Survival Of Chronic Lymphocytic Leukemia Patients Research Articles

T-bet suppresses proliferation of malignant B cells in chronic lymphocytic leukemia

AbstractThe T-box transcription factor T-bet is known as a master regulator of the T-cell response but its role in malignant B cells has not been sufficiently explored. Here, we conducted single-cell resolved multi-omics analyses of malignant B cells from patients with chronic lymphocytic leukemia (CLL) and studied a CLL mouse model with a genetic knockout of TBX21. We found that T-bet acts as a tumor suppressor in malignant B cells by decreasing their proliferation rate. NF-κB activity, induced by inflammatory signals provided by the microenvironment, triggered T-bet expression, which affected promoter-proximal and distal chromatin co-accessibility and controlled a specific gene signature by mainly suppressing transcription. Gene set enrichment analysis identified a positive regulation of interferon signaling and negative control of proliferation by T-bet. In line, we showed that T-bet represses cell cycling and is associated with longer overall survival of patients with CLL. Our study uncovered a novel tumor suppressive role of T-bet in malignant B cells via its regulation of inflammatory processes and cell cycling, which has implications for the stratification and therapy of patients with CLL. Linking T-bet activity to inflammation explains the good prognostic role of genetic alterations in the inflammatory signaling pathways in CLL.

Predicting the overall survival of patients with chronic lymphocytic leukemia in the dynamics of the disease

Relevance. The high prevalence of chronic lymphocytic leukemia (CLL) in elderly and senile patients of the European population dictates the need to find a publicly available solution to the problem of prognosis in this cohort of patients. Previously, a relationship was established between the survival of patients with CLL and the value of the glomerular filtration rate (GFR) at the time of diagnosis. The purpose of the study: to assess the relationship of GFR with the survival of CLL patients in the course of the disease. Materials and methods: 207 patients (98 men and 109 women) aged 45 to 80 years with CLL A-C according to Binet and known overall survival were studied. Annually from the 1st to the 5th year of the course of the disease, the maximum value of the patient's GFR was taken into account according to the CKD-EPI formula. The data was processed by data mining using a logical classification algorithm. Results. With a GFR value of more than 76 ml/min/1.73 m2 during the first year of the disease, more than 75 ml/min/1.73 m2 during the second year of the disease, more than 70 ml/min/1.73 m2 during the third year of the disease, more than 68 ml/min/1.73 m2 during the fourth and fifth years of the disease, the patient will overcome the calculated parameters of the median survival for the corresponding stage of CLL according to Binet. Otherwise, the overall survival of the patient will be less than the calculated median survival according to Binet. This article provides illustrative clinical examples. Conclusion. It was found that the value of GFR in CLL patients gradually decreases with the course of the disease, however, the value of this parameter in the dynamics of the disease can be considered as a significant factor in predicting the survival of patients with CLL. Based on the results, an application for an invention No. 2023108368 was filed.

A population level analysis of second hematological malignancies in chronic lymphocytic leukemia/small lymphocytic lymphoma survivors in the era of targeted therapies.

With improvement in survival after chronic lymphocytic leukemia (CLL) diagnosis, the real-world burden of second hematological malignancies (SHM) has not been comprehensively assessed in recent era. We analyzed risk, incidence, and outcomes of SHM in CLL patients between 2000 and 2019 using SEER database. CLL patients had greater risk for hematological malignancies than general population [SIR, standardized incidence ratio (95% CI):2.58 (2.46-2.70); p<0.05]. The risk for subsequent lymphoma increased by 1.75 folds in 2015-2019 compared to 2000-2004. The duration, after CLL diagnosis, of maximum risk for SHM decreased as 60-119months for time-period 2000-2004, 6-11months for 2005-2009 to 2-5months for 2010-2014 and 2015-2019. Incidence of SHM was 2.5% in CLL survivors (1736/70,346) with lymphoid SHM being more common than myeloid SHM, and DLBCL being the most common pathology (n=610, 35% of all SHM). Male sex, age ≤65years at CLL diagnosis, and chemotherapy treatment were associated with higher risk for SHM. The median gap between CLL and SHM diagnoses was 46months. The median survival for de-novo-AML, t-MN, CML, and aggressive NHL was 63, 86, 95, and 96months respectively. Although SHM remains rare, there is increased risk in recent era, likely due to improved survival in CLL patients, necessitating active surveillance strategies.

