Untreated Chronic Lymphocytic Leukemia Patients Research Articles

PS1134 CHRONIC LYMPHOCYTIC LEUKEMIA CELLS IMPAIR MITOCHONDRIAL FITNESS IN T CELLS AND IMPEDE CAR T CELL EFFICACY

Background:Chronic lymphocytic leukemia (CLL) patients acquire T cell dysfunction through a yet unresolved mechanism, impeding efficacy of immunotherapeutic strategies. As T cell function and development share an intricate relationship with T cell metabolism, we hypothesized that CLL cells impose a reduction in mitochondrial fitness and altered glucose metabolism on T cells, which may underlie the acquired T cell dysfunction.Aims:We aim to elucidate the underlying mechanism of T cell dysfunction in CLL and intend to resolve metabolic dysfunction via pharmacological intervention.Methods:We selected untreated CLL patients with a WBC greater than 20 × 109 cells/L. Age‐matched healthy donors (HD) were used as controls. CAR T cells were obtained from the University of Pennsylvania, generated from relapsed/refractory (R/R) CLL patients enrolled in two clinical trials of single‐agent CTL019 therapy (registered at clinicaltrials.gov; NCT01029366, and NCT01747486). We either directly analyzed T cells by flow cytometry or XF96 Seahorse, or after a 2 day culture with or without CD3 and CD28 antibodies.Results:We have previously shown that stimulated CLL derived CD8+ T cells had reduced expression of activation markers, increased expression of PD‐1 and reduced expression of glucose transporter GLUT‐1, coinciding with reduced glucose uptake. In addition we observed that mitochondrial biogenesis is impaired in CLL derived CD8+ T cells. Clinical significance of this data was further demonstrated by analyzing mitochondrial mass of CAR T cells from CLL patients showing complete response (CR) versus patients with no response (NR). Patients with CR had higher mitochondrial mass compared to NR. These findings support our hypothesis that CLL cells reprogram T cell metabolism, in particular mitochondrial fitness and biogenesis (Blood 2018, 132:235).In this follow up study we investigated (A) whether metabolic reprogramming is a pan T cell phenomenon or CD8 specific, (B) upstream signaling leading to metabolic reprogramming, and (C) if pharmacological intervention can boost T cell metabolism and thereby function. We found that (A) CD4 T cells share most metabolic alterations with CD8 T cells in CLL, and that (B) AKT signaling is disturbed in CLL T cells. For the metabolic interventions studies (C) we are currently exploring the effects of manipulating mitochondrial ROS or respiration, and blocking immune‐suppressive cytokines. Results of both approaches will be presented at the conference.Summary/Conclusion:We found that metabolic impairment in CLL T cells is not restricted to CD8 T cells, but also applies to CD4 T cells, and that impaired AKT signaling is at least part of the upstream bottleneck. We are currently investigating the use of compounds and metabolites in order to improve mitochondrial fitness of T cells for CLL patients to benefit autologous immunotherapies.

PB1914 PURGING WITH CHLORAMBUCIL TO PREVENT INFUSION-RELATED REACTIONS BEFORE OBINUTUZUMAB ADMINISTRATION: A MONOCENTRIC PILOT EXPERIENCE.

Background: The efficacy and safety of Obinutuzumab (G), in combination with chlorambucil (Clb-G) in patients with treatment-naïve chronic lymphocytic leukemia (CLL) and comorbidities was demonstrated in the CLL11 study by improved progression-free survival and overall survival compared with Clb plus rituximab (R) or Clb alone, leading to the FDA approval for its use in combination with Clb in patients with previously untreated CLL in 2013. Nevertheless, the Clb dose used in CLL11 was lower than the other European experiences of its use in associations with Rituximab, and, moreover, has emerged that the infusion of G was burdened by a high incidence of infusion-related reactions (IRRs). IRRs are among the worst complications of the frontline G-Clb treatment to deal with in urgency, complicating collectively up to 87% of the infusions (67% grade 1-2, 20% grade 3-4); reducing the disease burden before the administration of G could be a solution to prevent the occurrence of these adverse events (AE). Aims: Given the previous experience of our group, we used, in the same setting of patients as the CLL11, an increased dose of Clb in association with G, looking at safety and response. Methods: We retrospectively analysed a pilot cohort of ten untreated CLL patients (median age 73 years) with comorbidities, treated with G-Clb regimen. Chlorambucil 1 mg/Kg every 28 days was administered at a standard daily dose of 10 mg for 8 cycles, with 2 cycles of purging before the start of the G administration. Obinutuzumab was infused intravenously from the third cycle onwards according to the manufacturer. An independent-samples t-test was conducted to compare complete blood counts (CBCs) before and after the first 2 cycles of Clb. Results: At 2 months after the end of the therapy (median observation time of 10 months) the overall response rate (ORR) was 80%, with the partial responses (PR) accounting for the 60% and the complete responses (CR) for the remaining 20%. In the safety analysis, grade 3 or 4 treatment-emergent AE occurred in 50% of patients; most common were neutropenia (30%), thrombocytopenia (20%), infections (20%) and anaemia (10%). Three patients experienced grade 1-2 IRR (30%), but no grade 3-4 IRRs or clinical TLS have been detected in our cohort. One patient was hospitalized for AE (spondylodiscitis). When comparing CBCs before and after the first 2 cycles of Clb, we observed a statistically significant reduction of peripheral white blood cells and lymphocytes from a mean of 58900/mmc to 20270/mmc and 50310/mmc to 15710/mmc respectively (p = 0.034), while no significant differences were detected in haemoglobin levels, neutrophil or platelets counts. Two patients had to permanently cease treatment for AEs (pulmonary toxicity and spondylodiscitis). Two patients needed a dose reduction of Clb, and 1 both G and Clb. Median dose of Clb was 520 mg for each patient (210-640 mg) with median Clb of 78 mg for each cycle. Summary/Conclusion: Our real-life pilot analysis confirmed that the use of a Clb purging regimen is a valid option to minimize the IRR, since no grade 3-4 were observed. Haematological/extrahaematological toxicity and response rates were similar to already published experiences. These results are encouraging, but further studies with wider cohorts are needed to confirm them.

