bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic Disruption of □pdl and □pdlr Phospholipases in Mice Leads to Impaired Platelet Adhesion and Aggregation
Authors: Wang DingYan , Reheman Adili , Connelly Phil , Wang Youdong , Maguire Graham , He Feng , Keating Armand , Ni Heyu , Wen Xiao-Yan
Published: April 2020

Platelet adhesion and aggregation are critical events in thrombosis. Bioactive phospholipid LPA (lysophosphatidic acid) has been identified as an important agonist for platelet aggregation. Plasma LPA can be generated from phospholipid substrates by phospholipase A1 (PLA1) and phospholipase D (PLD). We previously identified two novel PLA1 enzymes, designated lpdl (lpd lipase) and lpdlr (lpdl related lipase). Together with phosphatidylserine phospholipase A1 (PS-PLA1), these three phospholipases form a unique PLA1 lipase subfamily. Phospholipids are important structural components of cellular membranes. Recent studies demonstrate that lipid raft microdomains on the platelet membrane contribute to platelet activation. The gathering of membrane lipid rafts is necessary for ADP-mediated platelet activation. Disruption of lipid raft results in a reduction of ADP-induced platelet aggregation. In addition, it is reported that alteration of membrane lipid composition also affects the function of platelet β3 integrin (GPIIbIIIa). Thus, phospholipases may play an important role in platelet function. However, the roles of lpdl and lpdlr in platelet activation and thrombosis are unknown. To study the function of lpdl and lpdlr, we have recently knocked out both lpdl and lpdlr genes in mice by deleting their exons I and II, including their translation start codon ATG. RT-PCR confirmed that neither lpdl nor lpdlr gene is expressed in homozygous lpdl−/− or lpdlr−/− mice but expressed in wild-type (WT) tissues, indicating successful knockout of these phospholipases. Next, optical platelet aggregometry was used to assess platelet aggregation in platelet rich plasma (PRP). Platelet aggregation was induced with 5, 10 and 20 μM ADP in pooled PRP (3×108 platelets/mL) from age- and sex-matched WT, heterozygous and homozygous mice. Our results demonstrated that both the lpdl−/− and lpdlr−/− mice have decreased platelet aggregation after ADP stimulation. We further studied thrombus formation in lpdl−/− mice using an ex vivo perfusion chamber model on collagen type I-coated rectangular glass microcapillary tubes. Our preliminary data showed that both platelet adhesion and aggregation were impaired in the lpdl−/− mice. We are repeating these experiments and are examining lpdlr−/− mice using the same perfusion chamber model. In vivo thrombosis models with intravital microscopy and the mechanisms of how lpdl and lpdlr enzymes regulate platelet activation will also be investigated. These studies could potentially identify a novel pathway for regulation of platelet function, which may lead to development of new diagnostic and/or therapeutic methods for atherothrombosis. (Drs. D. Wang and A. Reheman contribute equally to this work)
ATG Plus Cyclosporine-a Should Be Used as Immunosuppressive Therapy in Patients with Severe Aplastic Anemia-Systematic Review and Meta-Analysis
Authors: Gafter-Gvili Anat , Ram Ron , Gurion Ronit , Paul Mical , Yeshurun Moshe , Raanani Pia , Shpilberg Ofer
Published: April 2020

Background: Immunosuppressive therapy (IST) is the therapeutic alternative for patients ineligible for allogeneic transplant. Although the combination of anti-thymocyte globulin (ATG) and cyclosporine-A (CsA) is considered the “gold standard” for these patients, the essentiality of using both drugs can be challenged.

Objectives: This study aims to compare between treatment with ATG + CsA and ATG alone for patients with severe aplastic anemia (SAA), and with non-severe aplastic anemia (NSAA).

Methods: Systematic review and meta-analysis of randomized controlled trials of patients with aplastic anemia comparing ATG and CsA with ATG alone. The Cochrane Library, MEDLINE, conference proceedings and references were searched until 2008. Outcomes assessed were: all-cause mortality, overall hematological failure, refractory disease. Relative risks (RR) with 95% confidence intervals (CIs) were estimated and pooled.

Results: Our search yielded 4 trials. For patients with SAA there was a significant reduction in all-cause mortality at 3 months in the ATG+CsA arm compared with the ATG alone arm (RR 0.50 [95%CI 0.29–0.85]). This effect was also shown at 1 year (RR 0.54 [95%CI 0.30–0.99]) (Figure) and at 5 years (RR 0.58 [95%CI 0.36–0.93]). There was also a reduction in overall hematological failure (RR 0.67 [95% CI 0.49–0.90]) and in the number of patients with refractory disease (RR 0.51 [95%CI 0.33–0.81]) in the ATG+CsA arm.

In patients with NSAA, there was no difference in mortality at 6 months (RR 1.03 [95%CI 0.07–15.78]) and at 5 years (RR 1.03 [95%CI 0.07–15.78]) or in refractory disease (RR 0.89 [95% CI 0.40–1.99]), when ATG+CsA was compared to ATG alone. However, there was a reduction in overall hematological failure in the ATG+CsA arm (RR 0.70 [95% CI 0.42–0.88]).

Conclusions: Our review demonstrates that the combination of ATG with CsA significantly reduces short and long term mortality in patients with severe but not with non severe aplastic anemia. The combination of both drugs should therefore be considered the gold standard only for patients with SAA.

