scholarly journals High Aurora Kinase and Low Dido Levels Characterizes a Sub-Group of Chronic Lymphocytic Leukemia with Chromosomal Gains and High White Blood Cell Counts: Potential Inter-Regulatory Role of E2F1 and Mir-17-92 Cluster

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2029-2029
Author(s):  
Felipe C Souza ◽  
Josiane L Schiavinato ◽  
Antonio R. Lucena-Araujo ◽  
Fabio M Oliveira ◽  
Amelia G Araújo ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Chronic Lymphocytic Leukemia ◽  
Blood Cell ◽  
White Blood Cell ◽  
Chromosomal Instability ◽  
Regulatory Mechanism ◽  
Lymphocytic Leukemia ◽  
Transcript Levels ◽  
Expression Levels

Abstract During cell cycle division Aurora kinases (AURKA and AURKB) participate in the formation and control of mitotic spindle fibers, while, protein isoforms (DIDO1, DIDO2 and DIDO3), derived by alternative splicing of the DIDO gene, assist at the junction of microtubules to kinetochores. Thus, both are relevant to cell cycle maintenance. Interestingly, overexpression (or gain of function) of AURKs or low expression (or loss of function of DIDO) are both associated with centrosomal amplification and chromosomal instability (CIN), leading to aneuploidy. Among hematological diseases with CIN records, chronic lymphocytic leukemia (CLL) can display centrosome amplification and changes in AURKs expression levels leading to aneuploidy. The Despite this, there are no studies evaluating the potential association of these genes with CIN in CLL. By evaluating their gene expression levels in CLL samples from patients with or without chromosomal aberrations, we show that increased levels of AURKA and AURKB and, conversely, reduced levels of DIDO variants, are both significantly associated with chromosomal gains and with increased white blood cell (WBC) counts. Clearly, CLL samples without any cytogenetic abnormality had expression levels similar to samples mostly harboring non-numerical aberrations. The finding that the expression levels of AURKs and DIDO variants are completely opposed, showing a discrete inter-related pattern, led us to investigate the potential regulatory mechanism behind this. Given that other have previously shown that the oncogenic miR-17-92 cluster is significantly upregulated in purified CLL patient cells expressing unmutated IGHV genes (as compared to mutated patient cells), and that miR-17 is expressed at significantly higher levels in unmutated or ZAP-70 high cases (bad prognostic cases generally associated with chromosomal instability), we investigated the potential negative regulation of DIDO variants by microRNAs from this cluster. In addition, based on the already described regulatory mechanism by which AURKA overexpression induces the E2F1-mediated transcription upregulation of the miR-17-92 cluster (with an observed expression correlation of both proteins in cancer specimens); we decided to investigate this regulatory axis in CLL. Notably, we found that all DIDO variants are predicted to be heavily targeted by several miRs of this oncogenic cluster. We show that CLL samples with low DIDO expression, in addition to the already mentioned AURK high levels, displayed significant higher levels of the transcription factor E2F1 and of its transcriptional target, the miR-17-92 primary transcript (MIR17HG). Moreover, by using the NTERA-2 cell line as a model, we show that siRNA knockdown of AURKA (at the transcript and protein level, as confirmed by qPCR and western blot) is accompanied by a striking significant reduction of E2F1 and also of MIR17HG. Furthermore, transfection of NTERA-2 cells with synthetic mimics of the miR-17~92 cluster (namely, miR-19a, miR-20a and miR-92a) results in a clear and significant reduction in the transcript levels of all DIDO variants. Finally, specific siRNA inhibition of the DIDO3 variant (but not the others) led to a significant reduction in the transcript levels of all DIDO variants, indicating an additional mechanism contributing to the downregulation of DIDO transcripts. Altogether, our results demonstrate the existence of a potential interconnected regulatory mechanism between AURK and DIDO, associated with CIN and higher WBC counts in CLL. More importantly, the high expression levels of AURKs and the associated low levels of DIDO variants are specifically associated with cytogenetic abnormalities presenting chromosomal gains, highlighting the specific cellular mechanism underlying the CIN observed in this distinct CLL group. Given the central role of CIN in cancer genesis and progression, these findings will likely have an important impact on prognosis or treatment of CLL. Funded by: FAPESP, CNPq and CAPES. Disclosures No relevant conflicts of interest to declare.

