Stable Chronic Lymphocytic Leukemia Research Articles

Multiplatform Profiling Characterizes Functional Networks in Genomically Stable and Instable Chronic Lymphocytic Leukemia

Multiplatform Profiling Characterizes Functional Networks in Genomically Stable and Instable Chronic Lymphocytic Leukemia

False, Reversed but Not True: A Curious Case of Hyperkalemia

Falsely elevated potassium levels are common in routine laboratory tests and should be differentiated from true hyperkalemia. If the patient is inappropriately treated for hyperkalemia, the resulting hypokalemia can lead to life-threatening cardiac arrhythmias. We present the case of a 67-year-old woman with a past medical history of stable chronic lymphocytic leukemia, who presented for chest pain and had an elevated potassium level of 5.8 mEq/L, which, upon repeat laboratory testing, was then 6.7 mEq/L. She was initially treated for hyperkalemia. Laboratory test results showed creatine kinase levels at 43 U/L, lactate dehydrogenase levels at 177 U/L, phosphorus levels at 4.5 mg/dL, and uric acid levels at 6.4 mg/dL, indicating no evidence of tumor lysis syndrome. The patient was later diagnosed with reverse pseudohyperkalemia, indicated by falsely elevated plasma potassium levels in the presence of serum potassium levels within normal limits and venous blood gas samples.

Out of sight: a lesson in drug errors

A 76-year-old man developed recurrent encephalopathy, visual disturbance, myoclonus, generalised seizures and atonic drop attacks on a background of a gastrectomy for adenocarcinoma and stable chronic lymphocytic leukaemia. He presented...

Overexpression of SH2-Containing Inositol Phosphatase Contributes to Chronic Lymphocytic Leukemia Survival.

Balanced activity of kinases and phosphatases downstream of the BCR is essential for B cell differentiation and function and is disturbed in chronic lymphocytic leukemia (CLL). In this study, we employed IgH.TEμ mice, which spontaneously develop CLL, and stable EMC CLL cell lines derived from these mice to explore the role of phosphatases in CLL. Genome-wide expression profiling comparing IgH.TEμ CLL cells with wild-type splenic B cells identified 96 differentially expressed phosphatase genes, including SH2-containing inositol phosphatase (Ship2). We found that B cell-specific deletion of Ship2, but not of its close homolog Ship1, significantly reduced CLL formation in IgH.TEμ mice. Treatment of EMC cell lines with Ship1/2 small molecule inhibitors resulted in the induction of caspase-dependent apoptosis. Using flow cytometry and Western blot analysis, we observed that blocking Ship1/2 abrogated EMC cell survival by exerting dual effects on the BCR signaling cascade. On one hand, specific Ship1 inhibition enhanced calcium signaling and thereby abrogated an anergic response to BCR stimulation in CLL cells. On the other hand, concomitant Ship1/Ship2 inhibition or specific Ship2 inhibition reduced constitutive activation of the mTORC1/ribosomal protein S6 pathway and downregulated constitutive expression of the antiapoptotic protein Mcl-1, in both EMC cell lines and primary IgH.TEμ CLL cells. Importantly, also in human CLL, we found overexpression of many phosphatases including SHIP2. Inhibition of SHIP1/SHIP2 reduced cellular survival and S6 phosphorylation and enhanced basal calcium levels in human CLL cells. Taken together, we provide evidence that SHIP2 contributes to CLL pathogenesis in mouse and human CLL.

PF395 COMPLEX EVALUATION OF CELLULAR AND HUMORAL IMMUNITY IN PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA

Background:Chronic lymphocytic leukemia (CLL) is typically associated with a significant immune deficiency resulting in elevated risk of infections ‐ the leading cause of death in CLL patients (pts). However, many specific mechanisms contributing to suppression of immunity in CLL are not yet well understood.Aims:Comprehensive assessment of immunological parameters in patients with CLL.Methods:We prospectively enrolled 126 CLL pts: 42 with clinically stable disease not requiring treatment (median age, 65 years; males, 50%; low/medium/high modified Rai risk, 59/36/5%) and 84 with CLL progression indicated for therapy (median age, 68 years; males, 64%; low/medium/high modified Rai risk, 0/43/57%). In addition, parameters of immunity were compared before and after first‐line chemoimmunotherapy in 39 pts (FCR, n = 13; BR, n = 15; G‐CLB, n = 5; R‐CLB, n = 6). Control group consisted of 46 healthy blood donors (median age, 40 years; males, 59%). Cellular subpopulations (CLL cells, T cells, Th and Tc cells, NK cells and polyclonal B cells) were quantified by flow cytometry (cytometer Navios, Beckman Coulter). Humoral immunity was evaluated by quantification of serum immunoglobulin concentrations (total IgG, IgG subclasses 1–4, IgA 1–2 and IgM) using Immunochemistry Image System (Beckman Coulter). Activation of complement was assessed by Wieslab Complement System Screen (Euro Diagnostica). Serum concentrations of immunity‐regulating cytokines (IL‐4, IL‐5, IL‐6, IL‐10, IL‐17A, IL‐17F, IL‐33 and IFN‐γ) were determined by xMAP multiplex technology (Luminex).Results:Serum IL‐4, IL‐6 and IL‐10 were elevated in CLL compared to controls (all p <0.0001). Polyclonal B cells were decreased (p < 0.0001) whereas Tc cells were increased (p = 0.019 and p < 0.0001) in stable and progressive CLL in comparison to controls. Both CLL subgroups had significantly lower IgG, A and M when compared to controls (all p < 0.0001). Progressive CLL pts had significantly higher numbers of NK cells (p = 0.0003) and stable CLL pts significantly higher IFN‐γ (p = 0.01) vs controls. Pts with progressive CLL had significantly lower concentrations of IgG, A, and M (p = 0.0012, p < 0.0001 and p = 0.027) than stable pts. Chemoimmunotherapy resulted in significant decline of polyclonal B cells, NK cells, Th cells, Tc cells and T regs (all p < 0.0001) but serum IgA and complement activation (both classical and alternative pathway) increased after therapy (p = 0.048, p = 0.002 and p = 0.0017).Summary/Conclusion:Conclusions: The present study identified multiple defects in cellular as well as humoral immunity in CLL pts. Clinical progression of CLL was associated with further decline of immune parameters. Cytoreductive therapy resulted in significant decrease in all physiological lymphocyte subpopulations; however, several markers of humoral immunity improved after treatment. The enrollment into the study continues and further analyses are underway including assessment of association of immune defects with infections and changes in immunological parameters over longer follow‐up. Supported by project PROGRES Q40/08, by DRO Ministry of Health (UHHK, 00179906) and by League Against Cancer Prague.image

