Prognosis Of Chronic Lymphocytic Leukemia Patients Research Articles

Long-read transcriptome sequencing of CLL and MDS patients uncovers molecular effects of SF3B1 mutations.

Mutations in splicing factor 3B subunit 1 (SF3B1) frequently occur in patients with chronic lymphocytic leukemia (CLL) and myelodysplastic syndromes (MDS). These mutations have different effects on the disease prognosis with beneficial effect in MDS and worse prognosis in CLL patients. A full-length transcriptome approach can expand our knowledge on SF3B1 mutation effects on RNA splicing and its contribution to patient survival and treatment options. We applied long-read transcriptome sequencing (LRTS) to 44 MDS and CLL patients, as well as two pairs of isogenic cell lines with and without SF3B1 mutations, and found >60% of novel isoforms. Splicing alterations were largely shared between cancer types and specifically affected the usage of introns and 3' splice sites. Our data highlighted a constrained window at canonical 3' splice sites in which dynamic splice site switches occurred in SF3B1-mutated patients. Using transcriptome-wide RNA binding maps and molecular dynamics simulations, we showed multimodal SF3B1 binding at 3' splice sites and predicted reduced RNA binding at the second binding pocket of SF3B1K700E Our work presents the hitherto most complete LRTS study of the SF3B1 mutation in CLL and MDS and provides a resource to study aberrant splicing in cancer. Moreover, we showed that different disease prognosis most likely results from the different cell types expanded during carcinogenesis rather than different mechanisms of action of the mutated SF3B1 These results have important implications for understanding the role of SF3B1 mutations in hematological malignancies and other related diseases.

Elevated serum ELABELA and SERPINA3 as novel biomarkers to predict poor prognosis in Egyptian chronic lymphocytic leukemia patients

Abstract Background Chronic lymphocytic leukemia (CLL) is the most common kind of adult leukemia worldwide. Increased expressions of serpin peptidase inhibitor, clade A member 3 (SERPINA3) and apelin receptor early endogenous ligand (ELABELA) have been observed in various malignancies. However, there is little information on their clinical importance in CLL. Patients and methods The study included 67 newly diagnosed CLL patients and 66 healthy individuals as a control group. This study was conducted at the Oncology Center, Mansoura University, Mansoura, Egypt from April 2020 to March 2022. Serum levels of SERPINA3 and ELABELA were determined using an enzyme-linked immunosorbent assay. Results This study found that serum levels of SERPINA3 and ELABELA were significantly higher in CLL patients than in the control group with P less than 0.001 and P=0.001, respectively. SERPINA3 and ELABELA were found to be independent risk factors for CLL. In CLL patients, increased serum levels of SERPINA3 and ELABELA were associated with advanced staging, reduced progression-free survival, and overall survival. Conclusion Serum SERPINA3 and ELABELA could be used as prognostic biomarkers to predict poor prognosis in CLL patients. So, inhibition of SERPINA3 and ELABELA may be a promising therapeutic target for CLL treatment.

Real-World Clinical Outcomes and Adverse Events in Patients with Chronic Lymphocytic Leukemia Treated with Ibrutinib: A Single-Center Retrospective Study.

Background and Objectives: The treatment of chronic lymphocytic leukemia (CLL) has acquired new targeted therapies. In clinical trials, ibrutinib improved outcomes safely. Real-world data called for a reappraisal of ibrutinib strategies. We report on a single center's experience with ibrutinib monotherapy, aiming to explore the outcomes, tolerability, and prognosis of CLL patients in routine clinical practice. Materials and Methods: Data were collected from all CLL patients treated with ibrutinib at Fundeni Clinical Institute, Bucharest, Romania, between January 2016 and June 2021. Results: A total of one hundred twenty-three CLL adult patients were treated with ibrutinib. Of the patients, 87% had relapsed/refractory CLL. The median age at ibrutinib initiation was 65 years; 44.7% of patients were staged Rai III/IV. At 32-month median follow-up, the median progression-free survival (PFS) was 50 months, the overall survival (OS) was not reached, and the overall response rate (ORR) was 86.2%. The age or number of previous therapies did not impact outcomes or tolerability. An Eastern Cooperative Oncology Group performance status (ECOG PS) score ≥ 2 and shorter time from initiation of last therapy (TILT) before ibrutinib predicted inferior PFS. Baseline characteristics had no impact on the OS except for TILT in R/R CLL patients. Drug-related adverse events (AEs) of any grade and grade ≥ 3 AEs were reported in 82.1% and 30.9% of the patients, respectively. Infections were the most common AEs (29.3%). Drug discontinuation was permanent in 43.9% of patients, mainly due to disease progression (17.1%) and toxicity (8.9%). Patients with a Cumulative Illness Rating Scale (CIRS) score ≥ 6 had a higher risk for toxicity-related discontinuation. An ECOG PS ≥ 2 predicted an increased rate of permanent discontinuation and grade ≥ 3 AEs. Conclusions: The outcomes of this study align with the results from ibrutinib clinical trials. Our study demonstrated that poor patient fitness, early relapse before ibrutinib, and permanent ibrutinib discontinuation are essential outcome determinants. Patient comorbidity burden and fitness were significant predictors for ibrutinib intolerance.

