Blast Cells In Peripheral Blood Research Articles

Establishment of human acute monocytic leukemia model with systemic infiltration in NPG mice.

A model construction of systemic acute leukemia is challenging. Herein, we established a systemic leukemia mouse model using highly immunodeficient NPG mice without any immunosuppressive treatments. NPG mice received tail intravenous injection of SHI-1 cells at the concentration of 1×107 cells (group A) or 5×107 cells (group B) and randomly sacrificed each seven days post-inoculation. Tumor development was monitored using nested-PCR, peripheral blood-smear analysis, flow cytometry, pathological examinations, and immunohistochemistry. The median survival of mice in groups A and B were 33.0 and 30.0 days, respectively. Blast cells in peripheral blood appeared on day 14 in group B, and on day 21 in group A. In addition, SHI-1 cell specific MLL-AF6 mRNA was detected in both spleen and bone marrow on day 14 post-inoculation. 21 days after inoculation, we observed human CD45+CD33+ cells with an SH-1-immunophenotype in the peripheral blood, spleen, and bone marrow, as well as solid neoplasms in multiple organs. Moreover, the histologically infiltrated leukemic cells expressed CD45. In conclusion, the current study demonstrated the normal growth of SHI-1 cells in the NPG mice without immunosuppression, which caused systemic leukemia similar to that observed in acute leukemia patients. We developed an efficient and reproducible model to study leukemia pathogenesis and progression.

Analysis of Clinical Features, Prognostic Characteristics and Gene Mutations of the EB Subtype of Primary Myelodysplastic Syndrome Patients with Myelofibrosis

Objective ：This study aims to analyze the clinical features, prognostic characteristics and gene mutations of the primary myelodysplastic syndrome with myelofibrosis (MDS-MF) patients, to compare the differences and impact of MF on MDS-EB group and MDS-Non-EB group, ultimately to improve the cognition of MF on MDS-EB group. Methods ：From January 1, 2013 to February 1, 2020, 417 newly diagnosed primary MDS patients in the first affiliated hospital of Zhejiang University with bone marrow (BM) biopsy examination were included. They were divided into two groups (MDS-EB group and MDS-Non-EB group) according to 2016 WHO MDS classification.BM was reassessed on whether associated with fibrosis in grade MF0,1,2,3. (2005 European Myelofibrosis Network criteria). The clinical features, prognosis characteristics and gene alterations were analyzed between the MDS-EB group and MDS-Non-EB group retrospectively. Results ： ① MF was confirmed in 46.3％（193/417）cases of all the MDS patients, of which 66.3％（128/193）were MF-1 , 25.9％（50/193）were MF-2 and 7.8%(15/193) were MF-3 respectively. Compared with MDS-MF0 group, MDS-MF were significantly associated with worse OS(P=0.035) and worse PFS(p<0.001). ② There was 174 cases of MDS-EB and 243 cases of MDS-Non-EB. The proportion of MF in MDS-EB group was 54.0%(94/174) and significantly higher than in MDS-Non-EB group 40.7%（99/243）(p=0.010).The distribution of MF grade was also differently significant between the two groups(p=0.048). The clinical features analysis showed that there is higher level of C-reactive Protein (CRP), higher level of serum lactate dehydrogenase (LDH) and higher level of β2-microgram in the peripheral blood in the MF(MF1,2,3) group than in the MF0 group, regardless of whether it was in the EB group or the non-EB group. The difference was statistically significant (ALL p value<0.05). ③The impact of MF on MDS-EB patients and MDS-Non-EB patients has different significances concerning OS, PFS and t-AML. As far as the OS prognostic factor, no matter the comparison of MF0, 1,2,3 four groups, the comparison MF1,2,3 three groups, the comparison of MF0-1 with MF2-3, all showed that MF was an adverse prognostic factor in the EB-MF group (All p value <0.05), and it also showed the adverse impact related with the grade of MF, but none of any in the MDS-Non-EB group (All p value>0.05)(Figure1). Comes to the PFS, it showed the MF on MDS-Non-EB was an inferior factor when compared MF+(MF1,2,3) to MF-(MF0)(p=0.0097), but not between the comparison MF1,2,3 groups(p=0.96) and none of the MDS-EB groups（p=0.31 and 0.092 respectively）. The incidence of MF was related with a higher rate of t-AML not only in MDS-EB group but also in MDS-Non-EB group compared with MF0, no matter compared the MF+(MF1,2,3) with MF-(MF0) but also the comparison of MF0,1,2,3) (All P value < 0.001). ④ In the MDS-EB cohort, 66 patients in the MF-(MF-0) group completed Next-generation-sequencing (NGS) test, and 61 patients in the MF+ (MF1,2,3) group completed NGS testing. It seems that the incidence of gene mutation has no difference between the two groups. We conducted univariate COX regression analysis about the 30 genes (which were suggested in the NCCN guideline 2020) with OS, PFS and t-AML in the MDS-EB-MF cohort. The results showed that TP53, Nras, WT1 was related to the worse OS; U2AF1, Niras and TP53 was related to worse PFS and GATA2, IDH2, TP53, STAG2 was related to t-AML. Conclusion: MDS-MF has unique laboratory and clinical characteristics, act as an independent risk factor for shorter OS and worse PFS in the MDS-MF patients in our large cohort. In this study, we retrospectively analyzed 417 primary MDS cases in our hospital and analyzed the impact of MF on MDS-EB group and MDS-Non-EB group respectively. Our results showed the MF has a worse OS on the MDS-EB-MF group than the MDS-EB-MF0 group but no impact on MDS-Non-EB cohort. It was also consistent with the 5th edition of the WHO Classification of Haemato-lymphoid Tumors guidelines in which MDS-f was listed as a subtype concerning MDS with fibrosis concomitant with 5-19% blast cells in bone marrow or 2-19% blast cells in peripheral blood. The NGS sequencing showed TP53, Nras, WT1 was related to the worse OS; U2AF1, Niras and TP53 was related to worse PFS and GATA2, IDH2, TP53, STAG2 was related to t-AML.As far as we know, this is the first analysis of bone marrow fibrosis in MDS-EB patients. We need more research to help us understand the MDS-f subtype.