Assessing the potential of immunotherapy in treating chronic lymphocytic leukemia through meta-analysis.

7531 Background: Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults and has a heterogenous presentation. CLL is known to shape the immune response to survive. Studying these processes will help gauge the potential success of immunotherapy and point to therapeutic targets. Methods: We used our Search, Tag, Analyze, Resource platform to meta-analyze patient samples from Gene Expression Omnibus. We tagged peripheral B cells from 741 CLL patients and peripheral B cell samples from 150 healthy donors as a control. We also tagged and compared B cell samples from 84 CLL progressors to 91 patients with stable CLL. Lastly, we tagged peripheral T cells from 70 CLL patients and T cells from 35 healthy donors as a control. We then analyzed the signature in Ingenuity Pathway Analysis. Results: Analysis of CLL cell samples identified T cell exhaustion signaling as our top canonical pathway. IL2, IL5, and TGFB1 were top upstream regulators. We found upregulation of PDL1, CTLA4, and Lag3, known markers for immunosuppressive B cells. FMOD, which sequesters TGFB, was also upregulated along with molecules that modulate BCR signaling such as MIR155HG. EBF1, required for B cell differentiation, and the co-stimulatory molecule CD80 were downregulated. Analysis of progressing CLL versus stable CLL highlighted metabolic changes. S-adenosyl-L-methionine biosynthesis, methionine degradation to homocysteine, cysteine biosynthesis, and acetate conversion to acetyl-CoA were top canonical pathways. No difference was seen in PDL1, CTLA4, and Lag3 expression but EBF1 was upregulated. Lastly, our T cell analysis demonstrated NFAT in regulation of the immune response as the top canonical pathway. Conclusions: Our results reinforce the promise immunotherapy can have in treatment of CLL and suggests more aggressive cases of CLL are a function of metabolic changes as opposed to differences in immune escape. We also suggest a role of NFAT in T cell exhaustion in the context of CLL.

Cytotoxic CD4+ Cells in Chronic Lymphocytic Leukaemia: An Extended Immunophenotypic Analysis Examining Their Association with Cytomegalovirus Serostatus and Similarities with Cytotoxic CD8+ Cells

Introduction:CD4+ T lymphocytes play a fundamental role in the adaptive immune system and traditionally their principle function has been seen as one of a ‘helper’ cell, interacting with and activating fellow immune cells to orchestrate and regulate the degree of immune response. There is a subset of CD4+T lymphocytes, however, that have the ability to acquire a cytotoxic phenotype. These cytotoxic CD4+ cells have been shown to have anti-viral functions but also they have previously been reported to exhibit anti-tumour effects in murine models of solid organ malignancies e.g. melanoma.The aim of this research was to identify, quantify and characterise cytotoxic CD4+ cells (CD4+ CTLs) in patients with Chronic Lymphocytic Leukaemia (CLL), to examine their association with cytomegalovirus (CMV) and to investigate the phenotype differences of the CD4+ CTLs isolated from patients who were CMV seropositive and seronegative.Methods:The peripheral blood (PB) from 90 patients with stable CLL (that was not requiring therapy) was collected after ethical approval and consent had been obtained. Flow cytometry was performed to identify and quantify the level of cytotoxic CD4+ cells (as defined as CD4+ Granzyme B+ cells) and to characterise the extended immunophenotype of the cells including transcription factors (including GATA-3, FoxP3, Eomes and Tbet) and co-inhibitory and co-stimulatory molecules (including LAG-3, TIM3, CTLA-4, PD-1, ICOS, OX40 and 4-1BB).In a subset of patients, serial samples of PB were collected to assess whether the quantity of cytotoxic CD4 cells changed over time in untreated CLL patients.Mass cytometry was performed to detect a broad range of surface molecules and to compare the extended phenotype of CD4+ (including CD4+ CTLs) and CD8+ cells in patients who were CMV seropositive and CMV seronegative in 14 patients with untreated CLL.An analysis of the CLL tumour environment was performed using multi-colour immunohistochemistry to detect CD4+ CTLs in paired bone marrow trephine and lymph node biopsies from a subset of patients. The expression of co-inhibitory and co-stimulatory molecules on the CD4+ CTLs was also examined in the bone marrow and lymph node biopsies.Results:The median age of the patients was 67 years (range: 41-90 years) with 29 females and 61 males. Fourteen patients had previously required treatment for CLL but were not currently on treatment and 76 patients had never required treatment.There was a significant increase in the percentage of CD4+ CTLs in the PB of those patients who were CMV seropositive when compared to those who were CMV seronegative (P<0.0001). Forty-five patients had PB samples collected at two different time-points and the median time between the two samples was 189 days (range: 7-735 days). The median difference in CD4+CTL values between the 2 samples for this group of patients was 0.19% (range: -25 - 46%).The extended immunophenotyping of the CD4+ CTLs showed that they had a phenotype more similar to cytotoxic CD8+ cells than non-cytotoxic CD4+ cells, with respect to both their transcription factor and checkpoint molecule expression.The mass cytometry analysis showed that there were significant differences in the expression of surface markers on CD4+ CTLs present in the PB of patients who were CMV seropositive compared with those who were CMV seronegative.The CD4+ CTLs were identified in both the bone marrow and lymph node biopsies. There was a significantly higher percentage of CD4+CTLs in the bone marrow trephine biopsies than in the lymph node biopsies (P=0.0068).Conclusions:This research identifies the presence of CD4+ CTLs in the PB, bone marrow and lymph node biopsies of patients with CLL. The transcription factor profile of CD4+ CTLs was more similar to cytotoxic CD8+ cells than non-cytotoxic CD4+ cells. Although a significantly higher percentage of CD4+CTLs was identified in patients who were CMV seropositive, CMV seronegative patients still retained a measurable population of CD4+ CTLs. The CD4+ CTLs in patients who were CMV seronegative had phenotypic differences when compared with CD4+ CTLs isolated from CMV seropositive patients, which may indicate an alternative, non-anti-viral function. Further research is required to explore whether the alternative function of CD4+ CTLs in CLL is an anti-tumour effect and to investigate the potential to harness them for therapeutic benefit in anti-tumour immunotherapies. DisclosuresNathwani:Freeline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BioMarin: Consultancy, Patents & Royalties; UniQure: Patents & Royalties.