COMORBIDITY &amp; CLL DIFFERENT SCORES – DIFFERENT RESULTS

Introduction: Due to the implementation of the outpatient centres, our network provides care for 50% of the patients with hematological diseases in Vienna. Here we analyse the influence of our own CIRS Score on survival of CLL patients. Additionally we focus on renal comorbidities. Methods: 1150 CLL patients who were diagnosed, observed and treated between 2011 and 2021 within our haematological network were analysed. Study data was generated by reviewing electronic and paper medical records. Comorbidities were assessed with the conventional CIRS Score including utilizing our own Version where we did not focus on severity to detect more patients. We differentiated between 2 groups: low risk pts. without any or with just one existing co-morbidity and high-risk patients with 2 or more organ systems involved, regardless of the severity. Moreover we demonstrate a Composite Comorbidity Score “Severe4” with <3 or >3 points, validated in 4 Organ Systems as shown on ASH 2022 by Shouse et al. and the cut-off Score of 6 points as used by the German CLL Study Group. Furthermore the Cockcroft-Gault formula (GFR). Results: 55% of the patients were male. The median age at diagnosis was 68 years. IGHV was mutated in 53% of patients, del(17p) was present in 7,4% of patients. 27% of our 1150 patients received CLL specific treatment. As shown in Figure 1 our own interpretation of the CIRS-Score was able to identify 625 pts. classified as high risk with a significant lower OS. Regardless of the Severity, the involvement of 2 or more organ systems was more relevant than just one. Similarly to the Severe4 Score we could also show a significant OS difference in 167 pts. using a cut-off Score of 3 Points in at least one category. A highly significant OS difference can also be shown in 245 pts when using the established cut-off Score of 6 points. Emphasizing the weakness of the CIRS Score in detecting renal comorbidity due to the high threshold concerning the Serum Creatinine of 1,5 mg/dl we were able to demonstrate that 144 of our pts. had a GFR of <60 mL/min at time of diagnosis, which reflects Stage 3 CKD, resulting in a significant lower OS. When using the CIRS Score alone only 31 pts. would have been classified as renal impaired. Keyword: chronic lymphocytic leukemia (CLL) No conflicts of interests pertinent to the abstract.

CLL-035 Long-Term Survival After Fludarabine, Cyclophosphamide, and Rituximab Treatment in Previously Untreated Chronic Lymphocytic Leukemia Patients

Chronic lymphocytic leukemia (CLL) is characterized by a lower incidence rate in Iraq and Kurdistan as compared to Western countries. However, a good prognosis of CLL is dependable on diagnosis, risk stratification, and a better choice of an appropriate treatment regimen. To evaluate the effectiveness of fludarabine, cyclophosphamide, and rituximab (FCR) regimen in comparison to other chemotherapy regimens in the management of patients with CLL in Kurdistan, Iraq. A retrospective review study carried out in three cancer centers in the Kurdistan region of Iraq for a duration of 10 years through the period from January 1, 2010 to December 31, 2019, on 152 CLL patients. CLL was diagnosed according to the International Workshop on CLL. The treatment of CLL patients was either by FCR chemoimmunotherapy regimen or other chemotherapies. The FCR chemoimmunotherapy was the treatment of 38.8% of CLL patients, while 61.2% of CLL patients were treated by other chemotherapies. There was a significant association between younger age patients and the use FCR chemoimmunotherapy (P = 0.001). There was a significant association between a complete response and treatment by FCR chemoimmunotherapy (P = 0.02). The mean overall survival duration and progression free survival of CLL patients treated by FCR chemoimmunotherapy were significantly longer than the mean survival time of CLL patients treated by other chemotherapies (P = 0.01). Complete response and survival of CLL patients treated by FCR chemoimmunotherapy were better than the complete response and survival of CLL patients treated by other chemotherapies.

CLL-093 Long-Term Survival After Fludarabine, Cyclophosphamide, and Rituximab Treatment in Previously Untreated Chronic Lymphocytic Leukemia Patients

Chronic lymphocytic leukemia (CLL) is characterized by a lower incidence rate in Iraq and Kurdistan as compared to Western countries. However, a good prognosis of CLL is dependent on diagnosis, risk stratification, and a better choice of an appropriate treatment regimen. To evaluate the effectiveness of fludarabine, cyclophosphamide, and rituximab (FCR) regimen in comparison to other chemotherapy regimens in the management of patients with CLL in the Kurdistan region of Iraq. A retrospective review study was carried out in three cancer centers in the Kurdistan region of Iraq for a duration of 10 years from January 1, 2010, to December 31, 2019, on 152 CLL patients. CLL was diagnosed according to the International Workshop on CLL. The treatment of CLL patients was either by FCR chemoimmunotherapy regimen or other chemotherapies. The FCR chemoimmunotherapy was the treatment of 38.8% of CLL patients, while 61.2% of CLL patients were treated by other chemotherapies. There was a significant association between younger age patients and the use of FCR chemoimmunotherapy (P=0.001). There was a significant association between a complete response and treatment by FCR chemoimmunotherapy (P=0.02). The mean overall survival duration and progression-free survival of CLL patients treated by FCR chemoimmunotherapy were significantly longer than the mean survival time of CLL patients treated by other chemotherapies (P=0.01). The complete response and survival of CLL patients treated by FCR chemoimmunotherapy were better than the complete response and survival of CLL patients treated by other chemotherapies.

Distinct Age-Related Clinical Features and Risk Assessment in Chinese With Chronic Lymphocytic Leukemia.

The biological and clinical features of chronic lymphocytic leukemia (CLL) exhibited profound heterogeneity across Chinese and patients of predominately European descent. However, the age-related peculiarities and risk assessment of Chinese CLL patients remained ill-defined. The present study demonstrated that CLL patients were characterized by the earlier age at onset in China (median age at diagnosis: 63 years old) than in the United States (median age at diagnosis: 69 years old). Young patients from Shandong Provincial Hospital CLL database displayed prolonged overall survival than the Surveillance, Epidemiology, and End Results cohort. Furthermore, among Chinese CLL patients, young patients showed an increased relapse rate compared with elderly patients. To optimize the risk assessment of CLL patients, novel risk score models named PR-Score and HBG-Score were developed for predicting the outcomes of young and elderly CLL patients respectively. The neonatal survival prediction systems were superior to international prognostic index for CLL (CLL-IPI) and Binet stage in assessing the overall survival and progression free survival of CLL patients. The analyses highlighted refinement of risk evaluation for CLL patients in different age groups, providing insights into individualized diagnosis and treatment of CLL.