PF363 UNDETECTABLE EXPRESSION OF WNT5A, A GENE ENCODING A ROR1 LIGAND, DEFINES MEMORY B-CELL LIKE CHRONIC LYMPHOCYTIC LEUKEMIA

Background: Chronic lymphocytic leukemia (CLL) is a malignancy with highly variable disease course among patients. Although many approaches distinguishing patients with worse and better prognosis have been published, IGHV somatic hypermutation status remains one of the strongest prognostic predictors. However, even within prognostically favorable IGHV-mutated (M-CLL) group, there is a subset of “grey-zone” patients with worse prognosis. We have shown recently that expression of WNT5A, a gene encoding a ligand activating WNT/PCP signaling pathway via ROR1 receptor, distinguishes the cases with worse prognosis within M-CLL (Janovska, Clin Cancer Res 2016). Still, over 80% of M-CLL patients had undetectable WNT5A expression, suggesting possible silencing of the gene expression through its methylation. Aims: To analyze methylation status of WNT5A promoter in the context of WNT5A mRNA expression, IGHV somatic hypermutations and methylation-based CLL classification (Querios, Leukemia 2015). Methods: Using bisulphite sequencing we analyzed methylation status of 3 CpG islands (CGI) located in WNT5A promoter and 5 marker CpG sites distinguishing methylation-based prognostic subgroups of CLL (located in B3GNTL1, CTBP2, TNF, SCARF1 and intergenic region on chromosome 14). mRNA expression was analyzed with quantitative real-time PCR using TaqMan probes (ThermoFisher Scientific): WNT5A, and HPRT1, TBP were used as controls. Results: Thirty-nine previously untreated CLL patients were chosen for the analysis. They were equally distributed among 4 groups based on IGHV status and WNT5A expression [WNT5A- M-CLL (10), WNT5A+ M-CLL (9), WNT5A- IGHV-unmutated CLL (U-CLL; 10), and WNT5A+ U-CLL (10), respectively]. Within the first of the three CpG islands of the WNT5A promoter (CGI1), we identified three CpG sites whose methylation status significantly correlated with the WNT5A expression in M-CLL (P = 0,004; P = 0,004; P = 0,0268; Mann-Whitney test). Furthermore, complete demethylation of the three CpG sites was associated with high WNT5A expression in M-CLL. In fact, complete demethylation of the whole tested CGI1 region was noted in these patients. Methylation analysis of 5 marker CpGs (Querios, Leukemia 2015) enabled us to classify patients into three methylation-based prognostic groups: 9/10 WNT5A- M-CLL patients belonged to the memory B-cell like CLL (m-CLL) group, and 7/9 WNT5A+ M-CLL patients to the intermediate CLL (i-CLL). U-CLL patients irrespective of WNT5A expression were assigned as naive B-cell like CLL (n-CLL) in 19/20 cases.Summary/Conclusion: Within M-CLL, methylation of the 3 CpGs located in the CGI1 of WNT5A promoter correlated significantly with WNT5A expression. Higher WNT5A expression was associated with complete demethylation of CGI1 locus. Our classification based on WNT5A expression and IGHV status overlapped with the methylation-profile-based CLL classification (m-CLL, i-CLL, n-CLL). Furthermore, memory B-cell like CLL group was associated with undetectable WNT5A level in our cohort. Supported by grants: MUNI/A/1105/2018, CEITEC2020 LQ1601, MZCR-RVO 65269705. Spanish Ministry of Health, Plan Estatal de I + D + I, ISCIII, FEDER, FIS PI15/01763ISIII PI15/01763 and PI18/01484.

Impact of gene mutations and chromosomal aberrations on progression-free survival in chronic lymphocytic leukemia patients treated with front-line chemoimmunotherapy: Clinical practice experience

The impact of genetic aberrations on rituximab-based therapeutic regimens has been intensely studied in chronic lymphocytic leukemia (CLL). According to the current consensus chemoimmunotherapy consisting of rituximab and DNA-damaging drugs is not suitable for patients with TP53 defects. In our study, we focused on CLL patients with an intact TP53 gene and investigated four recurrently mutated genes in CLL, genomic aberrations by FISH, and IGHV status with the aim of analyzing their impact on progression-free survival (PFS) after front-line therapy with FCR (fludarabine, cyclophosphamide, rituximab) or BR (bendamustine, rituximab) regimens. Using next-generation sequencing, we analyzed 120 patients treated with FCR and 57 patients treated with BR at a university hospital. We used a 10% cut-off for variant allele frequency and recorded the following mutation frequencies in the pre-therapy samples: ATM 23%, SF3B1 20%, NOTCH1 19% and BIRC3 11%. The data on cytogenetic aberrations (11q22, 13q14, trisomy 12) and IGHV mutation status were also considered in PFS analyses. In univariate analyses, we observed a negative impact of BIRC3 mutations and 11q22 deletion in both regimens, while the unmutated IGHV status was associated with a significantly shorter PFS only in the FCR-treated cohort. In a multivariate analysis, only deletion 11q22 in both regimens, and the unmutated IGHV in the FCR cohort maintained an independent association with the reduced PFS. Notably, sole 11q22 deletion, without an ATM mutation on the other allele, manifested the shortest PFS of all analyzed markers. Deletion 11q22 and IGHV status predict PFS in previously untreated CLL patients.