scholarly journals Pseudohyperkalemia in Patients with Chronic Lymphocytic Leukemia

2011 ◽  
Vol 2011 ◽  
pp. 1-3 ◽  
Author(s):  
Stephen I. Rifkin
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Blood Cell ◽  
Mechanical Stress ◽  
White Blood Cell ◽  
Whole Blood ◽  
Correct Diagnosis ◽  
Plasma Potassium ◽  
Lymphocytic Leukemia ◽  
Blood Potassium

Pseudohyperkalemia occurs occasionally in patients with extreme leukocytosis. Increased white blood cell fragility coupled with mechanical stress is felt to be causal. Serum and plasma potassium levels have been both associated with pseudohyperkalemia. Whole blood potassium determination will usually verify the correct diagnosis. It is important to diagnose this condition early so that patients are not inappropriately treated. Two patients with chronic lymphocytic leukemia and extreme leukocytosis are presented, one with pseudohyperkalemia and one with probable pseudohyperkalemia, and diagnostic considerations are discussed

CD72 Expression Patterns in ZAP70 Positive and Negative Chronic Lymphocytic Leukemia: Potential Regulatory Role in BCR Signalling

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4159-4159
Author(s):  
Francisco P. Careta ◽  
Rodrigo A. Panepucci ◽  
Daniel M Matos ◽  
Rodrigo Proto-Siqueira ◽  
Wilson A. Silva-Junior ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Expression Patterns ◽  
Surrogate Marker ◽  
Lymphocytic Leukemia ◽  
Negative Regulator ◽  
Beta Catenin ◽  
Cell Surface Glycoprotein ◽  
Expression Levels

Abstract Introduction: Absence of mutations in IgVH genes or higher number of ZAP70+ cells (as a surrogate marker) in chronic lymphocytic leukemia (CLL) B-cells defines a patient group with a poorer clinical course. These features relate to the role of BCR signalling in the proliferation and survival of CLL B-cells, and establish a link between these markers and the biology of CLL prognostic subgroups. The identification of additional players in this context may help to better understand the molecular basis of this disease and contribute to develop new therapeutic approaches. A search for genes potentially related to BCR signalling, when comparing mutated and unmutated CLL cases using serial analysis of gene expression, revealed a 4-fold increase of CD72 tags in unmutated samples, a specific B cell surface glycoprotein known to transmit both positive and negative signals in BCR signalling. Objective: This finding lead us to explore the potential role of CD72 on BCR signalling in distinct CLL prognostic subgroups, as defined by ZAP70 expression. Methods: Percentage of ZAP70+ and CD72+ cells were evaluated by flow cytometry on gated CD19+CD5+ cells in 25 CLL samples. Positive cases for ZAP70 and CD72 were defined using a cut-off of 35% and 40% positive cells, respectively. Real time PCR was used to quantify the expression levels of 3 genes related to proliferation and survival, RELB, Beta-Catenin (CTNNB1) and AKT1, on 16 CD19+ enriched (purity > 90%) CLL samples. Results: Samples were classified as 11 ZAP70+ and 14 ZAP70−. Median percentage of CD72+ cells in ZAP70+ was significantly higher than for ZAP70− cases (82% compared to 39%, respectively, P=0.0029). Furthermore, percentages of CD72 and ZAP70 were positively correlated (r=0.5930 and P=0.0009). Interestingly, ZAP70+ cases were restricted to CD72+ cases (n=11, CD72+ZAP70+ [+/+]), whereas six CD72+ cases were ZAP70− (ZAP70−CD72+ [−/+]). Finally, there were 8 cases CD72−ZAP70− [−/−]. No differences among these 3 groups were observed in regard to laboratory parameters (white blood cells, total lymphocytes, lymphocyte percentage, haemoglobin, haematocrit and platelet number). Despite the reduced number of samples analysed (6 +/+, 6 −/− and 4 −/+), transcripts for RELB (P<0.05), CTNNB1 (P<0.05), and AKT1(P=0.057) were expressed at higher levels in ZAP70+CD72+ than in ZAP70−CD72+ samples. Additionally, the transcripts were expressed at higher levels in ZAP70−CD72− than in ZAP70−CD72+ samples, and this difference was statistically significant (P<0.05) for CTNB1 and AKT1, but not for RELB (P=0.054). Conclusion: Our data indicate that higher percentages of ZAP70+ cells are associated with higher expression levels of transcripts related to proliferation and survival of CLL B-cells. In the absence of ZAP70 expression, CD72 may act as a negative regulator of the BCR pathway, as indicated by the lowest levels of transcripts on ZAP70−CD72+ cases.