Figure Figure
Evaluation of Pegfilgrastim Efficacy in Peripheral Blood Stem Cell Mobilization of Patients with Lymphoma
Authors: Bouabdallah Krimo , Bijou Fontanet , Dilhuydy Marie-Sarah , Banos Anne , Milpied Noel-Jean
Published: April 2020

Twenty four patients (pts) with planned autologous stem cell transplantation for lymphoma diseases (Hodgkin’s disease=4; non-Hodgkin’s lymphoma=20) received chemotherapy (CT) (Induction CT=3 and salvage regimen= 21) followed by a fixed single dose (6 mg) administration of Pegfilgrastim (PF) after the last day of CT for peripheral blood stem cell collection (PBSC) (target cell dose of 3 2×106 CD34+/kg). Median age was 53 yrs (24–68) and median weight was 72, 5 kg (45–98). Among the 24 pts, 7 received more than 2 lines of CT regimens. The injection of PF was well tolerated. Median time interval between day 1(D1) of the cycle of CT mobilization and first leukapheresis session was 14 days (10–18) while the median time interval between injection of PF and first leukapheresis session was 9 days (6–13). Stem cell collection was started when the absolute number of circulating CD34+ cells was >10×106/L and performed with standard volume leukapheresis. Median CD34+ cells level at D1 of leukapheresis was 35, 5/mm3 (11–320) and interestingly, more than 35 % of pts could reach this median level of CD34+ early after PF injection (around D6). Notably, 22 pts reached the target cell dose in 2 sessions of leukapheresis or less (10 pts after 1 session, 10 other pts after 2 sessions, 2 pts after 3 and 4 sessions respectively). The median number of leukapheresis sessions was 2(1–4) and the median CD34+ cells harvested was 4×106/kg (0,8–26,6). Two pts (DLBCL = 1 and FL = 1) could not reach the level of CD34+ required to start leukapheresis and both became secondary refractory to CT. In univariate analysis, PBSC collection of > 4×106/kg was highly correlated with pts who started their collection at D9 of PF administration (P=0,01) and with those presenting a CD34+ cells level > 35.5/mm3 at D1 of leukapheresis (P=0,033). White blood cells level higher than 9 G/l was also predictive of circulating CD34+ cells >35,5/mm3 (P=0,033). These data suggest that PF may represent an attractive option for PBSC mobilization particularly for pts with lymphoma when optimal compliance of frequent sequential regimens of CT is required. We also emphasize that stem cell mobilization is effective even in pts in second or subsequent salvage CT regimen. Importantly, the circulating CD34+ count should be performed from D6 of PF administration. The presentation will include the updated data.
The Philadelphia Chromosome Is Not a Stable Structure and Is Vulnerable to Further Rearrangements during Disease Progression in CML
Authors: Virgili Anna , Brazma Diana , Chanalaris Anastasios , Grace Colin , Nacheva Elisabeth
Published: April 2020

Chronic myeloid leukaemia (CML) is a pluripotent haematopoietic stem cell disorder characterized by the expression of the BCR/ABL1 fusion gene, which commonly results from formation of the Philadelphia chromosome (Ph) after a t(9;22)(q34;q11) or related variant rearrangement. BCR/ABL1 is a constitutively activated tyrosine kinase and its amplification has been described in association with resistance to imatinib in CML patients. BAC array CGH analysis on CML patients and CML cell lines (Brazma et al., 2007) revealed unexpected genomic imbalances in form of duplications and high copy number gains affecting the region immediately downstream of the ABL1 gene at the Philadelphia (Ph) chromosome in patients at the blast crisis stage. We aimed to confirm and map these amplifications by fluorescence in situ hybridization (FISH) on 19 CML patients in accelerated phase/blast crisis and 10 CML cell lines (KU812, K562, MEG-01, MC3, BV173, EM-2, LAMA-84, KCL-22, JK-1 and CML-T1) with more than 1 copy of the BCR/ABL1 fusion gene. We used a range of BAC probes and 9q and 22q sub-telomeric probes in order to do the FISH mapping. While the majority of the analysed patients and cell lines (12/19 patients and 6/10 CML cell lines) had 2 identical Ph chromosomes, 2 main groups of abnormalities were identified. Firstly, gains of the Ph chromosome taking the form of ider(22)t(9;22) chromosome were detected in 1 or more copies in a subset of bone marrow cells of 5/19 patients and, secondly, high copy number gains were seen in 2/19 patients and 2/10 cell lines (K562 and KU812). The amplified region was variable in size spanning from 400 Kb up to 1.5 Mb downstream of the ABL1 gene. In 1 patient, 7 different cell sub-clones harbouring increasing levels of amplification were found. The gains resulted in formation of different chromosome structures-from an ider(22)t(9;22) to markers with tandem amplifications, which included the BCR/ABL1 fusion with variable in length sequences downstream of the ABL1. Duplication of some 571 Kb downstream of ABL1 was also detected in 1 of the 2 apparently normal Ph chromosomes in the MC3 cell line, while a larger duplication (5.16 Mb) was found in another cell line (MEG-01). These findings confirm the presence of duplications and high level amplifications at the der(22) t(9;22) in CML patients and that the sequences involved are variable in length, indicating that the Ph chromosome is an unstable structure and vulnerable to further rearrangements during disease progression.
Ikaros Is a Frequently Affected Hematopoietic Differentiation Factor in Pediatric Relapse-Prone Precursor B-Cell Acute Lymphoblastic Leukemia
Authors: Kuiper Roland P. , van Leeuwen Frank N. , van der Velden Vincent , van Reijmersdal Simon V. , de Vries Jeltje , Keijzers-Vloet Suzanne T.M. , Hehir-Kwa Jayne , van Wering E. , van Dongen Jacques JM , Hoogerbrugge Peter M. , van Kessel Ad Geurts
Published: April 2020

