FLT3-ITD Allele Frequency Is an Independent Prognostic Factor for Poor Outcome in FLT3-ITD-Positive AML Patients.

BACKGROUND: FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is a molecular genetic alteration significantly affecting the clinical outcome in patients with acute myeloid leukemia (AML). FLT3-ITD mutations are characterized by variable mutant-to-wild allelic ratios (ARs) and sizes of the duplicated sequences. The size of the inserted sequence may vary from a few to hundreds of nucleotides. The aim of this work was to determine the impact of FLT3-ITD ARs, FLT3-ITD allelic frequency (AF), and allele size in de novo AML. PATIENTS AND METHODS: We studied 117 patients with FLT3-ITD gene mutation-positive AML, dividing them into those with low ARs and those with high ARs (>0.64) and examined their prognostic impact. RESULTS: High FLT3-ITD AR ≥ 0.64 and AF ≥ 0.5 were significantly associated with a lower overall survival compared with lower AR (median 0.625 vs. 1.020 months, respectively; P = .041) and AF (median 0.493 vs. 0.954 months, respectively; P = .009). NPM1 mutation had no favorable impact on the low-level FLT3-ITD group. CONCLUSION: Initial high total leukocyte count, FLT3-ITD AF, and splenomegaly are independent prognostic factors for poor outcome in FLT3-ITD-positive AML.

Duplication 1q is highly correlated with poor prognosis in high hyperdiploid pediatric B-acute lymphoblastic leukemia.

BACKGROUND: The role of structural abnormalities in high hyperdiploidy (HeH) has been debatable, with few studies that addressed recurrent translocations with concurrent HeH (t-HeH). We aimed at the characterization of HeH cases in pediatric B-acute lymphoblastic leukemia (B-ALL) patients with special emphasis on the structural abnormalities including t-HeH. PATIENTS AND METHODS: Our study included all patients diagnosed with HeH over the period from January 2016 to April 2019 presenting to the Pediatric Oncology Department, National Cancer Institute, Cairo University. RESULTS: Among 480 de novo B-ALL pediatric patients, HeH was detected in eighty (16.7%) cases with a median age of 5 years. t-HeH was identified in 17/480 (3.5%) cases: 9(1.9%) with t(12;21), 7(1.5%) with t(9;22), and 1(0.2%) with t(4;11). Duplication (1q) was the most prevalent structural abnormality in c-HeH (hyperdiploidy without recurrent translocations) (n = 12,15%). Children ≥10 years or presenting with white blood cells (WBC) ≥50 × 109 /L) had an inferior 3 year-overall survival as compared to younger children (P = .003), and to lower WBC (P = .02). Duplication (1q) was an independent adverse parameter on the disease-free survival (DFS) of c-HeH patients (P = .004). CONCLUSIONS: Older age and WBC ≥ 50 × 109 /L were adverse prognostic factors. Duplication (1q) is correlated with lower DFS in c-HeH patients. t-HeH has distinct patterns of chromosomal gain.

No prognostic significance of normalized copy number of PML-RARA transcript at diagnosis in patients with acute promyelocytic leukemia.

BACKGROUND: Acute promyelocytic leukemia is a peculiar disease with few studies that have investigated the prognostic significance of PML/RARA transcript level at diagnosis. PATIENTS AND METHODS: This retrospective study included all cases diagnosed with acute promyelocytic leukemia over the period from June 2015 to March 2019. The normalized copy number (NCN) was tested by real-time polymerase chain reaction at diagnosis, and at the end of induction regimen. RESULTS: Our study included 83 de novo APL patients, 53 (63.9%) were adults and 30 (36.1%) were children. The median (range) age of our patients was 28.0 (1.0-70.0) years. The pediatric group had a significantly higher prevalence in males (p = 0.02), higher incidence of disseminated intravascular coagulopathy (p = 0.014), and high-risk groups (p = 0.017). At diagnosis, the median NCN (%) of the entire group at 22.5 was set as the cut off value. There was no significant association between NCN at diagnosis and other prognostic variables except for bone marrow promyelocytes (p = 0.006). High-risk group APL patients as well as those presenting with hemorrhage had an inferior overall survival (OS) (p = 0.007; p < 0.001) respectively. PML-RARA NCN at diagnosis did not have an impact on the OS or increased risk of relapse of our patients (p = 0.434; p = 0.721). CONCLUSION: the initial PML/RARA tumor burden is not a prognostic factor for APL. The initial TLC at 10x109/L cut off is the most important predictive for OS. Early detection and close monitoring are required to decrease the high rate of early deaths in developing countries.