scholarly journals Determine White Blood Cell Count (WBCC) Limits and ABL Ct Cut-Off with ABL Copy Number (CN) Calibration for the Xpert® BCR-ABL Ultra Assay

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5367-5367
Author(s):  
Huilin Wei ◽  
Krupa Shridhar ◽  
Gwo-Jen Day
Keyword(s):  
Blood Cell ◽  
White Blood Cell ◽  
Copy Number ◽  
Conflicts Of Interest ◽  
Regulatory Body ◽  
Rna Expression ◽  
Transcript Levels ◽  
Expression Levels ◽  
Standard Curve ◽  
Ct Value

Introduction Monitoring BCR-ABL transcript levels in peripheral blood of patients using real-time quantitative PCR is standard of care in the management of Chronic Myeloid Leukemia. Xpert BCR-ABL Ultra is an automated cartridge-based assay developed by Cepheid for sensitive assessment of BCR-ABL transcript levels. However, in clinical situations the total RNA isolated from white blood cell (WBC) in the patient's blood is insufficient to ensure assay LoD of 0.003% (IS)/MR4.52, or high WBCC in specimen can overload the Ultra cartridge, resulting in inaccurate test results. Therefore, it is necessary to determine proper WBCC input, to ensure accurate assay quantification results. Objectives The objective of this study was to determine the correlation between ABL copy number input and Ct value, determine lower and upper WBCC input limits for the Xpert BCR-ABL Ultra assay and establish the ABL Ct cut-offs. Methods To establish an upper ABL Ct limit that corresponds to a minimal ABL CN input of 32,000 for supporting LoD, a BCR-ABL plasmid DNA (pDNA, ERM-AD623) was serially diluted and tested in an EDTA whole blood background to make a calibration panel. A standard curve was generated against which the ABL Ct could be estimated as a function of ABL copy number. To estimate the lower WBCC limit, CML negative EDTA lysate with normal WBCC was serially diluted with WBC depleted EDTA lysate. To estimate the upper WBCC limit, EDTA lysate from a specimen with high WBCC (69.9×106cells/mL) was diluted with CML negative lysate with normal WBCC (3.5×106 cells/mL). To validate the WBCC limit, CML positive lysate with high WBCC (40×106cells/mL) was diluted with high WBCC (40×106cells/mL) CML negative lysate to produce five BCR-ABL levels within 0.03% (IS)/MR3.52 to 65% (IS)/MR0.19. Each level was further diluted with PBS lysate to achieve 8 levels of WBCC from 40×106 to 0.04×106 cells/mL. Results ABL Ct value of ~19 corresponding to a minimum ABL copy number input of 32,000 was established to support assay LoD of 0.003% (IS)/MR4.52 (Figure.1). To allow for a one Ct buffer, the upper ABL Ct cut-off was set at 18. The lower WBCC input limit, corresponding to the upper ABL Ct limit of ~18 with estimated ABL copy number greater than 64,000, was established to be 0.15×106 cells/mL (Figure.2) The upper WBCC input limit was established to be 30×106 cells/mL. This corresponds approximately to a Ct value of 10 (Figure.3). The Xpert BCR-ABL Ultra test monitors RNA expression levels of the BCR-ABL fusion transcript and the ABL transcript. Unlike a gene DNA marker, RNA expression levels are variable and can fluctuate. To accommodate for the variability in RNA expression levels and based on results from this study, the upper ABL Ct cut-off was lowered to 8. The lower and upper WBCC input limit, together with ABL Ct cut-off, were validated with CML patient specimens with WBCC input ranging from 0.04 to 40 × 106 /ml within IS% range of 0.03% to 65% (Table.1). The normal WBCC of 4 to 10 × 106/mL across all tested concentrations yielded ABL Ct range from 13.2 to 11.7 with estimated ABL copy number of 2.5×106 to 7.8×106. Conclusions A correlation between ABL copy number input and ABL Ct value was established with the standard curve generated from a pDNA calibration panel. The lower WBCC limit was set at 1.5×105 cells/mL and upper WBCC limit was set at 3.0×107 cells/mL, together with ABL Ct cut-offs of 18 and 8 and ABL copy number of 6.4×104 to 1.4×108, to support Xpert BCR-ABL Ultra assay performance. The normal WBCC of 4 to10×106/mL across all tested concentrations yielded an ABL Ct range from 13.2 to 11.7 with estimated ABL copy number of 2.5×106 to 7.8×106, indicating Xpert BCR-ABL Ultra assay normally provided more than 10 times the required minimum ABL Copy number to support the assay LoD claim at 0.003% (IS). *Product in development. Not for use in diagnostic procedures. Not reviewed by any regulatory body. Disclosures No relevant conflicts of interest to declare.