Activating MAPK Pathway Mutations Mediate Primary Resistance to PI3K Inhibitors in Chronic Lymphocytic Leukemia (CLL)

Resistance to PI3K inhibitors including idelalisib remains poorly understood. The present study was designed to identify mechanisms underlying PI3K inhibitor resistance in CLL through WES, RNAseq and biochemical confirmation. We performed WES on matched tumor and germline samples from 28 relapsed CLL patients treated with PI3K inhibitors (idelalisib, 79% (22/28), pilaralisib 10.7% (3/28), voxtalisib 10.7% (3/28)). The median number of prior therapies was 4.5 and the adverse cytogenetic aberrations del(17p) and del(11q) were seen in 21% and 32% respectively. A median of two longitudinal tumor samples were sequenced per patient (Nsamples= 68). An average of 23 ± 12 (range = 4-58) non-silent somatic mutations were identified in the initial sample. The non-silent mutation rate was 0.64 ± 0.32 per MB (range = 0.11-1.61) and typical of CLL. Significantly mutated genes (based on MutSig2CV) included SF3B1 36%, TP53 21%, DDX3X 14% and BRAF 11%. The cohort was sub-divided into responders (R) (Ncases= 18, Nsamples= 47) and non-responders (NR) (Ncases= 10, Nsamples= 21). Among the 10 NR, 7 had been treated with idelalisib and 3 with voxtalisib. Comparison of the mutational landscape between R and NR showed that, notably, mutations in BRAF(N=5), MAP2K1(N=2), KRAS(N=2),XPO1(N=1), PLEKHA1(N=1), INPPL1(N=1) and NXF1(N=1) were exclusively found in NR. Moreover, MAPK pathway genes (KRAS, BRAF and MAP2K1) were the only recurrent somatic mutations present exclusively in NR. We inferred the cancer cell fraction (ccf) for each somatic mutation using ABSOLUTE and found that the ccf values for MAP2K1 (p=0.01), XPO1 and PLEKHA1 mutations increased with time, indicating a positive selection pressure. Phylogic analysis of 6 R and 4 NR with at least 3 serial samples identified a median of 2 dynamic subclones (change of at least 10% between any two timepoints) and showed enrichment of the “Reactome_RAF_MAP_Kinase_Cascade” pathway in NR (p=3x10-4, q=0.04). In addition, Gene Set Enrichment Analysis comparing RNAseq samples drawn prior to or after PI3K therapy in five of the patients from our cohort (1 primary NR and 4 progressors after response) demonstrated activation of the “Reactome_Prolonged_ERK_Activation_Events” pathway at the later time-points (p=2.3x10-4, q= 0.03).Given these findings, we further investigated the role of the ERK/MAPK pathway in idelalisib resistance. Western blot analysis of ERK1/2 phosphorylation in ex vivo idelalisib treated PBMCs from an initially responding patient who acquired resistance later showed that idelalisib failed to inhibit ERK1/2 phosphorylation only at the resistant timepoint. This persistent ERK1/2 activation was inhibited by combination treatment with the MAP2K1 inhibitor CI-1040. We then profiled pAKT and pERK relative to total, by western blot on ex vivo idelalisib-treated samples (5 R, 5 NR). Idelalisib treatment ex vivo inhibited AKT phosphorylation in both R and NR, but no reduction in ERK phosphorylation was seen in NR, in contrast to R.Based on these findings we hypothesized that increased or persistent ERK activity confers resistance to PI3K inhibitors and tested the impact of MAP2K1 mutations on ERK activity. Among NR, we identified two different mutations in MAP2K1, E203K and Q56P (2/10 NR vs 0/18 R), both previously described as activating in solid tumors. In a separate cohort of multiply relapsed CLL patients, we found 4 cases of MAP2K1 F53L mutations, also previously reported in solid tumors. We studied the functional significance of these MAP2K1 mutations by generating stable MEC1 CLL cell lines expressing HA tagged E203K, F53L or Q56P MAP2K1 mutants. We found that all mutant cell lines had elevated basal ERK1/2 phosphorylation (E203K p<0.001; F53L p<0.001; Q56P p<0.001). Moreover, idelalisib did not inhibit IgM-induced ERK1/2 phosphorylation in the mutants when compared to the controls expressing wild type MAP2K1 (E203K p<0.001; F53L p<0.001; Q56P p<0.001). Preliminary results using the ERK1/2 inhibitor SCH772984 showed that the combination of SCH772984 along with idelalisib effectively decreased ERK1/2 phosphorylation in the mutant cell lines compared to idelalisib alone.Taken together, these data implicate the ERK pathway in idelalisib resistance and suggest that ERK1/2 inhibitors either combined with idelalisib at therapy start or added to idelalisib at early progression might sensitize patients to PI3K delta therapy. DisclosuresBrown:Acerta / Astra-Zeneca: Membership on an entity's Board of Directors or advisory committees; Invectys: Membership on an entity's Board of Directors or advisory committees; Sunesis: Consultancy; TG Therapeutics: Consultancy; Morphosys: Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Consultancy; Gilead: Consultancy, Research Funding; Celgene: Consultancy; Genentech: Consultancy; Pharmacyclics: Consultancy; Abbvie: Consultancy; Verastem: Consultancy, Research Funding; Beigene: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Sun Pharmaceutical Industries: Research Funding; Loxo: Consultancy; Boehringer: Consultancy.

