scholarly journals Risk of Serious Adverse Events, Infection and Sepsis in Patients with Relapsed and Refractory Chronic Lymphocytic Leukemia/ Small Lymphocytic Lymphoma Treated with Idelalisib

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5489-5489
Author(s):  
Jonathan Kopel ◽  
Sriman Swarup ◽  
Anita Sultan ◽  
Lukman Tijani ◽  
Ei Moe Phyu ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Adverse Events ◽  
Conflicts Of Interest ◽  
Group Versus ◽  
Lymphocytic Leukemia ◽  
Control Group ◽  
Study Group ◽  
Small Lymphocytic Lymphoma ◽  
Phase 3 ◽  
Serious Adverse Events

Introduction: Idelalisib is a first-in-class potent, oral, selective small-molecule inhibitor of δ isoform of phosphatidylinositol 3-kinase (PI3Kδ), which involves in the signaling of B-cell receptor pathways via activation of downstream serine threonine kinases AKT and mammalian target of rapamycin (mTOR), and has been implicated in the pathogenesis of chronic lymphocytic leukemia/ small lymphocytic lymphoma (CLL/SLL). Yet, there are considerable safety concerns. We undertook a systematic review and combined analysis of phase 3 randomized controlled trials to determine the risk of serious adverse events, infection and sepsis in patients with relapsed/ refractory CLL/SLL treated with idelalisib. Methods: We systematically conducted a comprehensive literature search using MEDLINE, EMBASE databases and meeting abstracts from inception through June 2019. Phase 3 RCTs utilizing idelalisib in patients with relapsed and refractory CLL/SLL that mention serious adverse events, infection and sepsis as adverse effects were incorporated in the analysis. Mantel-Haenszel (MH) method was used to calculate the estimated pooled risk ratio (RR) with 95% confidence interval (CI). Heterogeneity was assessed with Cochran's Q- statistic. Random effects model was applied. Results: Three phase 3 RCTs with a total of 892 patients with relapsed and refractory CLL/SLL were eligible for analysis. Studies compared ofatumumab vs idelalisib+ofatumumab, rituximab vs idelalisib+rituximab, bendamustine+ rituximab vs idelalisib+bendamustine+rituximab and ofatumumab vs duvelisib. The randomization ratio was 2:1 in Jones et al. study and 1:1 in other studies. The I2 statistic for heterogeneity was 83, suggesting moderate heterogeneity among RCTs. The incidence of serious adverse events was 341 (69.59%) in study group vs 177 (44.03%) in control group with RR of 1.50 (95% CI: 1.28-1.75; p < 0.0001). Pneumonitis was noted in 14 (2.86%) vs 1 (0.25%) in control group (RR, 5.42; 95% CI: 1.22-24.13; p = 0.03). The incidence of any-grade pneumonia was 78 (15.92%) in study group vs 45 (11.19%) in control group (RR, 1.38; 95% CI: 0.98 - 1.96; P = 0.07). High-grade pneumonia was reported in 59 (12.04%) in idelalisib arm versus 33 (8.21%) in control group with RR of 1.36 (95% CI: 0.82 - 2.27; P = 0.23). Pneumocystis jiroveci (PJP) pneumonia rate was 2.56% higher in study group compared to control arm (RR, 4.25; 95% CI: 1.10 - 16.34; P = 0.04). Febrile neutropenia was noted in 13.47% in study group versus 4.73% in control arm (RR, 2.39; 95% CI: 0.90-6.34; p = 0.08). Sepsis rate was 3.36% higher in idelalisib group compared to control arm and the pooled RR was statistically significant at 2.64 (95% CI 1.10-6.30; p = 0.03). Treatment-related deaths were 45 (6.92%) in idelalisib arm vs 21 (3.74%) in control arm according to analysis of 2 trials. The pooled RR was not statistically significant at 1.64 (95% CI: 0.99 -2.71; P = 0.06). Conclusion: Patients on idelalisib experienced higher risk of serious adverse events, pneumonitis, PJP pneumonia, and sepsis, with RR of 1.50 for serious adverse events, RR of 5.42 for pneumonitis, RR of 4.25 for PJP pneumonia and RR of 2.64 for sepsis respectively. Nevertheless, there was no significant increase in treatment-related deaths due to TRAE in the idelalisib group, compared to control arm. Preemptive measures with proper supportive care are required to reduce those toxicities which can ultimately improve patients' quality of life and may probably affect patients' compliance. Disclosures No relevant conflicts of interest to declare.

