Course Of Chronic Lymphocytic Leukemia Research Articles

THE EXPRESSION OF THE MAIN AND ALTERNATIVE TRANSCRIPT (SORL1 Delta2) OF THE SORL1 GENE IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS AFFECTED BY THE CHORNOBYL ACCIDENT.

to study clinical-hematological data and expression of the main and alternative transcripts of SORL1 genein chronic lymphocytic leukemia (CLL) patients affected by the Chornobyl catastrophe. Analysis was performed in the main group of 34 CLL patients irradiated due to the Chornobyl NPP acci-dent (30 clean-up workers, and 4 evacuees) and in the control group of 27 non-irradiated CLL patients. Groups ofpatients were comparable by age, sex, stage of disease, mutational status of IGHV genes. Expression of the main andalternative transcripts of SORL1 gene was evaluated by Quantitative Real-time polymerase chain reaction (PCR). TheIGHV gene mutational status, TP53 and SF3B1 mutations were studied by PCR followed by direct sequencing. Data wereanalyzed with the SPSS software package, version 20.0. Relative expression level of the main transcript of SORL1 gene was low (mean 1.71 ± 0.55, median 0.57),did not correlate with the IGHV gene mutational status, TP53 and SF3B1 mutations, stage of disease. The expressionof B transcript was not detected, F transcript was expressed at a very low level in 9 patients. The average relativeexpression level of SORL1-Δ2 transcript was 14.1 ± 6.04 (median 3.48; range 0.01-90.51). The expression of SORL1-Δ2transcript above the median was more frequent among patients on C stage (p = 0.001), and in patients with unmu-tated IGHV genes was associated with an extremely negative course of CLL (median of overall survival 9 months vs61 months at low expression). Relative expression levels of the main and alternative transcripts of SORL1 gene inpatients of the main and the control groups did not differ. Our preliminary data suggest that increased expression of SORL1-Δ2 transcript in CLL patients withunmutated IGHV genes can be considered as a negative prognostic marker.

A reduced panel of eight genes (ATM, SF3B1, NOTCH1, BIRC3, XPO1, MYD88, TNFAIP3, and TP53) as an estimator of the tumor mutational burden in chronic lymphocytic leukemia.

IntroductionMutational complexity or tumor mutational burden (TMB) influences the course of chronic lymphocytic leukemia (CLL). However, this information is not routinely used because TMB is usually obtained from whole genome or exome, or from large gene panel high‐throughput sequencing.MethodsHere, we used the C‐Harrel concordance index to determine the minimum panel of genes for which mutations predict treatment‐free survival (TFS) as well as large resequencing panels.ResultsAn eight gene estimator was defined encompassing ATM, SF3B1, NOTCH1, BIRC3, XPO1, MYD88, TNFAIP3, and TP53. TMB estimated from either a large panel of genes or the eight gene estimator was increased in treated patients or in those with a short TFS (<2 years), unmutated IGHV gene or with an unfavorable karyotype. Being an independent prognostic parameter, any mutation in the eight gene estimator predicted a shorter TFS better than Binet stage and IGHV mutational status among patients with an apparently non‐progressive disease (TFS >6 months). Strikingly, the eight gene estimator was also highly informative for patients with Binet stage A CLL or with a good prognosis karyotype.ConclusionThese results suggest that the eight gene estimator, that is easily achievable by high‐throughput resequencing, brings robust and valuable information that predicts evolution of untreated patients at diagnosis better than any other parameter.

Standard treatment approaches for relapsed/refractory chronic lymphocytic leukemia after frontline chemoimmunotherapy.

Despite the effectiveness of chemoimmunotherapy (CIT), in most cases the clinical course of chronic lymphocytic leukemia (CLL) is characterized by consecutive episodes of disease progression and need for therapy. Treatment possibilities for patients with CLL in whom CIT fails whose disease progresses after initial CIT include pathway inhibitors (PIs) and, for selected patients, cellular therapy (ie, allogeneic stem cell transplant, chimeric antigen receptor T cells). PIs (ie, Bruton tyrosine kinase inhibitors, phosphatidylinositol 3-kinase inhibitors, and BCL2 inhibitors) are revolutionizing the treatment of CLL. PIs have proved to be more effective than CIT, both as upfront therapy and for relapsed/refractory disease, largely because they may overcome the negative impact of adverse biomarkers (eg, TP53 aberrations, unmutated IGHV) on outcomes and because of their acceptable toxicity. In this article, the management of patients with relapsed/refractory CLL is discussed, with a particular emphasis on the role of PIs.

Next-Generation Sequencing Targeted Chronic Lymphocytic Leukemia Panel: A Pilot Study