RPS15 mutations Repress mRNA Translation in Chronic Lymphocytic Leukemia Cells

We and others recently reported mutations within the RPS15 gene, encoding a component of the 40S ribosomal subunit, in clinically aggressive chronic lymphocytic leukemia (CLL). RPS15 mutations resided within an evolutionary conserved region, alluding to an oncogenic rather than a tumor-suppressor role. Our pilot functional analysis revealed that, similar to other ribosomal proteins (RPs), RPS15 also binds MDM2 and may impact on the p53 response. Here, we performed ribosome profiling in order to gain global insight into changes in translation induced by RPS15 mutations in CLL cells. This technique involves measuring translational efficiency (TE), by comparing the levels of ribosome-associated mRNA footprints against the total mRNA for each gene. For 6 CLL cases bearing mutant (mut, n=3) or wildtype (wt, n=3) RPS15, we obtained both ribosome-protected footprints (RPFs) and matching mRNA sequencing data. In parallel, we created stable MEC1 CLL cell lines expressing an additional copy of wt or mut RPS15 (131S) by lentiviral transduction; validation of the transgene expression was performed by Sanger sequencing of amplified cDNAs. Ribosome footprinting and subsequent library preparation of RPFs and total mRNA for all samples was performed with the Illumina Truseq Ribo Profile Kit and all libraries were sequenced on a NextSeq500 instrument. Reads were aligned to the human hg19 genome using Bowtie2. SystemPipeR was used to determine the percentage of reads mapping to 5‘ UTRs, CDS, and 3‘ UTRs and triplet periodicity was assessed using RibORF. The RPFs were of high quality, as assessed by expected RPF size (28-30nt), CDS enrichment, and triplet periodicity.To determine differentially expressed genes between RPS15-mut vs RPS15-wt cases we used DESeq2 while, for differentially translated genes we used Xtail. Changes in transcription and translation between PRS15-wt vs RPS15-mut cases showed limited overlap in both primary CLL cells and cell lines (12.8% and 12.9%, respectively), indicating the potential of ribosome profiling to reveal additional information compared to RNA sequencing alone. In primary CLL cells, 474 genes showed differences only at the transcription level (log2FC mRNA>I1I, p<0.05), while 742 genes were modulated only at the translation level (log2FC RPF>I1I, p<0.05). We identified 322 genes with differential TE (log2FC TE<I1I, p<0.05) between PRS15-wt vs RPS15-mut CLL cases; 262/322 (81%) showed reduced TE in RPS15-mut versus RPS15-wt cases. Similar analysis for the stable MEC1 cell lines revealed 749 genes displaying differences only at the transcription level (log2FC mRNA>I1I), while 1859 genes were regulated only at the translation level (log2FC RPF>I1I). Overall, 771 genes displayed differential TE (log2FC TE<I1I, adjusted p<0.1) between PRS15-wt vs RPS15-mut MEC1 cell lines; 48% of the genes showed reduced TE in mut vs wt cell lines and the remaining 52% augmented TE. The slightly different results compared to those obtained from primary CLL cells, may be attributed to the following reasons: (i) MEC1 cells are TP53-aberrant; (ii) the PRS15-wt cell line overexpresses the RPS15 gene compared to primary CLL cells; and,( iii) the RPS15-mut cell line expresses both the wt and mut RPS15 mRNAs (22% of the mapped reads correspond to the mut RPS15 and 78% to the wt gene, respectively). Gene ontology analysis (Enrichr) of the genes showing differential TE, revealed that in both primary CLL cells and MEC1 cell lines a large fraction of the deregulated transcripts is implicated in RNA binding processes (adj-p=0.0001; adj-p=1.98X10^-13, respectively) which are known to induce translational repression. Interestingly, in primary CLL cells, amongst genes with reduced TE we identified genes implicated in tRNA biosynthesis, protein processing in the endoplasmatic reticulum and the Hippo signaling pathway (p<0.01). Additionally, enrichment analysis revealed that a proportion of genes with reduced TE were targets of the MYC transcription factor (adj-p=0.0005). RP genes, despite unchanged mRNA levels, showed changes in RPF levels and differential TE, suggesting that RPs are also deregulated at the translational level. In conclusion, we show that RPS15 mutations rewire the translation program of CLL cells by reducing the TE of critical molecules, including translation initiation factors and other regulatory elements. DisclosuresHadzidimitriou:Abbvie: Research Funding; Gilead: Research Funding; Janssen: Honoraria, Research Funding. Stamatopoulos:Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Gilead: Honoraria, Research Funding.

B cells and macrophages pursue a common path toward the development and progression of chronic lymphocytic leukemia.