Chronic Lymphocytic Leukemia (CLL): evaluation of AKT protein kinase and microRNA gene expression related to disease pathogenesis

The present study evaluated 56 patients diagnosed with Chronic Lymphocytic Leukemia (CLL) and a control group of 44 clinically healthy subjects with no previous history of leukemia. Genetic expressions of AKT and microRNAs were evaluated by quantitative PCR (qPCR). A significant increase in AKT gene expression in patients when compared to controls was observed (p = 0.017). When the patients were stratified according to Binet subgroups, a significant difference was observed between the subgroups, with this protein kinase appearing more expressed in the B+C subgroup (p = 0.013). Regarding miRNA expression, miR-let-7b and miR-26a were reduced in CLL patients, when compared to controls. However, no significant differences were observed in these microRNA expressions between the Binet subgroups (A versus B+C). By contrast, miR-21 to miR-27a oncogenes showed no expression difference between CLL patients and controls. AKT protein kinase is involved in the signaling cascade that occurs with BCR receptor activation, leading to increased lymphocyte survival and protection against the induction of cell death in CLL. Thus, increased AKT protein kinase expression and the reduction of miR-let-7b and miR-26a, both tumor suppressors, may explain increased lymphocyte survival in CLL patients and may be promising markers for the prognostic evaluation of this disease.

ІМУННІ ЦИТОПЕНІЇ У ХВОРИХ НА ХРОНІЧНУ ЛІМФОЦИТАРНУ ЛЕЙКЕМІЮ (ОСОБЛИВОСТІ ПЕРЕБІГУ, ПРОГНОСТИЧНІ МАРКЕРИ)

Background: Immune cytopenia (IC) is one of the major complications in chronic lymphocytic leukemia (CLL). The paper describes the peculiarities of different immune cytopenia in CLL patients and the importance of individual prognostic markers in the course of the disease. Methods: We observed 62 patients with CLL complicated by immune cytopenia. Among these patients 30 had autoimmune hemolytic anemia (AIHA), 18 experienced immune thrombocytopenia (ITP), 10 had Fisher-Evans syndrome (FES), 3 were diagnosed with partial red cell aplasia (PRCA), and immune neutropenia (IN) was revealed in 1 patient. In addition to general examination and laboratory studies, the following examinations were performed: immunophenotyping of peripheral blood lymphocytes, flow cytometry (CD5; CD19; CD20; CD23; CD38; ZAP70), Coombs test, a molecular cytogenetic study of peripheral blood lymphocytes using the FISH method with TP53 and ATM probes, the level of ß2-microglobulin. Results: It was established that the overall survival of CLL patients with IC depends on the form of the latter. The median overall survival in patients with Fisher-Evans syndrome was the shortest (75 months), slightly better survival was observed in patients with AIHA (median 80 months), the best survival was found in patients with ITP (median not reached). Among unfavorable markers of CLL with IC, there is the presence of del 11q22.3. Unfavorable prognostic markers were also the following: a positive Coombs test, high levels of ZAP 70 expression, and high levels of ß2-microglobulin

Prognostic significance of microRNA 17\u201392 cluster expression in Egyptian chronic lymphocytic leukemia patients

BackgroundAbnormal expression patterns of microRNAs (miRs) play an important role in the development and progression of malignancy. Identification of the clinical significance and prognostic value of these small molecules in chronic lymphocytic leukemia (CLL); a disease of heterogeneous biological landscape and clinical course, has always been of tremendous translational value.AimTo evaluate the prognostic value of microRNA17-92 cluster members in Egyptian CLL patients.MethodsThe expression levels of miR17-92 cluster members were evaluated by qRT-PCR, including miR17, miR18a, miR19a, miR19b-1, miR20a, and miR92a-1. Other investigations included serum LDH, serum β2 microglobulin (β2M), CD38 and ZAP70 expression by flow cytometry, fluorescence in situ hybridization (FISH) for 17p deletion, and imaging studies (computerized tomography (CT) scans of neck, chest, abdomen, and pelvis or PET-CT scans).ResultsOverexpression of all members of the miRNA17-92 cluster was detected in CLL patients compared to controls (p = < 0.001 for all miRs while p = 0.01 for miR19b-1). A significant positive correlation between Hb and miR17 and a significant negative correlation between Hb and miR19b-1 were observed (p = 0.041, 0.017 respectively). A statistically significant positive correlation between miR19b-1 expression and each of the WBCs and absolute lymphocytic count (ALC) was detected (p = 0.023, 0.022 respectively). Moreover, a statistically significant relation between miR19b-1 expression and advanced Binet stages was also found (p = 0.05). Regarding miR18a, a statistically significant positive correlation with LDH level was found (p = 0.003). We also found a significant positive correlation between miR92a-1 and β2M level (p = 0.005), as well as a significant relation between miR17 and negative CD38 expression (p = 0.034). However, no significant relationships between any of studied miRNA expression levels and 17p deletion or response to treatment were observed. Patients who expressed miR19b-1 were significantly indicated to start therapy at diagnosis (p = 0.05). The overall survival of CLL patients included in our study was 90.2% after 1 year from the time of diagnosis. Patients with high expression of miR19a had better OS than those with low expression (p = 0.04).ConclusionsOverexpression of all members of the miR17-92 cluster was detected in Egyptian CLL patients. MiR18a, miR19b-1, and miR92a-1 also have an adverse prognostic value while miR17 can be considered a good prognostic marker. High expression of miR19a is associated with better OS.