scholarly journals Incidence of Serious Adverse Events, Pneumonitis, Infection and Sepsis in Patients with Relapsed and Refractory Chronic Lymphocytic Leukemia/ Small Lymphocytic Lymphoma Treated with Phosphatidylinositol 3-Kinase (PI3K) Inhibitors

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 798-798
Author(s):  
Sriman Swarup ◽  
Donald P. Quick ◽  
Anita Sultan ◽  
Myint Aung Win ◽  
Ei Moe Phyu ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Adverse Events ◽  
Lymphocytic Leukemia ◽  
Control Group ◽  
Study Group ◽  
Phosphatidylinositol 3 Kinase ◽  
Small Lymphocytic Lymphoma ◽  
Phase 3 ◽  
Serious Adverse Events ◽  
Pi3k Inhibitors

Introduction: Phosphatidylinositol 3-kinase (PI3K) is a kinase protein involved in downstream signaling for multiple pathways in B cells and is important for B cell survival, proliferation and metabolism. Hence, PI3K inhibitors have become an attractive therapeutic option for treatment of B cell malignancies. Two prominent PI3K inhibitors, (PI3Kδ-selective inhibitor) and duvelisib (PI3Kδ/γ-combinatorial inhibitor), are currently being studied in relapsed and refractory chronic lymphocytic leukemia or small lymphocytic lymphoma (CLL/SLL) and have shown to improve survival in recent trials with notable toxicities. We analyzed phase 3 trials to assess the incidence of serious adverse events, pneumonitis, infection and sepsis associated with PI3K inhibitors in this susceptible population. Methods: We systematically conducted a comprehensive literature search using MEDLINE, EMBASE databases and meeting abstracts from inception through June 2019. Phase 3 RCTs utilizing PI3K inhibitors in patients with relapsed and refractory CLL/SLL that mention serious adverse events, pneumonitis, infection and sepsis as adverse effects were incorporated in the analysis. Mantel-Haenszel (MH) method was used to calculate the estimated pooled risk ratio (RR), and risk difference (RD) with 95% confidence interval (CI). Heterogeneity was assessed with Cochran's Q- statistic. Random effects model was applied. Results: Four phase 3 RCTs with a total of 1,216 patients with relapsed and refractory CLL/SLL were included in analysis. Studies compared ofatumumab vs idelalisib+ofatumumab, rituximab vs idelalisib+rituximab, bendamustine+ rituximab vs idelalisib+bendamustine+rituximab and ofatumumab vs duvelisib. The randomization ratio was 2:1 in Jones et al. study and 1:1 in other studies. The I2 statistic for heterogeneity was 49, suggesting some heterogeneity among RCTs. The incidence of serious adverse events was 479 (73.69%) in study group vs 252 (44.92%) in control group (RR, 1.58; 95% CI: 1.37 - 1.83; P &lt; 0.0001 and RD, 0.26; 95% CI: 0.13 - 0.40; P = 0.0002). Pneumonitis was noted in 20 (3.07%) vs 1 (0.18%) in control group with RR of 6.53 (95% CI: 1.74 -24.53; P = 0.005) and RD of 0.02 (95% CI: 0.01 - 0.04; P = 0.0004). The incidence of any-grade pneumonia was 107 (16.46%) in study group vs 54 (9.63%) in control group (RR, 1.61; 95% CI: 1.00 - 2.58; P = 0.05). High-grade pneumonia was reported in 81 (12.46%) in idelalisib arm versus 35 (6.24%) in control group with RR of 1.84 (95% CI: 0.82 - 4.13; P = 0.14). Pneumocystis jiroveci pneumonia (PJP) rate was 2.24% higher in study group compared to control arm (RR, 3.87; 95% CI: 1.22 - 12.29; P = 0.02). Any-grade upper respiratory tract infection (URTI) was 14% in study group versus 7.84% in control arm (RR, 1.65; 95% CI: 1.17 - 2.34; P = 0.005). Sepsis rate was 2.88% higher in idelalisib group compared to control arm and the pooled RR was statistically significant at 2.68 (95% CI: 1.19 - 6.04, P = 0.02). Treatment-related deaths were 64 (11.85%) in study arm vs 28 (6.17%) in control arm according to analysis of 3 trials. The pooled RR was also statistically significant at 1.87 (95% CI: 1.21 -2.88; P = 0.005). Conclusions: Our meta-analysis showed that the incidence of serious adverse events, pneumonitis, PJP pneumonia, any-grade URTI and sepsis was significantly higher in PI3K inhibitors group with RR of 1.58 for serious adverse events, RR of 6.53 for pneumonitis, RR of 3.87 for PJP pneumonia and RR of 2.68 for sepsis respectively. Moreover, patients on PI3K inhibitors experienced 5.68% higher incidence of treatment-related deaths with RR of 1.87, compared to control arm. Since treatment-related serious toxicities and deaths are higher amongst patients treated with these agents, extra caution should be observed and recommended with their use. Disclosures No relevant conflicts of interest to declare.