Relapse is the most common cause of treatment failure in childhood acute lymphoblastic leukemia (ALL), and is difficult to predict in the majority of cases. Here, we performed genome-wide copy number profiling of 34 paired diagnosis-relapse samples from children diagnosed with precursor-B cell ALL. The majority of the copy number abnormalities were preserved between matched diagnosis and relapse samples, but lesions unique in either of the two samples were observed in 82% of the cases. In 68% of the cases lesions present at diagnosis were no longer detected in relapse samples (including recurrent lesions affecting the PAX5, CDKN2A, and EBF genes), indicating that these lesions were secondary events, absent in the original therapy-resistant progenitor clone. Deletions in the IKZF1 gene encoding the hematopoietic differentiation factor Ikaros were observed in 38% of the diagnosis samples, which is >6-fold the amount detected in an unselected group of pediatric ALLs. Tiling-resolution oligo arrays were used to map the breakpoints, which demonstrated that the protein-coding exons 3–6, encoding the DNA-binding Zn-finger domains, were most commonly deleted. Sequence analysis revealed that point mutations in IKZF1 do occur but are less frequent. Furthermore, IKZF1 deletions were always preserved in relapse. Together, we conclude that IKZF1 deletions are frequent events in therapy-resistant clones of relapse-prone pediatric precursor B-ALL.
Circulating Angiogenic Cytokines in Chronic Lymphocytic Leukemia: Relationship to Modern Prognostic Factors and Effect of Fludarabine-Based Treatment
Authors: Smolej Lukas , Andrys Ctirad , Pekova Sona , Kucerova Lenka , Hrudkova Monika , Belada David , Maly Jaroslav
Published: April 2020

Angiogenesis has been studied in chronic lymphocytic leukemia (CLL) since mid 1990s. Given the extraordinary heterogeneity of clinical course of CLL patients, angiogenesis assessment may potentially improve prognostic stratification. There are numerous reports on increased markers of angiogenesis in CLL; however, data on possible association of angiogenic markers with modern prognostic factors are very scarce. Therefore, we evaluated prognostic significance of angiogenic cytokines, namely basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), endostatin, and angiopoietin-2 (Ang-2) in patients with untreated CLL. Plasma levels of angiogenic factors were quantified using enzyme-linked immunosorbent assay (bFGF, VEGF, n=106; endostatin, n=83, Ang-2, n=55). Eighty healthy donors served as control group. Relationship of angiogenic markers to modern prognostic factors (IgVH mutation status, cytogenetic aberrations detected by fluorescent in situ hybridization [FISH]) was also analyzed. Results: bFGF, VEGF, and endostatin were significantly elevated in CLL patients when compared to controls (bFGF, VEGF: p<0.0001, endostatin: p=0.039); Ang-2 levels were not different from the control (p=0.192). There were no differences in angiogenic factors with regard to gender, clinical course or modified Rai stage. Similarly, no association of angiogenic factors with IgVH or FISH was identified. Interestingly, in a pilot subgroup of 16 patients with serial measurements before and after successful fludarabine-based combination treatment, levels of bFGF as well as VEGF decreased significantly (p<0.0001 and p=0.0027, respectively). In conclusion, our study confirms that angiogenic signaling is intensive in CLL. Fludarabine-based treatment may have intrinsic antiangiogenic properties. Further research is needed for better understanding of angiogenic processes, especially with respect to possible refinement of individual patient’s prognosis in this disease.
Prognostic Value of β2-Microglobulin (β2M) Expression on Lymphocyte Surface in Chronic B-Lymphocytic Leukemia (B-CLL)
Authors: Volkova Maya , Molodyk Alvina A. , Bialik Tatiana E. , Frenkel Marina A.
Published: April 2020

Background: Modern therapy diminished prognostic significance of serum β2M level.

Aims: This study was designed to investigate the correlation between the number of lymphocytes expressing β2M, serum β2M level, and the results of treatment.

Methods: 45 patients (pts) were included (Binet stage B, 36 and C, 9). β2M expression on the lymphocyte surface was detected with immunocytochemical method by means of Dako-Cytomation LSAB2 System HRP, β2M serum level by ELISA.

Results: In 29 pts β2M was expressed on 80–100% of lymphocytes (group I), in 16 pts on 35–79% (group II). Number of lymphocytes expressing β2M did not correlate with lumphocyte dimension, form of nucleus, and presence of prolymphocytes in peripheral blood (PB). β2M serum level ranged from 3,0 to 10,5 mg/L. Number of leukocytes and lymphocytes in PB and bone marrow in both groups had no difference. Number of pts with normal β2M serum level (3,5 mg/L or less) was practically the same (group I, 13,8%; group II, 18,8%). Among pts with high β2M serum level (> 6 mg/L) there were 28% in group I and 69,0% in group II. Increased level of LDG (>450 MU/L) was detected in 10% of pts of group I and in 50% in group II. Treatment in both groups was similar: regimens with fludarabine–gr.I, 23 of 29 pts and gr.II, 12 of 16; R-COP–gr.I, 3 pts and gr.II, 2 pts. Two pts in gr.II received leukeran; 3 pts in gr.I didn’t need any treatment. Of 42 pts treated 20 pts achieved complete remission; 13, partial remission, and 9 pts failed. In gr.I complete remission was achieved in 17 of 26 treated pts (65,4%), in gr.II, in 3 of 16 (18,8%), p=0,0038. Partial remission was received in 6 pts of gr.I (23,1%) and in 7 pts (43,8%) of gr.II, p=0,14. Three pts of gr.I (11,5%) and 6 pts of gr.II (37,2%) failed (p=0,05).

Conclusion: Comparison of the therapy results showed that β2M expression on large number (80–100%) of lymphocytes in B-CLL can be considered as the favourable prognostic sign. It was associated with lower level (<6 mg/L) of serum β2M (in 71,2% vs. 31% of pts, p=0,009) and LDG (90% vs. 50% of pts, p=0,0051). Disclosure: No relevant conflicts of interest to declare.
Serum Folate Measurement by Chemiluminescence in Patients with Paraproteinaemia
Authors: Wakeman Lisa J , Lewis John O , Morris Keith , Benton Ann , Munro Roger C , Al-Ismail Saad
Published: April 2020

Introduction: Paraproteins cause interference in many assays systems due to increased viscosity and/or by non-specific binding to either analytes or reagents which may variably affect the results. There is abundant evidence to indicate that performance characteristics of automated, competitive protein-binding assays for folate assays are normally sound, but there is a paucity of data comparing folate assays in patients with paraproteinaemia compared to normals.