Background: Rapid progress in next-generation sequencing (NGS) technologies make it possible to spell out the mutational status, the genetic and epigenetic variability of chronic lymphocytic leukemia (CLL). The identification of driver mutations allows us to expand understanding of the pathogenesis of CLL, to identify prognostic groups and to select potential targets for therapy, contributing to the development and implementation of new targeted drugs and its combinations. Nevertheless, the course of CLL does not always correspond to the existing prognostic risk groups, assessed by "standard" cytogenetic and molecular genetic methods. The NGS technology admits establishing markers of an unfavorable course of the disease. Aim: To assess the mutational status of patients (pts) with CLL using the developed Lymphoid Targeted NGS Panel and to study the possible correlation of the mutational status with the clinical characteristics of the disease. Method: In this prospective study were included 24 pts with CLL: treatment-naïve (n=8) and relapsed/refractory (n=16). The diagnosis of CLL was established according to iwCLL criteria (iwCLL, Hallek et al., 2018) and show only typical immunophenotype. The pts were divided into three prognostic groups according to cytogenetic assay: favorable (n=14), neutral (n=2), and unfavorable (n=8). Although, 20/24 pts were divided into two prognostic groups taking into account the data on the mutational status of the immunoglobulin heavy-chain variable (IGHV) region: favorable (n=8) and unfavorable (n=12). Four patients have no available data on IGHV mutational status. All patients had indications for starting treatment: FCR (n=7), RB (n=6), ibrutinib (n=3), venetoclax (n=1), acalabrutinib (n=5), combination of venetoclax and acalabrutinib (n=2). DNA samples were extracted from peripheral B-cell lymphocytes via the standard phenol-chloroform method. Average reading depth of 1000x is produced on a MiSeq platform (Illumina, USA). The 2% threshold of allele frequency (VAF) was used. The clinical significance of mutations was established using the following databases: COSMIC, ClinVar, gnomAD with application in silico analysis (Cscape, Cancer Genome Interpreter, SNPs&amp;Go). The Lymphoid Panel includes 117 genes, part of which is involved in the main 8 cellular signaling pathways underlying the pathogenesis of CLL. We have completed a pilot NGS study using the developed Lymphoid Targeted Gene Panel on DNA samples of six treatment-naïve pts. Results: Genetic aberrations were identified in all DNA samples using NGS. Somatic mutations were detected in 82.9% of cases, in an amount from 15 to 37. In four pts (4/6) with an unfavorable prognosis (cytogenetics and unmutated IGHV), known pathogenic variants of mutations were identified: JAK3 V722L, NOTCH1 P2514fs*4, IDH2 T352P, TP53 Lys120Glu, BRAF D594G. The existing approach to the interpretation of the results does not allow making an unambiguous conclusion about the clinical significance of variants in the IDH2 and JAK3 genes, despite the known pathogenic effect of the variants. The detected variant of the mutation (Lys120Glu) in the TP53 gene is often associated with the presence of a 17p13 deletion, which was confirmed by the FISH assay and correlated with the unfavorable clinical course of the disease in patient CLL-024. Twenty-two mutations were identified, the pathogenicity of which has not yet been determined, in the amount of 2 to 5 (median=3.5) mutations per patient. It should be noted that two patients (CLL-023, CLL-024) with unfavorable prognosis had mutations both in BCR gene and in NOTCH2 gene of unknown significance. Conclusion: The data obtained from a pilot study demonstrate the possibility of using NGS technology in clinical practice. The assessment of the mutational status of pts with CLL using NGS correlates with the clinical parameters of pts. Considering that there is currently no information about prognostic significances of identified mutations, additional research is required. Disclosures Martynkevich: Pfizer: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau. Shuvaev:Pfizer: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau. Voloshin:Novartis: Honoraria, Speakers Bureau.

Comparison of Clinical and Laboratory Features, Drug Availability, and Outcomes of CLL Patients Treated in Public or in Private Hospitals in Brazil: A Retrospective Analysis of the Brazilian Registry of CLL

Introduction: Chronic lymphocytic leukemia (CLL) typically occurs in elderly patients and has a highly variable clinical course. It is important to understand the aspects that affect the outcomes of CLL in a real-world setting. Besides biological factors, other socioeconomic and health system factors may influence the clinical course of CLL. Hence, data from the Brazilian Registry of CLL was analyzed to compare clinical and treatment-related characteristics in patients with CLL treated in public or in private institutions in Brazil. Objective: To describe the outcomes of a series of CLL patients followed in public or in private hospitals in Brazil. Methods: The Brazilian Registry of CLL was started in 2004 as a prospective non-interventional data collection tool. Inclusion criteria for enrollment followed the IWCLL guidelines. For this analysis, we included all patients with minimum available data for analysis of patient and disease characteristics and survival. Results/discussion: From January 2004 to July 2020, 2927 patients from 37 centers met eligibility criteria for this analysis: 2324 (79%) were followed at public hospitals and 603 (21%) at private hospitals. The majority were male (57%), with median age of 65 years (ranging from 23 to 106). Binet stage was A in 1618 (58%) patients, B in 628 (23%) and C in 525 (19%). FISH for del(17p) was performed in only 479 patients (16%), while FISH for the most common aberrations [del(13q), +12, del(11q), and del(17p)] was performed in only 445 patients (15%). IGVH mutational status was performed in only 211 patients (7%), and karyotype in only 140 patients (5%). Comparing public and private hospitals, we observed that patients in public hospital are slightly older (median age 66 years vs. 64 years for private hospitals, P=0.04), had more advanced diseases at diagnosis (frequency of Binet B or C was 44% in public vs. 32% in private hospital, P&amp;lt;0.0001), and there were more patients with elevated creatinine levels (14% vs. 10%, P=0.03). All prognostic markers were significantly more available in private than in public hospitals: FISH for del17p (42% of cases vs. 10%, respectively, P&amp;lt;0.0001), IGVH mutational status (13% vs. 6%, respectively, P&amp;lt;0.0001) and karyotype (16% vs. 2%, respectively, P&amp;lt;0.0001). The frequency of del(17p) was similar between public and private hospitals (10% vs. 11%, P=NS), while the frequency of unmutated IGHV status was more common in private hospitals, although not statistically different (60% vs. 48%, P=0.09). Analyzing 2102 diagnosed after 2010, we observed that 864 patients (41%) were treated after a median time of 7 months (range: 0-267) after diagnosis. First line treatment was predominantly based chlorambucil (45%) or fludarabine (40%). Anti-CD20 monoclonal antibody was used in only 39% of cases: (rituximab in 35% and obinutuzumab in 4%). Novel agents were used in first line in only 2% of patients, and in most cases in the context of a clinical trial. Of note, most patients (86%) with del(17p) detected by FISH were treated with chemoimmunotherapy. When comparing treatments between public or private hospitals we observed striking differences: in public hospitals there were significantly less patients receiving fludarabine-base regimens (36% vs. 54%, P&amp;lt;0.0001), and anti-CD20 monoclonal antibodies (28% vs. 78%, P&amp;lt;0.0001). Overall survival at 6 years was significantly worse in public than in private hospitals (72% vs. 93%, respectively, P&amp;lt;0.0001). After a multivariate analysis, survival in patients from public hospitals remained significantly worse than in private hospitals (hazard ratio 3.4, 95% confidence interval 2.4 - 4.8), after correcting for age, Binet staging and renal function. Conclusion: Our data indicate that are striking differences between patients treated in public or private hospitals in Brazil. The lack of accessibility to basic laboratory tests for prognostic factors and adequate therapies probably explains the worse outcome of patients treated in public institutions. In fact, prognostic testing rates were poor in both contexts and most high-risk patients received chemoimmunotherapy first-line. Urgent strategies are needed to increase accessibility to prognostic testing and to novel agents for quality improvement in health care in CLL patients in Brazil. Disclosures No relevant conflicts of interest to declare.