Comparison of Characteristics of Chronic Myeloid Leukemia Combined with Different Degrees of Myelofibrosis : A Real-World Multicenter Retrospective Study from China

Chronic myeloid leukemia (CML) is a hematological malignancy characterized by excessive proliferation of myeloid cells, which belongs to the group of myeloproliferative neoplasms (MPNs). Myelofibrosis (MF) is a significant prognostic indicator of CML, and its discovery at the time of diagnosis aids in decisions regarding the patient's course of treatment, according to a descriptive cross-sectional study. Nevertheless, the influence of MF is not clear. Our study is to investigate clinical characteristics and prognosis of CML pantients with different degrees of myelofibrosis, exploring the influence of MF. We retrospectively analyzed 1757 CML patients with bone marrow biopsy results and compared the characteristics of CML combined with different degrees of MF in 44 Chinese institutions from January 2010 to October 2022(ChiCTR2200061208). A total of 1757 CML patients with MF, including 1090 males (62.0%) and 667 females (38.0%) with a median age of 49 (6-92) years. 1757 CML patients were treated with TKIs, as shown in the picture ( Figure A).The median WBC count was 119.8×10 9/L (0.1-910.8×10 9/L) in peripheral blood at diagnosis, the median HB level was 111.5 g/L (12.6-181 g/L), the median PLT count was 512.1×10 9/L (5-4384×10 9/L), the median EOS% was 3.3% (0.0-31.1%), the median BAS% was 4.3% (0.0-19.0%), the median spleen size was 4.10 cm (0.0-26.0 cm). Patients with MF were older (p<0.001); had higher leukocyte counts (p<0.001) and platelet counts (p<0.001); were more prone to anemia (p<0.001), acidophilia (p<0.001), alkalophilia (p<0.001), and splenomegaly (p<0.001); and a higher proportion of peripheral blood blast cells (p<0.001). There was no significant difference between the two groups in the male to female ratio. Patients without MF were more likely to reach 3-month EMR (p<0.001), 6-month BCR::ABL IS ≤ 1% (p<0.001), and 12-month MMR (p<0.001). The proportion of medium / high-risk patients with MF according to the Sokal score (p<0.001), Hasford score (p<0.001), ELTS score (p<0.001) and EUTOS score (p=0.002) was significantly increased ( Figure B). CML patients with MF had lower OS and PFS than patients without MF (p=0.007, p=0.006, Figure D, E). CML patients with MF were more prone to treatment failure with imatinib as the first-line treatment (p<0.001, Figure F). There was no statistically significant difference in treatment failure between first-generation TKI (1G-TKI) and second-generation TKI (2G-TKI) (p=0.222, Figure G).With increasing degree of MF, CML patients had higher leukocyte counts were more susceptible to anemia and splenomegaly, and had more peripheral blood blast cells. However, there was no significant difference in sex ratio, age, platelets, eosinophils, and basophils. CML patients without MF were more likely to achieve 3-month EMR, 6-month BCR::ABL IS ≤ 1%, and 12-month MMR. As the degree of MF increased, the proportion of intermediate / high risk patients according to Sokal score, Hasford score, and ELTS score increased significantly ( Figure C), OS and PFS of CML patients decreased (p<0.001, p<0.001, Figure H, I) and the failure rate of first-line treatment increased (p<0.001, p<0.001, Figure J, K). In conclusion, we performed 1757 CML patients with MF and compared the characteristics of CML combined with different degrees of MF. As the degree of MF increased, the proportion of intermediate / high risk patients according to Sokal score, Hasford score, and ELTS score increased, the efficacy of TKIs was lower, the prognosis of patients was worse. So MF is an independent risk factor affecting the prognosis of patients with CML. Acknowledgement: This research was funded by the Key R&D Program of Zhejiang, No. 2022C03137; Public Technology Application Research Program of Zhejiang, China, No. LGF21H080003; Zhejiang Medical Association Clinical Medical Research special fund project, No. 2022ZYC-D09. *Correspondence to: Dr Jian Huang, Department of Hematology, The First Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310003,Zhejiang, Peoples R China.E-mail: househuang@zju.edu.cn