Method: Serum samples were prepared from venous blood taken into Greiner Vacuette containers (code: 456018) in fifty patients with paraproteinaemia (IgG Kappa, n = 20: IgG Lambda, n = 9: IgM Kappa, n = 6: IgM Lambda, n = 4: IgA Kappa, n = 5: IgA Lambda, n = 3: multiclonal, n = 3). Serum folate assays were measured according to manufacturer’s instructions on Beckman-Coulter Access and Abbott Architect haematinics analysers on the day of sample collection.

Results: The ratio of means of serum protein concentrations in the paraprotein sub-groups compared to the mean values of age/gender related reference ranges were: IgG = 1.57: IgM = 7.8: IgA = 6.3. Serum albumin concentration was normal in all patients. Mean serum folate results (±1SD) for paraprotein patients and normals controls (laboratory personnel) are shown in the table below:

Serum Folate (μg/L) Access Architect paired t-test Normal controls (n = 50) 5.98 (±3.57) 5.01 (±3.14) p<0.05 Paraprotein patients (n = 50) 5.08 (±2.79) 5.01 (±3.03) p0.700

When analysed using the paired-t-test serum folate was significantly different in normal subjects using the Access compared to the Architect. However, no such significant difference between the two systems was observed in the paraproteinaemia group. Although the mean serum folate in normals was higher compared to the paraprotein group as analysed by the Access, this difference was not significant when analysed using the two-sample t-test. Interestingly the mean serum folate when analysed in the two groups using the Architect gave identical mean values. There were no statistically significant differences between the two methods in any of the paraprotein sub-types (p>0.05 in each case). Conclusions: The complex and variable mixture of proteins present in paraproteinaemias pose potential erroneous measurement in immunoassay systems. This is because they may interfere with the associated antigen-antibody reactions which are dependant on complimentary matching shapes being assumed by the antibody variable regions of the immunoglobulin. Despite this, our data using two distinct chemiluminescence analysers for folate measurement indicate no statistically significant difference for this parameter between patients with paraproteinaemia compared to normal controls. However, it has yet to be determined whether differing results between these two groups are generated using different analytical techniques.
Phase II Study of Dasatinib in Patients with Relapsed CLL
Authors: Garg Ravin Jain , Wierda William , Fayad Luis , Estrov Zeev , Bickel Susan M , O’Brien Susan
Published: April 2020

Preclinical studies have demonstrated that the Src-related tyrosine kinase Lyn is upregulated and constitutively activated in CLL cells. Lyn inhibitors have been shown to reduce survival of CLL cells in vitro and the Src inhibitor PPW results in the death of murine B cell leukemia. This has spurred interest in the role of Src inhibition as a treatment option for patients with CLL. Dasatinib is a dual Src- and Abl-kinase inhibitor that was recently approved by the FDA for the treatment of patients with CML failing imatinib. It has been shown to induce apoptosis in CLL cells in vitro.

The aim of this study was to investigate the activity of dasatinib in patients with relapsed CLL.

Patients with relapsed CLL, SLL, T-PLL and normal organ function were eligible. The dose of dasatinib was 50 mg PO bid continuously with possible escalation up to 70 mg bid. Each cycle was 4 weeks. Thirteen patients have been treated. The median age was 67 years (range, 42 to 83). The median number of prior treatments was 4 (range, 1 to 8). Five patients (38%) had Rai stage 0–1 disease, the other 8 (62%) had stage II–IV disease. Two patients were inevaluable for response; one changed his mind after only 2 days of treatment and the other patient was hospitalized with Aspergillus 2 weeks into treatment. Of the remaining 11 patients, 6 were treated for 3 cycles, 1 for 6 cycles, 1 is still on treatment, and 3 had therapy stopped after 2, 15, and 34 days for lip numbness, persistent headache, and thrombocytopenia/fatigue, respectively. Five patients had dose escalation after cycle 1, and 1 patient had escalation after cycle 3. The Grade 1 and 2 toxicities included 38% headache, 38% flushing, 46% fatigue, 46% anorexia/nausea, and 23% diarrhea. One patient (8%) had both Grade 3–4 headache and fatigue. One patient (8%) had Grade 3–4 diarrhea and another (8%) had a Grade 3 pleural effusion. Hematologic toxicities included neutropenia in 76%, (grade 3-4 63%), thrombocytopenia in 44% (grade 3–4 22%), and 80% with Grade 1–2 anemia. Eleven patients were evaluable for response. No patient achieved CR or PR based on NCI-WG Criteria. Several patients showed evidence of biologic activity of dasatinib including 1 PR in nodes but worsening anemia, 1 stable disease for 3 cycles, and 2 patients with significant reductions in ALC; one patient had a drop from 76,000/ml to 32,000/ml after only 2 days of dasatinib but stopped therapy because of headache, one had a reduction in WBC from 40,000 to 6,000. One patient continues on therapy. In this trial dasatinib produced no responses by NGI-WG criteria but showed evidence of biologic activity in a very heavily pretreated group of pts with CLL. Accrual continues.
Changes in Immunogenicity of Chronic Lymphocytic Leukemia Cells Mediated by Epigenetic Modifiers
Authors: Dubovsky Jason A , Powers John J. , Sotomayor Eduardo M. , Pinilla Javier
Published: April 2020