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

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

Expression of CTLA-4 and CD86 Antigens and Epstein-Barr Virus Reactivation in Chronic Lymphocytic Leukemia-Any Link with Known Prognostic Factors?

Simple SummaryChronic lymphocytic leukemia (CLL) accounts for one-third of all leukemias. The Epstein-Barr virus (EBV) has the ability to transform B-cells into cancer cells. A history of EBV infection increases the chances of acquiring CLL and it worsens the prognosis in CLL. We tried to assess whether EBV affects the course of CLL by deregulating the CTLA-4/CD86 pathway. The expression of CTLA-4 and CD86 on immune cells in patients with CLL has been evaluated and linked to indicators of EBV infection and clinical outcomes. Our studies have shown that anergy, which is expressed by inhibition through the interaction of CTLA-4 and CD86, is an important mechanism leading to the inhibition of the antitumor response and CLL progression.Infection with Epstein-Barr virus (EBV) worsens the prognosis in chronic lymphocytic leukemia (CLL), but the underlying mechanisms are not yet established. We intended to assess whether EBV affects the course of CLL by the deregulation of the CTLA-4/CD86 signaling pathway. We used polymerase chain reaction to measure the load of EBV DNA in the blood of 110 newly diagnosed patients with CLL. The expression of CTLA-4 and CD86 antigen on lymphocytes was assessed with flow cytometry. Additionally, CTLA-4 and CD86 serum concentrations were measured through enzyme-linked immunosorbent assays. Fifty-four percent of the patients had detectable EBV DNA [EBV(+)]. In EBV(+) patients the CTLA-4 and CD86 serum concentrations and their expressions on investigated cell populations were significantly higher than in EBV(−) patients. EBV load correlated positively with unfavorable prognostic markers of CLL and the expression of CTLA-4 on CD3+ lymphocytes (r = 0.5339; p = 0.027) and CD86 on CD19+ cells (r = 0.6950; p < 0.001). During a median follow-up period of 32 months EBV(+) patients were more likely to require treatment or have lymphocyte doubling (p < 0.001). Among EBV(+) but not EBV(−) patients, increased expressions of CTLA-4 lymphocytes were associated with elevated risks of progression. We propose that EBV coinfection may worsen prognosis in CLL patients, partly due to EBV-induced up-regulation of CTLA-4 expression.

Single-Cell RNA Sequencing Suggests Novel Drivers of Chronic Lymphocytic Leukemia Patients with Ibrutinib Resistance

Objective:Ibrutinib is currently the most widely used BTK inhibitor that approved for the treatment of both initially diagnosed and relapsed and refractory chronic lymphocytic leukemia (CLL) patients. Although ibrutinib shows high response rates in clinical practice, it has certain limitations. There are still a certain number of patients who have to discontinue treatment due to drug-resistance or side effects. The ibrutinib resistance of CLL patients has caused widespread concerns, necessitating the development of novel treatment strategies.Methods:Here, we examined the heterogeneity of peripheral blood mononuclear cells (PBMCs) from patients with ibrutinib-sensitive (IBS) and -resistant (IBR) CLL by analyzing bulk and single-cell level gene expression profiles, clinical features, biological properties, and phenotypes. Seven distinct ibrutinib-resistant subpopulations were identified and two candidate genes LGALS1 (galectin 1, Gal-1) and LAG3 (lymphocyte-activating gene 3, CD223) were screened that contribute toward ibrutinib-resistance and poor survival in CLL patients. These results were validated in primary cells from CLL patients and also in ibrutinib-resistant CLL cell line (MEC1-IR) which was generated by culturing the parental cell line in vitro with progressively increasing concentrations of ibrutinib. Marker-gene expression was detected using qRT-PCR, western blotting, and ELISA, while functional analyses including CCK8, flow cytometry and trypan blue staining were conducted with or without OTX008, a selective Gal-1 inhibitor.Results:ScRNA-seq revealed that cells from IBR and IBS samples were distributed in different clusters and suggested that IBR cells display a unique transcriptional pattern (Fig A). IBR-B cells have higher stemness scores and are enriched in some energy metabolismPathways (Fig B). According to the proportion of B cells from IBR samples, we classified each B-cell cluster into three main subgroups, i.e., IBR, IBS, and shared cluster (Fig C). IBR-B cells displayed more interactions with monocytes, NK, T, and dendritic cells than IBS B cells, suggesting that IBR B cells may actively build connections with other immune cells to reshape the protective niche (Fig D). A close correlation between LGALS1 and LAG3 expression was observed and both of them were found to be highly expressed in IBR CLL patients (Fig E), their expression level gradually increased along the trajectory of B cells from IBS to IBR (Fig F). Diagnosis and prognosis stratification of CLL with receiver operating characteristic (ROC) curves revealed that patients with higher expression of both LGALS1 and LAG3 showed the poorest overall survival, indicating that LGALS1 and LAG3 are associated with ibrutinib-resistance and poor prognosis in CLL (Fig G). Concordantly, acquired resistance following chronic exposure to ibrutinib leads to upregulation of LGALS1 and LAG3 (Fig H). LGALS1 inhibitor OTX008 effectively inhibits the growth of ibrutinib-resistant CLL cells, particularly for IBR patients (Fig I).Conclusion:In conclusion, our findings demonstrate that ibrutinib-resistant CLL cells exhibit a unique transcriptional pattern. The combination of LGALS1 and LAG3 expression could serve as an indicator of the sensitivity of ibrutinib and prognosis of CLL patients. LGALS1 inhibitor OTX008 helps to overcome ibrutinib-resistance of CLL cells. Our findings may expand the current knowledge regarding ibrutinib-resistant CLL patients, identify improved biomarkers for patient selection, and offer a promising combinatorial therapeutic strategy for IBR CLL patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Neutrophil to T-Lymphocyte Ratio (NLR) and Neutrophil to Monocyte Ratio (NMR) Are Promising Biomarkers in Chronic Lymphocytic Leukemia Prognostic Assessment