STIM1 at the plasma membrane as a new target in progressive chronic lymphocytic leukemia

BackgroundDysregulation in calcium (Ca2+) signaling is a hallmark of chronic lymphocytic leukemia (CLL). While the role of the B cell receptor (BCR) Ca2+ pathway has been associated with disease progression, the importance of the newly described constitutive Ca2+ entry (CE) pathway is less clear. In addition, we hypothesized that these differences reflect modifications of the CE pathway and Ca2+ actors such as Orai1, transient receptor potential canonical (TRPC) 1, and stromal interaction molecule 1 (STIM1), the latter being the focus of this study.MethodsAn extensive analysis of the Ca2+ entry (CE) pathway in CLL B cells was performed including constitutive Ca2+ entry, basal Ca2+ levels, and store operated Ca2+ entry (SOCE) activated following B cell receptor engagement or using Thapsigargin. The molecular characterization of the calcium channels Orai1 and TRPC1 and to their partner STIM1 was performed by flow cytometry and/or Western blotting. Specific siRNAs for Orai1, TRPC1 and STIM1 plus the Orai1 channel blocker Synta66 were used. CLL B cell viability was tested in the presence of an anti-STIM1 monoclonal antibody (mAb, clone GOK) coupled or not with an anti-CD20 mAb, rituximab. The Cox regression model was used to determine the optimal threshold and to stratify patients.ResultsSeeking to explore the CE pathway, we found in untreated CLL patients that an abnormal CE pathway was (i) highly associated with the disease outcome; (ii) positively correlated with basal Ca2+ concentrations; (iii) independent from the BCR-PLCγ2-InsP3R (SOCE) Ca2+ signaling pathway; (iv) supported by Orai1 and TRPC1 channels; (v) regulated by the pool of STIM1 located in the plasma membrane (STIM1PM); and (vi) blocked when using a mAb targeting STIM1PM. Next, we further established an association between an elevated expression of STIM1PM and clinical outcome. In addition, combining an anti-STIM1 mAb with rituximab significantly reduced in vitro CLL B cell viability within the high STIM1PM CLL subgroup.ConclusionsThese data establish the critical role of a newly discovered BCR independent Ca2+ entry in CLL evolution, provide new insights into CLL pathophysiology, and support innovative therapeutic perspectives such as targeting STIM1 located at the plasma membrane.

FBXW7 mutations reduce binding of NOTCH1, leading to cleaved NOTCH1 accumulation and target gene activation in CLL

AbstractNOTCH1 is mutated in 10% of chronic lymphocytic leukemia (CLL) patients and is associated with poor outcome. However, NOTCH1 activation is identified in approximately one-half of CLL cases even in the absence of NOTCH1 mutations. Hence, there appear to be additional factors responsible for the impairment of NOTCH1 degradation. E3-ubiquitin ligase F-box and WD40 repeat domain containing-7 (FBXW7), a negative regulator of NOTCH1, is mutated in 2% to 6% of CLL patients. The functional consequences of these mutations in CLL are unknown. We found heterozygous FBXW7 mutations in 36 of 905 (4%) untreated CLL patients. The majority were missense mutations (78%) that mostly affected the WD40 substrate binding domain; 10% of mutations occurred in the first exon of the α-isoform. To identify target proteins of FBXW7 in CLL, we truncated the WD40 domain in CLL cell line HG-3 via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9). Homozygous truncation of FBXW7 resulted in an increase of activated NOTCH1 intracellular domain (NICD) and c-MYC protein levels as well as elevated hypoxia-inducible factor 1-α activity. In silico modeling predicted that novel mutations G423V and W425C in the FBXW7-WD40 domain change the binding of protein substrates. This differential binding was confirmed via coimmunoprecipitation of overexpressed FBXW7 and NOTCH1. In primary CLL cells harboring FBXW7 mutations, activated NICD levels were increased and remained stable upon translation inhibition. FBXW7 mutations coincided with an increase in NOTCH1 target gene expression and explain a proportion of patients characterized by dysregulated NOTCH1 signaling.

Inferring clonal heterogeneity in cancer using SNP arrays and whole genome sequencing.

Clonal heterogeneity is common in many types of cancer, including chronic lymphocytic leukemia (CLL). Previous research suggests that the presence of multiple distinct cancer clones is associated with clinical outcome. Detection of clonal heterogeneity from high throughput data, such as sequencing or single nucleotide polymorphism (SNP) array data, is important for gaining a better understanding of cancer and may improve prediction of clinical outcome or response to treatment. Here, we present a new method, CloneSeeker, for inferring clinical heterogeneity from sequencing data, SNP array data, or both. We generated simulated SNP array and sequencing data and applied CloneSeeker along with two other methods. We demonstrate that CloneSeeker is more accurate than existing algorithms at determining the number of clones, distribution of cancer cells among clones, and mutation and/or copy numbers belonging to each clone. Next, we applied CloneSeeker to SNP array data from samples of 258 previously untreated CLL patients to gain a better understanding of the characteristics of CLL tumors and to elucidate the relationship between clonal heterogeneity and clinical outcome. We found that a significant majority of CLL patients appear to have multiple clones distinguished by copy number alterations alone. We also found that the presence of multiple clones corresponded with significantly worse survival among CLL patients. These findings may prove useful for improving the accuracy of prognosis and design of treatment strategies. Code available on R-Forge: https://r-forge.r-project.org/projects/CloneSeeker/. Supplementary data are available at Bioinformatics online.

