-kB. On the contrary, the higher long term viability of M CLL cells was paralleled by a maintained Bcl-2 and NF-kB expression. IL-4 and CD40L, used alone or in combination, as well as murine and human BMSC were capable of rescuing UM tumor cells from apoptosis. The pro-survival effect of these stimuli was exerted through the upregulation of Bcl-2 and was totally independent from the recovery of NF-kB nuclear translocation. We observed that UM CLL cells were less susceptible to spontaneous apoptosis when cultured as unfractionated peripheral blood mononuclear cells (PBMC) as compared to purified leukemic cells. This higher cell viability was associated with a retained expression of Bcl-2 and of the nuclear form of NF-kB, thus suggesting the presence of a pro-survival element in the peripheral blood of these patients. The extremely low numbers of NLCs generated from PMBC of UM patients ruled out a role for these cells in supporting the survival of unpurified leukemic cells. Conversely, a pro-survival effect on UM CLL cells was exerted by autologous T cells. Indeed, a significant reduction in the apoptotic rate of leukemic cells was observed when purified UM cells were cultured in the presence of autologous peripheral blood T cells (PBT). The prosurvival effect of circulating T cells was particularly evident at high T:B ratio, did not require a cell-cell contact and was mediated by the upregulation of Bcl-2 and the activation of NF-kB in leukemic cells.These data indicate that the survival of UM tumor cells is highly dependent on the action of multiple microenviromental stimuli. Conversely, M cells are intrinsically more resistant to apoptosis and minimally influenced by the local microenviroment. The higher dependency of UM CLL cells from extrinsic signals might be exploited to develop new therapies targeting the tumor microenvironment and to improve the outcome of more aggressive CLL. Disclosures:No relevant conflicts of interest to declare.

Dichotomy in NF-κb Signaling and Chemoresistance in IgVH Mutated Versus Unmutated CLL Cells Revealed by CD40Ligand/CpG Stimulation

Introduction and aim IgVH mutated (M) and unmutated (UM) Chronic Lymphocytic Leukemia (CLL) cells differ in their response to the TLR-9 ligand CpG. Whereas CpG induces proliferation in UM CLL it induces apoptosis in M CLL cells obtained from peripheral blood (PB). Yet, PB CLL cells are generally not representative of the cycling fraction which resides in lymph node (LN) proliferation centers. In vitro CD40 stimulation of CLL cells enhances NF-κB activity, upregulates anti-apoptotic proteins Bcl-XL and Bfl-1 and results in chemoresistance. These characteristics are highly similar to, and provide a model for, the situation in LN. In the present study we analyzed the effect of CD40 ligation on CpG responses of both IgVH M and UM CLL cells.Methods An in vitro co-culture system of PB CLL cells with 3T3 fibroblasts expressing human CD40L was employed to which soluble CpG was added. Cells, RNA and protein fractions were analyzed with respect to:apoptosis, proliferation and drug sensitivity andNF-κB signaling and downstream targets.Results In both prognostic subgroups 72 hours of CD40 stimulation results in chemoresistance to different classes of drugs (fludarabine, velcade and roscovitine), as we reported before. However, an important difference in chemoresistance was seen when soluble CpG was added to the culture; UM CLL cells remained drug resistant, but M CLL cells now became sensitive. A corresponding dichotomy in NF-κB signaling occurred upon stimulation with CD40L/CpG compared to CD40L alone. M CLL cells showed gradually declining classical and alternative NF-κB activation and the downstream target Bcl-XL, whereas in UM CLL cells NF-κB and Bcl-XL levels remained equal. Interestingly, after treatment with ABT-737, a BH3 mimetic which is very effective against Bcl-XL, also UM CLL cells became sensitive for cytostatic drugs underscoring the importance of anti-apoptotic Bcl-XL. Induction of TNF-α secretion, also a downstream effect of NF-κB activation, took place similarly in both subgroups upon stimulation with CD40L and CpG. We hypothesize that the source of the dichotomy NF-κB activation lays in upstream signaling determinants such as cIAP1/2, NIK and TRAF family members.Conclusions Our data uncover a novel distinction in NF-κB activation and drug susceptibility in mutated versus unmutated CLL cells in a relevant model for LN proliferation centers. Together, they provide a rationale for the development of novel drug regimens targeting the NF-κB pathway to treat UM CLL patients.