Clinical features, laboratory characteristics, and outcome of ETP and TCRA/D aberrations in pediatric patients with T-acute lymphoblastic leukemia

BackgroundT-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy with few accepted prognostic factors that limit the efficiency of therapy. The aim of the current study was to assess the clinical and laboratory features of T-cell receptor (TCR) aberrations and early T-cell precursor (ETP) subtype as well as their outcome to therapy.MethodsSixty-three newly diagnosed pediatric T-ALL patients were assessed for the ETP status using immunophenotyping. Screening of TCRA/D aberrations was done by fluorescent in situ hybridization (FISH). The data were correlated to the patients’ clinical features, response to treatment, and survival rates.ResultsSeven patients (11%) had ETP-ALL. The ETP-ALL patients were older (P = 0.013), presented with lower white blood cell (WBC) count (P = 0.001) and lower percentage of peripheral blood (PB) blast cells (P = 0.037), more likely to have hyperdiploid karyotype (P = 0.009), and had been associated with TCRA/D gene amplification (P = 0.014) compared to other T-ALL patients. Of note, the same associations had been significantly observed in patients with TCRA/D gene amplification. Patients with TCRA/D amplification frequently coincided with TCRβ aberrations (P = 0.025). TCR-β aberrations were significantly associated with negative MRD at the end of induction compared to TCR-β-negative patients. There was a nonsignificant trend of ETP-positive cases to have lower overall survival (OS) (P = 0.06). Patients with TCR aberrations had no significant differences regarding disease-free survival (DFS) or OS rates compared to those with normal TCR.ConclusionETP-ALL patients tend to have increased mortalities. There was no significant impact of TCR aberrations on the survival rates of the patients.

A short review about chronic myeloid leukemia

Chronic myeloid leukemia (CML) develops as a result of a clonal process in a pluripotent stem cell. Anemia, granulocytosis, basophilia, thrombocytosis and splenomegaly are some of the symptoms of the condition. According to clinical findings, the illness progresses through three stages, each of which is identified by a rise in number of the blast cells in peripheral blood or bone marrow: chronic (10%), accelerated (10-19%) and acute leukemia-like blast crisis (20%). Most CML cases could be preliminary diagnosed by the presence of splenomegaly in addition to mutation in the BCR-ABL gene as well as complete blood count (CBC) test. Moreover, a bone marrow biopsy can provide a major confirmation of the disease process and staging. Definitive diagnosis of the disease can be either achieved through fluorescence in situ hybridization (FISH) or polymerase chain reaction PCR technique.

Point Mutations in the FLT3-ITD Region Are Rare but Recurrent Alterations in Adult AML and Associated With Concomitant KMT2A-PTD.

FLT3-ITD mutations are common druggable alterations in patients with acute myeloid leukemia (AML) and associated with poor prognosis. Beside typical ITD mutations, point mutations and deletions in the juxtamembrane domain (JMD) have been observed. However, due to the low frequency of these alterations, there is only limited information on molecular and clinical associations. To evaluate the prognostic impact of non-ITD mutations in the FLT3 JMD region, we analyzed a large cohort of 1,539 adult AML patients treated in different protocols of the Study Alliance Leukemia, using next-generation sequencing. Non-ITD point mutations and deletions within the FLT3 JMD were identified with a prevalence of ~1.23% (n = 19). Both FLT3-ITD and non-ITD mutations were associated with a higher rate of NPM1 (42%–61%; p < 0.001) and DNMT3A mutations (37%–43%; p < 0.001), as well as an increased percentage of peripheral blood (54%–65%) and bone marrow blast cells (74%; p < 0.001), compared to FLT3-wild-type patients. Most significantly, AML patients with FLT3 non-ITD mutations had a higher rate of concomitant KMT2A-PTD mutations (37.5%; p < 0.001) as compared to FLT3-ITD (7%) or FLT3-wild-type cases (4.5%). In a multivariable analysis, FLT3 non-ITD mutations were not an independent prognostic factor. However, patients with dual FLT3 non-ITD and KMT2A-PTD mutations showed a trend for inferior outcome, which points at a functional interaction in this subset of AML.