scholarly journals First-line treatment of chronic lymphocytic leukemia with ibrutinib plus obinutuzumab versus chlorambucil plus obinutuzumab: final analysis of the randomized, phase 3 iLLUMINATE trial

Haematologica ◽  
2022 ◽  
Author(s):  
Carol Moreno ◽  
Richard Greil ◽  
Fatih Demirkan ◽  
Alessandra Tedeschi ◽  
Bertrand Anz ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Final Analysis ◽  
Lymphocytic Leukemia ◽  
Small Lymphocytic Lymphoma ◽  
First Line ◽  
Open Label ◽  
Phase 3 ◽  
First Line Therapy ◽  
Line Therapy ◽  
Open Label Phase

iLLUMINATE is a randomized, open-label phase 3 study of ibrutinib plus obinutuzumab (n=113) versus chlorambucil plus obinutuzumab (n=116) as first-line therapy for patients with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma. Eligible patients were aged ≥65 years, or

Analysis of Stereotyped IGHV Distribution In a Series of 1133 Chronic Lymphocytic Leukemia Patients: The Experience of a Multicenter Italian Study Group

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2423-2423
Author(s):  
Francesco Maura ◽  
Giovanna Cutrona ◽  
Massimo Gentile ◽  
Serena Matis ◽  
Monica Colombo ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Heavy Chain ◽  
Conflicts Of Interest ◽  
Pairwise Alignment ◽  
Immunoglobulin Heavy Chain ◽  
Lymphocytic Leukemia ◽  
Study Group ◽  
Data Set ◽  
Italian Study ◽  
Mutational Status