Development of targeted drugs produced a significant changes in the management of patients with Chronic Lymphocytic Leukemia (CLL). The recent advances in molecular diagnostic techniques has provided a better prediction of disease progression but they are not easily accessible to all Institutions.CLL cells proliferation and survival are sustained by a complex microenvironmental network of cells interactions that are at least partially mediated through B cell receptor signaling.An increased number of studies describe the role of neutrophils and monocytes as markers of the inflammation and antitumor immunity that support tumorigenesis. Recently, authors published the increased NLR is associated to a prognosis in different cancer type.In our study we retrospectively evaluated the prognostic significance of peripheral blood neutrophils, monocytes and non-neoplastic lymphocytes in a unicentric, unselected, CLL cohort of 400 CLL patients at diagnosis (median age 64; stage Binet A 76%,B 18%,C 6%).Using blood count and the flow cytometric analysis reports, absolute neutrophil count (ANC), absolute monocyte count (AMC) and absolute T-lymphocyte count (ALC-CD3+) were evaluated and the neutrophil to T-lymphocyte ratio (NLR), the monocyte to T-lymphocyte ratio (MLR) and the neutrophil to monocyte ratio (NMR) were calculated at diagnosis, follow up and at relapse. Clinical and biological data from all patients were also retrieved to make a better comparison with the major clinical and prognostic markers commonly used.Compared to normal controls, CLL patients showed an increase AMC value (788±65 vs 469±51 cells/μL, p=0.005) and a slightly increased in the ALC-CD3+ value (2514±63 vs 2189±45 cells/μL, p=0.04). No difference in the ANC. The median NLR ratio was higher in CLL patients compared to healthy individuals (2.32 vs 1.7, p=0.02) and appeared more increased in early stage (2.65, 2.14, 1.95 mean value in Binet A, B, C respectively, p=0.0015). NMR median value was also significantly higher in early stage compared to advanced stage (8.0, 6.7, 5.1 in Binet A, B, C, p=0.0008).Conversely, MLR showed the opposite trend resulting more elevated in advanced stage at diagnosis, although with a low statistical significance.We did not found any correlation between these ratio and the IGVH status or genetic abnormalities.NLR and NMR higher level are associated with the absence of serum prognostic markers, such as CD38, and CD49d (p=0.002).160 out of 400 patients of our CLL cohort received a treatment during the course of disease. In our analysis, NLR and NMR are significantly higher in untreated patients than treated ones (median NLR 2.42 vs 1.95, p=0.0021; NMR 8.0 vs 7.0, p=0.0002). Among treated patients NLR and NMR significantly correlate with the time to treatment initiation (TTFT): lower NLR and NMR ratio correspond to a shorter TTFT (NLR p=0.03; NMR p=0.0035). For MLR the trend is opposite.A subgroup analysis conducted only on Rai 0/Binet A patients (n 219, 55%) showed that NLR and specially NMR are significantly associated with the risk of progression: lower NLR and NMR ratio correspond to an increased risk to symptomatic progression (p=0.0017), strengthening the hypothesis that high ratios are associated with a more indolent form of disease.In the 0/A population, analysis showed that a higher AMC and at a lesser extend a high ALC-CD3+ correlate with the progression rate and treatment initiation (Kaplan-Meier survival curve, p= 0.0001).Univariate analysis suggested that NLR ≥ 3.0, NMR ≥ 8.0 and AMC <900/mmc predicted a good prognosis in CLL patients. However, only NMR and AMC appeared as independent prognostic factors in multivariable analysis in Rai 0/Binet A patients.Our in vitro experiments supported these finding confirming: the non “immunosuppressive” role for neutrophils in CLL and the increased ability for monocytes to suppress T-lymphocytes activation and expand T-regulatory cell.In our study NLR ratio showed a positive prognostic role in CLL. A strongest protective meaning is displayed by NMR value. These combined value and AMC count provide more prognostic information at diagnosis and are associated with clinical outcome in CLL patients.These simple, cheap and reliable markers could be easily integrated into clinical practice reflecting both inflammatory status, immune response and microenviroment that contribute to disease progression. DisclosuresNo relevant conflicts of interest to declare.