Performance evaluation and validation of automated digital image analysis in peripheral blood cells morphology examination

Objective: To verify and evaluate the performance of automated digital image(DIA) for peripheral blood cell morphology examination. Methods: Three hundred and seventy-nine routine peripheral blood smears and 18 plasmodium positive peripheral blood smears were collected. Blood smears were made and stained by Wright -Giemsa method.White blood cell (WBC) differentiation of blood smears were pre-classified by DIA (DIA direct classification), re-classified (manually reviewed after DIA classification), and artificially classified under microscope. the inter-assay and intra-assay coefficients of variation (CV) of DIA were respectively calculated for repeatability verification. Taking the artificial microscopy as the gold standard, the sensitivity、specificity and accuracy of DIA were calculated. The DIA ability of peripheral blood blast cell morphological count, platelet (PLT) morphological count and morphological examination of plasmodium were also verified. Results: Except for eosinophils and basophils, the inter-assay and intra-assay CV of WBC classification by DIA in normal samples were < 10%. The CV of WBC classification in abnormal samples increased with the decrease of cell percentage. The sensitivity, specificity and accuracy of DIA pre-classification were 90.5%, 99.2%, 98.2%. Through pre-classification and re-classification by DIA,the results of the blood smears which triggered blast cell alarm had a good correlation with manual classification(r=0.812, 0.983, both P<0.01). The PLT morphological count by DIA had high correlation with hematology analyzer (r=0.946, P<0.01). The deviation absolute value of two methods of PLT count was < 15%, while in PLT aggregation or giant thrombocytosis samples,the deviation absolute value of PLT count by two methods was > 15%. After image acquisition by DIA, 17 plasmodium trophozoites were detected in 18 plasmodium-positive peripheral blood smears, and the images were clear. Conclusions: The DIA system has good repeatability, high sensitivity, specificity and accuracy in peripheral blood WBC classification. Its pre-classification and re-classification results have high correlation with the manual classification results.

Роль молекулярно-генетичних транслокацій у первинній відповіді на лікування дітей, хворих на гостру лімфобластну лейкемію, за програмою ALLIC BFM 2009

The correlation analysis between the number of blast cells in bone marrow and peripheral blood was performed, and the dependence of blast percentage on the presence of molecular genetic translocations (AF4/MLL, BCR/ABL1, TEL/AML, E2A/PBX1) in patients with acute lymphoblastic leukemia (ALL) under the conditions of ALLIC-BFM 2009 cytostatic therapy was researched. The purpose of the study was to establish a relationship between the number of blast cells in bone marrow and peripheral blood depending on the presence of molecular genetic translocations for early detection of induction treatment by ALLIC BFM 2009. Materials and methods. The survey group consisted of 105 children aged 12 months to 16 years (median age was 6 years). Among those surveyed were 62 boys (59.0%) and 43 girls (41.0%). All patients were diagnosed with acute lymphoblastic leukemia. Results and discussion. Correlation analysis revealed a high degree of correlation between the number of blast cells in the bone marrow and peripheral blood, as the correlation coefficient (r) is 0.87. It is shown that the increase in the number of blast cells depends on the presence of chromosomal translocations. The highest number of blasts was observed in patients with BCR/ABL1 and E2A/PBX1 translocations, in whom the content of blasts in bone marrow was 97 and 96%, respectively, and in peripheral blood - 67 and 73%, respectively. It was found that treatment under the ALLIC BFM 2009 program leads to a decrease in the number of blast cells in the bone marrow and blood with minimal values on the 33rd day of treatment. It has been shown that the highest levels of blast cells during chemotherapy are observed in patients with chromosomal translocations BCR/ABL1 and E2A/PBX1. In patients with AF4/MLL translocation, the efficacy of therapy was the highest because no blast cells in the bone marrow were visualized on day 33 of treatment. The study of the primary response of patients with acute lymphoblastic leukemia to induction treatment according to the ALLIC BFM 2009 program revealed the dependence of the level of blast cells of bone marrow and blood on the type of chromosomal aberration. Patients with BCR/ABL1 and E2A/PBX1 have the highest resistance to chemotherapy with molecular genetic translocations, and patients with AF4/MLL and TEL/AML have the lowest resistance, as evidenced by the presence and absence of blast cells in the peripheral blood, respectively. Conclusion. Establishing the relationship between cytogenetic and molecular genetic features of the tumour clone will help determine the degree of malignancy of the process, as well as the risk group for the course of the disease. Determining the dependence of acute leukemia on molecular genetic translocations will make it possible to further modify the treatment program