The development and progression of chronic B-cell tumors depend on a complex microenvironmental network of cells that include monocyte-derived macrophages. In chronic lymphocytic leukemia (CLL) the survival of malignant cells is supported in vitro by nurse-like cells, which differentiate from CD14+ monocytes and have been identified as tumor-associated macrophages (TAMs). The role of the monocyte/macrophage lineage in CLL has been extensively studied in vitro, but only recently has been investigated in in vivo models. We here discuss how the cellular and molecular interactions that physiologically occur between B cells and macrophages can be subverted in chronic B lymphoid malignancies. Clinical approaches for the therapeutic targeting of TAMs are under evaluation. Promising strategies, along with a direct impact on the malignant cells, affect crucial pathways involved in the interaction of leukemic cells with TAMs. As an example, ibrutinib reduces CLL cell chemoattraction by inhibiting macrophage secretion of CXCL13. Lenalidomide and trabectedin prevent TAM recruitment mainly through CCL2 blockade. Most advanced strategies aim at depleting macrophages by targeting the CSF1/CSF1R pathway, which is fundamental for TAM survival. Of note, CSF1 transcripts are significantly more abundant in progressive CLL patients when compared with stable CLL and the frequency of CSF1R+ TAMs correlates with poor survival in hematological malignancies. The successful combination of CSF1R inhibition with currently available agents targeting malignant cells might represent the next therapeutic frontier in CLL. Conceivably these approaches may become applicable to numerous chronic B lymphoid malignancies.

Haemophagocytosis and encephalitis in chronic lymphocytic leukaemia

A 77-year-old man was hospitalized with progressive confusion and gait disorder of 3 weeks duration. He had been diagnosed with quiescent chronic lymphocytic leukaemia (CLL) 5 years previously without having required treatment. Examination showed fever, confusion, a right hemiparesis and ataxia. A full blood count showed: leucocytes 24·3 × 109/l (neutrophils 1·4 × 109/l, lymphocytes 22·6 × 109/l), haemoglobin concentration 110 g/l and platelet count 57 × 109/l. Serum ferritin was increased to 669 μg/l (normal < 400). Other laboratory tests, including fibrinogen concentration, triglycerides, soluble CD25, lactate dehydrogenase and transaminases levels, were normal. Magnetic resonance imaging (MRI) of the brain showed multiple hyperintensities on fluid-attenuated inversion recovery (FLAIR) sequences spanning the periventricular white matter, corpus callosum, basal ganglia, left internal capsule, midbrain and cerebellum (left). There was a subtle perivascular enhancement on gadolinium. Cerebrospinal fluid (CSF) analysis showed pleocytosis (140 cells/μl) with 85% lymphocytes, a high protein of 0·74 g/l and normal glucose. Despite an extensive workup, no infectious aetiology was identified on CSF testing. Blood cultures were repeatedly negative. Epstein‒Barr virus (EBV) polymerase chain reaction (PCR) on peripheral blood showed 1477 copies/ml and a low-grade EBV reactivation was diagnosed. PCR on the CSF for other herpes virus, flavivirus, enterovirus and EBV was negative. A bone marrow aspirate was hypercellular with a normal number of megakaryocytes and stable CLL. Histiocytes were seen (<0·5% of cells), of which 80% were observed to have engulfed mainly numerous CLL lymphocytes (top right), but also erythroid cells (bottom right). Further diagnostic images are shown in Appendix S1. Whole body positron emission tomography‒computerized tomography (PET-CT) scanning showed no change in the number, size or metabolic activity of lymph nodes or spleen. Vaccination against influenza 1 week before the clinical onset led us to consider a diagnosis of acute disseminated encephalomyelitis (ADEM). The patient was started on intravenous corticosteroids then intravenous immunoglobulin, with progressive clinical, CSF and MRI improvement and normalization of platelet count within 2 weeks. Histiocytes engulfing numerous lymphocytes is an unusual observation. It has rarely been associated with haemophagocytic lymphohistiocytosis (HLH) in CLL. However, the findings of this patient do not meet the diagnostic criteria of HLH. ADEM is a potentially severe inflammatory demyelinating immunomediated cerebral condition triggered by infection or vaccination. ADEM must be differentiated from infectious encephalitis as the treatment is different. CLL is associated with various immunological complications but the coincidence of haemophagocytosis and ADEM is rare. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

To treat or not to treat: metabolomics reveals biomarkers for treatment indication in chronic lymphocytic leukaemia patients.

In chronic lymphocytic leukaemia (CLL), the clinical course of patients is heterogeneous. Some present an aggressive disease onset and require immediate therapy, while others remain without treatment for years. Current disease staging systems developed by Rai and Binet may be useful in forecasting patient survival time, but do not discriminate between stable and progressive forms of the disease in the early stages. Recently ample attention has been directed towards identifying new disease prognostic markers capable of predicting clinical aggressiveness at diagnosis. In the present study serum samples from stable (n = 51) and progressive (n = 42) CLL patients and controls (n = 45) were used with aim to discover metabolic indicators of disease status. First an LC-MS based metabolic fingerprinting method was used to analyse selected samples in order to find a potential markers discriminating aggressive from indolent patients. Ten of these discovered markers were validated on the whole set of samples with an independent analytical technique. Linoleamide (p = 0.002) in addition to various acylcarnitines (p = 0.001–0.000001) showed to be significant markers of CLL in its aggressive form. Acetylcarnitine (p = 0.05) and hexannoylcarnitine (p = 0.005) were also distinguishable markers of indolent subjects. Forming a panel of selected acylcarnitines and fatty acid amides, it was possible to reach a potentially highly specific and sensitive diagnostic approach (AUC = 0.766).