Abstract Abstract 2423 Chronic lymphocytic leukemia (CLL) is characterized by an extremely variable clinical course. Mutational status of the immunoglobulin heavy-chain variable (IGHV) region defines two disease subsets with different prognosis. A fraction of CLL cases carries highly homologous B-cell receptors (BCR), i.e. characterized by non-random combinations of immunoglobulin heavy-chain variable (IGHV) genes and heavy-chain complementarity determining region-3 (HCDR3). We performed sequence analysis to characterize IGHV regions in a panel of 1133 CLL patients investigated by a multicenter Italian study group. A total of 1148 rearrangements were identified; the analysis of stereotyped subsets was performed based on previously reported criteria (Messmer et al, J Exp Med 2004; Stamatopuolos et al, Blood 2007). Specifically, we compared all our sequences with those found in three different publicly available data sets (Stamatopoulos et al, Blood 2007; Murray et al, Blood 2008 and Rossi et al, 2009 Clin Cancer Res). In addition, a pairwise alignment within all sequences was performed in order to discover novel potential subsets (HCDR3 identity > 60%). Based on the 2% cut-off used to discriminate between Mutated (M) and Unmutated (UM) cases, 777 sequences (67.59%) were classified as M, while 371 sequences (32.3%) as UM. The most represented IGHV genes within mutated cases were IGHV4-34 (104/118) and IGHV3-23 (85/96), whereas IGHV1-69 (97/112) was the most frequently used in the UM group. Interestingly, the IGHV3-21 gene, reported to be frequently expressed in CLL patients from Northern Europe, was present in only a small fraction of cases (24; 2.07%), confirming a previous finding reported by Ghia et al (Blood 2005) in a smaller panel. In our series, stereotyped HCDR3 sequences were found in 407/1148 (35.45%) patients, 177 of whom were M and 230 were UM cases. Overall, we observed that stereotyped sequences were significantly associated with UM IGHV status (Fisher's exact test, P<0.0001). Among the 407 stereotyped HCDR3 sequences, 345 belong to the clusters reported by Murray et al and 14 to those described by Rossi et al., 2009 Clin Cancer Res. The most frequent stereotyped subsets identified in our panel were #1 (35 cases), #7 (28 cases), #4 (24 cases), #3 and #9 (16 cases), #28 (13 cases), and #2 (12 cases), together with subsets #5, #8, #10, #12, #13, #16 and #22 (all ranging from 6 to 9 cases). Finally, we were able to identify by auto-matching analysis 48 sequences potentially specific for 23 novel putative stereotype subsets. In our series we identified 407/1148 (35.45%) stereotyped HCDR3 sequences. The percentage was higher than that reported by Stamatopoulos et al and Murray et al. This discrepancy may partially be due to the different approach used in our analysis, namely the matching to a general data set including all published stereotyped subsets instead of the auto-matching performed by those Authors. We demonstrated a significant association between IGHV status and stereotyped sequences and confirmed the finding that #1 is the most frequent subset identified so far. Finally, we were able to identify a series of 23 novel putative subsets that will require further confirmation. Disclosures: No relevant conflicts of interest to declare.

Ibrutinib (PCI 32765) Rapidly Improves Platelet Counts in Chronic Lymphocytic Leukemia / Small Lymphocytic Lymphoma (CLL/SLL) Patients and Has Minimal Effects On Platelet Aggregation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1789-1789 ◽  
Author(s):  
Mohammed Farooqui ◽  
Jay Nelson Lozier ◽  
Janet Valdez ◽  
Nakhle Saba ◽  
Ajunae Wells ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Chronic Lymphocytic Leukemia ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Lymphocytic Leukemia ◽  
Significant Activity ◽  
Small Lymphocytic Lymphoma ◽  
Open Label ◽  
Platelet Counts ◽  
Pre Treatment