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.

Prognostic Value of Myocardial Enzymes and Therapeutic Potential of Cardioprotective Agents in Chronic Lymphocytic Leukemia Patients with Cardiovascular Comorbidities

Introduction: Chronic lymphocytic leukemia (CLL) is the most common leukemia among adults in the western world. Cardiovascular disease (CVD), with a high incidence among comorbidities, is associated with significant morbidity and inferior quality of life in cancer patients. In CLL, comorbidities are associated with increased mortality from infections, second cancers and CVD. However, previous studies have not uncovered the relationships between cardiac biomarkers and the prognosis of CLL patients. In addition, whether cardioprotective drugs affect the effect of therapy in CLL patients remains undefined.Methods: Five hundred and ten CLL patients diagnosed in Shandong Provincial Hospital affiliated to Shandong University from October 2010 to July 2020 were enrolled in our study. The Kaplan-Meier curves were used to estimate survival rates, while the log-rank tests were used for comparison. Univariate and multivariate logistic regression analysis was used to verify prognostic independence. The available expression data and survival information of CLL samples and normal samples were extracted from 2 databases. The expression level of genes involved in the KEGG lipid and atherosclerosis pathway was analyzed to evaluate the prognosis of CLL patients. GSEA enrichment analysis was performed to explore the potential function of TNFRSF1A. Cell proliferation assay with the Cell Counting Kit-8 (CCK-8) was performed to detect the therapeutic potential of the common cardioprotective drugs atorvastatin, metoprolol and losartan in CLL.Results: Among the cohort, 30% CLL patients were complicated with CVD. Compared with those without CVD, CLL patients with CVD were of significantly advanced age (p<0.001), larger proportion with diabetes (p=0.003), as well as higher levels of N-terminal brain natriuretic peptide precursor (NTpro-BNP; p=0.016) and myohemoglobin (MYO; p=0.005). The Kaplan-Meier curves revealed that Binet stage (χ2=3.863, p=0.049), creatine kinase isoenzyme (CK-MB; χ2=4.335, p=0.037) and MYO (χ2=4.010, p=045) were related to the OS time of CLL patients with CVD (shown in Figure 1). The univariate logistic analysis showed that sex (p=0.031), Rai stage (p=0.036), Binet stage (p=0.017), β2-MG (p=0.004), creatine kinase (CK; p=0.017), α-hydroxybutyrate dehydrogenase (α-HBDH; p=0.017), MYO (p=0.025) and C-reactive protein (p=0.007) were associated with the outcomes of CLL patients with CVD. The multivariate logistic analysis confirmed that α-HBDH (p=0.046) and MYO (p=0.013) were independent prognostic biomarkers for the outcomes of CLL patients with CVD.To further evaluate the prognosis of CLL patients and CVD, the expression of genes involved in the KEGG lipid and atherosclerosis pathway were assessed. Most of them were dysregulated (shown in Figure 2A-B). The genes which were differential expression were performed with Kaplan-Meier analysis and univariate and multivariate Cox analysis subsequently. TNFRSF1A was upregulated among CLL cells and was independent prognostic biomarkers of both OS (shown in Figure 2C) and time to first treatment (TTFT) in CLL patients (shown in Figure 2D). GSEA enrichment illuminated that TNFRSF1A was related to the lipid catabolic and metabolic process, as well as other metabolic processes (shown in Figure 2E-F).To investigate the effect of cardioprotective drugs on chronic lymphocytic leukemia patients, cell proliferation assays were performed. Atorvastatin, metoprolol and losartan decreased the proliferation of CLL cell lines with IC50 values of 94.19 uM, 123.60 uM and 411.80 uM in EHEB cells, and 79.20 uM, 138.60 uM and 318.5 uM in MEC-1 cells, respectively (shown in Figure 3).Conclusions: In conclusion, we demonstrated CLL patients with CVD were of older age, larger proportion of patients with diabetes, higher levels of NTpro-BNP and MYO compared those without CVD for the first time. α-HBDH and MYO were independent prognostic biomarkers for survival in CLL patients with CVD. TNFRSF1A was an independent prognostic biomarker of OS and TTFT in CLL patients. Furthermore, atorvastatin, metoprolol and losartan exhibited inhibitory effects in CLL cell lines. The evaluation of cardiac biomarkers is beneficial to predict the clinical outcomes of CLL patients. CLL patients may benefit from the application of cardioprotective agents. Strengthening the evaluation of CVD risk and preventing CVD may improve the prognosis of CLL patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Survival of patients with chronic lymphocytic leukemia before and after the introduction of chemoimmunotherapy in Germany