Prognostic Implications of PIM-2 Expression in Samples from Patients with Chronic Lymphocytic Leukemia and Impact in the Sensitivity to the Pan-PIM Kinase Inhibitor PIM447

Background and objectives. PIM kinases (PIM1, PIM2, PIM3) are proteins known to be overexpressed in several hematological malignancies. In particular, in chronic lymphocytic leukemia (CLL) they are involved in cell survival, resistance to apoptosis (especially PIM2 and PIM3) and interactions with the microenvironment (PIM1).The aim of this study was dual: I) to evaluate the preclinical efficacy of PIM447, a pan PIM kinase inhibitor, in CLL and to study potential synergies with other drugs; and II) to evaluate the expression of PIM-kinases in different stages of the disease and correlate it with the prognosis and the sensitivity to the drug.Methods. Peripheral blood samples from untreated patients with different stages of the disease (monoclonal B lymphocytosis (MBL), stable CLL not requiring treatment (sCLL), and active CLL requiring treatment (aCLL)) were collected after informed consent. The ex vivo efficacy of PIM447 was analyzed by flow cytometry with annexin V in these samples. Moreover, PIM447 efficacy was also analyzed in two cell lines (MEC-1 and JVM-2) by MTT assay. Synergy with other drugs effective in CLL (bendamustine and fludarabine) was evluated with the calcusyn software. Protein levels of PIM Kinase proteins were evaluated by capillary electrophoresis immunoassay (WESTM ProteinSimple) in monoclonal B cells purified by CD19 selection with anti-CD19 magnetic microbeads and the autoMacs Cell separator (both from Miltenyi Biotec) from a subset of patients.Results. The pan PIM inhibitor, PIM447 was active in both cell lines tested, MEC-1 (IC50 5μM) and JVM2 (IC50 7μM), and also in monoclonal B cells from freshly isolated patients samples (sCLL=11; aCLL=5), with no difference in sensitivity between the different stages of the disease (IC50 of 4,8 μM and 4,7 μM for sCLL and aCLL respectively).There was a clear therapeutic window as treatment with PIM447 at doses toxic for monoclonal B cells, preserved T lymphocytes (figure 1) (median % of apoptosis for B cells and T lymphocytes respectively of 23 vs 20 at 5μM and 87 vs 35 at 10 μM).Moreover, PIM447 demonstrated to potentiate the activity of both bendamustine and fludarabine, being especially synergistic with this last one (combination index 0.1-0.6).A second objective was to analyze PIM2 protein expression by western blot in monoclonal B cells from these samples and correlate it with clinical and biological features. Up to now, it has been evaluated in 18 samples (MBL=4; sCLL=8; aCLL=6,). All of them expressed PIM-2. Expression levels of this protein were significantly higher in active CLL as compared with indolent stages of the disease (p=0,012). Patients with an unmutated IGHV status also displayed higher levels of PIM2 (p=0,01). Finally, samples with high PIM2 levels were slightly more resistant to PIM447 as compared with samples with lower protein levels (IC50 of 7,7 μM vs 5 μM, respectively).We are currently completing the analysis of the PIM2 levels of remaining samples and we are also measuring the levels of PIM1 protein, what will be available at the meeting.Conclusions: PIM-Kinase inhibition with PIM447 is effective in vitro in CLL cell lines and ex vivo in samples from patients. It synergizes with other agents especially fludarabine. PIM2 protein levels correlated with the clinical activity of CLL and with the mutational state of IGHV. Although all patients appear sensitive ex vivo to PIM447, further work is required to define PIM2 expression as a marker of sensitivity. [Display omitted] DisclosuresOcio:Array BioPharma: Consultancy, Research Funding; Celgene: Honoraria, Research Funding; Amgen/Onyx: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Consultancy; Mundipharma: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; MSD: Research Funding; Pharmamar: Consultancy, Research Funding; Jassen: Honoraria.

To Treat or Not to Treat: Metabolomics Reveals Biomarkers for Treatment Indication in Chronic Lymphocytic Leukaemia Patients

In chronic lymphocytic leukaemia (CLL) clinical course of patients is heterogeneous. Some present an aggressive disease onset and require immediate therapy, while others remain without treatment for years. Current disease staging systems developed by Rai and Binet may be useful in forecasting patient survival time, but do not discriminate between stable and progressive forms of the disease in the early stages. Recently ample attention has been directed towards identifying new disease prognostic markers capable of predicting clinical aggressiveness at diagnosis. In this research we reached for LC-MS metabolic fingerprinting method to analyse serum of stable (n=51) and progressive (n=42) CLL patients and controls (n=45) with aim to discover metabolic indicators of disease status. A panel of markers discriminating aggressive from indolent patients was discovered. Ten of them were selected for validation on larger population (45 controls and 92 CLL) with an independent analytical technique. Linoleamide (p=0.002) in addition to various acylcarnitines (p=0.001-0.000001) showed to be significant markers of CLL in its aggressive form. Acetylcarnitine (p=0.05) and hexannoylcarnitine (p=0.005) were also distinguishable markers of indolent subjects. Forming a panel of selected acylcarnitines and fatty acid amides, it was possible to reach a highly specific and sensitive diagnostic approach (AUC=0.766). DisclosuresNo relevant conflicts of interest to declare.

Protein profiles distinguish stable and progressive chronic lymphocytic leukemia

Patients with a stable chronic lymphocytic leukemia (CLL) double their blood lymphocyte count in >5 years, but may develop progressive disease with lymphocytes doubling in <12 months. To identify a protein signature for progressive CLL, whole cell extracts of peripheral blood mononuclear cells from patients with CLL (n = 27) were screened using iTRAQ (isobaric tags for relative and absolute quantification) analysis. A total of 84 differentially abundant proteins were identified from patients with stable and progressive CLL. Subsequently, 32 of these proteins were quantified by SRM (selected reaction monitoring) using extracts of purified CD19+ CLL cells from patients (n = 50). Hierarchical clustering of these protein profiles showed two clusters of patients that correlated with progressive and stable CLL, providing signatures that should be useful for triaging patients. Some of the proteins in the progressive cluster have not been linked with CLL, for example, glutamate dehydrogenase 1 and transcription intermediary factor 1-beta.