scholarly journals Risk Factors of White Blood Cell Progression Among Patients With Chronic Lymphocytic Leukemia at Felege Hiwot Referral Hospital, Bahir Dar, Ethiopia

2022 ◽  
Vol 21 ◽  
pp. 117693512110699
Author(s):  
Gedam Derbew Addisia ◽  
Awoke Seyoum Tegegne ◽  
Denekew Bitew Belay ◽  
Mitiku Wale Muluneh ◽  
Mahider Abere Kassaw
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Blood Cell ◽  
White Blood Cell ◽  
Blood Cells ◽  
White Blood Cells ◽  
Lymphocytic Leukemia ◽  
Bahir Dar ◽  
Felege Hiwot Referral Hospital ◽  
Wbc Count

Background: Leukemia is a type of cancers that start in the bone marrow and produce a serious number of abnormal white blood cells. Bleeding and bruising problems, fatigue, fever, and an increased risk of infection are among symptoms of the disease. The main objective of this study is to identify the determinant of the progression rate of white blood cells among patients with chronic lymphocytic leukemia at Felege Hiwot Referral Hospital (FHRH), Bahir Dar, Ethiopia. Methods: A retrospective study design was conducted on 312 patients with chronic lymphocytic leukemia at FHRH, Bahir Dar, Ethiopia under treatment from 1 January 2017 to 31 December 2019. A linear mixed-effects model was considered for the progression of the white blood cell data. Results: The estimated coefficient of the fixed effect intercept was 84.68, indicating that the average white blood cell (WBC) count of the patients was 84.68 at baseline time by excluding all covariates in the model ( P-value <.001). Male sex ( β = 2.92, 95% confidence interval [CI] 0.58, 0.5.25), age ( β = .17, 95% CI 0.08, 0.28), widowed/divorced marital status ( β = 3.30, 95% CI 0.03, 6.57), medium chronic lymphocytic leukemia (CLL) stage ( β = −4.34, 95% CI −6.57, −2.68), high CLL stage ( β = −2.76, 95% CI −4.86, −0.67), hemoglobin ( β = .15, 95% CI 0.07, 0.22), platelet ( β = .09, 95% CI 0.02, 0.17), lymphocytes ( β = .16, 95% CI 0.03, 0.29), red blood cell (RBC) ( β = .17, 95% CI 0.09, 0.25), and follow-up time ( β = .27, 95% CI 0.19, 0.36) were significantly associated with the average WBC count of chronic lymphocytic leukemia patients. Conclusions: The finding showed that age, sex, lymphocytic, stage of chronic lymphocytic leukemia, marital status, platelet, hemoglobin, RBC, and follow-up time were significantly associated with the average WBC count of chronic lymphocytic leukemia patients. Therefore, health care providers should give due attention and prioritize those identified factors and give frequent counseling about improving the health of chronic lymphocytic leukemia patients.