Chronic lymphocytic leukemia (CLL) is the most common leukemia of adults in western countries. Therapy is indicated in symptomatic and advanced stages and has changed fundamentally since 2010 when rituximab, an anti-CD20 antibody, has been approved for treatment of CLL. Until then therapy had been based on chemotherapy drugs. This study investigates whether survival in CLL patients improved at the population level after the introduction of combined chemoimmunotherapy. Data from the cancer registry North-Rhine Westphalia was used to calculate relative survival (RS) by applying period analyses. Age-standardized 5-year RS increased from 79% in 1998–2002 (75% in 2003–2007) to 81% in the calendar period 2008–2012 and 88% in 2013–2016 for men and continuously from 71% in 1998–2002 to 92% in 2013–2016 for women. In CLL patients aged 15–69 years 5-year RS increased from 83% to 90% for men and from 82% to 94% for women after adding an anti-CD20-antibody to chemotherapy while in the older age group of 70–79-year-old CLL patients an increase by 20 percentage points was observed. These findings show marked improvements in the survival of CLL patients at the population level subsequently to the approval of anti-CD 20 antibodies like rituximab, ofatumumab or obinutuzumab for CLL treatment.

Survival trends in chronic lymphocytic leukemia in the era of oral targeted therapies in the United States: SEER database analyses (1985 to 2017).

7524 Background: The survival of chronic lymphocytic leukemia (CLL) patients has progressively improved after the approval of new targeted therapy for first-line treatment and relapsed disease. We performed a corresponding analysis from the U.S. population-based SEER database (1973–2017) to explore the trend of survival and the effect of advanced CLL treatment on overall survival in CLL patients. Methods: Data were extracted from SEER*Stat for all patients 15 years or older with a primary diagnosis of CLL with or without subsequent cancers. A period analysis was performed to estimate the 5- and 10-year relative survival rates for patients diagnosed (dx) during different calendar periods from 1985 to 2017, based on gender and age at time of diagnosis (15–44, 45–54, 55–64, 65–74, 75–84, 85 years or older). A mixture cure model was used to examine the proportion of long-term survivors per gender and age category among CLL patients diagnosed between 1985 and 2015. Cox proportional hazard modeling was used to calculate the hazard ratios (HRs) of death adjusted for gender and age at diagnosis for two cohorts: (a) diagnosed in 2000–2003 and followed to 2012; (b) 2004–2007 and followed to 2015. Results: For males, the 5-year age-adjusted relative survival rate improved progressively from 72.0% (dx 1985-1989) to 88.2% (dx 2010-2014); for females, from 76.8% (dx 1985-1989) to 90.8% (dx 2010-2014). The corresponding 10-year age-adjusted relative survival rates were 47.3% (dx 1985-1989) and 72.5% (dx 2005-2009) for males; and 58.2% (dx 1985-1989) and 78.7% (dx 2005-2009) for females. The table below shows the proportions of long-term survivors for the 1985–2017 cohort as estimated in the mixed cure model. The HRs (95%CI) of death for cohort (b) in comparison to cohort (a) were 0.58 (0.43–0.78), 0.58 (0.48–0.70), 0.57 (0.49–0.67), 0.68 (0.54–0.85); and 0.83 (0.68–1.02) for age categories of 45–54, 55–64, 65–74, 75–84, and 85 years or old. Conclusions: Survival is significantly improved by calendar period among patients diagnosed after 2004 and treated in the era of advanced therapies. Females and younger patients had a higher probability of long term survival. Future studies should consider such covariates as treatment type, disease stage and genetics.[Table: see text]

MiR-29 modulates CD40 signaling in chronic lymphocytic leukemia by targeting TRAF4: an axis affected by BCR inhibitors

B-cell receptor (BCR) signaling and T-cell interactions play a pivotal role in chronic lymphocytic leukemia (CLL) pathogenesis and disease aggressiveness. CLL cells can use microRNAs (miRNAs) and their targets to modulate microenvironmental interactions in the lymph node niches. To identify miRNA expression changes in the CLL microenvironment, we performed complex profiling of short noncoding RNAs in this context by comparing CXCR4/CD5 intraclonal cell subpopulations (CXCR4dimCD5bright vs CXCR4brightCD5dim cells). This identified dozens of differentially expressed miRNAs, including several that have previously been shown to modulate BCR signaling (miR-155, miR-150, and miR-22) but also other candidates for a role in microenvironmental interactions. Notably, all 3 miR-29 family members (miR-29a, miR-29b, miR-29c) were consistently down-modulated in the immune niches, and lower miR-29(a/b/c) levels associated with an increased relative responsiveness of CLL cells to BCR ligation and significantly shorter overall survival of CLL patients. We identified tumor necrosis factor receptor–associated factor 4 (TRAF4) as a novel direct target of miR-29s and revealed that higher TRAF4 levels increase CLL responsiveness to CD40 activation and downstream nuclear factor-κB (NF-κB) signaling. In CLL, BCR represses miR-29 expression via MYC, allowing for concurrent TRAF4 upregulation and stronger CD40–NF-κB signaling. This regulatory loop is disrupted by BCR inhibitors (bruton tyrosine kinase [BTK] inhibitor ibrutinib or phosphatidylinositol 3-kinase [PI3K] inhibitor idelalisib). In summary, we showed for the first time that a miRNA-dependent mechanism acts to activate CD40 signaling/T-cell interactions in a CLL microenvironment and described a novel miR-29–TRAF4–CD40 signaling axis modulated by BCR activity.