The success of immunotherapeutic treatments for B-cell chronic lymphocytic leukemia (B-CLL) hinges upon the circumvention of tolerogenic mechanisms which inhibit the formation of a robust anti-cancer immune response. Recent evidence suggests that DNA methylation inhibitors and histone deacetylase inhibitors can have lasting cancer-specific effects on the expression of highly immunogenic cancer-testis antigens (CTAs) as well as modulating costimulatory and major histocompatability molecule (MHC) surface expression. To investigate the potential efficacy of a combined therapy of methylation (5-aza-2′ytidine) and histone deacetylase (LAQ) inhibitors in human B-CLL we evaluated the expression of CTAs, costimulatory molecules (CD40, CD80, CD86), and MHC molecules (HLA-A, B, C, and HLA-DR) in both cell lines and primary cells from patients with CLL. In addition, we investigated the overall functional immunogenicity of treated B-CLL cells using mixed leukocyte reactions. Results showed that many highly immunogenic CLL-specific CTAs (SSX family, MAGE family, NY-ESO-1, GAGE-2, and NXF2) are significantly upregulated after treatment with both inhibitors. Our data also indicates that the combined effect of both inhibitors was capable of inducing a more immunogenic phenotype (increased CD86, CD80, CD40, HLA-A,B,C, and HLA-DR) in CLL cells consistently, while either drug alone was incapable of producing this effect. In addition treatment with both inhibitors was capable of increasing IFNγ release mediated by allogenic T-cells. Taken together these data indicate that a combined therapy with histone deacetylase and methylation inhibitors, possibly in combination with antigen-specific vaccine immunotherapy, may effectively induce cancer-specific antigen expression and increase costimulatory and antigen presenting capacity of the B-CLL cell.
The Stem Cell Factor-cKIT Pathway Must Be Inhibited to Enable Apoptosis Induced by Bcr-Abl Inhibitors in Chronic Myelogenous Leukaemia Cells
Authors: Belloc Francis , Airiau Kelly , Augis Vanessa , Jeanneteau Marie , Garcia Melissa , Guerin Estelle , Lippert Eric , Moreau-Gaudry François , Mahon Francois-Xavier
Published: April 2020

Imatinib is an effective first line therapy for early chronic phase chronic myelogenous leukaemia (CML) that acts by targeting the tyrosine kinase activity of BCR-ABL. However, mutations occurred that procure imatinib resistance and, to overcome this, second generation inhibitors of BCR-ABL have been developed. Among these, nilotinib is significantly more potent against BCR-ABL than imatinib but is used in the same order of concentration as imatinib in vivo. Moreover, nilotinib is more specific towards BCR-ABL than imatinib which, at efficient concentration, also inhibits c-KIT. In the current study an in vitro flow cytometry assay to analyze imatinib- and nilotinib-induced apoptosis in CML cells has been developed. Both drugs induced significant apoptosis in CD34 positive cells from 36 CML bone marrow samples collected at diagnosis while CD34 positive cells from 12 other myeloproliferative disorders samples were unaffected, confirming the high specificity of both inhibitors on BCR-ABL positive cells (p<10−9). Nilotinib was confirmed to be 100 times more potent than imatinib to induce apoptosis of CD34 positive cells. When the experiments were performed in the presence of a cocktail of cytokines, inhibitions of 26% for spontaneous apoptosis, 22% for imatinib-induced apoptosis (n=20, p<0.005) and 71% for nilotinib-induced apoptosis (p<10−6) were observed. The inhibition essentially occurred in CD117+/CD34+ cells. This differential inhibition by cytokines was confirmed on K562 cells (34% for imatinib and 71% for nilotinib). A blocking anti-CD117 antibody alleviated the anti-apoptotic effect of cytokines against nilotinib. Moreover, using shRNA against BCR-ABL, we demonstrated that K562 cells were not dependant of BCR-ABL expression as long as the SCF receptor pathway was activated. We conclude that the c-KIT pathway may substitute for BCR-ABL tyrosine kinase to activate survival signals and that c-KIT must be inhibited beside BCR-ABL to allow apoptosis of CML cells.
Imatinib Mesylate Induces BCR-ABL Independent Inhibition of Proteasomal Activity in CML Cells
Authors: Held Stefanie A.E. , Brossart Peter
Published: April 2020

Imatinib mesylate (IM; Gleevec) is currently used in the treatment of CML and GIST. IM has been described to influence the function and differentiation of antigen presenting cells, to inhibit the effector function of T lymphocytes and to decrease the immunogenicity of CML cells by downregulation of tumor associated antigens. In the present study, we analyzed the possible effects of IM on antigen processing and presentation. To accomplish this we employed active site labelling of proteasomal subunits to measure proteasomal activities in cells treated with IM and other tyrosine kinase inhibitors (TKIs). Proteasomal activity was determined in cell lysates from established CML cell lines and primary cells from CML patients using a biotinylated active site-directed probe, which, covalently binds and labels proteasomal subunits beta-1, beta-2 and beta-5 depending on their activity. In addition, in order to analyze a possible direct effect of IM, isolated 20S and 26S proteasomes were examined. We found that treatment with IM led to a concentration-dependent decrease of proteasomal activity in BCR-ABL positive cells. In line with these results IM reduced the expression of HLA-class I molecules in HLA-A2 transfected K-562 cells. This inhibitory effect was independent of protein expression of proteasome subunits as analyzed by western blotting and it was not due to the induction of apoptosis as only samples with nuclei fragmentation below 40% were utilized. Furthermore, these effects were not inhibited by addition of zVAD, a pan-caspase inhibitor of apoptosis. The inhibition of proteasomal activity was independent of BCR-ABL as analyzed using IM resistant CML cell lines including BA/F-T315I cell line which is transfected with the multiresistent T315I mutation. Application of BCR-ABL specific siRNA to knock down its expression had no significant influence on proteasomal activity. Accordingly, incubation of isolated 20S and 26S proteasomes with IM resulted in a profound inhibition of their activity as determined by reduced proteolytic cleavage of fluorogenic substrates. Interestingly, this inhibition of the proteasomal activity was not IM specific as nilotinib, a TKI with a higher BCR-ABL affinity had similar effects. Furthermore, the multikinase inhibitor sorafenib but not sunitinib reduced the proteasomal activity in cell lysates and isolated proteasomes. Incubation of cells with the mTOR inhibitor rapamycin inhibited the activity of proteasomal subunits while the PI3 kinase inhibitor LY-294002 that acts upstream of rapamycin had no effect. Our results demonstrate that IM and several other TKIs currently applied in the treatment of patients can affect the immunogenicity of malignant cells by a direct inhibition of the proteasomal activity.
CCN3, a Novel Growth Inhibitory Factor for Chronic Myeloid Leukemia
Authors: Lu Wanhua , McCallum Lynn , Perbal Bernard , Lazar Nourreddine , Irvine Alexandra
Published: April 2020