The effects of sildenafil citrate on malignant B-cells in patients with chronic lymphocytic leukemia

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 7076-7076
Author(s):  
B. A. McGregor ◽  
A. Gorrebeeck ◽  
E. Struble ◽  
A. Harroff
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Blood Cell ◽  
Cell Count ◽  
White Blood Cell Count ◽  
White Blood Cell ◽  
Lymphocytic Leukemia ◽  
Western Hemisphere ◽  
Blood Cell Count ◽  
Apoptosis Rate

7076 Background: Chronic lymphocytic leukemia (CLL) is the most common form of leukemia in the Western Hemisphere, with over 10,000 cases diagnosed annually in the United States. It is characterized by progressive accumulation of functionally incompetent long-lived lymphocytes, shown to be secondary to a defect in programmed cell death or apoptosis. The phosphodiesterase inhibitor sildenafil induces capsase dependent apoptosis of malignant B lymphocytes in vitro. This study will test the hypothesis that sildenafil reduces the expression of BCL-2 and increases the spontaneous apoptosis rate of malignant B-cells in patients with CLL. Methods: Thirteen patients with Rai Stage 0 CLL were enrolled. Nine of the patients were aged sixty-five years or older and received sildenafil 25 mg weekly; four patients were under the age of sixty-five and received sildenafil 25 mg weekly. All patients took the medication for a total of three months. Lymphocyte counts, BCL-2 expression, caspase 3 activity, and apoptosis rate were monitored on enrollment and monthly for duration of the study. Results: The median age of patients enrolled in the study was 74 with a median white blood cell count of 18 x103/mL. Twelve of the 13 patients completed three months of therapy. While one patient withdrew due to blehparitis, not felt to be a side effect of sildenafil, all other patients tolerated the medication well without any adverse effects. There was no significant decrease in white blood cell count or Bcl-2 expression; capsase 3 activity and apoptosis rates remained undetectable on presentation and throughout treatment. Conclusions: At a dose of 25 to 50 mg weekly, sildenafil does not appear to have any effects on the malignant B cells in CLL. While this dose may not produce a measurable clinical or cellular response, higher doses may still have an effect on the malignant B cells of CLL. The dose of sildenafil was based on a case series of patients with Viagra who had decreases in IgM levels while taking sildenafil 25–50 mg weekly. Subsequent studies in have shown a greater reduction in IgM in patients taking sildenafil 100 mg daily and patients did not report any significant side effects. A follow-up study using sildenafil 100 mg daily is warranted. No significant financial relationships to disclose.

scholarly journals Mutations at 3' Untranslated Region (3'UTR) of NOTCH1 Are Associated with Low CD20 Expression Levels in Chronic Lymphocytic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 306-306
Author(s):  
Tamara Bittolo ◽  
Federico Pozzo ◽  
Riccardo Bomben ◽  
Tiziana D'Agaro ◽  
Vanessa Bravin ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Chronic Lymphocytic Leukemia ◽  
Untranslated Region ◽  
Lymphocytic Leukemia ◽  
Transcript Levels ◽  
Expression Levels ◽  
Cd20 Expression ◽  
Trisomy 12 ◽  
Anti Cd20 ◽  
Notch1 Mutations