Leczenie wenetoklaksem młodego pacjenta z przewlekłą białaczką limfocytową z delecją 17p i nawrotem choroby po pierwszej linii leczenia — opis przypadku i przegląd literatury

Chronic lymphocytic leukaemia (CLL) is the commonest B-cell malignancy and leukaemia in western countries. It mainly affects the elderly population with a median age of 72. Overall survival varies from 2 to 15 years and depends on clinical and pathological risk factors like age, clinical stage and the presence of genetic mutations with TP53 aberrations conferring the worst prognosis. Indications for therapy include active and symptomatic disease and advanced clinical stage assessed in the Rai or Binet staging system. CLL therapy has been revolutionized in the last years due to the introduction of novel agents — small molecule inhibitors interfering with signalling pathways. These agents proved to be of greater efficacy than immunochemotherapy in both untreated and relapsed patients and led to the significant improvement in survival of CLL patients. These novel agents have been incorporated into new recommendations for CLL treatment. They have also improved the survival of high-risk CLL patients with TP53 aberrations and complex karyotype although their prognosis is still inferior as compared to other patients’ subgroups. Questions concerning optimal sequencing novel agents and the timing of allogeneic bone marrow transplantation remain still open for high-risk group CLL patients.

Telomere Length and CD49d Cooperate with IGHV Gene Status As Predictors of Long-Term Progression-Free Survival in CLL Patients Treated with FCR-Based Regimens

Background - Although there has been a revolution in the treatment of chronic lymphocytic leukemia (CLL), the challenge remains to identify the right drugs for the right patients. It is widely accepted that CIT, including the 'gold standard' fludarabine, cyclophosphamide and rituximab (FCR), is contraindicated for patients with TP53 disruption and, more recently, unmutated IGHV genes. Also patients with short, dysfunctional telomeres were shown to have inferior outcomes when treated with FCR-based regimens. To date, a role for CD49d in this setting has not been established. Aims - Here we evaluated the ability of telomere lenght (TL) and CD49d to cooperate with IGHV gene status to predict progression-free survival (PFS) in patients treated with FCR-based regimens in the frontline setting in three UK trials, ARCTIC, ADMIRE and CLL4. Methods - The study included a discovery cohort of 245 CLL treated with FCR/FCR-like regimens according to the two UK trials ARCTIC and ADMIRE. As there was no significant difference in PFS between the three arms of the study (P = 0.97), analysis was performed on the combined cohort. The median follow-up was 77.5 months with 157 progressions and 76 deaths. Twenty-nine patients were TP53 deleted and/or mutated, with shorter PFS compared to cases without TP53 disruption (final cohort, 216 TP53 wild-type CLL). The validation cohort was composed of 119 CLL samples derived from patients randomised to receive fludarabine, cyclophosphamide (FC) from the UK CLL4 trial. The median follow-up was 67.2 months with 99 progressions and 77 deaths. Fifteen CLL were TP53 mutated/deleted, with shorter PFS compared to cases without TP53 disruption (final cohort, 104 TP53 wild-type CLL). TL was measured using the high-throughput STELA assay and patients were bifurcated into two groups with either short telomeres inside the fusogenic range (TL-IFR) or long telomeres outside the fusogenic range (TL-OFR). CD49d was measured by flow cytometry and dichotomized as CD49dpos and CD49dneg based on the established 30% cut-off. For IGHV gene status, the 2% cutoff was used to split patients in mutated (IGHV-M) and ummutated (IGHV-UM). Results - In the 216 CLL with wild-type TP53 status from the ARCTIC/ADMIRE trials, CD49d expression was a predictor of PFS (P=0.02; HR=1.46 [1.03-2.06]). In keeping with previous reports, patients with IGHV-UM genes (P&amp;lt;.0001; HR=2.53 [1.79-3.58]) or TL-IFR (P=0.0002; HR=1.97 [1.30-2.98]) showed also significantly shorter PFS. This data was used as a starting point for a risk-stratification algorithm (Figure 1A). IGHV-UM cases could not be further dissected by TL or CD49d expression (P=0.76 log-rank test), their 8-year PFS being just 19.0%; (HR=5.58 [3.70-8.42]). In contrast, the IGHV-M group could be stratified by TL and CD49d expression (P&amp;lt;0.001 log-rank test). In particular, 13/84 (15.5%) of IGHV-M CLL with TL-IFR showed a median PFS of 3.0 years with a 8-year PFS of 15.4% (HR=6.45 [1.84-22.58]), similar to IGHV-UM cases (P = 0.19). Patients with IGHV-M genes and TL-OFR could be further stratified by CD49d expression into categories with different 8-year PFS: 43.1% in IGHV-M/TL-OFR/CD49dpos cases (HR=2.52 [1.08-5.89]), and 75.5% in the IGHV-M/TL-OFR/CD49dneg reference group (Table 1). Figure 1B shows the overlaid Kaplan-Meier curves for this hierarchical stratification. The proposed algorithm was then evaluated in the UK CLL4 trial. In keeping with the known inferiority of FC when compared with FCR, the 8-year PFS was just 5.2% in the IGHV-UM subset, irrespective of TL or CD49d expression. In concordance with the discovery cohort, the IGHV-M subset with TL-IFR had a similar median PFS to IGHV-UM cases (P=0.84). Furthermore, CD49d expression was able to stratify cases with TL-OFR; in particular, 18/104 (17.3%) were IGHV-M/TL-OFR/CD49dneg CLL which had a 8-year PFS of 77.8% (summary in Table 1 and Figure 1C). Conclusion - Our analysis shows that only IGHV-M/TL-OFR/ CD49dneg patients may benefit from CIT; this group represented just 56/321 cases (17.4%) of the combined cohort, suggesting that approximately 82% of patients should be considered for alternative therapies. Incorporation of this algorithm into clinical trial design and real-world practice would enable rational, risk-adapted, clinical management with the aim of treating all CLL patients with the optimal therapeutic regimen in the frontline setting. Disclosures Norris: TeloNostiX Ltd: Current equity holder in private company, Patents &amp; Royalties. Hillmen:Gilead: Other: Financial or material support, Research Funding; Alexion: Consultancy, Research Funding, Speakers Bureau; Apellis: Consultancy, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Speakers Bureau; Acerta: Other: Financial or material support; Roche: Consultancy, Other: Financial or material support, Research Funding, Speakers Bureau; AbbVie: Consultancy, Other: Financial or material support, Research Funding, Speakers Bureau; Pharmacyclics: Other: Financial or material support, Research Funding; Janssen: Consultancy, Other: Financial or material support, Research Funding, Speakers Bureau. Rawstron:BD Biosciences (Intrasure): Patents &amp; Royalties. Baird:TeloNostiX Ltd: Current equity holder in private company, Patents &amp; Royalties. Fegan:TeloNostiX Ltd: Current equity holder in private company, Patents &amp; Royalties. Pepper:TeloNostiX Ltd: Current equity holder in private company, Patents &amp; Royalties.