Clinical Staging and Flowcytomteric CD38 and Zap 70 Prognostic Indicators in Sudanese Patients with Chronic Lymphocytic Leukemia

Background: The clinical course of chronic lymphocytic leukemia is highly variable. The determination of ZAP70 and CD38 is increasingly utilized as prognostic factor for chronic lymphocytic leukemia. The aim of conducting this study was to investigate the frequency of CD38 and ZAP70 expression among Sudanese Chronic lymphocytic leukemia (CLL) patients and to relate them to the Binet and Rai clinical staging systems.&#x0D; Method: A total of 93 patients (mean age; 62.29 ± 11.68, sd) were enrolled in this cross-sectional study. CD38 and ZAP70 expression levels were measured with four color flowcytometry using the cut-off values of 20% for ZAP70 and 30% for CD38 expression. Staging was assessed by using clinical examination and CBC for all patients. Data were analyzed using the Statistical Package for Social science for Windows (SPSS), version 22.&#x0D; Results: There were 93 CLL patients and the median age of the group was 63 years (36–95 years). About 71% of the patients presented with lymphadenopathy, 53.8% with splenomegaly, 73.1% with anemia, and 45.2% with thrombocytopenia. There was higher frequency of Binet stage C and Rai stage IV (62 [66.6%] patients and 34 [36.5%] patients, respectively). In addition, CD38 and ZAP70 showed higher frequency among Binet and Rai advance stages. ZAP70 and CD38 positivity were detected in 21 patients (22.6%) and 31 patients (33.3%), respectively. There was no statistically significant association between ZAP70 and CD38 and clinical staging systems (P-value &gt; 0.05).&#x0D; Conclusion: No significant association was observed between Flowcytometric (CD38 and Zap70) Prognostic Indicators and clinical staging systems.&#x0D; Keywords: chronic lymphocytic Leukemia, Flowcytometry, ZAP70, CD38, clinical staging systems

A Glance to Infectious Complications During the Course of Chronic Lymphocytic Leukemia: Single Center Experience

A Glance to Infectious Complications During the Course of Chronic Lymphocytic Leukemia: Single Center Experience

Prognostic impact of NOTCH1 and MYD88 mutations in chronic lymphocytic leukemia patients

Background. Chronic lymphocytic leukemia (CLL) is one of the most common types of leukemia in adults with highly heterogeneous clinical course of the disease. Currently available prognostic factors are not fully efficient in predicting the course of CLL. New molecular mutations such as NOTCH1 and MYD88 could partly explain the CLL heterogeneity and help in identifying clinically relevant groups of patients. Material and methods. NOTCH1 c.7544_7545delCT (n = 200) in PEST domain (exon 34) and MYD88 L265P (n = 60) mutations was investigated by amplification refractory mutation system (ARMS). Expression of MYD88 in CLL was assessed in peripheral blood (PB) (n = 60) and bone marrow (BM) (n = 92) of CLL patients and 25 healthy volunteers (HVs) using qRT-PCR. Results. NOTCH1 mutation occurred in 18/200 (9.0%) CLL patients. Patients harboring NOTCH1 mutations prevalently belonged to aggressive cases, i.e. cases with an unmutated IGVH gene status, expression of CD38 and ZAP-70. MYD88 mutation occurred in 2/60 (3.3%) CLL patients. MYD88 mutations were strikingly enriched among patients expressing mutated IGVH genes. Our study demonstrated significantly higher PB MYD88 expression than in HVs and relevantly higher PB MYD88 expression in comparison with BM (respectively p < 0.0001 and p = 0.0015). There was no correlation between MYD88 expression in PB and BM and expression of ZAP-70, CD38 and IGVH mutational status. Conclusions. NOTCH1 mutations are more frequently detected in cases with unfavorable biological markers and seem to be independent predictive markers for worse outcome in CLL patients. Further collaborative studies in CLL are obligate to study the prognostic and predictive relevance of MYD88 mutations and expression.