Minimal Residual Disease (MRD) and T/NK Cell Dynamics during Fludarabine, Cyclophosphamide Plus Rituximab (FCR) Followed by Fludarabine Plus Rituximab (FR) and Remission Maintenance Therapy with Rituximab in Previously Untreated B-Chronic Lymphocytic Leukemia (B-CLL): Riskfactor Stratification in the Chairos Study

Based on the efficacy of Fludarabine, Cyclophosphamide plus Rituximab (FCR) as first-line therapy for chronic lymphocytic leukemia (CLL), we conducted a non-randomized multi-center phase II trial with early brief intensive induction with FCR for 3 cycles followed by fludarabine/rituximab for 3 cycles. In addition patients received a 2 year 3-monthly rituximab maintenance regimen. The trial recruited 42 patients. Clinical baseline data, clinical response results and toxicity of a planned first interim analysis were reported previously (Egle et al, ASH 2007). We now present the data from flow cytometric minimal residual disease (MRD) monitoring during the study in combination with the performed risk factor analyses and report on the findings of detailed analysis of T-cell and NK cell dynamics during the induction phase of the trial. From the previously reported high number of CRs (94% after complete 6 cycle induction) 64% were MRD negative by flow cytometric definition (less than 10−3 G/L CLL cells). All non CR patients were correctly called as MRD positive. Less than 0.1 G/L CLL cells after 3 cycles of FCR identified all patients achieving MDR negativity after the complete induction regimen. CD30 risk, as well as age, FISH cytogenetic analysis and analysis of IgVH mutation status were analysed for their impact on MRD dynamics. We could not find any difference in MRD responses between CD38 high and low risk groups or between age groups (> and <65a). Neither did abberations found in FISH cytogenetics predict the MRD responses and all CLL patients harboring an 11q deletion achieved MRD negativity. Interestingly, we found the only predictive factor to be the IgVH mutation status with 15 unmutated CLL patients significantly less likely to achieve MRD negativity after induction treatment (p=0,025). Especially, three patients with rearranged IgVH 3–21 did poorly, suggesting that these patients may need a different therapeutic approach. Six months of Rituximab maintenance therapy were able to convert 37% of patients from MRD positive state after remission induction to a MRD negative state. T and NK cell numbers and ratios were followed during therapy. In addition T cell subsets and their CD38 expression status were analysed. While absolute T and NK/NKT cell numbers decreased dramatically during treatment (CD4 and CD8 cell numbers decreased to medians of less than 100/μl after 6 cycles of induction treatment), a count of less than 200 CD4 cells/μl after 3 cycles of FCR was found to be predictive of MRD negativity. In addition, we observed an earlier recovery of NK and NKT cells after the end of induction treatment leading to an inverted NK(T) to T cell ratio early during maintenance treatment. This may have implications for NK-dependent ADCC during rituximab maintenance therapy. Regarding the CD4 and CD8 compartment we observed in addition to the reduction in absolute numbers a marked shift of T cell subsets. Naïve T cells were more completely depleted by FCR/FR treatment than T cells from the memory compartment, with central memory (CM) CD4 cells increasing from a median of 49% to 79%. This was less pronounced in the CD8 compartment. CD38 expression on T cells was monitored, because our group found that this was predictive of CLL course (Tinhofer Blood 2006). Interestingly we found a marked increase of the proportional representation of CD38+ CD8 and CD4 T cells in the T cell compartment with a more pronounced effect observed in the CD8 compartment. CD8 T cell counts were also quicker to recover during rituximab maintenance therapy. While these data point to essential caveats regarding the immunosuppressive toxicity of the treatment, they may also point to a window of opportunity regarding the modification of T cell responses during the phase of immune reconstitution after FCR/FR therapy. In conclusion we find that only IgVH mutation status was predictive of achievement of MRD negativity and we could show significant skewing of T cell and NK(T) cell subsets during chemoimmunotherapy. A better understanding of these T/NK cell dynamics may lead to the development of improved targeting of the immune recovery phase after chemotherapy using immunomodulating maintenance strategies.

Phenotypic and Functional Features of Vγ9/Vδ2 T Cells in Chronic Lymphocytic Leukemia Are Strongly Correlated with the Mutational Status of the Immunoglobulin Heavy Chain Variable Region (IgVH) and Metabolic Activity of the Mevalonate Pathway in Tumor Cells.