Prognostic Significance of bcr-1 and bcr-3 Isoforms of PML-RARA and FLT3-ITD in Patients With Acute Promyelocytic Leukemia.

BACKGROUND: Acute promyelocytic leukemia (APL) has a characteristic peculiar morphologic and genetic features as well as a more favorable outcome. We studied the differential effect of bcr-1 and bcr-3 isoforms of the promyelocytic leukemia/retinoic acid receptor alpha (PML-RARA) transcript together with FLT3 internal tandem duplication (FLT3-ITD) mutation status on the outcome of newly diagnosed de novo APL patients. PATIENTS AND METHODS: This cohort study included all patients diagnosed with APL at outpatient medical and pediatric oncology clinics of the National Cancer Institute, Cairo University, Cairo, Egypt, from May 2012 to January 2018. RESULTS: The study included 118 patients with APL, 71 adults (60.2%) and 47 children (39.8%). Median (range) age was 25 (1.5-70) years. Children had significantly higher total leukocyte count (≥10 × 109/L), disseminated intravascular coagulation (DIC), and thrombocytopenia (< 40 × 109/L) than adults (P = .04, .03, and .04, respectively), while the latter group had significantly higher hemorrhage than children (P = .04). FLT3-ITD mutation was detected in the whole group, children, and adults in 23.7%, 30.6%, and 24.6%, respectively. FLT3-ITD mutation was significantly associated with leukocytosis in the whole group (P = .039). bcr-3 was significantly associated with FLT3-ITD mutation in the whole APL cohort and in adults (P = .011, P = .022). All children (both bcr-1 and bcr-3) and all adult patients with bcr-3 experienced CR, while 22 (78.5%) of 28 patients with bcr-1 experienced CR (P = .04). APL patients with DIC and hemorrhage had significantly lower overall survival (P = .002 and < .001, respectively). Overall survival for APL in children was significantly better than in adults (P = .005). Multivariate analysis indicated that age was an independent prognostic variable affecting survival (hazard ratio = 2.6; 95% confidence interval, 1.3-5.3; P = .007) (adults had hazard ratio of death 2.6 times higher than children). DIC and FLT3-ITD were independent prognostic variables affecting survival in children with APL (hazard ratio = 12.3; 95% confidence interval, 1.46-104.61; P = .021; and hazard ratio = 5.2; 95% confidence interval, 1.01-26.95; P = .048, respectively). CONCLUSION: Age is an independent prognostic factor for APL. bcr-3 is significantly associated with FLT3-ITD in adults with APL. DIC and FLT3-ITD are adverse prognostic factors in children with APL. Despite children being at higher risk, outcome is better than in adults.

Low expression of miR-204 is associated with expression of CD34 and poor performance status in denovo AML.