Age-related changes in the BACH2 and PRDM1 genes in lymphocytes from healthy donors and chronic lymphocytic leukemia patients

BackgroundAge-related genetic changes in lymphocyte subsets are not currently well documented. BACH2 is a transcription factor that plays an important role in immune-mediated homeostasis by tightly regulating PRDM1 expression in both B-cells and T-cells. BACH2 gene expression is highly sensitive to DNA damage in aged mice. This concept led us to investigate the variation in BACH2 and also PRDM1 expression in major lymphocyte subsets with age.MethodsLymphocyte subsets from 60 healthy donors, aged from 20 to 90 years, and 41 untreated chronic lymphocytic leukemia patients were studied. BACH2 and PRDM1 gene expression was analyzed by real-time quantitative PCR. BACH2 gene expression was correlated with its protein expression. Lymphocyte apoptosis was evaluated after intracellular oxidative stress-inducing etoposide treatment of T and B cells.ResultsOur analysis shows BACH2 mRNA downregulation with age in healthy donor CD4+, CD8+ T-cells and CD19+ B-cells. Decreased BACH2 expression was also correlated with an age-related reduction in CD8 + CD28+ T-cells. We found a strong correlation between age-related BACH2 downregulation and decreased CD4+ T-cell and CD19+ B-cell apoptosis. PRDM1, as expected, was significantly upregulated in CD4+ T-cells, CD8+ T-cells and CD19+ B-cells, and inversely correlated with BACH2. A comparison of untreated chronic lymphocytic leukemia patients with age-matched healthy donors reveals that BACH2 mRNA expression was further reduced in CD4+ T-cells, CD8+ T-cells and leukemic-B cells. PRDM1 gene expression was consequently significantly upregulated in CD4+ and CD8+ T-cells in chronic lymphocytic leukemia patients but not in their leukemic B-cells.ConclusionOverall, our data suggest that BACH2 and PRDM1 genes are significantly correlated with age in human immune cells and may be involved in immunosenescence.

IGH translocations in chronic lymphocytic leukemia: Clinicopathologic features and clinical outcomes.

The prevalence, clinicopathologic correlates, and outcomes of previously untreated chronic lymphocytic leukemia (CLL) patients with IGH-BCL2 and IGH-BCL3 translocations are not well known. Using the Mayo Clinic CLL database, we identified patients seen between March 1, 2002 and September 30, 2016 who had FISH testing performed within 3 years of CLL diagnosis. The prognostic profile, time to first therapy (TTT), and overall survival (OS) of patients with IGH-BCL2 and IGH-BCL3 translocation were compared to patients without these abnormalities (non-IGH group). Of 1684 patients who met the inclusion criteria, 38 (2.2%) had IGH-BCL2, and 16 (0.9%) had IGH-BCL3 translocation at diagnosis. Patients with IGH-BCL3 translocation were more likely to have high and very-high CLL-International Prognostic Index, compared to patients with IGH-BCL2 translocation and the non-IGH group. The 5-year probability of requiring therapy was significantly higher for IGH-BCL3 compared to IGH-BCL2 and non-IGH groups (84% vs 33% vs 29%, respectively, P < 0.0001). The 5-year OS was significantly shorter for IGH-BCL3 compared to IGH-BCL2 and non-IGH groups (45% vs 89% vs 86%, respectively, P < 0.0001). On multivariable analyses, IGH-BCL3 translocation was associated with a shorter TTT (hazard ratio [HR] = 2.7; P = 0.005) and shorter OS (HR = 5.5; P < 0.0001); IGH-BCL2 translocation did not impact TTT and OS. In conclusion, approximately 3% of all newly diagnosed CLL patients have either an IGH-BCL2 or IGH-BCL3 translocation. Patients with IGH-BCL3 translocations have a distinct prognostic profile and outcome. These results support the inclusion of an IGH probe during the routine evaluation of FISH abnormalities in newly diagnosed CLL.

Chronic Lymphocytic Leukaemia Is Associated with a Broad Platelet-Function Defect Which Is Exacerbated By Ibrutinib and Acalabrutinib