Myelodysplastic Syndrome/Myeloproliferative Neoplasms with Ringed Sideroblasts and Thrombocytosis (MDS/MPN RS-T)–A Case Report with Literature Review on Diagnosis and Management

Myelodysplastic syndrome/Myeloproliferative neoplasms with ringed sideroblasts and thrombocytosis (MDS/MPN RS-T) is a rare disorder with mixed features of dysplasia and myeloproliferation. This is a relatively new independent entity included in the 2016 WHO classification as MDS/MPN with RS-T. The diagnostic criteria include erythroid lineage dysplasia, ≥15% Ringed Sideroblasts (RS), &lt;1% blast cells in peripheral blood, &lt;5% blast cells in the bone marrow, persistent thrombocytosis with platelet count ≥450×109/L, presence of SF3B1 mutation, absence of BCR-ABL1 gene fusion and rearrangement of PDGFRA, PDGFRB or FGFR1 or PCM1-JAK2. In MDS/MPN RS-T, two mutations are commonly seen JAK 2, which promotes myeloid proliferation, and SF3B1 gene, which causes myelodysplasia with ringed sideroblasts commonly observed in this syndrome. We present a relatively young 59-year-old woman diagnosed with MDS/ MPN RS-T based on the above guideline criteria. She has low-risk MDS, which favors a good prognosis; however, the presence of Essential Thrombocythemia (ET) favors a poor prognosis. Currently, there is no consensus on the specific management of this entity, given its rarity. She was referred to an allogeneic hematopoietic stem cell transplant center for curative treatment since she had become transfusion dependent. Before curative treatment, the patient was initiated on ruxolitinib as a bridging therapy to bone marrow transplant.

Dysregulation of CCAAT/enhancer binding protein-alpha (CEBPA) expression in the bone marrow of acute myeloid leukemia patients

BackgroundAcute myeloid leukemia (AML) is a heterogeneous malignant disease characterized by accumulation of different types of mutations commonly the CCAAT/enhancer binding protein-alpha (CEBPA). However, the dysregulations of CEBPA expression in AML is still a debatable issue. The aim of the current study was to assess CEBPA gene expression in bone marrow (BM) aspiration specimens of 91 AML patients, compared to 20 control donors of bone marrow transplantation (BMT), using RT-PCR. Data were correlated with patients’ clinico-pathological features, response to treatment, progression-free survival (PFS), and overall survival (OS) rates.ResultsThere was overexpression of CEBPA gene in AML patients compared to normal control [1.7 (0.04–25.6) versus 0.17 (0–4.78), respectively, P < 0.001]. Upregulation of CEBPA expression associated significantly with increased BM hypercellularity, total leucocyte counts, peripheral blood blast cell count, and poor PFS (P < 0.001, 0.002, 0.001, and 0.013, respectively). There was no significant association between CEBPA expression and any other relevant clinico-pathological features or OS rates (P = 0.610) of the patients. ROC analysis for biological relevance of CEBPA expression with AML showed that sensitivity and specificity of CEBPA expression at a cut-off value of 0.28 are 92.3% and 78.6%, respectively (P < 0.001). All patients who had CEBPA overexpression and mutant FLT3 showed BM hypercellularity, adverse cytogenetic risk, increased TLC, and PB blast cells count (P = 0.007, P < 0.001, 0.016, and 0.002, respectively).ConclusionCEBPA overexpression could be used as a genetic biological marker for AML diagnosis, as well as a poor prognostic factor for disease progression. It has no impact on OS rates of the patients.

Clinical features and outcome of pediatric acute lymphoblastic leukemia with low peripheral blood blast cell count at diagnosis.

Peripheral blood (PB) blast cell count on day 8 of prednisone therapy has been considered one of the strongest predictors of outcome in children with acute lymphoblastic leukemia (ALL). However, little is known about the clinical features and prognostic impact of PB blast cell count at diagnosis in these patients. The aim of this study was to evaluate the relationship between initial PB blast cell count and clinical prognosis of pediatric ALL.The study comprised 367 patients with ALL, aged 0 to 14 years, enrolled and treated using the Chinese Children's Leukemia Group-ALL 2008 protocol between 2011 and 2015. The majority (91.6%) of patients were B-cell precursor ALL (BCP ALL), and 8.4% were T-cell ALL (T-ALL).Patients with BCP ALL in the low PB blast cell count group (<1 × 109/L) had significantly superior survival rates to those in the high count group (≥30 × 109/L). In T-ALL, the low count group showed significantly inferior survival rates compared to both the intermediate count group (1–29.9 × 109/L) and high count group. Multivariate analysis revealed that the initial white blood cell count and minimal residual disease at the end of induction therapy were independently predictive of BCP ALL outcome, while risk stratification was shown to be an independent prognostic factor for T-ALL outcome.These results indicated that low blast cell count in PB at diagnosis was associated with different clinical outcomes in patients with BCP ALL and T-ALL, although it was not an independent outcome predictor by multivariate analysis.