Targeting Inhibition of N6-Methyladenosine Demethylase Fto Displays Potent Anti-Tumor Activities in Chronic Lymphocytic Leukemia

Introduction Accumulating evidence indicates that Fat mass and obesity-associated protein (FTO), a N6-methyladenosine (m6A) RNA demethylase exerts crucial roles in oncogenesis. FB23-2, a novel inhibitor selectively targeting FTO m6A demethylase activity displayed promising potency in acute myeloid leukemia. Yet, no literature has been reported regarding the effects of FTO and FB23-2 in the tumorigenesis and development of chronic lymphocytic leukemia (CLL). Hence, the aim of this study was to investigate the clinical significance and mechanisms of FTO regulation in CLL. Methods Peripheral blood samples from 55 de novo CLL patients (36 males and 19 females; age range 32-82 years, median 62 years) were collected with informed consents in Shandong Provincial Hospital. CD19+ B cells were isolated with informed consents from healthy donors. Expression levels of FTO mRNA and protein in CLL cells were determined by quantitative RT-PCR and western blotting. Methylated RNA immunoprecipitation sequencing (MeRIP-Seq) and RNA sequencing (RNA-seq) were conduct to profile RNA m6A methylation and expression of CLL cells. Lentiviral vectors were constructed to stably silence and overexpress FTO in CLL cells. Besides, cell viability, apoptosis and cell cycle were assessed by cell counting kit-8, annexin V-PE/7AAD and PI/ RNase staining, respectively. Results Aberrantly increased expression of FTO was observed in CLL patients and CLL cell lines at mRNA and protein level compared with normal B cells from healthy donors (Figure 1A-B). Clinical correlation analyses suggested FTO high expression was significantly associated with 11q23 deletion (p=0.012; Figure 1C). Furthermore, Keplan-Meier plot indicated that elevated FTO expression predicted adverse outcome in CLL patients (HR=1.758, p=0.019; Figure 1D). ROC curve confirmed the prognostic value of FTO in survival of CLL patients (AUC=0.600, p=0.018; Figure 1E). To explore the potential role of FTO in CLL tumorigenesis, CLL cells were transfected with lentiviral vectors to stably silence and overexpress FTO. CLL primary cells and MEC1 cells with silence of FTO exhibited attenuated cell proliferation, increased fast-onset apoptosis (Figure 2A-D). Western blotting assay suggested significant down-regulated Bcl-2, enhanced cleaved-PARP and BAX expression in FTO-deficient CLL cells. Whereas, gain-of-function assay showed promoted cell survival in FTO-overexpressed CLL cells (Figure 2F-G). Additionally, serial dilution of FTO inhibitor FB23-2 decreased viability of MEC1 and primary CLL cells in time-dependent manner, and displayed rare cytotoxicity in normal B cells (Figure 3A-B). Besides, annexin V-PE/7AAD and western blotting assay indicated obvious apoptosis was induced with treatment of FB23-2 in CLL primary cells from 9 de novo CLL patients (Figure 3C-D). Importantly, obvious G2/M phase arrest and enhanced sensitivity to Venetoclax were also detected in FTO-reduced CLL cells. Furthermore, interactive MERIP-seq and RNA-seq of CLL cells with control and deleted FTO expression were performed to investigate the m6A Methylation-Mediated mechanism of FTO regulation of CLL pathogenesis. A total of 573 significantly changed peaks, of which 301 were significantly up-regulated and 272 peaks were significantly down-regulated (Figure 4A). Differentiated peaks were located in 3'UTR (42.58%) and 5'UTR (24.43%). Annotations of bioinformatics analyses indicated that FTO was functionally enriched in cell apoptosis in CLL progression (Figure 4B). Western blotting assay suggested significant down-regulated p-CHK2, c-myc, p-p53, cyclinD1 and enhanced p-H2AX expression in FTO-deficient CLL cells, indicating FTO accelerated CLL cell survival via DNA damage pathway (Figure 4C). Conclusion Taken together, our investigations identified for the first time the oncogenic role of FTO in CLL tumorigenesis and regulatory mechanism of FTO inhibitor FB23-2 in CLL cells by MERIP sequencing and ex vivo evaluation. Expression of FTO was upregulated and associated with inferior prognosis of CLL patients. FB23-2 exerted potent therapeutic potential in abrogating cell survival and inducing cell cycle arrest via m6A methylation. This study provides a rationale on evaluation of FTO-targeted intervention formulating a novel treatment paradigm in progressed CLL that warrants clinical investigation. Disclosures No relevant conflicts of interest to declare.