INTRODUCTIONBleeding from thrombocytopathy is a common complication of advanced chronic lymphocytic leukaemia (CLL). In addition to disease-related thrombocytopenia, the presence of the CLL clone and/or therapeutic interventions may further impair platelet function. In particular, the BTK inhibitors ibrutinib and acalabrutinib are known to inhibit platelet glycoprotein VI (GPVI)-mediated platelet aggregation. We compared platelet function and markers of GPVI activation between untreated CLL patients, ibrutinib-treated CLL patients and healthy controls, and studied the in vitro effects of ibrutinib and acalabrutinib on clinically utilised platelet function assays to assess their impact on GPVI-mediated as well as non-GPVI-mediated platelet activation pathways.METHODSBlood samples from 17 healthy volunteers and 8 untreated CLL patients were spiked with vehicle or comparable plasma concentrations of ibrutinib (0.3µM, 1.0µM) and acalabrutinib (1.8µM, 6.0µM) attainable during the treatment of CLL. Additional samples were obtained from 5 CLL patients undergoing ibrutinib treatment. Platelet function was evaluated using whole blood multiple electrode aggregometry (MEA - Multiplate®) and light transmission aggregometry (LTA - AggRAM®) in response to varying concentrations of aggregation-inducing reagents (collagen, CRP-XL, ADP, TRAP, ristocetin, arachidonic acid, and adrenaline). Shear-induced platelet adhesion was assessed using PFA-100®. Soluble GPVI plasma levels were assessed by ELISA.RESULTSIn the absence of treatment, CLL patients exhibited significant platelet defects on whole-blood platelet function analyses in response to various agonists including ADP, ristocetin, TRAP and collagen (MEA) and prolongation of PFA-100® collagen/epinephrine closure time. This impairment was not replicated in assays using platelet-rich plasma (LTA). Ibrutinib-treated CLL patients demonstrated an additive impairment of platelet function, especially in regards to collagen-mediated activation by MEA or PFA-100®. There was no significant difference in soluble GPVI levels between normal, untreated or ibrutinib treated CLL patients.Addition of clinically-attainable concentrations of ibrutinib and acalabrutinib in vitro produced similar concentration-dependent inhibition of platelet function in healthy controls, with inhibition of aggregation evident in response to various agonists including collagen, CRP-XL, ristocetin and ADP but not arachidonic acid or TRAP. Ibrutinib also impaired aggregation in response to epinephrine, and caused selective prolongation of the PFA-100® collagen/epinephrine closure time, an effect not observed with acalabrutinib. MEA appears more sensitive and reproducible than LTA to describe the various inhibitory effects on platelet aggregation. Similar concentration-dependent inhibition of platelet function was observed by adding ibrutinib and acalabrutinib in vitro to blood samples from untreated CLL patients.CONCLUSIONSCLL is associated with a broad platelet function defect, which can be exacerbated by BTK inhibitors. Acalabrutinib induces a platelet function defect similar but less potent to that observed with ibrutinib, with the exception of shear-induced platelet adhesion (PFA-100®) which was only abnormal with ibrutinib. Routine platelet function assays are capable of quantifying BTK inhibitor-induced platelet dysfunction in CLL patients, with the most sensitive and reproducible measure being collagen-induced aggregation by MEA. There was no evidence for BTK-dependent platelet GPVI cleavage. Whole-blood platelet function assays may have utility in managing CLL patients presenting with bleeding or requiring urgent surgery during therapy with BTK inhibitors. DisclosuresMcGregor:Pfizer: Other: Conference travel support; Bristol-Myers Squibb: Other: Conference travel support; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria. Baker:CSL Behring: Research Funding; Biogen Idec: Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Research Funding; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi Sankyo: Research Funding; Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Other: Conference travel support, Research Funding; Shire: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Conference travel support; Roche: Other: Conference travel support; Novo Nordisk: Other: Conference travel support.

Real Life Use of Bendamustine Plus Rituximab Versus Chlorambucil Plus Rituximab As Front-Line Therapy for Elderly CLL Patients. Retrospective Multicenter Study in the Lazio Region

Real Life Use of Bendamustine Plus Rituximab Versus Chlorambucil Plus Rituximab As Front-Line Therapy for Elderly CLL Patients. Retrospective Multicenter Study in the Lazio Region

Enhanced Expression of Beta-Catenin and Axl Receptor Tyrosine Kinase (RTK) in Chronic Lymphocytic Leukemia (CLL) B-Cells with Co-Culture on Marrow Stromal Cells: Implications for Leukemic Cell Drug Resistance

IntroductionCLL remains an incurable disease and represents a significant health problem in the western world. Increasing evidence highlights that the impact of marrow stromal cells is a key component influencing CLL B-cell survival. We have utilized an in vitro bone marrow stromal cell (BMSC) model system and found unique alterations in CLL B-cells with BMSC co-culture that point to previously unidentified biologic changes in the CLL B-cells that may influence CLL B-cell signaling and drug resistance.MethodsPurified primary CLL B-cells (n= 39) from previously untreated CLL patients were cultured alone or co-cultured with primary BMSCs from either normal individuals (n=26) or CLL patients (n=17) at a 50:1 ratio in AIMV medium. After 48 hours, separated CLL B-cells or BMSCs were examined by immunoprecipitation/Western blot analyses and where needed real time PCR was done to assess the presence of intracellular proteins. In separate experiments to assess CLL B-cell killing, purified CLL B-cells were treated with TP-0903, fludarabine, chlorambucil and ibrutinib as single agents with or without BMSC co-culture.ResultsWe observed significant increases in expression of Axl for both mRNA and protein levels in CLL B-cells co-cultured with BMSCs compared to CLL B-cells cultured alone. We also detected significantly increased expression of β-catenin at the protein level in CLL B-cells co-cultured with BMSC. But, we did not see any significant change in β-catenin or Axl protein expression in BMSCs co-cultured with CLL B-cells. Co-culturing of CLL B-cells with BMSCs using transwells confirmed that the upregulation of both Axl and β-catenin is dependent on the direct contact of CLL B-cells with BMSCs. The CLL B-cells from co-culture also had upregulation in phosphorylated (P)-ERK-1/2 but no change in P-AKT(Ser473). High nuclear β-catenin and P-ERK-1/2 levels were also detected in co-cultured CLL B-cells. ERK associates with and inactivates GSK-3β resulting in the up-regulation of β-catenin. We next checked for P-GSK-3β (Ser9) in co-cultured CLL B-cells. Upregulation in P-GSK-3β (Ser9) detected in co-cultured CLL B-cells suggests inactivation of GSK-3β and increasing β-catenin accumulation in co-cultured CLL B-cells. Moreover, inhibition of P-ERK-1/2 with inhibitor PD98059 in CLL B-cells cultured with BMSCs inhibited β-catenin as well as Axl expression levels. We also determined the phosphorylation status of Axl in CLL B-cells in co-culture with BMSC but found no change either at Y702 (Axl kinase domain) or total tyrosine phosphorylation levels for Axl in CLL B-cells. Thus, we assume that the role of Axl in co-cultured leukemic B-cells is independent of its kinase activity. Next we determined the effect of the highly specific Axl inhibitor TP-0903 on CLL B-cell status of Axl and b-catenin while in BMSC co-culture. Interestingly, both Axl and β-catenin protein expression levels were found to be further upregulated in CLL B-cells exposed to sub-lethal doses of TP-0903 in co-culture with BMSC. Treatment with chemotherapeutic or targeted therapy drugs, (i.e. fludarabine, chlorambucil or ibrutinib) also led to increase in expression levels of both β-catenin and Axl CLL B-cells co-cultured with BMSC. Of interest CLL B-cells were less sensitive to the chemotherapy drugs in presence of BMSCs, suggesting a role for both Axl and β-catenin in stromal mediated CLL B-cell drug resistance to these agents. This was not true for the Axl inhibitor as TP-0903 was able to induce robust cell death by targeting P-Axl and overcome BMSC mediated protection even in the presence of increased Axl and b-catenin. We also found that TP-0903 decreased P-Axl as well as the Axl downstream mediator, P-Akt(S473) and reduced Mcl-1 expression in CLL B-cells in BMSC co-culture.ConclusionsHere we show that marrow stromal cell mediated increased expression in both β-catenin and Axl in CLL B-cells is associated with leukemic B cell survival and drug resistance. The mechanism for this may in part be via activated ERK levels that also occur when CLL B-cells contact BMSC. The BMSC resistance appears to be more profound for chemotherapeutic agents since Axl inhibitor can still induce CLL B-cell killing with BMSC co-culture. These studies suggest that a further understanding of the roles of Axl and β-catenin in the resistance status of CLL B-cells mediated by contact with BMSC are warranted. [Display omitted] DisclosuresWarner:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals, Inc: Employment. Kay:Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees.