Musashi 2 influences chronic lymphocytic leukemia cell survival and growth making it a potential therapeutic target

Progression of chronic lymphocytic leukemia (CLL) results from the expansion of a small fraction of proliferating leukemic B cells. When comparing the global gene expression of recently divided CLL cells with that of previously divided cells, we found higher levels of genes involved in regulating gene expression. One of these was the oncogene Musashi 2 (MSI2), an RNA-binding protein that induces or represses translation. While there is an established role for MSI2 in normal and malignant stem cells, much less is known about its expression and role in CLL. Here we report for the first time ex vivo and in vitro experiments that MSI2 protein levels are higher in dividing and recently divided leukemic cells and that downregulating MSI2 expression or blocking its function eliminates primary human and murine CLL and mature myeloid cells. Notably, mature T cells and hematopoietic stem and progenitor cells are not affected. We also confirm that higher MSI2 levels correlate with poor outcome markers, shorter time-to-first-treatment, and overall survival. Thus, our data highlight an important role for MSI2 in CLL-cell survival and proliferation and associate MSI2 with poor prognosis in CLL patients. Collectively, these findings pinpoint MSI2 as a potentially valuable therapeutic target in CLL.

Unraveling KDM4 histone demethylase expression and its association with adverse cytogenetic findings in chronic lymphocytic leukemia.

The acquisition of complex karyotypes is related to the progression of chronic lymphocytic leukemia (CLL) and patients with this condition have a poor prognosis. Despite recent advances in the classification of prognosis in CLL patients, understanding of the molecular mechanisms that lead to genomic instability and progression of this disease remains inadequate. Interestingly, dysregulated expression of KDM4 members is involved in the progression of several cancer types and plays a role in the DNA damage response; however, the gene expression profile and the importance of KDM4 members in CLL are still unknown. Here, we assessed the gene expression profile of KDM4A, KDM4B, and KDM4C in 59 CLL samples and investigated whether these histone demethylases have any influence on the prognostic markers of this leukemia. KDM4A gene expression was higher in CLL patients as compared with control samples. In contrast, CLL samples showed decreased levels of the KDM4B transcript in relation to control cases, and no difference was detected in KDM4C expression. Furthermore, patients with positive expression of ZAP-70 had lower expression of KDM4B and KDM4C as compared with ZAP-70-negative patients. More importantly, patients with low expression of these histone demethylases had higher leukemic cell numbers and displayed adverse cytogenetic findings and the acquisition of a complex karyotype. The present data clearly show that the expression of KDM4 members is dysregulated in CLL and impact the prognosis of this leukemia. These findings are useful for a better understanding of the impact of epigenetics on CLL progression.

TLR2 Expression on Leukemic B Cells from Patients with Chronic Lymphocytic Leukemia

Antigenic stimulation is considered as a possible trigger of neoplastic transformation in chronic lymphocytic leukemia (CLL). B-cell receptor plays a key role in the interactions between the microenvironment and leukemic cells; however, an important role has also been attributed to Toll-like receptors (TLRs). It is believed that disorders of TLR expression may play a part in the pathogenesis of CLL. In this study, we investigated the potential role of TLR2 in CLL by analyzing its expression on leukemic B cells in correlation with clinical and laboratory parameters characterizing disease activity and patients’ immune status. We assessed the frequencies of TLR2+/CD19+ cells by the flow cytometry method in peripheral blood of 119 patients with CLL. The percentage of TLR2+/CD19+ cells was significantly lower in patients with CLL as compared to the healthy volunteers. There was also a lower percentage of TLR2+/CD19+ cells in CLL patients with poor prognostic factors, such as ZAP70 and/or CD38 expression, 17p and/or 11q deletion. On the other hand, among patients with del(13q14) associated with favorable prognosis, the percentage of TLR2+/CD19+ cells was higher than among those with del(11q22) and/or del(17p13) as well as in the control group. We found an association between low percentage of CD19+/CD5+/TLR2+ cells and shorter time to treatment. We also demonstrated the relationship between low percentage of CD19+/CD5+ TLR2-positive and overall survival (OS) of CLL patients. CLL patients with a proportion of 1.6% TLR2-positive B CD5+ cells (according to the receiver operating characteristic curve analysis) or more had a longer time to treatment and longer OS than the group with a lower percentage of TLR2 positive cells. To sum up, the results of the study suggest that low TLR2 expression is associated with poor prognosis in CLL patients. The monitoring of CD19+/CD5+/TLR2+ cells number may provide useful information on disease activity. Level of TLR2 expression on leukemic B cells may be an important factor of immunological dysfunction for patients with CLL. Our study suggests that TLR2 could becomes potential biological markers for the clinical outcome in patients with CLL.