Safety and Efficacy of Bendamustine Monotherapy in the First-Line Treatment of Patients with Chronic Lymphocytic Leukemia: Polish Lymphoma Research Group Real-Life Analysis

Bendamustine is a cytostatic drug with a unique structure, combining the features of purine nucleoside analogs and alkylating agents. In patients with chronic lymphocytic leukemia (CLL) it is commonly used in combination with rituximab (BR protocol) both in the first-line as well as subsequent lines of therapy, and in clinical trials it is often combined with new targeted therapies. Therefore, the data on its real-life safety and efficacy are of clinical significance. As the Polish Lymphoma Research Group (PLRG), we retrospectively analyzed the efficacy and tolerability of bendamustine monotherapy in 96 patients with CLL. The median number of bendamustine cycles was 5, and 44 patients did not complete the planned 6 cycles (46%). Among the adverse events associated with the earlier termination of bendamustine treatment, infections were the most common (20.5%), followed by neutropenia (15.9%) and thrombocytopenia (15.9%). Dose reductions and/or delays occurred in 31% of treatment cycles (132 of 425) with neutropenia (17.9%) as the most frequent cause. Efficacy analysis showed an overall response rate of 88.2% with complete remission and partial remission achieved in 43.8 and 41.7% of patients, respectively. At the 24th month of follow-up, progression-free survival was 52% and overall survival was 69.7%. Bendamustine in monotherapy was found to be safe and efficacious, at least in terms of early response. Special attention should be paid to infectious complications, and especially that immune disorders are characteristic in the clinical course of CLL. Our observations suggest efforts must be made to ensure the proper timing and proper dose in the administration of the drug, and to avoid the premature termination of the treatment.

Examination of clonal evolution in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is one of the most frequent lymphoproliferative diseases. CLL is characterized by unusual heterogeneity, which probably reflects its biological and genetic lack of homogeneity. Clonal chromosome aberrations belong to the most important prognostic and predictive factors in CLL. This research was aimed at observing clonal evolution in CLL at the chromosomal level, and assessing its clinical significance in relation to selected prognostic factors. The study involved 72 untreated patients with CLL. The preliminary investigations using cytogenetic banding analysis (CBA) and FISH were performed at the time of diagnosis, and again after about 24 months to observe clonal changes (clonal evolution). In addition, other parameters were evaluated, i.e., the expression of ZAP-70 kinase, CD38 antigen, and the mutation statuses of IGVH and NOTCH1 genes. Classic prognostic factors, i.e., categorized ZAP70 and CD38 expressions as well as mutations in IGVH and NOTCH1 genes did not influence the course of clonal evolution in the examined group of patients. Clonal evolution was detected in 45.8% of patients by means of CBA, and in 19.4% patients with FISH. Analysis of chromosomal aberrations in the examined group of patients showed that the incidence of 17p deletions and translocations in karyotypes has a negative impact on overall survival. CE was found to be a risk factor for the occurrence of disease progression (OR = 2.22). Our observations indicate that combined CBA and FISH are the most optimal techniques for monitoring clonal evolution in the course of CLL.

Programmed cell death 1 expression and Epstein-Barr virus infection in chronic lymphocytic leukaemia: a prospective cohort study.

PurposeInfection with Epstein-Bar virus (EBV) is associated with an unfavourable prognosis in chronic lymphocytic leukaemia (CLL), but the underlying mechanisms remain unknown. We aimed to establish whether EBV worsens the course of CLL by up-regulating the programmed cell death 1 expression.Patients and methodsUsing polymerase chain reaction, we measured EBV DNA in the blood of 110 newly diagnosed, treatment-naïve patients with CLL. We used flow cytometry to measure the expression of programmed cell death protein 1 (PD-1) and programmed cell death protein 1 ligand (PD-L1) on CD4+, CD8+, and CD19+ cells. Additionally, PD-1 and PD-L1 serum concentrations were measured using enzyme-linked immunosorbent assays. We related the expressions of PD-1 and PD-L1 to EBV DNA load and clinical outcomes.ResultsFifty-nine (54%) patients had detectable EBV DNA [EBV(+)], and these patients had more advanced disease at baseline than the rest. PD-1 and PD-L1 serum concentrations and their expressions on all cell populations were higher in EBV(+) than EBV(-) patients. EBV load correlated positively with unfavourable clinical markers of CLL and the expression of PD-1 and PD-L1 on CD4+ and CD8+ cells (rho =0.42–0.75; p<0.001). EBV(+) patients had increased risks of treatment initiation and lymphocyte doubling during a median follow-up period of 32 months (p<0.001). Among EBV(+), but not EBV(-), patients, higher expressions of PD-1 and PD-L1 on CD4+ and CD8+ cells were associated with higher risks of treatment initiation and lymphocyte doubling (p≤0.020).ConclusionEBV-induced up-regulation of PD-1-PD-L1 expression is associated with worse outcomes in CLL.

PB2067 PROGNOSTIC ROLE OF ACTIVE AND LATENT TRANSFORMING GROWTH FACTOR β1 LEVELS IN BLOOD OF PATIENTS WITH NON‐HODGKINS LYMPHOMA AND CHRONIC LYMPHOCYTIC LEUKEMIA