INTRODUCTION: Acute myeloid leukemia (AML) is the most common acute leukemia in adults. There is growing evidence that microRNAs (miRNAs) provide prognostic information in AML. MiR-204 has a tumor suppressor function, and several studies have proven its role in solid cancers. The aim of this work is to evaluate the level of expression of miR-204 in adults newly diagnosed with AML with normal karyotype and to correlate its level of expression with disease outcome and different prognostic factors. PATIENTS AND METHODS: The study included 87 adult patients newly diagnosed with AML. Detection of miR-204 was done using RT-PCR in patients and seven age-matched controls. RESULTS: Acute myeloid leukemia patients showed significantly lower miR-204 expression, compared to control group (P = .029). Low miR-204 expression was significantly associated with positive CD34 (P = .017), with poor performance status (PS) (P = .009), and with the presence of diabetes mellitus (DM) (P = .014). Low expression of miR-204 was also significantly associated with shorter overall survival (OS) (P = .020) and disease-free survival (DFS) (P = .013). Low miR-204 expression was identified as an independent prognostic factor for prediction of shorter OS (P = .034) and DFS (P = .027) in AML. CONCLUSION: To the best of our knowledge; this is the first time to prove the correlation between miR-204 expression and CD34 expression. Further study of this correlation is needed to confirm the role of miR-204 in CD34-positive cells, including leukemic stem cells. This correlation may have therapeutic implications. MiR-204 can be used as a biomarker for PS in AML patients.

Detection of KIT mutations in core binding factor acute myeloid leukemia.

We have investigated the frequency and the effect of KIT mutations on the outcome of patients with CBF-AML. 69 patients (34 pediatrics and 35 adults) with CBF-AML were enrolled in the study. The frequency of KIT mutations was higher in adults compared to pediatrics (22.9% and 14.7%, p = 0.38) respectively. Leukocytosis ≥ 20 × 109 /L was significantly associated with pediatrics compared to adults. t(8;21)(q22;22) was significantly associated with thrombocytopenia in adults. We conclude that no significant difference is found between KIT mutated and unmutated CBF-AML in adults and pediatrics. Children with CBF-AML present with leukocytosis. t(8;21) is associated with thrombocytopenia.

Enhanced transdermal delivery of ondansetron using nanovesicular systems: Fabrication, characterization, optimization and ex-vivo permeation study-Box-Cox transformation practical example.

This study aimed to formulate suitable nanovesicles (NVs) for transdermal delivery of Ondansetron. It also illustrated a practical example for the importance of Box-Cox transformation. A 23 full factorial design was used to enable testing transfersomes, ethosomes, and transethosomes of Ondansetron simultaneously. The independent variables (IVs) studied were sodium taurocholate amount, ethanol volume in hydration medium and sonication time. The studied dependent variables (DVs) were: particle size (PS), zeta potential (ZP) and entrapment efficiency (EE). Polynomial equations were used to study the influence of IVs on each DV. Numerical multiple response optimization was applied to select an optimized formula (OF) with the goals of minimizing PS and maximizing ZP absolute value and EE. Box-Cox transformation was adopted to enable modeling PS raised to the power of 1.2 with an excellent prediction R2 of 1.000. ZP and EE were adequately represented directly with prediction R2 of 0.9549 and 0.9892 respectively. Response surface plots helped in explaining the influence of IVs on each DV. Two-sided 95% prediction interval test and percent deviation of actual values from predicted ones proved the validity of the elucidated models. The OF was a transfersomal formula with desirability of 0.866 and showed promising results in ex-vivo permeation study.

Tri-block co-polymer nanocarriers for enhancement of oral delivery of felodipine: preparation, in vitro characterization and ex vivo permeation.

This study aimed to prepare, optimize and characterize novel felodipine-loaded polymeric nanomicelles, using a pluronic mixture of F127 and P123. Thin-film hydration method was adopted for the preparation of different polymeric nanomicelles (T1-T12) according to a 41.31 full factorial design. Factors studied were: Pluronic®:drug ratio (P:D ratio) (10, 20, 30 and 40 w/w) and percent of hydrophilic polymer (F127%) (33.33%, 50% and 66.67% w/w). Optimization criteria were to maximize transmittance percent (T%) and entrapment efficiency percent (EE%) and to minimize particle size (PS) and polydispersity index (PDI). The optimized formulation was further characterized by DSC, FTIR and 1H NMR studies. It was also subjected to stability testing and ex vivo permeation using rabbit intestines. Spherical nanomicelles of particle size ranging from 26.18 to 87.54 nm were successfully obtained. The optimized formulation was found to be the already prepared formulation T12 (P:D ratio of 40 and 66.67% F127) with suitable T% and EE% of 95.12% and 91.75%, respectively. DSC, FTIR and 1H NMR studies revealed felodipine (FLD) incorporation within T12 nanomicelles. T12 enhanced the ex vivo intestinal permeation of FLD when compared to a drug suspension and showed good stability. Therefore, pluronic nanomicelles could be promising for improved oral delivery of FLD.