Expression of miRNA-155 in Uygur patients with chronic lymphocytic leukemia and its clinical significance

Objective To explore the expression of miRNA-155 (miR-155) in CD19+ B lymphocytes in Uygur patients with chronic lymphocytic leukemia (CLL), and to analyze its clinical significance. Methods Peripheral blood (10-15 ml) samples of 30 Uygur CLL patients, 20 Han CLL patients, 10 Han healthy people and 10 Uygur healthy people were collected. Mononuclear cells were isolated and B lymphocytes were purified by using CD19+ magnetic-bead system. Total RNA was extracted from CD19+ B lymphocytes. The expression of miR-155 was measured by using TaqMan probe method. All the results were analyze combined with the clinical data of patients. Results The expression level of miR-155 in Han and Uygur CLL patients had no statistical difference (5.994±1.156 vs. 5.856±1.359, t= 0.556, P= 0.710), which was significantly higher than that in the healthy controls (t= 18.612, P 0.05). The expression level of miR-155 had a significant statistical difference between ZAP-70 negative group and ZAP-70 positive group (5.7 ± 1.1 vs. 6.5 ± 1.4, t= 1.575, P= 0.029). The expression level of miR-155 also had a statistical difference between mutated immunoglobulin heavy chain variable region (IGHV) and unmutated IGHV (5.3 ± 1.1 vs. 6.5 ± 1.1, t= 0.210, P= 0.001). The expression level of miR-155 was 5.4 ± 1.2, 5.7 ± 0.7, and 6.6 ± 1.5 in the favorable prognosis group, moderate prognosis group and poor prognosis group respectively (F= 3.609, P= 0.035); The expression levels of miR-155 had significant differences between moderate group and poor prognosis group (P= 0.032), favorable group and poor prognosis group (P= 0.007). Conclusions The expression of miR-155 in Han and Uygur CLL patients has no significant difference. High-level expression of miR-155 is associated with the poor prognosis of CLL patients. Key words: Leukemia, lymphocytic, chronic; MicroRNAs; MicroRNA-155; Minority groups

EBV infection leads to activation of chronic lymphocytic leukemia (CLL) cells and worsen clinical prognosis in CLL patients

Event Abstract Back to Event EBV infection leads to activation of chronic lymphocytic leukemia (CLL) cells and worsen clinical prognosis in CLL patients Jacek Rolinski1, Ewelina Grywalska1*, Agnieszka Bojarska-Junak1 and Piotr Grabarczyk2 1 Medical University of Lublin, Department of Immunology and Immunotherapy, Poland 2 Institute of Haematology and Transfusion Medicine, Department of Immunohaematology, Poland Chronic lymphocytic leukemia (CLL) is a heterogenic disease in clinical outcome, but the reason of heterogeneity remains not understood. The role of chronic immune stimulation, leading to the activation of B lymphocytes is under investigation. Preliminary observations indicate a possibility of facilitate clonal expansion and leukemic transformation of B cells by exoantigens. The aim of the study was an assessment of the influence of Epstein-Barr virus (EBV) infection on activation of CLL cells and determination of the relationship between the presence of viral DNA and CLL clinical outcome. The assessment of the immunophenotype of peripheral blood (PB) lymphocytes, focusing on lymphocyte activation markers, such as CD25 and CD69, with the use of flow cytometric method, and the assessment of the presence of the EBV DNA with the use of RT-PCR method, was conducted in blood samples (PBMC) of 80 untreated CLL patients. Significantly higher percentage of activated B cells CD19+CD25+ and T cells CD3+CD25+ was found in PB of those CLL patients in whom DNA EBV was found in PBMC (p<0.0000001 and p=0.024, respectively). Positive correlation between the number of copies DNA EBV and the percentage of CD19+CD25+ B cells (r=0.502, p=0.033), CD19+ZAP-70+ B cells (r=0.751, p=0.0003), and CD19+CD38+ B cells (r=0.476, p=0.038). Moreover, those patients whose lymphocyte counts doubled in less than 12 months had more DNA EBV copies (p=0.0003). Obtained results suggest that EBV plays an important role in the stimulation of CLL lymphocytes and may induce B-CLL cells proliferation, leading to the fatal outcome of the disease. Keywords: Epstein-Barr virus, chronic lymphocytic leukemia, activation, RT-PCR, Flow Cytometry Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Host-pathogen interactions Citation: Rolinski J, Grywalska E, Bojarska-Junak A and Grabarczyk P (2013). EBV infection leads to activation of chronic lymphocytic leukemia (CLL) cells and worsen clinical prognosis in CLL patients. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.01073 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 28 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Ewelina Grywalska, Medical University of Lublin, Department of Immunology and Immunotherapy, Lublin, 20-093, Poland, ewelina.grywalska@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Jacek Rolinski Ewelina Grywalska Agnieszka Bojarska-Junak Piotr Grabarczyk Google Jacek Rolinski Ewelina Grywalska Agnieszka Bojarska-Junak Piotr Grabarczyk Google Scholar Jacek Rolinski Ewelina Grywalska Agnieszka Bojarska-Junak Piotr Grabarczyk PubMed Jacek Rolinski Ewelina Grywalska Agnieszka Bojarska-Junak Piotr Grabarczyk Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Chronic lymphocytic leukemia: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up