Background:Transforming growth factor β1 (TGFβ1), member of TGFβ‐ superfamily, is a potent inhibitor of hematopoietic cells proliferation which is synthesized as an inactive protein, latent TGFβ1. Active form of TGFβ1 appears in microenvironment under the influence of various physiological activators such as thrombospondin‐1, plasminogen and released via proteolytic cleavage. In vitro an active form owns growth‐inhibitory activity in relation to leukemia cells. However, in vivo TGFβ1 is often involvement in the pathological conditions and his behavior sometimes is complex and paradoxical. Dysregulation of the TGFβ1 pathway has been implicated in course of lymphoid malignancies. According to published data TGFβ1 concentration in blood usually is determined by the immunological method (ELISA) which detects total cytokine level including inactive epitopes of TGFβ1 molecules and its inability to measure biologically active TGFβ1.Aims:The aim of this study was to determine the prognostic role of active and latent TGFβ1 levels in blood of patients with non‐Hodgkins lymphoma (NHL) and chronic lymphocytic leukemia (CLL).MethodsThe TGFβ1 concentration was determined by bioassay using cells line CCL64, mink lung epithelial cells. The measure both of active and latent TGFβ1 levels has been applied in plasma and supernatants (SP) of mononuclear cells from peripheral blood (MCPB) of 43 untreated of non‐Hodgkins (NHL) patients (pts) and 63 pts with chronic lymphocytic leukemia (CLL). The results were compared with respective indexes in 15 healthy donors. For active TGFβ1 assay CCL64 cells were incubated with native plasma or with SP of MCPB. For total, latent TGFβ1 assay respective samples had been initially treated by 1 N HCl‐acid and than 1 N NaOH. Whereafter 3H‐methylthymidine was added to CCL64 cells after 24 h incubation with samples. The concentration of TGFβ1 was determined using a calibration curve based on the results of rhTGFβ1 influence in various doses on CLL64 cells.Results:It was discovered that latent concentration of TGFβ1 in plasma both of NHL (4,918 ± 1,240 ng/ml) and CLL pts (3,53 ± 0,40 ng/ml) was significantly higher in comparison with donors (2,11 ± 0,38 ng/ml; p &lt; 0,001 and p &lt; 0,05, respectively). Especially high level of latent TGFβ1 was detected in plasma of pts with aggressive NHL (4,60 ± 0,18 ng/ml) comparing with indolent NHL (2,91 ± 0,58 ng/ml; p &lt; 0,001). Latent TGFβ1 levels in SP MCPB of NHL (7,06 ± 1,23 ng/ml) and CLL pts (2,78 ± 0,78 ng/ml) significantly exceeded (p &lt; 0,001 in both cases) respective contents of TGFβ1 active form. Concentration of an active form of TGFβ1 in SP MCPB of CLL pts was significantly higher (1,32 ± 0,38 ng/ml) than in NHL pts (0,42 ± 0,28 ng/ml; p &lt; 0,001), which may some relation to the indolent course of CLL. In the group of NHL pts with complete remission latent level of TGFβ1 in plasma (1,72 ± 0,83 ng/ml) was significantly lower than in pts with partial remission (3,283 ± 0,570 ng/ml) or without remission (4,66 ± 1,25 ng/ml).Summary/Conclusion:An increase concentration of TGFβ1 in blood of pts with NHL or CLL can arise up through making of latent form by leukemia cells and to influence on the course of these neoplasms. Simultaneous measurement of both active and latent TGFβ1 levels would help us understand the mechanism of progressive lymphoid malignancies. Obtained results may allow to recommend determination both of active and latent TGFβ1 levels in plasma and in SP MCPB in order to predict the course of NHL and CLL.

Growth dynamics in naturally progressing chronic lymphocytic leukaemia.

How the genomic features of a patient's cancer relate to individual disease kinetics remains poorly understood. Here we used the indolent growth dynamics of chronic lymphocytic leukaemia (CLL) to analyse the growth rates and correspondinggenomic patterns of leukaemia cells from 107 patients with CLL, spanning decades-long disease courses. We found that CLL commonly demonstrates not only exponential expansion but also logistic growth, which is sigmoidal and reaches a certain steady-state level. Each growth pattern was associated with marked differences in genetic composition, the pace of disease progression and the extent of clonal evolution. In a subset of patients, whose serial samples underwent next-generation sequencing, we found that dynamic changes in the disease course of CLL were shaped by the genetic events that were already present in the early slow-growing stages. Finally, by analysing the growth rates of subclones compared with their parental clones, we quantified the growth advantage conferred by putative CLL drivers in vivo.

Evaluation of the prognostic and predictive value of free light chains in patients with chronic lymphocytic leukemia – preliminary results

AbstractIntroductionκ and λ serum free light chains (sFLCs) are produced during physiological lymphopoesis by plasmocytes and B lymphocytes in a constant ratio related to heavy chains. The measurement of sFLC plays an important role in the diagnosis and monitoring of patients with multiple myeloma (MM). The first reports suggested that sFLC disturbances might have prognostic value also in patients with chronic lymphocytic leukemia (CLL). Aim of the study: The aim of the study was to evaluate the relationship between sFLC concentration and recognized prognostic factors and clinical course of CLL. Materials and methods: The sFLC concentration was measured using a latex-enhanced immunoassay in 59 patients with newly diagnosed CLL. The relationship between sFLC concentration and time to start of the treatment (TFT), the response rate to therapy (ORR) and overall survival (OS) was assessed.ResultsA significant correlation was found between sFLC κ concentration and the clinical stage of leukemia according to Rai classification, β-2 microglobulin concentration, LDH activity, CD38 expression, as well as between sFLC λlevel and β-2 microglobulin concentration and platelet count (PLT ). There was also a correlation between the values of summated κ and λ and the clinical stage of disease according to Rai classification, β-2 microglobulin concentration, CD38 expression, white blood cells count (WBC), lymphocyte count (ALC) and hemoglobin (Hgb) concentration. The κ/λ ratio (FCLR) values were significantly different in the CD38+ and CD38- population.SummarySimple and reproducible clonality index, which constitutes the sFLC concentration assessment, can be an attractive, potential prognostic marker in patients with CLL, however further studies are needed on a larger group of patients especially in relation to the predictive value of sFLC.