Single Cell Transcriptomic Characterization of the Immune Microenvironment in Naturally Progressing Chronic Lymphocytic Leukemia (CLL)

The genetic changes occurring during natural progression and following therapy in CLL have recently been characterized. However, the role of co-existing immune cell populations during disease evolution has not been systematically evaluated. New single cell transcriptome sequencing (scRNA-seq) technologies provide a powerful approach for dissecting the composition and state of complex heterogeneous cell populations such as immune cells. We hypothesized that changes of specific immune features underlie the kinetics of progression of CLL.To this end, we selected a discovery set of CLL samples from 6 patients previously genetically characterized by bulk whole-exome sequencing (WES). Three patients were “slow-progressors” (median time to first treatment [TTFT]: 10.7 years (yrs), range: 7.7-12.2), while the other three were “fast-progressors” (median TTFT: 1.9 yrs, range: 1.3-2.4). We processed paired samples obtained close to the time of diagnosis (T1; median yrs from diagnosis: 2.54, range 0.6-4.2) and close to first treatment (T2; median yrs to TTFT: 0.22, range 0-5.9). Non-CD19+CD5+ cells were isolated from peripheral blood mononuclear cells by flow cytometry for scRNA-seq using the 10X Genomics Chromium Controller platform. Replicates were available in 8 of 12 samples, for a total of 20 specimens. Dimensionality reduction and clustering was then performed by a novel pipeline called MUDAN (multi-sample unified discriminant analysis). We used a mixed-effects model to identify trends in cell type-specific gene expression and proportions over time across patients.We observed changes in the composition of immune cells over time across these patients. In total, 57,069 cells were analyzed (median number of cells per sample: 4,394, range 774-8657) and the mean number of genes per cell was 906 (SD: 116.7). We identified a total of 13 immune cell types, consistent with diverse subsets of T and NK cells, myeloid cells, and hematopoietic stem cells. At T1, fast-progressors had a higher proportion of myeloid cells (dendritic cells, classical and non-classical monocytes, p<0.0001, p=0.004 and p<0.0001, respectively) as compared to slow-progressors, which showed a higher proportion of naïve and memory CD4+ T cells (p=0.001 and p=0.016, respectively). Over the course of disease, both fast and slow-progressors converged into a decrease in the lymphoid:myeloid ratio (L:M ratio in T1: 10.1 vs. T2: 7.3, p=0.023), due in part to a proportional increase in non-classical monocytes (p=0.004) and a decrease in CD4+ T cells (p=0.003).Over time, we observed a broad upregulation of gene expression across all cell types in both fast and slow-progressors. Within the lymphoid compartment, we observed an upregulation of cell activation (CD3E, CD69, and GITR), chemotaxis (CCL3, CCL4, and XCL2), cytotoxicity (GZMK, GZMA, and NKG7) and pro-inflammatory (INFG, ISG15, and TNF) markers, especially in memory CD4+ T cells. In contrast, memory CD8+ T cells, which were marked by the inhibitory molecule KLRB1(CD161) in our cohort, showed the lowest increase in these gene signatures. TIGIT followed by LILRB2, CD160 and LAG3 were the most upregulated immune checkpoints across time, especially in memory CD4+ and naïve CD8+ T cells, and resting NK cells. LAG3 was revealed to be the only of these immune checkpoints associated with fast-progressors (p<0.05). No significant changes in the expression of PDCD1, HAVCR2 (TIM3) or TNFRSF14 (HVEM) were observed over time. Within the myeloid compartment, we observed a lack of upregulation of cell activation markers along with a marked downregulation of chemoattractants (XCL1, CXCR5 and CXCR4) and antigen presentation molecules (HLA-DOA, HLA-E, and PSME1) over time, especially in classical monocytes. Further analyses correlating CLL genetics to immune kinetics and validating these results in a larger patient cohort are ongoing.Altogether, these findings provide fresh insights of the evolving immune microenvironment during CLL progression. Despite differences in the immune composition at the time of diagnosis, fast and slow-progressors converged into an immune activation state, mostly dominated by CD4+ T cells, ineffective cytotoxic cells and poor immunogenic phagocytic cells that ultimately contributed to the progression of the disease. These results provide a rationale for new immunotherapeutic strategies in previously untreated CLL patients. DisclosuresKipps:Verastem: Membership on an entity's Board of Directors or advisory committees; F. Hoffmann-La Roche Ltd: Consultancy, Research Funding; Celgene: Consultancy; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Genentech Inc: Consultancy, Research Funding. Wu:Neon Therapeutics: Equity Ownership.