Chronic Myeloid Leukaemia (CML) is characterized by expression of the constitutively active BCR-ABL tyrosine kinase. Previously, we identified down-regulation of the negative growth regulator, CCN3, as a result of BCR-ABL kinase activity. Reduced CCN3 expression is a prominent feature in both primary human CML cells and cell lines. We now show that CCN3 is growth inhibitory and enhances imatinib induced growth inhibition.

To evaluate the biological consequence of CCN3 expression in CML, K562 cells were stably transfected with a construct containing CCN3 (pCMV82-23) and growth characteristics and activation of signaling pathways were compared to cells transfected with empty vector (control). CCN3 expression was undetected by Real-time PCR in control cells whilst pCMV82-23 cells expressed 2.25 × 106 copies per 50ng of cDNA. pCMV82-23 cells showed reduced colony formation capacity (p=0.003) and reduced cell growth over a period of five days (p=0.005). Investigation of cellular signaling showed CCN3 expression resulted in significant down-regulation of three major signaling pathways and demonstrated reduced phosphorylation of ERK (p=0.002), pAKT (p=0.017) and pSTAT5 (p=0.005) compared to control cells. Protein levels for total ERK, AKT and STAT5 were unaffected by CCN3 expression. Flow cytometry showed that sustained CCN3 expression resulted in an accumulation of cells within the subG0 stage of the cell cycle (11.4% ± 3 (p=0.040)).

To determine if CCN3 expression could influence sensitivity to the BCR-ABL kinase inhibitor, imatinib, pCMV82-23 cells and control cells were treated with imatinib (5uM) for 48h. Control cells treated with imatinib showed moderate growth inihibition (19.6% ± 2.5) compared to untreated control. pCMV82-23 cells showed a significant increase in the magnitude of imatinib induced growth inhibition (63.3% ± 10.5 (p=0.043)). This was associated with an increased accumulation of cells in the subG0 area of the cell cycle, 34.6% ± 5 for pCMV82-23 cells compared to control cells (21.7% ± 8) in response to imatinib treatment (p=0.006). To then determine if these effects could be reproduced using recombinant CCN3 (rCCN3), K562 cells were treated with imatinib (5uM) alone or in combination with rCCN3 (10nM) for 48h. K562 cells treated with the combination of rCCN3 and imatinib showed enhanced growth inhibition (71.8% ± 7.9) compared to cells treated with imatinib alone (81.1% ± 9.2 (p=0.008)).

Loss of CCN3 is consistent with properties associated with the CML phenotype. Sustained expression of CCN3 in K562 cells restores growth control and re-establishes induction of apoptosis. Both increased expression of CCN3 or addition of the recombinant protein provide additional benefit for imatinib induced growth inhibition thus providing a novel avenue for therapeutic intervention.
Synergistically Cooperation of Bortezomib and Arsenic Trioxide on Chronic Myelogenous Leukemia
Authors: Yan Hua , Li Dao , Chen Li , Wu Ying-Li , Li Jun-Ming , Chen Guo-Qiang
Published: April 2020

Chronic Myelogenous Leukemia (CML) is one of myeloproliferative disorders characterized by the chromosome translocation (9; 22), which causes the generation of the Bcr-Abl fusion protein. Latter activates a number of signal transducers and transcription factors, thus leading to multi-resistance to chemotherapeutic agents. Introduction of imatinib mesylate (Gleevec) significantly increased CML patients’ response but still some of patients got disease progression. Therefore, it still remains a high priority to develop new approaches to treat Bcr-Abl+ leukemia. Aim: We aim to investigate the synergy of bortezomib and arsenic trioxide on inducing the apoptosis on Bcr-Abl+ K562-s as well as K562-r cells, which was sensitive or resistant to imatinib treatment respectively. Material and methods: Bcr-Abl+ K562-s and K562-r cells were chosen to evaluate the drugs’ effect. We observed the inhibition of cell growth and cell viability after bortezomib and/or arsenic treatment. Flow cytometric evaluation and western blot analysis were performed to detect the development of apoptosis, the changeable expression of Bcr-Abl protein and the apoptosis-related proteins. Results: K562-s and K562-r cells had different response to imatinib treatment. As expected, 0.25μM of imatinib can inhibit half of K562-s cell growth whereas 10–15 more times of concentration of imatinib were necessary to reach the same inhibitory effect in K562-r cells. Combined bortezomib at 12nM with 1μM of ATO, the concentration inducing 50% of K562-s growth arrest, synergistically induced apoptosis in K562-s cells after 48hrs of cotreatment, confirmed by notable elevation of Annexin V+ cells through flow cytometric analysis, when compared to those in the control or single treatment (p<0.01), and immunoblotting detection of caspase-3 cleavage, degradation of poly-adenosine diphosphate-ribose polymerase (PARP), and decreased expression of Bcr-Abl protein. More surprisingly, 24nM concentration of bortezomib and 2μM of ATO, 2 times of concentration needed for 50% of K562-s cell growth inhibition respectively, inhibited 50% of K562-r cells growth, far less than the increased proportion needed during imatinib treatment. And more of important, although single treatment of bortezomib or ATO had no or little effect on inducing apoptosis, combined treatment significantly induced the apoptosis in K562-r cells (p<0.05), associated with activation of apoptosis-relevant proteins and obvious degradation of Bcr-Abl protein.