Distribution of Some Functional Molecules on T-Lymphocytes in Chronic Lymphoblastic Leukemia

T-lymphocytes, their subpopulations, and natural killer cells (NCs) were characterized according to the distribution of membrane molecules involved in intercellular interactions and signaling (CD45, CD3, CD4, CD8, CD16, CD56) in patients with chronic lymphocytic leukemia at the stage of disease diagnosis. Current methodological approaches to determine the studied parameters were used, taking into account the characteristics of the lymphoid pool of blood in patients with chronic lymphocytic leukemia. It has been established that the population of T-lymphocytes of the patient peripheral blood is characterized by decreased content of CD45 and CD3 surface receptors. The average level of the membrane CD4 receptor was reduced in lymphocytes belonging to the T-helper subpopulation. The CD8 receptor remained unchanged in subpopulation of cytotoxic T-lymphocytes. CD8 and CD16 molecules were distributed on the surface of NC cells of patients with chronic lymphocytic leukemia at a normal level, while the expression of CD56 was significantly less. The data facilitate understanding of the energy of immune system cells and can be used to predict the course of chronic lymphocytic leukemia.

Richter syndrome epidemiology in a large population based chronic lymphocytic leukemia cohort from Norway

BackgroundTransformation to aggressive lymphoma (Richter syndrome, RS) occurs in a substantial subset of patients who must discontinue targeted therapy for chronic lymphocytic leukemia (CLL). RS has an extremely poor prognosis. MethodsUsing the nation-wide database of The Cancer Registry of Norway of 7664 CLL patients registered between 1953–2012, we identified 107 patients experiencing RS. ResultsSeventy seven (72%) of RS patients were identified among 2631 CLL patients diagnosed between 2003–2012; diffuse large B-cell lymphoma (DLBCL) was identified in 65 (84%), Hodgkin lymphoma (HL) in 12 (16%) patients and the diagnosis was confirmed in 50 (65%) available biopsy specimens. The incidence rate in this period was 4.7/1000 person-years (95% CI: 3.8–5.9). The median survival from CLL diagnosis was 1.7 years (95% CI: 0.34–2.3) for RS patients while it was 10.3 years (95% CI: 9.5–10.9) for the remaining CLL patients. Male gender predominated among RS patients (69%) compared to CLL population (58%) and RS patients were diagnosed with CLL at a significantly younger age than the remaining patients (65 vs. 72 years). Median time from diagnosis of CLL to RS was 2 years (Range, 0–13 years). No CLL treatment was administered in 25 (33%) patients prior RS diagnosis; a median of 1 treatment line was administered to pretreated patients. The median duration of survival after RS diagnosis was 27 months (95% CI; 9–88). ConclusionsCollectively, RS was a rare complication of CLL in the chemoimmunotherapy era, occurred early in the CLL course in younger, and both treatment naïve and pretreated patients, and shortened survival substantially.

Expression of the PIM2 gene is associated with more aggressive clinical course in patients with chronic lymphocytic leukemia.

The PIM2 gene belongs to the PIM family, which encodes serine/threonine kinases involved in cell survival and apoptosis. The relation between the expression of the PIM2 gene and the course of chronic lymphocytic leukemia (CLL) has not been fully determined. The aim of the study was to evaluate the role of the PIM2 gene as a marker of CLL malignancy and its importance as a predictive and prognostic factor. Sixty-seven patients, 35 females and 32 males, aged 49-90 years, with de novo CLL, and 14 healthy individuals were enrolled in the study. Expression of the PIM2 gene was analyzed using TaqMan RQ-PCR assay and western blot test. Median PIM2 gene expression in CLL patients was higher than in controls. Patients with high expression of the PIM2 gene had shorter progression-free survival and time to first treatment than patients with low PIM2 expression. It was found that patients with CR had lower expression of the PIM2 gene than patients without complete remission (CR). Notably, associations between high PIM2 expression and rapid lymphocyte doubling time, the percentage of malignant lymphocytes with ZAP70 expression and the Rai stage were revealed. We found that the PIM2 gene is associated with a more aggressive clinical course of CLL.

The Risk of Venous Thromboembolism Is High in Chronic Lymphocytic Leukemia Because of Additional Cancers: A Danish National Cohort Study