Abstract Background. In chronic lymphocytic leukemia (CLL), NOTCH1 mutations associate with clinical resistance to anti-CD20 immunotherapy in FCR combination (Stilgenbauer et al., Blood, 2014, Dal Bo et al., AHO, 2014), that can be ascribed to a NOTCH1 mutation-driven repression of CD20 levels by HDACs (Pozzo et al., Leukemia, 2016). Recently, novel recurrent mutations have been identified in the 3'untranslated region of NOTCH1 (3'UTR NOTCH1 mutations), determining a novel splicing event within the last NOTCH1 exon (Puente et al., Nature, 2015), leading to an impaired degradation of NOTCH1 protein. Aim. To determine if 3'UTR NOTCH1 mutations associate with low CD20 levels. Methods. NOTCH1 mutations were screened by next-generation sequencing (NGS) in exon 34 and part of 3'UTR with at least 1000X coverage. NOTCH1 (transmembrane, TM, or intracytoplasmic domain, NICD) protein levels were investigated by western blot (WB). CD20 expression was investigated by flow cytometry with a FITC-conjugated anti-CD20 antibody (L27 clone). MS4A1 (encoding for CD20 protein) transcript levels were investigated by qRT-PCR. Susceptibility to anti-CD20 rituximab and ofatumumab was investigated by complement-dependent cytotoxicity (CDC) assay. NOTCH1 signaling was inhibited by gamma-secretase inhibitor (GSI). Results. i) NOTCH1 mutational screening. In 649 CLL, NOTCH1 mutations were detected in 115 cases (17.72%), overall accounting for 127 mutations (73 c.7541-7542delCT, 11 other frameshift, 17 nonsense, 1 missense, and 25 3'UTR mutations). For analysis purposes, the 115 mutated cases were subdivided in cases with NOTCH1 coding mutations (coding NOTCH1-mut, 90 cases) and cases with 3'UTR NOTCH1 mutations (3'UTR NOTCH1-mut, 25 cases). Four cases with concomitant 3'UTR NOTCH1 mutation and coding NOTCH1 mutation were assigned to the 3'UTR group according to the substantially higher mutational burden detected for the 3'UTR mutation. ii)NOTCH1protein expression. In keeping with alternative splicing events causing an impaired NOTCH1 protein degradation, 3'UTR NOTCH1-mut cases showed higher levels than NOTCH1 wild-type (NOTCH1-wt) cases of both NOTCH1 TM and NICD by WB, and similar to coding NOTCH1-mut cases (Fig. 1a). iii) CD20 expression levels. According to FISH classification, variable CD20 levels were found by flow cytometry, with the highest levels in trisomy 12 CLL. Of note, 3'UTR NOTCH1-mut cases expressed lower CD20 levels than NOTCH1-wt cases in both trisomy 12 CLL (mean MFI in 9 3'UTR NOTCH1-mut cases = 2446.11 vs mean MFI in 66 NOTCH1-wt cases = 8254.20; p<0.0001) and non-trisomy 12 CLL (mean MFI in 16 3'UTR NOTCH1-mut cases = 2033.50 vs. mean MFI in 468 NOTCH1-wt cases = 3294.07; p=0.0001), and comparable to coding NOTCH1-mut cases (trisomy 12 CLL, mean MFI in 34 coding NOTCH1-mut cases = 2570.73; p=0.8530; non-trisomy 12 CLL, mean MFI in 56 coding NOTCH1-mut cases = 2538.13; p=0.1500) (Fig. 1b). Similarly, in both trisomy 12 CLL and non-trisomy 12 CLL categories, MS4A1 transcript levels were lower in 3'UTR NOTCH1-mut cases than in NOTCH1-wt cases (p=0.0274 and p=0.0072, respectively), again with expression levels comparable with coding NOTCH1-mut cases (p=0.2874 and p=0.9610, respectively, Fig. 1c). iv) Susceptibility to anti-CD20 in vitro. In keeping with CD20 levels, 3'UTR NOTCH1-mut cases showed lower relative lysis induced by rituximab or ofatumumab, in CDC assay, than NOTCH1-wt cells (7 3'UTR NOTCH1-mut cases, 9 NOTCH1-wt cases; mean relative lysis upon rituximab: 2.67% vs. 25.56%; p=0.0349; mean relative lysis upon ofatumumab: 39.26% vs. 60.64%; p=0.0286). In the same manner, coding NOTCH1-mut cases showed lower relative lysis induced by rituximab or ofatumumab than NOTCH1-wt cases (9 coding NOTCH1-mut cases, 9 NOTCH1-wt cases; mean relative lysis upon rituximab: 2.53% vs. 25.56%; p=0.0339; mean relative lysis upon ofatumumab: 30.60% vs. 60.64%; p=0.0114; Fig. 1d). v) Modulation of CD20 expression by NOTCH1 inhibition. GSI treatment increased both CD20 protein and transcript levels in 3'UTR NOTCH1-mut cases, as previously reported for coding NOTCH1-mut cases (p=0.0376 and p=0.0326, respectively; Fig. 1e,Pozzo et al., Leukemia, 2016). Conclusions.In keeping with an impaired NOTCH1 protein degradation, 3'UTR NOTCH1-mut cases had low CD20 expression levels, suggesting the introduction of 3'UTR NOTCH1 mutation evaluation in CLL patients undergoing anti-CD20 immuno-chemotherapy. Disclosures No relevant conflicts of interest to declare.