Effectiveness of the Combination of Rituximab and Standard Chemotherapeutic Regimens in Previously Untreated Patients with Chronic Lymphocytic Leukaemia in Real-Life: Results from a Noninterventional Study (CILI Study)

Chronic lymphocytic leukemia (CLL) is one of the most common haematological malignancies exhibiting remarkable heterogeneity in clinical course. Rituximab added to standard chemotherapy has been proven to increase response rate and eventually survival among previously untreated CLL patients. CILI was an open-label, non-randomized, single arm, multicentric, observational study aimed to collect real-life effectiveness data for rituximab used according to the current label in combination with standard chemotherapy in previously untreated CLL patients. Overall response rates (ORR) in the entire study population as well as in various subgroups were estimated. Adverse events were recorded during the entire course of the study. A total number of 150 patients were enrolled by 15 Hungarian study sites. Out of these, 82 patients received 6 cycles of rituximab containing treatment. Overall response rates of 88.24% (CI95%: 81.6–93.12%) and 94.59% (CI95%: 86.73–98.51%) were recorded in the intent-to-treat (ITT) and per-protocol (PP) populations, respectively. In both study populations, somewhat higher ORR was observed in patients aged ≥65 years. Subgroups defined according to either chromosomal aberrations (presence of 11q and 17p deletions) showed apparently high ORRs, though these rates were most probably biased by low patient numbers. 144 adverse events were reported during the study, of which 15 AEs were considered to be related to the administration of rituximab. Analyses of the efficacy variables have revealed comparable results to those previously reported by controlled clinical trials.

Bone marrow hematopoietic dysfunction in untreated chronic lymphocytic leukemia patients.

The consequences of immune dysfunction in B-chronic lymphocytic leukemia (CLL) likely relate to the incidence of serious recurrent infections and second malignancies that plague CLL patients. The well-described immune abnormalities are not able to consistently explain these complications. Here, we report bone marrow (BM) hematopoietic dysfunction in early and late stage untreated CLL patients. Numbers of CD34+ BM hematopoietic progenitors responsive in standard colony-forming unit (CFU) assays, including CFU-GM/GEMM and CFU-E, were significantly reduced. Flow cytometry revealed corresponding reductions in frequencies of all hematopoietic stem and progenitor cell (HSPC) subsets assessed in CLL patient marrow. Consistent with the reduction in HSPCs, BM resident monocytes and natural killer cells were reduced, a deficiency recapitulated in blood. Finally, we report increases in protein levels of the transcriptional regulators HIF-1α, GATA-1, PU.1, and GATA-2 in CLL patient BM, providing molecular insight into the basis of HSPC dysfunction. Importantly, PU.1 and GATA-2 were rapidly increased when healthy HSPCs were exposed in vitro to TNFα, a cytokine constitutively produced by CLL B cells. Together, these findings reveal BM hematopoietic dysfunction in untreated CLL patients that provides new insight into the etiology of the complex immunodeficiency state in CLL.

Chronic lymphocytic leukemia treatment algorithm 2018

The treatment landscape for patients with chronic lymphocytic leukemia (CLL) has changed considerably with the introduction of very effective oral targeted therapies (such as ibrutinib, idelalisib, and venetoclax), and next-generation anti-CD20 monoclonal antibodies (such as obinutuzumab). These agents lead to improved outcomes in CLL, even among patients with high-risk features, such as del17p13 or TP53 mutation and unmutated immunoglobulin heavy chain (IGHV) genes. Each of these treatments is associated with a unique toxicity profile; in the absence of randomized data, the choice of one type of treatment over another depends on the co-morbidities of the patient. Chemoimmunotherapy still plays an important role in the management of previously untreated CLL patients, particularly among young fit patients who have standard risk FISH profile and mutated IGHV genes. Richter’s transformation of CLL remains a difficult complication to treat, although therapy with programmed death 1 inhibitors such as pembrolizumab and nivolumab has shown impressive responses in a subset of patients. Our ability to risk stratify CLL patients continues to evolve; the CLL-International Prognostic Index (CLL-IPI) is the best validated tool in predicting time to first therapy among previously untreated patients. This review summarizes the current approach to risk stratification and management of CLL patients.

ANALYSIS OF THE 3΄UTR REGION OF THE NOTCH1 GENE IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS

Detection of mutations in the 3'UTR region of the NOTCH1was performed by direct sequencing in 87previously untreated CLL patients (from the total group of 237CLL patients) with unmutated immunoglobulin heavy-chain variable (UM IGHV) genes and without mutations in hotspot regions of TP53, SF3B1, and exon 34of NOTCH1genes. Mutations in the 3'UTR region of the NOTCH1were revealed in three of 87CLL patients (3.4%). Two cases with non-coding mutations were related to subset #1of stereotyped B-cell receptors, and one case belonged to stereotyped subset #28a. Analysis with inclusion of 30UM IGHV cases with previously detected c.7544_7545delCT mutations revealed that the frequency of UM IGHV genes of I phylogenetic clan (except IGHV1-69) was significantly increased, and the frequency of UM IGHV3and IGHV4genes, on the contrary, was reduced in NOTCH1-mutated cases comparing with NOTCH1-unmutated cases (p = 0.002) and the general group (p = 0.013). SNP rs3124591did not affect the risk of CLL and survival parameters of the patients. At the same time, differences were found in the frequency of IGHV gene usage and in the structure of HCDR3in carriers of individual genotypes. The frequency of NOTCH1mutations in 3'UTR region was low. Our findings confirmed current data on the association between the structure of the B-cell receptor and the appearance of NOTCH1mutations. Some features of HCDR3structure were identified in carriers of TT and CC genotypes of rs3124591.