Factors Influencing Outcome in De Novo Myelodysplastic Syndromes Treated by Allogeneic Bone Marrow Transplantation: A Long-Term Study of 71 Patients

YELODYSPLASTIC syndromes (MDS) are a group M of clonal hematologic disorders characterized by progressive cytopenia. Some patients subsequently develop acute myeloid leukemia (AML), especially if they have an excess of marrow blast cells at diagnosis.',' Factors associated with poor survival include an advanced French-American-British (FAB) subtype or a high percentage of bone marrow (BM) blast cells, the presence of peripheral blood (PB) blast cells, cytopenia, an abnormal karyotype and advanced age. The combination of these different factors has been used by several authors to define scoring systems, which can segregate patients into subgroups with survival ranging from several months to several The median survival of patients with profound cytopenia or an excess of marrow blast cells does not exceed 2 years, even among young patients capable of tolerating intensive chemotherapy.6 In addition, when therapy-related, MDS and AML are rapidly life-threatening.' Intensive cytoreductive chemotherapy, low-dose cytarabine, differentiating agents such as retinoic acid, and growth factors have proven incapable of preventing death, although transient corrections of cytopenia have been reported.6.8-'0 Allogeneic BM transplantation (BMT) is the only treatment known to cure such patients, provided they are young (<50 years) and have a compatible donor. Several teams have published disease-free survival (DFS) rates of 35% to ~~ ~

Reductive regulation of BECN1 gene in adult Egyptian patients with do novo AML

BackgroundAcute myeloid leukaemia (AML) is a clonal haematopoietic disease characterized by the proliferation of immature blast cells in the bone marrow and peripheral blood. Autophagy is an inherent cellular route by which waste macromolecules are engulfed within autophagosomes prior to their fusion with cytoplasmic lysosomes for degradation. The BECN1 gene encodes the Beclin-1 protein, which regulates autophagy. Few reports have investigated BECN1 gene expression and its value in AML patients.ResultsThis randomized case-control study included 50 newly diagnosed AML patients, in addition to 20 subjects as a control group. BECN1 gene expression was assessed using real-time quantitative polymerase chain reaction (qRT-PCR).The median level of BECN1 gene expression in AML patients was 0.41 (IQR 0.29–1.03) in comparison to 1.12 (IQR 0.93–1.26) in the control group (P = 0.000). Seventy-two percent of AML patients showed reduced BECN1 gene expression, which was highly significantly associated with intermediate and adverse cytogenetic risk. Reduced BECN1 gene expression was associated with older age, higher total leukocyte counts, the presence of peripheral blood blast cells, a higher percentage of bone marrow blast cells, and higher expression of CD34 and CD117. FLT3-ITD mutation was detected in 14 patients (38.9%), all of whom showed reduced BECN1 gene expression (P = 0.006). BECN1 gene expression was also reduced in non-responder AML patients, with a highly statistically significant difference (P = 0.002).ConclusionA reduction in BECN1 gene expression might indicate a poor prognosis in adult Egyptian patients with de novo AML. Decreased BECN1 gene expression is associated with a higher risk of resistance to treatment. Targeting autophagy pathways may help in the treatment of AML patients.

Combined Expression of CD34 and FLT3-Internal Tandem Duplication Mutation Predicts Poor Response to Treatment in Acute Myeloid Leukemia.

BackgroundAcute myeloid leukemia (AML) is a common hematological malignancy associated with different cytogenetic and genetic abnormalities.MethodsFLT3-internal tandem duplication (FLT3/ITD) mutation and CD34 expression levels were assessed in the bone marrow (BM) aspirates of 153 de novo AML patients. Data were correlated with relevant clinic-pathological features of the patients, response to treatment, disease-free survival (DFS), and overall free survival (OS) rates.ResultsFLT3-ITD mutation was detected in 27/153 (17.6%) AML patients (P=0.001), and CD34 was expressed in 83/153 (54.2%) patients (P=0.293) compared to those with wild FLT3 and CD34− expression, respectively. Patients with FLT3-ITD mutation showed increased peripheral blood and BM blast cells, abnormal cytogenetics, poor DFS and OS compared to those with wild FLT3 (P=0.013, P<0.001, P=0.010, P=0.008 and P=0.004, respectively), while there was no significant association with response to treatment (P=0.081). There was no significant association between CD34 expression and response to treatment, DFS, and OS (P>0.05). FLT3-ITD mutation and FAB subtypes were independent prognostic factors for DFS. Older age ≥39 years, HB <7 mg/dL PB blast ≥54%, and FLT3-ITD mutation were independent prognostic factors for poor OS in AML patients. The presence of both FLT3-ITD mutation and CD34 expression associated significantly with resistance to therapy (P=0.024), short DFS and OS rates (P=0.006, P=0.037, respectively).ConclusionCombined expression of both FLT3-ITD mutation and CD34 expression is an important prognostic and predictive factor for poor disease outcome in AML patients.