Conclusion: Taken together, these findings indicate that bortezomib combined with ATO could be developed into a novel therapeutic strategy for CML. More study will be needed to further explore its potent mechanism involved during cotreatment.
Oligoclonal Vβ21 with Different Vα Partner in T Cells Associated with CML Cell Antigens
Authors: Li Yangqiu , Zha Xianfeng , Chen Shaohua , Yang Lijian , Lu Yuhong , Yu Zhi
Published: April 2020

Chronic myeloid leukemia (CML) is one of most common leukemia, although target therapy with imatinib is significant improved the survival rate, disease relapse and drug resistant with abl gene mutant are still the major clinical problem, improvement strategy to reverse the outcome of CML is the key point for the researchers. T-cell immunodeficiency was suggested in tumor patients for many years, understanding T cell immune status in leukemia patients might suggest treatment strategies to enhance immune competence in all. In order to investigate the clonal expansion and the feature of CDR3 sequence of T cells in patients with CML, the CDR3 of TCR Vα 29 and TCR Vβ 24 subfamily genes were amplified in peripheral blood mononuclear cells (PBMCs) from two case with CML using RT-PCR, the PCR products were further analyzed by genescan technique to evaluate clonality of the detectable TCR Vα and Vβ subfamilies. The oligoclonal PCR products were analyzed by sequencing to define the sequence of CDR3. Oligoclonal Vβ21 T cells could be identified in both CML cases, which might partner with different oligoclonal Vα subfamilies, including Vα7, Vα13 or Vα18. The CDR3 sequences from two Vβ21 and three Vα oligoclonal TCR genes were identified in Vβ21NDβNJβ1.1, Vβ21NDβNJβ2.7, Vα7NJα23, Vα13NJα49, Vα18NJα5 respectively. The length and motifs of CDR3 seem different in all five TCR genes, however, the motifs in both Vβ21 genes (GAV or LRV) share the valine as last residue of CDR3 motif, and all of three Vα gene (Vα7:G; Vα13:GDEAD or Vα18:GG ) share the Glycine as first residue in the CDR3 motif. All of five TCR sequences were already submitted to the Genbank (Accession No: EU395806, EU379940, FJ009444, EU589347 or EU770971 respectively). The results indicated that oligoclonal Vβ21 genes with different Vα partners which share part of CDR3 motifs might recognize the similar CML associated antigen epitope in CML patients. Farther investigation will be constructed the recombination vector containing the antigen specific TCRs for gene transfer to establish TCR modified-T cells, which was expected to employ for specific immunotherapy in CML.
Outcome of Patients with Chronic Myeloid Leukemia with T315I BCR-ABL Mutation
Authors: Pagnano Kati a BB , Silveira Rosana A , Nardinelli Luciana , Conchon Monika , De Souza Carmino , Llacer Pedro ED , Lorand-Metze Irene , Funke Vaneuza , Pasquini Ricardo , Mascarenhas Cintia C , Zanichelli Maria Aparecida , Clementino Nelma CD , Bendit Israel
Published: April 2020

The most frequent causes of imatinib resistance are BCR-ABL mutations, usually selected during treatment. Most of them destabilize the inactive conformation necessary for imatinib binding. The T315I mutation, located at the ATP binding pocket is resistant to imatinib and other available kinase inhibitors and is associated with a poor outcome. The aim of this study was to evaluate the frequency of T315I mutation in patients with CML resistant to imatinib and to describe their outcome. Mutation analysis was performed in two institutions, through direct automated sequencing. Total RNA was extracted from peripheral blood or bone marrow and amplification of the kinase domain of ABL from BCR/ABL was performed, using a semi-nested RT-PCR, to cover amino acids 244–486. PCR product was submitted to direct automated sequencing and compared with normal sequences of BCR-ABL gene (M14752, GenBank). A total of 173 resistant imatinib resistant patients were analyzed. BCR-ABL mutations were detected in 47 out of 173 (27%) patients. Among these patients, 14 (30%) presented T315I mutation. The mutation was found in ten patients during imatinib treatment, in one before start of imatinib, in three after dasatinib (2) and nilotinib (1) failure. Three patients have more than one mutation analysis, performed during different time-points of treatment: imatinib resistance, dasatinib resistance (3) and bone marrow transplantation relapse (1). Overall survival from diagnosis was 39,5% and 17,5% from mutation detection, in a median time of 69 and 13 months respectively. After imatinib resistance, eight patients were treated with dasatinib, one with nilotinib and had no hematological response; tree were treated with Hydrea and two were submitted to allogeneic bone marrow transplantation. Five patients died due to disease progression in median time of 19 months after mutation detection. One patient died after bone marrow transplantation, due to GVHD and infection. Eight patients are alive, three in CP, two in BC, two in hematological response and one in complete cytogenetic response after bone marrow transplantation. In conclusion, in this study we confirmed the high frequency of T315I mutation in imatinib resistant patients and their poor prognosis. Most of them started imatinib in a late chronic or advanced phase, with exception of two patients which started imatinib 800mg in early chronic phase, suggesting that there was inhibition of Ph positive sensitive cells and selection of resistant clones.
Myelodysplasia and Acute Myeloid Leukemia during Treatment of Chronic Myeloid Leukemia with Dasatinib
Authors: Conchon Monika , Bendit Israel , Ferreira Patricia , Dias Lucia , Kumeda Cristina , Chamone Dalton , Dorlhiac-Llacer Pedro
Published: April 2020