Chronic lymphocytic leukemia: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up

Abstract 4051: MiR-181b expression levels decreases during the progression of the Chronic Lymphocytic Leukemia: a new potential prognostic tool

Abstract In patients with Chronic Lymphocytic Leukemia (CLL) the prognosis is extremely variable, ranging from a very short to a normal lifespan. Classic clinical stages, based on biologic and clinical parameters, have been the most important factor for assessing prognosis in CLL patients; however, they are not devoid of limitations. Therefore, investigations are ongoing for searching new parameters in an attempt to add prognostic power to clinical stages. In this study we performed a genome-wide microRNA expression profiling to investigate the role of microRNA genes in the progression of CLL and to develop better prognostic indicators of these disease. We compared paired samples from CLL patients having an indolent and stable disease with their progressed aggressive form. We found that miR-181b was consistently down-regulated in the aggressive form of the disease compared to its previous stable counterpart. Further studies on serial time points from the same patient demonstrated that the strong decrease of the miR-181b expression levels occurs before the disease is clearly aggressive Moreover, we observed that the miR-181b expression remain constant over the time in two time points from CLL patients having a stable course; entailing a decreased levels of the miR-181b in the aggressive form of the CLL as a specific effect due to the progression of the disease. Finally in this study we investigate the biological role of the putative prognostic indicator related to the TCL1A, which is a critical gene in the pathogenesis of the CLL. In conclusion our data hint a prognostic role of the miR-181b in CLL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4051.

CD49d, Zap‐70, and CD38 Expression in Chronic Lymphocytic Leukemia

Zeta‐chain (TCR) associated protein kinase 70kDa (Zap‐70), and CD38 expression are predictors of IgVH mutation and prognosis in chronic lymphocytic leukemia (CLL). Previous studies indicate that Zap‐70 and CD38 are usually positive in cases of CLL with non‐mutated immunoglobulin variable region genes (IgVH), and may be used to predict IgVH mutation status and prognosis. Usually, cases of CLL positive for Zap‐70 or CD38 indicate a worse prognosis. In the present investigation, 62 cases of CLL were evaluated for CD38 expression, and 32 cases were evaluated for both Zap‐70 and CD38 expression. Of the 62 cases, 40% positively expressed CD38. Of the 32 cases evaluated for Zap‐70, 65% were positive, while only 35% were positive for CD38 expression; the remaining cases were negative for CD38. More recently CD49d, an integrin molecule, has been added as a predictor of prognosis in CLL. The results of this study show that Zap‐70 expression may be a better indicator of the mutational status of IgVH and prognosis of CLL than CD38 expression, and that CD49d represents an improved prognostic capability in CLL cases. In addition, CD38 negativity does not necessarily indicate that IgVH mutation has occurred. These data point to the need for a broader study to evaluate the significance of Zap‐70 and CD38 expression as indicators of IgVH mutation status, and of CD49d in the prognosis of CLL patients.