Background: Venous thromboembolism (VTE) is a frequent yet potentially preventable complication that increases morbidity and mortality among cancer patients. Chronic Lymphocytic Leukemia (CLL) is a heterogeneous hematological cancer. Patient specific factors (age, gender, performance status) and CLL specific markers (e.g. IgHV mutational status, Binet stage, and beta 2-microglobulin (B2M) levels) influence the clinical course of CLL. A considerable proportion of CLL patients are diagnosed with additional cancer after the CLL diagnosis. The risk of VTE in CLL is higher than the background population throughout the clinical course of CLL in contrast to solid tumors where VTE typically develops within the first few months after cancer diagnosis.Aim: To investigate the risk of VTE in CLL patients according to patient specific factors, CLL specific markers, CLL treatment and additional cancer after the CLL diagnosis.Methods: The Danish National CLL Registry provided prospectively collected clinical data from all CLL patients diagnosed in Denmark since 2008. These data were linked at person level by the unique civil registration number to the Danish National Patient Registry for information on VTE and additional cancers, to the Danish National Prescription Database for information on prescribed and reimbursed anticoagulation treatment and to the Danish Civil Registration System for information on vital status and emigration. Study entry was date of CLL diagnosis; subjects were followed until VTE, death, emigration or administrative censoring. Cumulative incidence of VTE according to Binet stage, B2M levels, IgHV mutational status and additional cancers diagnosed later than CLL were calculated. Additional cancer after the CLL diagnosis was defined as a time-varying exposure. Subjects contributed to the number being at risk of VTE in the “no additional cancer” group until the possible date of additional cancer at which they shifted to the “additional cancer after CLL” group. Death and additional cancer were treated as competing risks by use of the Aalen-Johansen estimator. In Cox proportional hazard models, we estimated the hazard ratios (HR) of VTE; WHO-performance score, previous VTE and sex were included in the models as categorical variables, age was included as a continuous variable and anticoagulation treatment and additional cancer were included as time-varying covariates in the appropriate models.Results: From 2008 through 2015, 3609 subjects were diagnosed with CLL, 60.1% were males and the median age at CLL diagnosis was 70.4 years. Median follow-up was 2.6 years. During the study period, 12.7% (461) were diagnosed with an additional cancer and 19.9% (721) died. Anticoagulation treatment was given to 6.3% (227) of the CLL patients in the study period. The majority of the CLL patients had mutated IgHV, and B2M levels below 4mg/L (Table 1). A VTE was registered in 92 CLL patients during the study period. The cumulative incidence of VTE was highest in CLL patients exposed to additional cancer followed by CLL patients with B2M levels above 4 mg/L and unmutated IgHV. (Figure 1). CLL patients with additional cancer after the CLL diagnosis had a 3-4 fold higher risk of VTE compared with CLL patients without additional cancer. Previous VTE increased the risk of VTE after the CLL diagnosis by 5 fold. This was to some extent because a larger proportion of CLL patients with previous VTE were exposed to an additional cancer after the CLL diagnosis (23.9%) compared with the total study population (12.8%). The HR of VTE in case of previous VTE however, remained high after adjustment for additional cancer cancer and other risk factors (Table 1). Unmutated IgHV and B2M level >4 mg/L increased the risk of VTE, these associations persisted after adjustment for additional cancer (Table 1). CLL treatment tended to increase the risk of VTE, patient related factors and CLL specific markers did not affect the association markedly.Conclusion: The risk of VTE was especially high in CLL patients with additional cancer. Previous VTE was also a substantial (expected) risk factor whereas high age and CLL specific markers (B2M level > 4 mg/L, unmutated IgHV) were significant but less strong risk factors of VTE. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Targeted Next-Generation Sequencing of TP53, BIRC3, NOTCH1, SF3B1, and ATM Identifies a Significant Proportion of Lower-Risk Chronic Lymphocytic Leukemia with Unfavorable Prognostic Indicators

IntroductionThe clinical course of chronic lymphocytic leukemia (CLL) is heterogeneous, ranging from indolent disease with essentially normal life expectancy to rapidly progressive disease with significantly decreased survival. Traditional risk stratification is dependent in large part on cytogenetic analysis by fluorescence in situ hybridization (FISH), with del(13q) as a sole abnormality associated with a favorable prognosis, trisomy 12 or normal cytogenetics associated with an intermediate prognosis, and del(11q) or del(17p) associated with an unfavorable prognosis.1 In the last few years, recurrent mutations in key genes including TP53, BIRC3, NOTCH1, SF3B1, and ATM have also been identified as unfavorable prognostic indicators in CLL.2-4 In a study by Rossi et al, integration of mutations and cytogenetic abnormalities was found to significantly improve the accuracy of survival prediction in CLL beyond that of FISH alone.2 The aim of this study is to evaluate the frequency of these unfavorable mutations in an unselected cohort of CLL patients seen in the community setting, and to determine the percentage of lower-risk patients with a worse than expected prognosis who may potentially benefit from earlier and/or novel therapy.MethodsAn unselected cohort of 633 CLL patients seen in the community setting was included in this study. Demographic data included a mean age of 69 years, male to female ratio of 1.8:1, and mean WBC count of 48 g/dL. DNA sequences of key exons in TP53, BIRC3, NOTCH1, SF3B1, and ATM were determined from peripheral blood or bone marrow aspirates using a clinically validated next-generation sequencing (NGS) assay with a 5% lower limit of detection for single nucleotide variants. Results were compared between duplicate samples and annotated using software that queried databases containing known somatic mutations and germline variants. FISH analysis utilized probes specific for 11q22.3 (ATM), chromosome 12, 13q14.3/13q34, and 17p13.1 (TP53). CLL diagnoses were confirmed by flow cytometry, with FISH for t(11;14)(IGH-CCND1) also performed to exclude mantle cell lymphoma. The percentage of CLL cells by flow cytometry ranged from 18-98%, with a mean of 69%.ResultsCytogenetic abnormalities were detected in 85% of CLL patients in this study, with del(13q) the most frequent (65%), followed by trisomy 12 (19%), del(11q) (17%), and del(17p) (10%). Mutations in any one of the 5 genes were detected in 37% of patients, with TP53 mutation the most frequent (12%), followed by mutations in NOTCH1 (11%), SF3B1 (10%), ATM (7%), and BIRC3 (3%). TP53 disruption as a whole was found in 14% of patients, consisting of del(17p) only (2%), TP53 mutation only (5%), and biallelic TP53 disruption (7%). ATM disruption as a whole was found in 21% of patients, consisting of del(11q) only (14%), ATM mutation only (4%), and biallelic ATM disruption (4%). Lower-risk patients, as defined by FISH showing either isolated del(13q), trisomy 12, or normal results, comprised 75% of patients, with 29% showing one or more unfavorable mutation: NOTCH1 (12%), SF3B1 (8%), TP53 (6%), ATM (4%), and BIRC3 (3%).ConclusionsMutations in TP53, BIRC3, NOTCH1, SF3B1, and ATM have previously been shown to be indicators of unfavorable prognosis in CLL. Using a clinically validated NGS assay, 37% of the CLL patients in our cohort were shown to have a mutation in any one of the 5 genes. In patients with lower-risk disease as defined by FISH, 29% showed one or more unfavorable mutation, including 6% with a TP53 mutation. Mutational profiling using NGS can thus help identify a significant proportion of lower-risk CLL patients with a worse than expected prognosis who may potentially benefit from earlier and/or novel therapy.ReferencesDohner H, et al. N Engl J Med 2000;343:1910-1916Rossi D, et al. Blood 2013;121:1403-1412Austen B, et al. J Clin Oncol 2007;25:5448-5457Skowronska A, et al. J Clin Oncol 2012;30:4524-32 DisclosuresNo relevant conflicts of interest to declare.