The incidence and significance of Philadelphia negative aberrant clones in patients treated with the second generation of tyrosine kinase inhibitors, including dasatinib, are infrequent. Here we report 2 patients with myelodysplastic syndrome associated with monosomy 7 in Philadelphia negative cells during consecutive imatininb and dasatinb therapy. No previous exposure to chemotherapeutic agents was reported in both patients. In case 1, retrospective assessment of the bone marrow sample collected prior to treatment with dasatinib did not show any abnormal clone, which became evident only after dasatinib was commenced. In case 2, monsomy 7 were found already in 15% Philadelphia negative cells during imatinib treatment and persisted during dasatinib therapy. In the 2 cases described, we assumed that the consecutive imatinib/dasatinib therapy, could abrogate the proliferation of the Philadelphia positive clone and allow pre-existing monosomy 7 clone to emerge.
Frequency and prognosis of Clonal Chromosomal Abnormalities Following Stem Cell Transplantation (SCT) in Acute Myeloid Leukemia (AML)
Authors: Grau Javi er , Cisneros Adela , Zamora Lurdes , Sancho Juan Manuel , Granada Isabel , Ruiz-Xivillé Neus , Xandri Marisol , Guardia Ramon , Fernandez Cristalina , Santafé Encarnación , Villena Carmen , Cabezón Marta , Sancho Esther , Xicoy Blanca , López Laia , Rodriguez Inés , Moreno Miriam , Orna Elisa , Ferra Christelle , Batlle Montserrat , Roncalés Francisco Javier , Flores Alonso , Juncà Jordi , Navarro José Tomás , Ribera Josep Maria , Feliu Evarist , Millá Fuensanta
Published: April 2020

Objective. To study the incidence, type and prognostic significance of clonal chromosomal abnormalities following SCT in patients with AML.

Patients and methods. From February 2000 to March 2008, 74 patients were studied. Karyotypes were analysed by G-banded chromosomes obtained from 24 hours bone marrow cultures, and were described according to ISCN 2005.

Results. Clonal abnormalities were observed in 17/74 patients (23%). Median follow-up was 11 months (range 5–47). Initial diagnosis: AML2 (6 cases), AML5a (3), AML6 (3), AML with inv(16)(p13;q22) (1), AML with multilineage dysplasia (1), AML5b (1), AML1 (1) and AML0 (1). Treatment before SCT: idarubicin, cytarabine and etoposide (16), FLAG-ida (1). All patients presented a normal karyotype at the time of SCT. SCT type: autologous (12 patients), allogeneic (3), allogeneic of reduced intensity (2). Conditioning regimen: TBI and cyclophosphamide (14 cases), busulfan and cyclophosphamide (1), fludarabine and busulfan (2). The median time between SCT and the appearance of clonal abnormalities was 8 months (range 3–36). At the time of clonal abnormality detection, 16 patients were in cytological and/or clinical relapse and 1 in complete remission. In 4 cases the initial clone reappeared, 1 showed the initial abnormalities with an acquired abnormality and 12 presented de novo clonal abnormalities. The median survival from the appearance of clonal abnormalities was 2 months (range 1–40). At the time of the analysis all patients had died.

Conclusions.

The appearance of clonal abnormalities following SCT in patients with AML is frequent. Time between SCT and the appearance of clonal abnormalities is short. The majority of patients presented de novo clonal abnormalities with cytological and/or clinical relapse and poor prognosis.
Haematopoietic Stem Cell Transplantation in Thalassaemic Patient with Renal Failure on Hemodialysis
Authors: Argiolu Francesca , Sanna Maria Adele , Bartoli Antonella , Ferrara Rocco , Piroddi Antonio , Addari Carmen , Orofino Maria Grazia , Rizzo Fabiana , Cossu Fausto , Scalas Maria Rosa , Broglia Monica , Giagu Nicolina , Galanello Renzo
Published: April 2020

Until now few patients with renal failure on hemodialysis have undergone haematopoietic stem-cell transplantation (HSCT), and none with thalassaemia. Case report. Patient of 45 years, UPN 168, with β Thalassaemia major, genetic compound β0 39 C-->T/β0 6-A, non transfusible due to red blood cell immunization (positive DAT, Hb 5–7 g/dl), under hemodialytic treatment for three years for bilateral focal nephrosclerosis. He suffered from cardiopathy (atrial thrombosis, pulmonary hypertension, atrial and ventricular dilatation), hypothyroidism, hepatopathy (HCV positive with grade II haemosiderosis). Quality of life was much harmed by the severe chronic anaemia and nephropathy. In December 2007 we performed HSCT from his HLA identical brother. Conditioning regimen included: Busulfan (Bu) IV (Busilvex Pierre Fabre Médicament) in single daily dose (3.5 mg/kg/day) for 4 days, Cyclophosphamide 40 mg/kg/day for 3 days. GvHD prophylaxis consisted of Cyclosporin 3 mg/kg/day IV (days -2 to +1), 2 mg/kg/day IV (days +2 to +42), changed to oral administration of 4 mg/kg/day from day +43. Hemodialysis was performed every other day in sterile room. Nucleated marrow cells infused: 4.4 × 108/kg. A mixed chimerism (VNTR more than 90% donor) was documented at day +19. ANC >0.5 × 109/L at day +21. PLT >20 × 109/L at day +15. Eight months after transplantation, patient is in good general condition, with stable mixed chimerism (more than 95% donor), haemoglobin above 11 g/dl, leucocytes and platelets in normal range. Substantial improvement of chronic anaemia ameliorated patient’s quality of life and now patient can be considered a kidney transplant candidate. This single case report does not allow definitive conclusions to be drawn regarding HSCT suitability of patients with chronic renal failure on hemodialysis. We can observe that once-daily intravenous busulfan administration (after the hemodialysis session) has made it possible to keep therapeutic range, as has been ascertained by pharmacokinetic study of busulfan.