Abstract Abstract 1789 INTRODUCTION: Ibrutinib (PCI 32765) is an orally administered covalent inhibitor of Bruton's Tyrosine Kinase (BTK). Ibrutinib has significant activity in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and is typically well tolerated (Byrd ASCO 2011, O'Brien ASH 2011). Rarely serious bleeding in patients concurrently on oral anticoagulation has been reported but was not related to thrombocytopenia (O'Brien ASH 2011). However, grade 1 or 2 ecchymosis/contusion is a frequent adverse event in patients on ibrutinib. In addition to being essential for B cell receptor signaling BTK is also involved in the signaling of the glycoprotein (GP)VI and GPIV von Willebrand (vW) receptors (Liu, Blood 2006). Thus, it is possible that ibrutinib could increase the bleeding risk by interfering with thrombus formation. In addition, lymphoproliferative disorders and some drugs have been associated with acquired vW-disease (AvWD). METHODS AND PATIENTS: In an ongoing single center, open label phase II trial we treat CLL/SLL patients with ibrutinib 420 mg daily on 28 day cycles (NCT01500733). We measured platelet (PLT) function on the PFA-100 instrument, vW-factor (vWF) antigen levels and activity (vWF-Ag/vWF-Act), and factor VIII (FVIII) on baseline, days 2 and 28. Here we report on effects of ibrutinib on platelet counts and function in 25 patients who completed >2 cycles. RESULTS: PLT counts prior to treatment ranged from 36 k/μl to 256 k/μl with a median of 102 k/μl. Twelve (48%) patients had a pre-treatment PLT count <100 k/μl. Median PLT counts for days 14, 28, and 56 increased to 140, 137, and 135 k/μl, respectively (P<.01). 76% of patients showed an increase after only 2 weeks on drug (median increase 25 k/μl (range 4–183 k/μl) that was sustained at subsequent timepoints. On day 14, 6 patients (24%) had a decrease in PLT count by a median of 13 k/μl from baseline; of these, 3 had a pre-treatment PLT count of <100 k/ul and 1 developed grade III thrombocytopenia (42 k/μl) that resolved to >100 k/μl by day 56. 20% (5 of 25) of patients reported grade 1 spontaneous ecchymosis with no correlation to platelet count, PFA testing, or vWF measurements. Of note we performed lymph node core biopsies in 35 patients taking ibrutinib with minimal bruising. Only 2 patients had more extensive local bruising/ecchymosis at the biopsy site. In 19 patients PFA-100 measurements of epinephrine (EPI) and adenosine diphosphate (ADP) stimulated platelet aggregation times were available (test requires PLT count >100 k/μl). Median changes in closure times with EPI and ADP on treatment were not significantly different from baseline (See table). Four (21%) patients started with abnormally prolonged EPI closure times (one on aspirin, one on ibuprofen; discontinued with the start of ibrutinib) which resolved by day 28 in 3 and decreased in 1. Three (16%) patients had a prolongation of EPI closure times on day 2 that resolved by day 28 in 2 and decreased in 1. All closure times on ADP were low or normal. No patients with abnormal PFA testing demonstrated spontaneous ecchymosis. From baseline to day 28 vWF-Act, vWF-Ag and FVIII decreased (P<0.05; n=24). All 3 values were high normal to elevated prior to treatment and decreased to normal on treatment. CONCLUSION: This preliminary report does not identify any significant ibrutinib effect on platelet function. PLT counts improved rapidly in the majority of patients and when seen transient decreases have been minimal. Three patients (16%) developed an abnormal reading in PLT function tests on treatment but none developed spontaneous echymosis or bleeding. The observed normalization of mildly elevated baseline levels of vWF and FVIII seems most consistent with a reduction in acute phase reactants and there was no evidence for AvWD on ibrutinib. The apparent functional tolerance of BTK inhibition in platelets is likely attributable to redundancy in the affected signaling pathways. This work was supported by the Intramural Research Program of NHLBI, NIH. We thank our patients for participating in these research studies. Disclosures: No relevant conflicts of interest to declare.

Tyro3, Axl Ve Mer (TAM) Receptors On Surfaces of Mononuclear Cells in Patients with B-Cell Chronic Lymphocytic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4588-4588
Author(s):  
Dilvin Guney ◽  
Aysin Tulunay ◽  
Funda Pepedil ◽  
Isik Kaygusuz ◽  
Cafer Adiguzel ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Gradient Centrifugation ◽  
Lymphocytic Leukemia ◽  
Control Group ◽  
Ligand Molecule ◽  
Tam Receptors ◽  
Cell Chronic Lymphocytic Leukemia

Abstract Abstract 4588 Background: Tyro 3 (Sky), Axl, and Mer receptors are members of the family of tyrosine kinases and Gas6 is their ligand molecule. In some types of cancer, upregulation of Axl/Gas6 indicated a worse prognosis, but an opposite situation was observed in renal “cell” carcinoma. This contradiction may suggest that Axl/Gas6 pathway varies depending on the type of cancer. The objective of this study is to investigate TAM receptors on surfaces of mononuclear cells in patients with B-Cell chronic lymphocytic leukemia (B-Cell-CLL). Material & Methods: B-Cell-CLL patients (grade 0–1, according to the classification of RAI), who were not on a drug treatment, were recruited in this study (n= 20; 9 female, 11 male). Their ages were 44 to 74 (mean: 63), and the control group consisted of 13 healthy volunteers (5 female, 8 male), whose age range is 20–89 (mean: 36). Mononuclear cells were isolated by density gradient centrifugation, and then surface TAM receptors were detected by flow cytometry. Mononuclear cell were stained with the primary antibodies against Tyro3, Axl and Mer. Results: The percentage of the surface TAM receptors on mononuclear cells from the patient group (25–75% interquartile range): Tyro 3= 25.50 (4.2– 45.62); Axl= 17/55 (5.57– 36.32), and Mer= 19.90 (1.92– 37.55). In the control group the following values were obtained: Tyro 3= 2.60 (1.35–3.25); Axl= 0.9 (0.4–2.6), and Mer= 2.50 (0.35–3.65). The percentage of three of them was significantly higher in the B-Cell-CLL group than those in the control group (P<0.01). Conclusion: In conclusion, this preliminary study showed that TAM receptors on surfaces of mononuclear cells are higher in patients with B-Cell-CLL patients than the control group. Gas6/TAM signaling may play a potential role in the pathogenesis of B Cell-CLL. Further studies are required to elucidate the actual role of Gas6/TAM signaling in B-Cell-CLL. Gas6/TAM signaling might be a new strategic goal for the treatment of B-Cell-CLL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Safety findings from CENTURION, a phase 3 consistency study of lasmiditan for the acute treatment of migraine