Studies on the morphology of leukaemic blast cells in relation to haematological parameters

A combination of haematological parameters with morphological evaluation of peripheral blood and bone marrow blast cells is crucial for leukaemia diagnosis. FAB (French– American–British) classification is a simple and powerful diagnostic tool for leukaemia in developing countries like India. Differentiation block in the early stages of haematopoiesis and morphological characteristics of leukemic blast cells are directly related to haematological parameters. The present study is an approach to increase understanding of the simple morphological FAB classification of leukaemia in relation to haematological parameters. The present study revealed that Chronic Myeloid Leukaemia (CML) was the most common type of leukaemia , followed by Acute Myeloid Leukaemia, Acute Lymphoid Leukaemia (ALL), and Chronic Lymphoid Leukaemia (CLL) in Nagpur. Most of the cases of Acute Leukaemia had severe anaemia and thrombocytopenia. Highest variation was found in Total WBCs count of different types of leukaemia , particularly in different subtypes of AML. The present study also suggested that FAB classification is not outdated, but it does require continuous revalidation and other procedures for refinement.

PB1713 NEW METHODS FOR ASSESSING THE GENOTOXICITY OF CHEMOTHERAPY DRUGS IN ACUTE MYELOID LEUKEMIA

Background:This abstract presents the experience of cultivating the peripheral blast cells of a patient with resistant acute myeloid leukemia (AML) and testing 2 methods for assessing the effects of chemotherapy drugs on them.Aims:Suitability examination of cell sorting (CS) and micronucleus test (MT) for assessing the genotoxicity of chemotherapy drugs.Methods:Blast cells were obtained by taking blood from a patient with multi‐resistant AML. On sampling days, the percentage of blasts in the patient's peripheral blood ranged from 50% to 75%. In total, 3 in vitro experiments were conducted.In experiment №1, 520650 CD34+ cells were sorted into 2 tubes, which were incubated for 4 days (37 °C, 5% CO2) in 5 ml of complete nutrient medium (CM): 80% RPMI‐1640, 10% fetal calf serum, 10% of the patient's original serum, 10 μl of phytohaemagglutinin and 100 U/ml penicillin.After the first day, decitabine was added to one of the samples at a concentration of 1160 ng/ml. To assess the genotoxicity of the drug at the end of cultivation, we determined the number of cells that retained viability by evaluating the results of labeling free DNA.In experiments №2 and №3, 0.5 ml of blood was cultured with 5 ml of CM. After 24 hours, various concentrations of decitabine (290 ng/ml, 580 ng/ml, 1160 ng/ml) were added to the samples of experiment №2. Daunorubicin (3400 ng/ml) was added to the samples of experiment №3, as well as its combination with interferon alpha‐2a in the calculation of 3600 IU/ml. Next, samples were cultured for 48 hours. To assess the genotoxicity in these experiments MT was used.Results:The results of experiment №1 are presented in Figure 1, №2 in table 1, №3 in table 2.Summary/Conclusion:1. CS allows selection of the required number of cells with the required phenotype and assess their viability after cultivation with high accuracy. In the sample with the addition of decitabine, living blast cells turned out to be almost 2 times less than in the control sample.imageimage2. MT can be used to assess the genotoxicity of chemotherapy drugs for peripheral blood blast cells. From the results of experiment №2, it follows that the concentration of the drug has a direct correlation with the level of its genotoxicity on blast cells. In experiment №3, the combination of daunorubicin with interferon showed much stronger genotoxic effects on blast cells compared to daunorubicin without interferon.