Real Life Dates of Chronic Lymphocytic Leukemia (CLL) in Germany. Interim Analysis of 234 Consecutively New Diagnosed Patients in a German Epidemiological-Clinical Register

Background:Therapy studies yield important insights into clinical features and therapeutic options of CLL. However studies in Germany represent less then 20% of all CLL patients (pts); pts >75 years (yrs) are included in 3% only. Furthermore initial phases are not reflected in multicenter studies and data representing real life situation are scarce.Methods:Out of 1.443 patients consulting our center because of leucocytosis (>10 Gpt/l) between 2003/07/01 and 2018/06/30 we diagnosed CLL consecutively in 234 pts (16,2%): 143 male (m), 91 female (f). Median age at diagnosis is 71,4 yrs. Median leucocyte count is 18,0 Gpt/l. BINET - Stages are: A in 82%, B in 11% and C in 7% of pts. Diagnoses are strictly based on WHO definition: Pts with small lymphocytic lymphomas (sLL, n=11) are included but those with monoclonal lymphocytosis of unknown significance (MLUS) or monoclonal B - lymphocytoses with uncertainly flow cytometry (FC) results are excluded. Histopathology, FC and genetics have been performed by external hematological reference laboratories. All investigations followed the rules of best clinical and laboratory practice. Dates of death are given by the record sections of the involved communities (deadline 2018/06/30).To address different questions we defined 3 groups of pts: A: “collective group”, (n=234), i.e. all pts.diagnosed from 2003/07/01 to 2018/06/30. B: “epidemiological group” (n=129), i.e. pts from 3 communities (113.000 inhabitants - inc - in 2009/12/31) in close proximity referred to our center. C: “genetic features group” (n=99), i.e. pts diagnosed continuously between 2012/07/01 and 2018/06/30 with systematically performed genetics, (i.e. at least 80% of pts in this group. Genetics are: Cytogenetic (banding) n=88; FISH (del 6q21/6q, del 11q22.3, +12/+12q, del 13q14/ 13q34, del 17p13.1, 14q32) n=85. PCR (IgVH status - mutated vs. unmutated; TP53, NOTCH1, SF3B1 mutation) n=82.Results:“Collective group” (A): 7,5 years OS is 72% and 15 years OS is 33% respectively (KAPLAN-MEIER). Age of pts acts as a predominant factor for long term OS: 77% in pts < 60 yrs vs. 40% in pts ≥60 to <75 yrs vs. 16% in pts ≥75 yrs. 15 yrs OS of treated pts is 32% (n=78, median age at diagnosis: 73,3 yrs, m : f = 1,50); 15 yrs OS of untreated patients is 33% (n=146, median age at diagnosis: 71,0 yrs, m : f = 1,56). Mean time to 1st treatment was 16,0 month (0,1 to 132,6 months).“Epidemiological group” (B): Raw incidence is 7,56/105 inh. (m: 9,13, f: 6,06), age adjusted incidence is 10,3/105 inh (m: 12,5, f: 8,3), with highest rate of age between 75 to < 80 yrs. Standard incidence is calculated as 6,27/105 (BRD 1987), 4,78/105 (Europe) and 5,06/105 (USA 2000). Prevalence could be measured directly (2017-12-31): 79,1/105 inh.“Genetic features group” (C): IgVH mutation status is hypermutated in 48 pts. vs. unmutated in 39 pts. Results of FISH analyses (n = 85) are: del 6: 5,5%, del 11: 17,6%, +12: 3%, del 13q: 70,3%, del 17p: 8,8%; normal status: 13,2%. PCR revealed mutations of NOTCH1 in 7%, of SF3B1 in 10,5% and of TP53 in 8,6% of 82 pts, respectively. Combined del17p by FISH and molecular TP53 mutations at diagnosis are found in 3 of 81 pts (3,7%).Cytogenetic findings different to results of FISH and/or PCR were found in 36 of 82 pts (43,9%).Calculated 5 yrs freedom of therapy is 75% in IgVH hypermutated pts vs. 45% in unmutated pts (p = 0,0005; log rank test); calculated 5 yrs OS is 92% vs. 59% (p=0,023).Special situations in CLL: 13 of 234 pts (5,5%) have one parent or sibling with low grade NHL, mostly CLL. In contrast there is non spouse suffering of CLL / low grade NHL. Deficiency in Immunoglobuline G (Ig G < 4,0 g/l) was found in 17 of 199 pts at diagnose (8,5%), 29 pts (12,4%) were substituted with Ig G's in the Course of CLL. In 14 (6,0%) pts. we diagnosed a 2nd hematologic malignancy (3x cMPN, 1x CML, 3x MDS, 3x AML, 1x FL, 1x MCL, 1x MZL, 1x DLCBL-independent of CLL; monoclonal gammopathies are not considered). RICHTER syndrome was found in 1 case, but progression to plasmocytic/plasmoblastic disease was seen in 3 cases. Li FRAUMENI Syndrome was revealed in 1 male patient, he has been in stable disease for 13 yrs.Conclusion:CLL seems to be more frequent than yet considered. IgVH mutation status seems to be very important as a single prognostic factor concerning time of 1st therapy as well as OS. Clinical features of CLL are very different and impressing. DisclosuresBöttcher:Genentech: Research Funding; Janssen: Honoraria; AbbVie: Honoraria, Research Funding; Celgene: Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.