TP53 Disruption and Telomere Instability In Chronic Lymphocytic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4135-4135
Author(s):  
Mélanie Pagès ◽  
Lauren Veronese ◽  
Patricia Combes ◽  
Romain Guièze ◽  
Gwendoline Soler ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Chromosomal Instability ◽  
Tp53 Mutation ◽  
Tp53 Gene ◽  
Lymphocytic Leukemia ◽  
Prognostic Impact ◽  
Wild Type ◽  
Expression Levels ◽  
The Impact ◽  
Tp53 Status

Abstract Background Disruptions of the TP53 tumor suppressor pathway by TP53 gene mutations and/or by17p deletions resulting in loss of the TP53 locus are clearly associated with poor survival and chromosomal instability in chronic lymphocytic leukemia (CLL). Chromosomal instability is promoted by telomere dysfunction, and the TP53 pathway is involved in the monitoring of telomere integrity. Aim The purpose of the present study was to describe the changes in main telomeric parameters, which can affect telomere integrity, associated with TP53 mutations and 17p deletions and to evaluate their potential prognostic value. Patients and Methods We performed a comparison between a group of TP53 disrupted CLL patients (n= 24) and a group of TP53 wild-type CLL patients (n= 61). Median age was 70 [39-89] years, M/F ratio was 2.4/1. At the time of sampling, 35 patients were in Binet stage A, 19 patients in stage B and 31 patients in stage C. A total of 50 patients were subsequently treated by immunochemotherapy (FCR n= 30, other n= 8), alemtuzumab (n= 8) or alkylating agent-based regimens (n= 4). TP53 gene mutation screening was performed using Sanger sequencing of the entire coding region (exons 2–11). Cytogenetic analysis (karyotype and FISH) were performed to evaluate the chromosomal instability and detect the marks of telomeric fragility. Quantitative PCR approaches were used to measure the relative telomere length and to evaluate expression levels of shelterin genes (TRF1, TRF2, POT1, RAP1, TPP1, and TIN2) and telomerase (hTERT). Results The patients showing a TP53 mutation without a 17p deletion and the patients carrying a 17p deletion with or without TP53 mutation appear to display the same level of telomeric instability. These cases are characterized by significant telomeric erosion (P<10-7), hTERT over expression (P<10-4) and decreased TRF1 (P<0.04) and TPP1 (P<0.016) expression levels. The impact of TP53 status on telomeric shortening is independent on Binet stages and classical markers of CLL progression (IgVH status, CD38 expression, lymphocyte doubling time). Moreover, TP53 disruption is strongly associated with telomere deletions, end-to-end fusions and chromosomal aberrations. Multivariate analysis on time to progression (TTP) including TP53 status and telomere characteristics revealed that two shelterin genes TTP1 (HR 7.1; 95% CI 2.0, 25.0; P= 0.0026) and RAP1 (HR 3.7; 95% CI 1.4, 9.6; P= 0.0088) have a significant prognostic impact which was independent on that of TP53 status (HR 6.9; 95% CI 2.3, 20.5; P= 0.0053). Low TPP1 and high RAP1 expression identify subsets of TP53 wild type patients with significantly shorter TTP as illustrated in Figure 1 and 2. Conclusion These results suggest that TP53 disruption may play a major role in telomeric and chromosomal instability in CLL. Shelterin gene expression may help to identify a subset of TP53 wild-type patients with adverse prognosis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals CTP Synthase 2: A Novel Prognostic Biomarker and Potential Therapeutic Target in Chronic Lymphocytic Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 14-15
Author(s):  
Ya Zhang ◽  
Xinting Hu ◽  
Yang Han ◽  
Xiangxiang Zhou ◽  
Na Chen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Regression Analysis ◽  
Chronic Lymphocytic Leukemia ◽  
Cox Regression ◽  
Lymphocytic Leukemia ◽  
Potential Mechanism ◽  
Cox Regression Analysis ◽  
Ctp Synthase ◽  
Binet Stage