Influence of Retronectin-Mediated T-Cell Activation on Expansion and Phenotype of CD19-Specific Chimeric Antigen Receptor T Cells.

Enhanced in vivo expansion, long-term persistence of chimeric antigen receptor T (CART) cells, and efficient tumor eradication through these cells are linked to the proportion of less-differentiated cells in the CART cell product. Retronectin is well established as an adjuvant for improved retroviral transduction, while its property to enrich less-differentiated T cells is less known. In order to increase these subsets, this study investigated the effects of retronectin-mediated T-cell activation for CD19-specific CART cell production. Peripheral blood mononuclear cells of healthy donors and untreated chronic lymphocytic leukemia (CLL) patients without or with positive selection for CD3+ T cells were transduced with a CD19.CAR.CD28.CD137zeta third-generation retroviral vector. Activation of peripheral blood mononuclear cells was performed by CD3/CD28, CD3/CD28/retronectin, or CD3/retronectin. Interleukin-7 and -15 were supplemented to all cultures. Retronectin was used in all three activation protocols for retroviral transduction. Expansion was assessed by trypan blue staining. Viability, transduction efficiency, immune phenotype, and cytokine production were longitudinally analyzed by flow cytometry. Cytotoxic capacity of generated CART cells was evaluated using a classical chromium-51 release assay. Retronectin-mediated activation resulted in an enrichment of CD8+ cytotoxic CART cells and less-differentiated naïve-like T cells (CD45RA+CCR7+). Retronectin-activated CART cells showed increased cytotoxic activity. However, activation with retronectin decreased viability, expansion, transduction efficiency, and cytokine production, particularly of CLL patient-derived CART cells. Both retronectin-mediated activation protocols promoted a less-differentiated CART cell phenotype without comprising cytotoxic properties of healthy donor-derived CART cells. However, up-front retronectin resulted in reduced viability and expansion in CLL patients. This effect is probably attributed to the retronectin-mediated activation of B cells with prolonged CLL persistence. Consequently, CART cell expansion and generation failed. In summary, activation with retronectin should be performed with caution and may be limited to patients without a higher percentage of tumor cells in the peripheral blood.

Resistance to complement activation, cell membrane hypersialylation and relapses in chronic lymphocytic leukemia patients treated with rituximab and chemotherapy.

The anti-CD20-specific monoclonal antibody rituximab (RTX), in combination with chemotherapy, is commonly used for primary treatment in chronic lymphocytic leukemia (CLL). However, relapses remain important and activation of the complement pathway is one of the mechanisms by which RTX generates the destruction of B cells directly by complement-dependent cytotoxicity (CDC), or indirectly by antibody-dependent cellular phagocytosis. In this study, the RTX capacity to induce CDC was established in 69 untreated CLL patients, this cohort including 34 patients tested before the initiation of RTX-chemotherapy. In vitro CDC-resistance to RTX predicts lower response rates to RTX-chemotherapy and shorter treatment free survival. Furthermore, the predictive value of CDC-resistance was independent from the clinical, cytogenetic and FcγR3A V158F polymorphism status. In contrast, CLL cell resistance to CDC predominates in IGHV unmutated patients and was related to an important α2-6 sialyl transferase activity, which in turn increases cell surface α2-6 hypersialylation. Suspected factors associated with resistance to CDC (CD20, CD55, CD59, factor H, GM1, and sphingomyelin) were not differentially expressed or recruited between the two CLL groups. Altogether, results provide evidence that testing RTX capacity to induce CDC in vitro represents an independent predictive factor of therapeutic effects of RTX, and that α2-6 hypersialylation in CLL cells controls RTX response through the control of the complement pathway. At a time when CLL therapy is moving towards chemo-free treatments, further experiments are required to determine whether performing an initial in vitro assay to appreciate CLL CDC resistance might be useful to select patients.

Clonal diversity predicts adverse outcome in chronic lymphocytic leukemia.

Genomic analyses of chronic lymphocytic leukemia (CLL) identified somatic mutations and associations of clonal diversity with adverse outcomes. Clonal evolution likely has therapeutic implications but its dynamic is less well studied. We studied clonal composition and prognostic value of seven recurrently mutated driver genes using targeted next-generation sequencing in 643 CLL patients and found higher frequencies of mutations in TP53 (35 vs. 12%, p < 0.001) and SF3B1 (20 vs. 11%, p < 0.05) and increased number of (sub)clonal (p < 0.0001) mutations in treated patients. We next performed an in-depth evaluation of clonal evolution on untreated CLL patients (50 "progressors" and 17 matched "non-progressors") using a 404 gene-sequencing panel and identified novel mutated genes such as AXIN1, SDHA, SUZ12, and FOXO3. Progressors carried more mutations at initial presentation (2.5 vs. 1, p < 0.0001). Mutations in specific genes were associated with increased (SF3B1, ATM, and FBXW7) or decreased progression risk (AXIN1 and MYD88). Mutations affecting specific signaling pathways, such as Notch and MAP kinase pathway were enriched in progressive relative to non-progressive patients. These data extend earlier findings that specific genomic alterations and diversity of subclones are associated with disease progression and persistence of disease in CLL and identify novel recurrently mutated genes and associated outcomes.