Vγ9/Vδ2 (γδ) T cells have an important effector and regulatory role in innate and adaptive immunity against microbes, stressed cells and tumor cells. They represent less than 5% of peripheral blood lymphocytes, but can be activated and expanded in vitro by aminobisphosphonates (ABP) like zoledronic acid (Zol), as surrogates of their natural ligands. In this study, we have investigated the proliferative response of γδ T cells to Zol in 59 patients with chronic lymphocytic leukemia (CLL) at diagnosis. Proliferation of γδ T cells was observed in 30 patients (51%)(responders, R), whereas 29 patients (49%) were non-responders (NR). γδ T-cell subset distribution [e.g., naive (TN), central memory (TCM), effector memory (TEM), and terminally differentiated effector memory (TEMRA)] was well balanced in R patients, whereas TEM and TEMRA were largely predominant in NR patients. TEMRA of NR patients had a unique profile of NK receptors expression, characterized by the expression of the ILT4 inhibitory receptor, whereas R patients tended to have an higher expression of the costimulatory molecule NKG2D. Notably, HLA-G, a negative prognosticator in CLL and a well known ILT4 ligand, was more expressed in tumor cells of NR patients. This correlation prompted us to determine whether other tumor cell features were different in R and NR patients. Indeed, the latter showed significantly higher tumor cell counts, higher frequency of poor-risk cytogenetic abnormalities, and a significantly shorter median time to first treatment. Lastly, a very significant association was observed with the mutational status of the tumor immunoglobulin heavy chain variable region (IgVH), which was mutated (M) in 96% of R patients, and unmutated (UM) in 74% of NR patients. To gain further insight into this association, we evaluated the activity of the mevalonate pathway in tumor cells of M and UM patients. This pathway, that can be targeted by Zol, generates the phosphoantigens naturally recognized by γδ T cells. Bioinformatic and biochemical approaches showed that this pathway is more active in tumor cells of UM than M patients. Based on these data, we propose that tumor cells of UM CLL patients can more easily engage γδ T cells and, in the long run, exhaust their immune surveillance potential by driving their differentiation into functionally impaired TEMRA. In conclusion, we have identified a novel mechanism of immune escape which can contribute to the disease progression in UM CLL patients.

Rituximab Infusion Two Months after Nonmyeloablative Transplantation Maintains B-Cell Disease Control with Minimal GVHD.

B cells are implicated in the pathophysiology of chronic GVHD, as evidenced by the association of Ab production against minor histocompatibility Ags with chronic GVHD, and rituximab efficacy in chronic GVHD. We hypothesize that anti-B cell therapy beginning 56 days after nonmyeloablative total lymphoid irradiation (TLI) and anti-thymocyte globulin (ATG) transplantation reduces chronic GVHD. Cognizant of possible GVL loss with B cell depletion, we treated patients with chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) because any loss of GVL may be offset by rituximab's direct anti-B cell tumor efficacy.Patients were conditioned with TLI (80 cGy in 10 fractions, d-11 to d-1) and ATG (1.5mg/kg/day, d-11 to d-7, total 7.5mg/kg). PBSC were infused on day 0. Primary GVHD prophylaxis was MMF from day 0 until d28 for MRD, d100 for URD and cyclosporine (CSA) tapered off by 6 months. To diminish donor B cell alloimmunity, rituximab (375 mg/m2) was infused on days 56, 63, 70, and 77 post-transplant.Fourteen patients have received HCT, and 11 (median age 57, range 46–65 yrs) have received four rituximab infusions (5 fludarabine-refractory, unmutated immunoglobulin heavy-chain variable region gene (IgVH) CLL; 6 MCL). Median follow-up is 290 days (108–381 days). No infusion related toxicities occurred. Transient leukopenia in all patients and frank neutropenia in one patient was noted as early as 30 days post-rituximab. There was one graft loss at day 50 in a patient with MCL progression. Only 3/11 patients had peripheral blood CD3 donor chimerism >95% before rituximab infusion day 56, but 6/7 patients achieved full donor chimerism post-rituximab day 180, showing rituximab has no detrimental effect on engraftment. Peripheral blood CD19+ B cells were undetectable in all patients days 90–270, and 2/2 evaluable patients at one year now have CD27+ CD19+ donor B cell reconstitution. Infections post-transplant include early CMV reactivation (5), influenza B (1), aspergillus sinusitis (1), ecthyma gangrenosum (1), VZV (1). All infections resolved. Non-relapse mortality at 100 days and currently is 0%. Four of the 5 unmutated IgVH CLL patients with diffuse lymphadenopathy and marrow involvement by 4-color flow and IgVH quantitation prior to HCT have achieved molecular CR by IgVH PCR. One CLL patient progressed 9 months after HCT and has received DLI. Four MCL patients were transplanted in PR; one achieved CR, 3 progressed before rituximab infusion and 2 subsequently died. The third progressed MCL patient responded to salvage chemotherapy with development of chronic GVHD and achieved CR. No patients developed acute GVHD. One patient developed chronic GI GVHD that resolved with six weeks of steroids.Rituximab infused 56–77 days after TLI-ATG is well tolerated and able to modulate donor B cell reconstitution without detrimental effect on engraftment or infection incidence. No acute GVHD occurred. This novel approach of rituximab infusion two months after TLI-ATG HCT provides safe in vivo peripheral donor B cell depletion with low chronic GVHD incidence thus far. Importantly, GVL is maintained with four CLL and two MCL patients achieving molecular CR after HCT.CLL/MCLMed AgeMed f/uMRD/URDGVHDCD3>95% Donor ChimerismDisease status pre-HCT»last f/u5/657 yrs290 days9/2aGVHD- none cGVHD- one,d257D56 pre-rituximab: 3/11 pts>95%; D180: 6/7 pts>95%CLL: 4 PR»CR; 1PR»PD–DLI; MCL: 1PR»CR; 3PR»PD-2 deaths; 2CR»CR