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
C Tassorelli ◽  
S Bragg ◽  
JH Krege ◽  
EG Doty ◽  
PA Ardayfio ◽  
...  
Keyword(s):  
Adverse Events ◽  
Acute Treatment ◽  
Motor Vehicle ◽  
Specific Treatment ◽  
Study Drug ◽  
Control Group ◽  
Phase 3 ◽  
Serious Adverse Events ◽  
Double Blind ◽  
Vehicle Accidents

Abstract Background Lasmiditan (LTN) is a selective 5-HT1F receptor agonist for the acute treatment of migraine in adults. We present detailed safety findings from the placebo-controlled, double-blind Phase 3 study, of LTN treatment across 4 attacks (CENTURION). Methods Patients were randomized 1:1:1 to LTN 200 mg (LTN200), LTN100, or a control group that received placebo for 3 attacks and LTN50 for either the 3rd or 4th attack (1:1). Safety analyses were conducted for patients who took ≥1 dose of study drug and, in some cases, those who took all 4 doses. Results Overall, 1471 patients treated 4494 attacks. The incidences of treatment-emergent serious adverse events (SAEs) were - placebo, n=2 (0.4 %); LTN100, n=1 (0.2 %); LTN200, n=2 (0.4 %); no specific treatment-emergent SAE was reported in more than one patient. The most common treatment emergent adverse events (TEAEs) with lasmiditan were dizziness, paresthesia, fatigue, nausea, vertigo, and somnolence; the vast majority were mild or moderate in severity. The incidences of these TEAEs were highest during the first attack and decreased during subsequent attacks. For patients who experienced a common TEAE with the first attack, less than 45 % experienced the same event in subsequent attacks. Patients who did not experience an event in the 1st attack infrequently experienced the same event in subsequent attacks. The time of onset of the common TEAE ranged from ~40 min to 1 h (dependent upon TEAE) and, for individual TEAE, the onset was similar across attacks. Duration was dependent upon TEAE and attack. It was shortest for paresthesia (< 2 h for all attacks); it ranged from 1.8 to 5.5 h for other common TEAEs and was generally similar across attacks. Serotonin syndrome was reported for 2 patients post LTN dosing; there were no meaningful differences across treatment groups in suicidality; there was no evidence of an increase in motor vehicle accidents. Conclusion In this blinded, controlled, multiple-attack study, LTN was associated with generally mild or moderate CNS-related TEAEs of short duration. TEAEs tended to decrease in frequency across the 4 attacks. Trial registration NCT03670810

scholarly journals Mutations in the NOTCH1 PEST Domain Enhance the Expression of CCL17 By Displacing the Transcription Factor HDAC1 in Chronic Lymphocytic Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4408-4408
Author(s):  
Zhenshu Xu ◽  
Shifen Wang ◽  
Xiuli Chen ◽  
Xueting Liang ◽  
Huixin Liang ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Transcription Factor ◽  
Chronic Lymphocytic Leukemia ◽  
Cd4 T Cells ◽  
Conflicts Of Interest ◽  
Premature Stop Codon ◽  
Lymphocytic Leukemia ◽  
Control Group ◽  
Multiple Point