Comprehensive Profiling of the Epitranscriptomic N6-Methyladenosine RNA Methylation in Chronic Lymphocytic Leukemia

Introduction N6-methyladenosine (m6A) is the most prevalent post-transcriptional modification of eukaryotic mRNA. Accumulating evidence suggests that RNA m6A methylation exerts crucial roles in oncogenesis. However, the mRNA m6A methylation pattern in chronic lymphocytic leukemia (CLL) has not been investigated. Hence, the aim of this study is to perform a comprehensive profiling to identify distinct m6A methylation signatures in CLL patients. Methods Peripheral blood samples from de novo CLL patients were collected with informed consents at the Department of Hematology in Shandong Provincial Hospital Affiliated to Shandong First Medical University. CD19+ B cells were isolated with informed consents from healthy donors. Methylated RNA immunoprecipitation sequencing (MeRIP-Seq) was conduct to profile mRNA m6A methylation of CLL-B cells and normal CD19+ B cells at Novogene (Beijing, China). The library preparations were sequenced on an Illumina Novaseq platform with a paired-end read length of 150 bp according to the standard protocols. The sequencing was carried out with 3 independent biological replicates. After mapping reads to the reference genome, exomePeak R package was used for the m6A peak identification in each anti-m6A immunoprecipitation group with the corresponding input samples serving as a control, and q-value threshold of enrichment of 0.05 was used for all data sets. The m6A-enriched motifs of each group were identified by HOMER. Differential peak calling was performed using exomePeak R package with parameters of p-value &amp;lt; 0.05 and fold_change &amp;gt; 1. Functional enrichment analyses of gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) of differentiated peaks associated genes were performed. All investigators comply with the guiding principles for experimental procedures found in the Declaration of Helsinki of the World Medical Association. Results By MeRIP sequencing, significant distribution of methylation peaks were detected in 5'UTR, 3'UTR and CDS regions of CLL primary cells (Figure 1A) and normal B cells (Figure 1B). Figure 1C-D illustrated the percentage of methylation peaks in the five regions, suggesting distinct m6A patterns in CLL cells. Moreover, Figure 1E-F revealed the positions of m6A methylation peaks in chromosomes of CLL and normal B cells. Furthermore, the compared distributions of m6A methylation peaks in CLL and normal B cells were presented in Figure 2A. Besides, the bean plot visibly displayed the obvious differentiation of methylation peaks in CLL group and normal B cells in read density (Figure 2B). Importantly, a total of 1836 significantly changed peaks, of which 1519 were significantly up-regulated and 317 peaks were significantly down-regulated (p&amp;lt;0.05, |log2Foldchange|&amp;gt;1; Figure 2C). These m6A peaks were located across 1850 genes. Functional enrichment analyses identified that differentiated peaks associated genes were potentially regulate RNA metabolic process via oncogenic pathways in CLL pathogenesis (Figure 3A-B). In addition, HOMER analysis identified 38 significant de novo m6A peak motifs, top 10 most significant peak motifs of which were presented (Figure 3C), illuminating potential detailed mechanism of m6A RNA methylation in the tumorigenesis and progression of CLL. Conclusion Taken together, our investigations explored for the first time the m6A methylation pattern of mRNA in CLL. m6A modifications play crucial roles in the progression and survival of CLL patients,highlighting m6A modifications-targeted intervention formulating a novel treatment paradigm in progressed CLL that warrants clinical investigation. Disclosures No relevant conflicts of interest to declare.

WNT3 and LEF1 as markers for diagnosis and survival prediction in chronic lymphocytic leukemia patients

The Wnt pathway is aberrantly activated in chronic lymphocytic leukemia (CLL) and contributes to the antiapoptotic and mitogenic characteristics of CLL cells. Lymphoid enhancer-binding factor‑1 (LEF1) acts as a mediator and key transcription factor of the Wnt/β-catenin pathway. LEF1 helps to regulate important genes involved in tumor cell death mechanisms. To analyze the expression levels of Wnt signaling pathway member (WNT3) and its key mediator LEF1 in Egyptian CLL patients and to detect the potential use of these genes as markers of CLL outcome. We quantified the expression levels of Wnt3 and LEF1 in peripheral blood mononuclear cells of 30 untreated CLL and 19 healthy controls by qRT-PCR (quantitative real time polymerase chain reaction). Our study demonstrated significant upregulation of both Wnt3 and LEF1 in CLL (P 0.9). Upregulation of WNT3 and LEF1 could be used as helpful and specific markers to distinguish our CLL patients. Low WNT3 and LEF1 expression is associated with shortened survival in CLL patients. These results indicate that WNT3 and LEF1 represent an attractive therapeutic target for future therapies in Egyptian CLL patients.