Transcriptomic analysis of monocytes and macrophages derived from CLL patients which display differing abilities to respond to therapeutic antibody immune complexes.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia. While therapeutic antibodies show clinical activity in CLL patients, resistance inevitably develops resulting in treatment failure. Identifying mechanisms of antibody resistance and methods to reduce resistance would be valuable in managing CLL. Monocyte derived cells (MDCs), also known as nurse like cells (NLCs) in CLL [1], [2], are known to be crucial components of the CLL microenvironment network and following “maturation” in in vitro culture systems are able to provide support for the survival of the malignant B cells from CLL patients. In addition to their protective role, MDCs are key effector cells in mediating responses to therapeutic antibody therapies [3]. We have determined that macrophages from patients with early stable CLL are able to elicit superior cytotoxic response to therapeutic antibodies than macrophages derived from patients with progressive CLL. We have exploited this unique finding to gain insight into antibody resistance. Thus, we have profiled monocytes on day 0 and MDCs on day 7 from antibody sensitive and antibody resistant CLL patients (GEO accession number GEO: GSE71409). We show that there are no significant differences in transcriptomes from the monocytes or MDCs derived from sensitive or resistant patient samples. However, we show that MDCs acquire an M2-like macrophage transcriptomic signature following 7 days culture regardless of whether they were derived from sensitive or resistant patient samples.

Increased chronic lymphocytic leukemia proliferation upon IgM stimulation is sustained by the upregulation of miR-132 and miR-212.

To assess the involvement of microRNAs (miRNAs) in B-cell receptor (BCR) stimulation, we first evaluated miRNA profiling following IgM cross-linking in chronic lymphocytic leukemia (CLL) cells and in normal B lymphocytes. Second, we combined miRNA and gene expression data to identify putative miRNA functional networks. miRNA profiling showed distinctive patterns of regulation after stimulation in leukemic versus normal B lymphocytes and identified a differential responsiveness to BCR engagement in CLL subgroups according to the immunoglobulin heavy chain variable region mutational status and clinical outcome. The most significantly modulated miRNAs in stimulated CLL are miR-132 and miR-212. Notably, these miRNAs appeared regulated in progressive but not in stable CLL. Accordingly, gene profiling showed a significant transcriptional response to stimulation exclusively in progressive CLL. Based on these findings, we combined miRNA and gene expression data to investigate miR-132 and miR-212 candidate interactions in this CLL subgroup. Correlation analysis pointed to a link between these miRNAs and RB/E2F and TP53 cascades with proproliferative effects, as corroborated by functional analyses. Finally, basal levels of miR-132 and miR-212 were measured in an independent cohort of 20 unstimulated CLL cases and both showed lower expression in progressive compared to stable patients, suggesting an association between the expression of these molecules and disease prognosis. Overall, our results support a model involving miR-132 and miR-212 upregulation in sustaining disease progression in CLL. These miRNAs may therefore provide new valuable strategies for therapeutic intervention.

Role of Sprouty2 in the Pathogenesis of Chronic Lymphocytic Leukemia

INTRODUCTION: Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in adults representing about 35% of all adult leukemias in the United States. CLL is a B-cell neoplasm which is characterized by accumulation of long lived mature B-cells in peripheral blood (PB), bone marrow (BM) and lymph nodes (LN). We and others have reported that CLL cells in tissue microenvironments have constitutively activated MAPK-Erk and AKT signaling pathways, which promotes their proliferation and survival. However, the molecular mechanisms and the gene(s) responsible for the activation of these pathways in CLL are not clearly understood. We performed gene expression profiling of CLL cells from PB, BM and LN and transcriptome analyses of CLL cells from PB from good and poor prognosis patients. Our studies identified Sprouty 2 (Spry2), a negative feedback regulator as one of the key genes involved in the regulation of MAPK-Erk and Akt pathways in CLL. We observed that Spry2 levels were down-regulated in poor prognosis CLL cells. Interestingly, Spry2 is epigenetically silenced in most human and mouse lymphomas and ectopic expression of Spry2 induces apoptosis in mouse B cells. We categorized CLL patients into good and poor prognosis to further evaluate Spry2 in CLL biology. Poor prognosis patients were defined by unmutated immunoglobulin variable heavy chain (IgVH), 11q22 deletion, 17p deletion, trisomy 12 or high CD38 and Zap70 expression. Good prognosis patients were defined as having mutated IgVH, 13q14 deletions or low CD38 and Zap70 expression.RESULTS: We found that Spry2 expression was significantly decreased by 3.2 log2-fold, (p=0.0001) at transcript and protein levels, respectively, in CLL cells from patients with poor prognosis compared to patients with good prognosis. Spry2 is a negative feedback regulator which attenuates cell proliferation, migration and survival. To determine the effect of Spry2 on CLL cells we transfected Mec-1 cells (stable human CLL cell line) and primary human CLL cells with a Spry2 cDNA containing vector. Ectopic expression of Spry2 induced spontaneous apoptosis in Mec-1 cells and primary CLL cells from poor prognosis patients. These results indicate that Spry2 negatively regulates the survival of human CLL cells. CLL cells have been shown to have active B-cell receptor (BCR) signaling promoting proliferation in vivo. To study the effect of BCR stimulation on Spry2 we stimulated BCR on Mec-1 and primary CLL cells by BCR cross-linking. Unexpectedly, the rate of proliferation of Mec-1 cells decreased upon BCR stimulation. We were able to demonstrate a transient increase in Spry2 levels that peaked at 12 hours post BCR stimulation, which we suspected was mediating the decrease in proliferation observed. We therefore repeated the BCR stimulation experiment using siRNA to inhibit the transient increase in Spry2 in primary CLL cells from seven good prognosis patients and in the Mec-1 cells. As predicted, we observed a significant increase in proliferation in all primary CLL samples and in the Mec-1 cells upon Spry2 knock down following BCR stimulation. Spry2 attenuates ligand induced MAPK-Erk and AKT signaling leading to suppression of proliferation and survival. We also examined the effect of Spry2 on MAPK-Erk and Akt signaling in CLL cells. Upon induction of exogenous Spry2 expression in Mec-1 cells there was a significant decrease in p-Akt and p-Erk levels. Conversely, Spry2 knock down resulted in an increase in p-Erk and p-Akt and enhanced cell proliferation.CONCLUSIONS: Spry2 is significantly down-regulated in the B-cells from patients with poor prognosis CLL. Down-regulation of Spry2 in CLL cells leads to hyperactivation of MAPK-Erk and Akt signaling and exogenous and endogenous expression of Spry2 leads to spontaneous apoptosis in human CLL cells. These results identify Spry2 as a negative regulator of CLL cell survival and proliferation and provide a potential molecular target for therapeutic intervention. DisclosuresBociek:Seattle Genetics, Inc.: Research Funding.