Abstract The NOTCH1 is a ligand dependent transcription factor that plays an important role in lymphocyte differentiation and apoptosis . NOTCH1 dysfunction is closely related to the proliferation, differentiation, and apoptosis of tumor cells in chronic lymphocytic leukemia (CLL). With the popularization of next-generation sequencing technology the relationship between NOTCH1 mutations and disease progression in CLL, has attracted increasing attention. Here, we investigated whether the loss of a 2 bp frame shift deletion mutation influences the NOTCH1 pathway and whether this mutation alters the NOTCH1 nuclear mechanism in CLL. The ICN plasmid was engineered by cloning the ICN coding sequence into a pmax-Clover vector. The c.7541-7542delCT mutation (CTdel) was generated by site-specific mutagenesis. The BaF3 cells were transfected with Amaxa Nucleofector technology then sorted. The NOTCH1 protein expression was evaluated by Western blotting using an anti-NOTCH1 antibody, which showed compatible with the ICN. The CTdel mutation resulted in a lower molecular weight band, consistent with the presence of a premature STOP codon. Results from qRT-PCR showed elevated mRNA expression of NOTCH1 in the groups transfected with ICN and CTdel genes. An immunofluorescence assay showed that NOTCH1 was distributed in both the nuclei and cytoplasm in the control cells, while it was located in the nucleus of the cells of the ICN and CTdel groups. Compared with the control group, the activity of the reporter genes in both the ICN and CTdel groups increased, with the highest increase in the CTdel group, as reported by the two-fluorescent enzyme reporting system assay. These results determine the presence or absence of a NOTCH1 mutation, the ICN protein is located in the nucleus, and show that the NOTCH1 pathway is enhanced and the function is more stable in the presence of a NOTCH1 mutation. From the RNA-seq results we found that RT-PCR showed transcription levels of CCL17 in the ICN and CTdel groups were higher than those in the control group, and that CCL17 in the CTdel group was significantly higher than in the ICN group. We collected the culture supernatant of CLL cells for an ELISA assay and found that CCL17 was significantly elevated in the CTdel group, but CCL17 was not detected in the control group and the ICN group. In order to verify the CCL17 function of in CLL with the NOTCH1 mutation, we performed a transwell experiment to detect the ability of mediating activated CD4+ T cell migration by CCL17. The results showed that the number of CD4+ T-cells in the CTdel group that migrated in response to CCL17 was more than in the ICN group. In order to verify that the NOTCH1 mutation changed the ICN binding function, we performed a CO-IP experiment. The results showed that ICN had an interaction with MTA2/HDAC1, but this interaction was weakened with CTdel. Mass spectrometry (MS) analysis suggested that ICN was combined with MTA2 while CTdel peptides were not detected in MTA2 samples. In order to verify the negative regulatory effect of MTA2 and HDAC1 on the NOTCH1 mutation induced by C-terminal truncation in CCL17 transcription, we conducted a CHIP experiment on the nuclear pyrolysis of ICN/CTdel.The results showed that the combination of MTA2/HDAC1 and the promoter of CCL17 and ICN/CTdel was weaker than that of the control group. Because of the multiple-point binding characteristics of transcription factors on gene expression regulation, it can be concluded that CTdel DNA binding is weaker than the binding of ICN. As a result, in the presence of the NOTCH1 protein C-terminal truncation, which has lost MTA2/HDAC1 binding, its inhibition is reduced and the CCL17 expression becomes significantly elevated. In conclusion, it is suggested that the NOTCH1 mutation found in CLL stimulates the NOTCH1 pathway, and is related to the high expression of CCL17. The chemokine CCL17 can cause the migration of CD4+ T-cells and change the microenvironment to favor tumor cell survival. ICN in the nucleus combines with CSL to form activating complexes or recruits transcription factor MTA2/HDAC1 to form inhibiting complexes, and constitutes the balance between the promotion and the inhibition for the downstream gene expression. The NOTCH1 mutation with CTdel could result in loss of this balance, and activate the expression of downstream genes, such as CCL17. Key words: chronic lymphocytic leukemia; NOTCH1; mutation; HDAC1; CCL17; chemokine Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document