VEGF Expression as Prognostic Factor in Early Clinical Stages of Chronic Lymphocytic Leukemia Patients

Introduction: Chronic Lymphocytic Leukemia (CLL) is the most frequent leukemia in adults of western countries. Its clinical course is variable with some patients presenting an indolent disease while others have an aggressive disease requiring prompt treatment. Biomarkers to identify patients with poor prognosis, especially in early clinical stages, are being studied. Increase of angiogenesis, as an apoptosis deregulator, has been associated to poor prognosis in some malignant diseases such as Multiple Myeloma and Non-Hodgkin Lymphoma. Expression of Vascular Endothelial Growth Factor (VEGF) in CLL cells seems to have an unfavorable impact on prognosis, even though only a few studies have addressed this issue.AIMS: to analyze the expression of VEGF and its relation to Binet clinical stage, mutation status of IgVH and event free survival.Patients and Methods: 52 CLL patients diagnosed at UNIFESP and HSPE were studied. VEGF expression was evaluated on blood samples stained with Biotinylated Human VEGF (R&D Systems) monoclonal antibody and acquired with FACScalibur® and analyzed CellQuest® software. The expression of VEGF was considered positive when its mean fluorescence intensity was >200. IgVH mutation of 25 cases status was also studied by sequencing of the amplified samples by RT-PCR using VH family specific primers. The sequences were analysed with lg Blast® software and considered mutated (M) when there was a germline homology of 98% or greater and those displaying homology <98% were classified as unmutated (UM). Fisher's exact test was used to compare results and Kaplan Meyer analysis to evaluate progression free survival (PFS). Differences were considered significant if p<0.05.Results: Of the 52 CLL patients (28M/24F, median age of 67 years varying between 38–78 years) 29 (56%) were Binet A, 17 (33%) Binet B and 6 (11%) Binet C. Twenty nine were VEGF+ and 23 VEGF−. Binet A patients were more frequently VEGF− than Binet B+C ones (p=0,002). In the whole population, VEGF+ patients presented shorter PFS when compared to VEGF− (p=0.004). Additionally, even early clinical stage patients (A+B), VEGF+ patients had an adverse prognosis with shorter PFS than those who were VEGF− (p=0.023). As for IgVH mutation status, of the 25 analyzed patients VEGF− were predominantly M while VEGF+ NM (p=0.043), and this difference was also seen when A+B patients were analysed (p=0.031).Conclusion: Our study shows that VEGF expression is associated with an adverse prognosis in CLL patients, even in those in early clinical stages (A+B). FC analysis of VEGF showed to be feasible and easily executable and could be evaluated as surrogate of IgVH mutation status in additional studies with a greater series of patients. (support: FAPESP proc.no.05/57792-0).

LPL and ADAM-29 Gene Expressions Stratify Prognosis in Clinical Stage BinetA and B Chronic Lymphocytic Leukemia Patients

Introduction: Chronic Lymphocytic Leukemia (CLL) is the most frequent adult leukemia in western countries. It is known to have a heterogeneous clinical course, some patients presenting an indolent disease while others have an aggressive disease requiring prompt treatment. Therefore, an individualized approach, especially in early clinical stage patients is necessary. Recent studies suggest that the biological markers LPL and ADAM-29 could be useful to predict prognosis in CLL patients: LPL being associated with an unfavorable prognosis while ADAM-29 to favorable one.Aims: to evaluate the expression of LPL (L), ADAM-29 (A) and the L/A ratio in CLL patients in regard to Binet clinical stage and progression free survival (PFS).Patients and Methods: thirty CLL patients followed at UNIFESP/EPM and HSPE were studied. RNA extraction was done by the TRIZOL method followed by c-DNA synthesis. c-DNA was amplified by PCR using LPL and ADAM-29 specific primers. T-Student's exact test was used to compare the genes frequencies and Kaplan Meyer analysis to evaluate PFS. The results were considered statistically significant when p<0.05.Results: Of the 30 CLL patients studied (16M/14F, median age of 67 years, varying from 38 to 64 years) 19 (63,3%) were early clinical stage (Binet A), 8 (26,7%) Binet B and 3 (10%) Binet C. In the Binet A group LPL was negative in 52.63% of the patients and ADAM-29 positive in 63.15% with relation LPL-/ADAM29+ (or L/A=0) in 52.63% (10/19). In contrast among Binet B and C patients 90% (10/11) presented relation LPL+/ADAM-29- (or L/A=1). Those who were LPL+ or ADAM-29- or L/A=1 had a shorter PFS when compared to those LPL-or ADAM-29+ or L/A=0 (p<0.05). Patients, even those in early clinical stages (Binet A and B), when LPL+, ADAM- and L/A=1 constitute a group with an unfavorable prognosis with a short DFPS (p<0.05).Conclusion: Our study shows that patients with a positive expression of LPL, negative for ADAM-29 and with L/A=1 have unfavorable prognosis independent of clinical stage. Analyzing Binet A and B together, we identified a subgroup LPL+, ADAM- and L/A=1 with a poor outcome. Thus, the expression profile of these genes could be useful to identify Binet A and B patients with an unfavorable prognosis, contributing to the decision making process to begin therapy in an individualized manner. (Support–FAPESP proc n. 05/57792-0)