Tumoral Maximum Standardized Uptake Value > 10 Measured on 18 f-FDG-PET: A New Valid Marker to Discriminate Richter Syndrome

Background:One of the main complications in CLL is Richter syndrome (RS). RS derives from the rare transformation of chronic lymphocytic leukemia (CLL) into an aggressive lymphoma, most commonly of the diffuse large B-cell lymphoma (DLBCL) type. RS occurs in 2.2% to 8% of patients with CLL and the prognostic is poor with a median survival from 5 to 8 months. Detection of RS by imaging has resulted in conflicting and non-significant results.Aim:The objective of this study was to validate recent findings correlating FDG/PET imaging and histological features in CLL and to demonstrate that a tumoral maximum standardized uptake value > 10 measured on 18F-FDG-PET is a new valid marker to discriminate RS.Results:From June 2006 through December 2012, 240 patients from the Division of hematology of Centre Hospitalier Lyon Sud and Créteil and from the Mayo Clinic Rochester have been analyzed with a mean age of 62 years (21-91). Clinical, histological (confirm by biopsy) and biological parameters have been identified with 10% of the patients as having RS, 34% stable CLL disease; 42% of rapid CLL progression (histological features of progression defined as increased large cell number, large confluent proliferation centers or high proliferation rate assessed by Ki-67 but not meeting criteria for diffuse large B-cell lymphoma/RS) and 14% with others diseases (e.g. infection and or cancers). For patients with stable CLL disease, the median tumoral SUV max was 2 (range: 0-2.4). Among patients with a rapid progression of CLL, 90% had a tumoral SUV max <10 but greater than the liver median SUV max (4.5 range: 1-11). In contrast, 90% of patients with RS had a median tumoral SUV max >10 (12.9; range: 5-27). A statistically significant difference between SUV max of CLL patients with stable disease and RS was observed (2.2 vs. 12.9; p< 0.0001) and similarly for SUV max of CLL patients with rapid disease progression and RS (4.5 vs 12.9; p< 0.0001). Regardless of the RS prevalence (2.2% to 8%), statistical tests identified a threshold of tumor SUV max > 10 as the more discriminating cut off. Using this threshold, the sensitivity and specificity of PET to identify RS in our cohort are 91% and 95% respectively. Assuming an RS prevalence of 2.2%, positive predictive value (PPV) and negative predictive value (NPV) using the >10 threshold were 28.7% and 99.8%; for an 8% prevalence of RS, the PPV and NPV are 60.8 and 99.2% respectively.The proportion of correctly classified patients with RS is more accurate using a threshold of tumoral SUV max > 10 than 5 (2.2% RS prevalence: 94.8% versus 71.8%; 8% RS prevalence: 94.6% versus 73.5%). Finally analysis of the area under the curve (AUC) reveals a value of 0.95 (95% confidence interval: 0.89- 0.99).Recently, Falchi et al, reported on 332 patients with CLL classified as 95 RS, 117 rapid progression and 120 stable disease and demonstrated a strong correlation between histological features and PET imaging. A SUV max ≥10 strongly correlated with a shorter overall survival. Similarly, in our study we have shown excellent sensitivity and negative predictive value estimated at 100%, revealing the ability of 18F-FDG-PET-CT to rule out the diagnosis of RS if the tumor SUV max is less than 10.Conclusion:18F-FDG-PET-CT is a valuable tool in the diagnostic evaluation of RS for patients with CLL. It is not only useful to identify RS syndrome and guide the site of biopsy but also to identify CLL patients who will experience more rapid disease progression. An SUV max > 10 is the optimal threshold to distinguish RS in CLL. DisclosuresNo relevant conflicts of interest to declare.

Cell surface phenotype profiles distinguish stable and progressive chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is clinically heterogeneous. While some patients have indolent disease for many years, 20–30% will progress and ultimately die of their disease. CLL may be classified by the Rai or Binet staging system, mutational status of the immunoglobulin variable heavy-chain gene (IGVH), ZAP-70 overexpression, cytogenetic abnormalities (13q−, + 12, 11q−, 17p−) and expression of several cell surface antigens (CD38, CD49d) that correlate with risk of disease progression. However, none of these markers identify all cases of CLL at risk. In a recent review, we summarized those CD antigens known to correlate with the prognosis of CLL. The present study has identified surface profiles of CD antigens that distinguish clinically progressive CLL from slow-progressive and stable CLL. Using an extended DotScan™ CLL antibody microarray (Version 3; 182 CD antibodies), and with refined analysis of purified CD19 + B-cells, the following 27 CD antigens were differentially abundant for progressive CLL: CD11a, CD11b, CD11c, CD18, CD19, CD20 (two epitopes), CD21, CD22, CD23, CD24, CD25, CD38, CD40, CD43, CD45, CD45RA, CD52, CD69, CD81, CD84, CD98, CD102, CD148, CD180, CD196 and CD270. The extensive surface profiles obtained provide disease signatures with an accuracy of 79.2%, a sensitivity of 83.9% and a specificity of 72.5% that could provide the basis for a rapid test to triage patients with CLL according to probability of clinical progression and potential earlier requirement for treatment.