Development and Validation of Eco-Friendly Liquid Chromatographic and Spectrophotometric Methods for Simultaneous Determination of Coformulated Drugs: Phenylephrine Hydrochloride and Prednisolone Acetate.

Five simple, sensitive, and eco-friendly LC and UV spectrophotometric methods have been developed for the simultaneous determination of phenylephrine hydrochloride (PHE) and prednisolone acetate (PRD) in their combined dosage form. The first method was reversed-phase (RP) LC using methanol-water-heptane-1-sulfonic acid sodium salt (75 + 25 + 0.1, v/v/w) as a mobile phase. Separation was achieved using an XSelect HSS reversed-phase C18 analytical column (250 × 4.6 mm, 5µm). The flow rate was 1.0 mL/min and UV detection was done at 230 nm. Quantification was achieved over the concentration ranges of 5-50 µg/mL for PHE and 2-90 µg/mL for PRD. Four spectrophotometric methods were proposed, namely dual wavelength, first derivative of ratio spectra, ratio difference, and mean-centering of ratio spectra. Linearity was observed in the concentration ranges of 10-120 and 5-35 µg/mL for PHE and PRD, respectively, for the spectrophotometric methods. Green solvents were used in the proposed methods because they play a vital role in the analytical methods' influence on the environment. The suggested methods were validated regarding linearity, accuracy, and precision according to the International Conference on Harmonization guidelines, with satisfactory results. These methods could be used as harmless substitutes for routine analysis of the mentioned drugs, with no interference from excipients.

Prognostic value of IDH1 mutations identified with PCR-RFLP assay in acute myeloid leukemia patients.

BACKGROUND: Somatic mutations in isocitrate dehydrogenase 1 (IDH1) gene occur frequently in primary brain tumors. Recently theses mutations were demonstrated in acute myeloid leukemia (AML). So far, assessment of these mutations relied on the DNA sequencing technique. AIM OF THE WORK: The aim of this study was to detect somatic mutations in IDH1 gene using mismatched primers suitable for endonuclease based detection, without the need for DNA sequencing, and to estimate its prognostic value, on patients with de novo AML. METHODS: Residual DNA extracted from pretreatment bone marrow (BM) samples of 100 patients with de novo AML was used. The polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP) was adapted to IDH1gene, codon 132 mutations screening. RESULTS: The frequency of IDH1 mutations was 13%. In the non-acute promyelocytic leukemia group (non-APL), IDH1 mutations were significantly associated with FLT3-ITD negative patients (p=0.03). Patients with IDH1 mutations did not achieve complete remission (CR). There was a trend for shorter overall survival (OS) in patients with IDH1 mutation compared to those with wild type (p=0.08). CONCLUSION: IDH1 mutations are recurring genetic alterations in AML and they may have unfavorable impact on clinical outcome in adult AML. The PCR-RFLP method allows for a fast, inexpensive, and sensitive method for the detection of IDH1 mutations in AML.

The importance of rapid aneuploidy screening and prenatal diagnosis in the detection of numerical chromosomal abnormalities.