Zap-70 and CD38 as predictors of IgVH mutation in CLL

Zeta-chain (TCR)-associated protein kinase 70 kDa (Zap-70) and CD38 expression may be of prognostic significance in chronic lymphocytic leukemia (CLL). Previous studies indicate that Zap-70 and CD38 are usually positive in cases of CLL with unmutated immunoglobulin variable region genes (IgVH) and may be used to predict IgVH mutation status and prognosis. Usually cases of CLL positive for Zap-70 or CD38 indicate a worse prognosis. In the present investigation, 47 cases of CLL were evaluated for CD38 expression, and 17 cases were evaluated for both Zap-70 and CD38 expression. Of the 47 cases, 19 (40.4%) positively expressed CD38. Of the 17 cases evaluated for Zap-70, 11 (64.7%) were positive for Zap-70, while only 6 (35.3%) were positive for CD38 expression; the remaining cases were negative for CD38. The results of this study show that Zap-70 expression may be a better indicator of the mutational status of IgVH and prognosis of CLL than CD38 expression. In addition, CD38 negativity does not necessarily indicate that IgVH mutation has occurred. These data point to the need for a more extensive study to evaluate the significance of Zap-70 and CD38 expression as indicators of IgVH mutation status and prognosis of CLL patients.

Predictive Value of Gene Expression with Respect to IgVH Mutational Status and Survival in Chronic Lymphocytic Leukemia.

Background: Chronic Lymphocytic Leukemia (CLL) has a variable clinical course, of which IgVH mutational status of the malignant B cells is a strong independent predictor for outcome.Objective: We studied the expression of 5 different genes for their value to predict IgVH mutational status and overall survival (OS).Methods: mRNA levels were quantified by real time PCR in lymphoprep separated but unsorted blood samples from 110 morphologically and fenotypically characterised CLL patients for the following genes: ZAP70, lipoprotein lipase (LPL), ADAM29, KIAA0610 and nRIP1. Expression levels were compared wih respect to IgVH mutational status (mutation = % germline < 98%), using logistic regression and with respect to OS (= time from day of laboratory tests to death due to any cause), using Cox regression analysis.Results: The predictive value of the expression of these 5 genes for IgVH mutational status is shown in table 1. Each of these genes showed to be an independent marker for the prediction of IgVH mutational status. LPL as a single marker was the best predictor for mutation. These factors taken together had a high predictive value towards mutational IgVH status (area under ROC curve = 0.93).table 1NOdds ratioP valueZAP70no851(>5.2)yes250.0980.002LPLno731(>0.04)yes370.029<0.001ADAM29no731(>30)yes376.610.012KIAA0610no811(>0.25)yes290.190.045NRIP1no881(>10)yes2248.730.008The predictive value of IgVH mutation and the expression of these 5 genes for OS is shown in table 2. From the six parameters tested, IgVH mutational status and LPL were predictors for OS at a significant level of 5%, while ZAP70 was of borderline significance.table 2Conclusion: measured in unsorted CLL samples, all genes, and especially LPL, had predictive value towards the presence of IgVH mutation. Expression of LPL and absence of mutation were adverse prognostic factors for survival. Thus, the replacement of the assessment of both IgVH mutational status and ZAP70 by assessment of LPL levels for the prediction of prognosis in CLL patients may be considered.Nsurvival at 24monthsP valuemutationno3670%yes4493%0.041ZAP70no6687%yes1870%0.078LPLno5594%yes2966%0.001ADAM29no5784%yes2784%0.486KIAA0610no6388%yes2171%0.133NRIPno7181%yes13100%0.167

Refining prognostic factors in chronic lymphocytic leukemia.

Recent knowledge in the understanding of biology and natural history of chronic lymphocytic leukemia (CLL), along with the emerging of new and more effective treatments have changed the way the CLL patients are studied and treated. Most of the patients are now being diagnosed in early stages and at much younger age than previously, resulting in longer overall survival but also posing special problems in respect to their clinical management. Moreover, in individual patients the prognosis is extremely variable, ranging from a very short to a normal lifespan. Thus, as a requisite for an optimal choice of therapy, prognostic assessment should be as precise as possible. Classic clinical stages, based on easily obtainable biologic and clinical parameters, have been the most important factor for assessing prognosis in CLL patients. However, although easy to apply, they are not devoid of limitations. For these reasons, recent investigations are searching new parameters in an attempt to add prognostic power to clinical stages. The prognostic impact of these new parameters, usually based on important biologic discoveries, is under investigation in prospective trials. Meanwhile, the classic prognostic factors ‐ particularly clinical stages ‐ should continue to be the basis for assessing prognosis, as well as forming the benchmark against which new prognostic parameters should be measured.