Acute lymphoblastic leukemia segmentation using local pixel information

The severity of acute lymphoblastic leukemia depends on the percentages of blast cells (abnormal white blood cells) in bone marrow or peripheral blood. The manual microscopic examination of bone marrow is less accurate, time-consuming, and susceptible to errors, thus making it difficult for lab workers to accurately recognize the characteristics of blast cells. Researchers have adopted different computational methods to identify the nature of blast cells; however, these methods are incapable of accurately segmenting leukocyte cells due to some major disadvantages, such as lack of contrast between objects and background, sensitivity to gray-scale, sensitivity to noise in images, and large computational size. Therefore, it is indispensable to develop a new and improved technique for leukocyte cell segmentation. In the present research, an automatic leukocyte cell segmentation process was introduced that is based on machine learning approach and image processing technique. Further, the characteristics of blast cells were extracted using 4-moment statistical features and artificial neural networks (ANNs). It was found that the proposed method yielded a blasts cell segmentation accuracy of 97% under different lighting conditions.

MiR-183-5p Inhibits Occurrence and Progression of Acute Myeloid Leukemia via Targeting Erbin.

Erbin has been shown to have significant effects on the development of solid tumors. However, little is known about its function and regulatory mechanism in hematological malignancies. The biological function of Erbin on cell proliferation was measured in vitro and in vivo. The predicted target of Erbin was validated by dual-luciferase reporter assay and rescue experiment. We found that overexpression of Erbin could inhibit the cell proliferation and promote the cell differentiation of acute myeloid leukemia (AML) cells, whereas depletion of Erbin could enhance the cell proliferation and block the cell differentiation in AML cells in vitro and in vivo. Besides, miR-183-5p was identified as the upstream regulator that negatively regulated the Erbin expression. The results were confirmed by dual-luciferase reporter and RNA pull-down assay. Furthermore, we found that miR-183-5p negatively regulated Erbin, resulting in enhanced cell proliferation of AML cells via activation of RAS/RAF/MEK/ERK and PI3K/AKT/FoxO3a pathways. The activation of RAS/RAF/MEK/ERK and PI3K/AKT/FoxO3a pathways was mediated by Erbin interacting with Grb2. These results were also validated by rescue experiments in vitro and in vivo. All above-mentioned findings indicated that the miR-183-5p/Erbin signaling pathway might represent a novel prognostic biomarker or therapeutic target for treatment of AML.

Prognostic Value of Transferrin Receptor-1 (CD71) Expression in Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) is the commonest childhood cancer. Transferrin receptor 1 (CD71) is a trans-membrane glycoprotein which has important role in iron homeostasis by acting as a gatekeeper regulating iron uptake from transferrin and is an attractive target for anti-cancer agents, particularly those that aim to induce lethal iron deprivation in malignant hematopoietic cells. To assess the prognostic value of Transferrin receptor -1 (CD71) in children with newly diagnosed ALL. This study was carried out on 75 patients with newly diagnosed ALL. Transferrin receptor-1 expression was analyzed on the bone marrow blasts by flow cytometry at time of diagnosis with positive CD71 expression is considered when ≥20% of malignant cells express this marker while negative expression is considered when <20% of malignant cells express this marker. Transferrin receptor-1 positive expression was detected in 45 patients (60%) while negative expression was found in the remaining 30 patients (40%). CD71 expression was significantly higher on T- ALL patients compared with B-ALL patients. Positive CD71 expression at diagnosis was significantly associated with bad clinical and laboratory prognostic factors as lymphadenopathy, higher white blood cell count, higher hemoglobin level, lower platelets count, and higher blast cells in peripheral blood and bone marrow and higher lactate dehydrogenase levels'. There were significant differences in disease free survival (DFS) and overall survival (OS) between positive and negative CD71 expression groups with significantly shorter DFS and OS in positive CD71 expression group compared to negative group. 'Positive Transferrin receptor -1 (CD71) expression in patients with ALL is adverse prognostic factor and should be taken in consideration in designing future therapeutic strategies based on patient- specific risk factors'.

Immunophenotypic Characteristics of T-Acute Lymphoblastic Leukemia in Omani Patients: A Correlation with Demographic Factors.

To study and classify the immunophenotypic characteristics of Omani patients diagnosed with T-cell acute lymphoblastic leukemia (T-ALL) and to correlate the results with age and gender as well as biological factors (peripheral and bone marrow blast cells percentage). Fifty cases from both genders and of all ages who fulfilled the inclusion criteria with a diagnosis of T-ALL were included in the study. Correlation of T-ALL subtypes with age, gender, and initial bone marrow and peripheral blood blast cells percentage was assessed using ANOVA. Among the 50 T-ALL patients analyzed, 44 were male and six were female giving a male-to-female ratio of 7:1 (p = 0.007). The average age of patients was 19.2 years with no significant differences in the three disease subtypes. No significant association was seen between the peripheral or bone marrow blast cell percentage and the differentiation stages of the neoplastic clone of T-ALL. All female patients were found to express an immature T-ALL phenotype. This study reports the subtypes of T-ALL in Oman for the first time. It is hoped that this will lead to a better understanding of the disease outcomes.