Introduction CTP synthase 2 (CTPS2) is a critical regulator in lymphocytes proliferation and nucleotides synthesis. Yet, the role of CTPS2 has not been explored in chronic lymphocytic leukemia (CLL). Hence, the aim of this study was to investigate the clinical significance and functional mechanism of CTPS2 regulation in CLL pathogenesis and progression. Methods In the present study, 1030 clinically annotated CLL patients from multiple cohorts were enrolled with informed consents. Peripheral blood samples from 66 de novo CLL patients were collected at the Department of Hematology in Shandong Provincial Hospital. Expression levels of CTPS2 in CLL cells were determined by quantitative RT-PCR and western blotting. Lentiviral vectors were utilized to stably silence CTPS2. RNA-sequencing and functional enrichment analysis were performed. Besides, cell viability, apoptosis and cell cycle were assessed by cell counting kit-8, annexin V-PE/7AAD and PI/RNase staining, respectively. All investigators comply with the guiding principles for experimental procedures found in the Declaration of Helsinki of the World Medical Association. Results Aberrantly increased expression of CTPS2 was detected in CLL primary cells and cell lines (MEC1 and EHEB) at mRNA and protein level compared with normal B cells (p&lt;0.001; Figure 1A). To validate the altered pattern of CTPS2 expression in CLL, we further investigated and found CTPS2 over-expression in CLL patients in 3 independent microarrays (p&lt;0.01; Figure 1B-C). In addition, we observed elevated CTPS2 expression in significant correlation with advanced Binet stage (p&lt;0.001; Figure 1D), unmutated IGHV (p=0.007; Figure 1E), and deletion of 11q23 (p&lt;0.001; Figure 1F). Kaplan-Meier curves showed stratified CTPS2high patients were observed with significantly shorter overall survival versus the CTPS2low group in 2 independent cohorts (cohort 1, HR=4.488, p=0.001, Figure 2A; cohort 2, HR=1.614, p=0.049, Figure 2B). Besides, enhanced expression of CTPS2 were revealed to predict inferior treatment-free survival (cohort 1, HR=2.715, p=0.003, Figure 2C; cohort 2, HR=1.909, p=0.008, Figure 2D). Univariate cox regression analysis suggested that CTPS2 high expression predicted adverse survival (HR=1.785, p=0.003). Moreover, multivariate cox regression analysis confirmed the prognostic value of CTPS2 overexpression in CLL patients independent of age and Binet stage (HR=1.724, p=0.007; Figure 2E). To investigate the biological processes of CTPS2 involving in CLL progression, functional assays were performed. CLL cells with CTPS2 silencing exhibited attenuated cell proliferation, increased fast-onset apoptosis and induced G2/M phase arrest (Figure 3A-E). Additionally, down-regulated Bcl-2 expression as well as promoted cleaved-PARP, Bax and p21 expression were observed in CTPS2 knock-down transfected CLL cells (Figure 3F). To further explore the mechanism of CTPS2 regulation in the tumorigenesis of CLL, RNA-sequencing was conducted in CLL transfected cells. Annotations of differentiated genes implicated that CTPS2 was functionally enriched in cell cycle, DNA replication activation and oncogenic pathways (Figure 4A). Accordantly, activation of p-ATM, p-BRAC1, p-H2AX were visibly elevated, illuminating the potential mechanism of CTPS2 regulating DNA damage in CLL pathogenesis (Figure 4B). Collectively, the interactive network of CTPS2 and its down-stream targets was established, illuminating the potential mechanism of CTPS2 regulation in CLL progression (Figure 4C). Conclusion Taken together, the present study was the first investigation on the role of CTPS2 in CLL tumorigenesis by in silico analysis and ex vivo evaluation. CTPS2 was up-regulated and conferred independent inferior prognostic significance, highlighting the effectiveness and potential of CTPS2 in risk stratification and targeted strategy in CLL patients. Disclosures No relevant conflicts of interest to declare.

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