OBJECTIVES: Evaluation of Fluorescent in situ hybridization (FISH) as a tool for rapid aneuploidy screening (RAS) of high risk pregnancies, before its approval in the national antenatal screening and genetic diagnosis program in Egypt. METHODS: The cytogenetic data of prenatal specimens, and results of FISH of 100 patients performed between, January 2009 and December 2009, at the Medical Genetics Center (MGC) laboratory were retrieved and reviewed. AneuVysion Assay kit was used for detection of 13, 21, X, Y, 18 aneuploidies. RESULTS: Maternal age varied from 21 to 44 years (mean was 35.6 year). Ninety percent of pregnancies had normal chromosomes and 10% of the cases had numerical chromosomal abnormalities. Trisomy 21 was the most frequent chromosomal disorder across all indications (5%), followed by Turner syndrome (2%), trisomy 18 (2%), and trisomy 13 (1%). When comparing the FISH data with karyotype results for chromosomes 13, 18, 21, X, and Y in the 83 individual tested, no false positive or negative results were detected by the FISH assay. The result obtained by FISH and the banding cytogenetic were in complete accordance. CONCLUSION: This study supports the integration of amniotic fluid (AF) FISH as a RAS test, in to routine antenatal practice for identification of chromosome aneuploidies. There are trends towards delayed childbearing and most cases of Down Syndrome (DS) are currently detected post-nataly in the Egyptian population. Consequently, the live birth prevalence of DS has increased, which might lead to a serious negative public health effects.

Expression of P-glycoprotein, Cyclin D1 and Ki-67 in Acute Lymphoblastic Leukemia: Relation with Induction Chemotherapy and Overall Survival.

Previous studies showed that non-cycling cells have a higher multidrug resistance (MDR) expression, which may be down-regulated by proliferation induction. Triggering these cells into proliferation down-regulates high MDR expression. The aim of this study was to determine the expression of P-glycoprotein (PGP) and cell cycle parameters (cyclin D1 and Ki-67) in acute lymphoblastic leukemia (ALL) at diagnosis, and to evaluate the correlation between the expressions of each marker, and the clinical significance of such expression with response to induction chemotherapy and overall survival. A total of 78 newly diagnosed ALL patients were enrolled in our study. PGP, cyclin D1 and Ki-67 were determined by flow cytometry. PGP expression was encountered in 10/78 (12.8%) of ALL cases. Cyclin D1 and Ki-67 were expressed in 16/77 (20.6%) and 27/76 (34.6%) of ALL cases, respectively. None of the parameters were associated with response to induction chemotherapy and overall survival. Based on the current analysis, we conclude that a joint immunophenotypic evaluation of PGP and cell cycle parameters like that adopted in this study is unlikely to reveal mechanisms of multidrug resistance associated with the clinical outcome.

Phenotype of apoptotic lymphocytes in children with Down syndrome.

BACKGROUND: Down syndrome (DS) is the most common and best-known chromosomal disorder and is associated with several other pathologic conditions including immunodeficiency which makes a significant contribution to morbidity and mortality. Various immunological theories and observations to explain the predisposition of individuals with DS to various infections have been published, one of which is increased apoptotic cells. AIM: The aim of this study was to identify the effect of apoptosis on both types of cells of specific immune response (T and B lymphocytes) in children with DS using Annexin V staining of phosphatidyserine (PS) as a specific marker of early apoptosis. SUBJECTS AND METHODS: The study included 17 children with karyotypically ascertained DS (7 males and 10 females). Their ages ranged from 4 months to 14 years with mean age of 5.7 +/- 4.35 years. Seventeen age and sex matched healthy children were included in the study as controls. Patients or controls with infections were excluded from the study. Complete blood picture, immunophenotyping, analysis of apoptosis using Annexin V was done at National cancer Institute to all children included in this study. RESULTS: Although CBC, differential count, relative and absolute number of CD(3+) and CD(16+) did not show significant differences between DS children and control group, the relative and the absolute size of apoptotic CD(3+) T lymphocytes, and the relative size of apoptotic CD(19+) B lymphocytes were significantly higher in DS children than in controls. On the other hand, no significant difference was detected as regards the absolute size of CD(19+) B lymphocytes in DS children and in controls CONCLUSION: our finding of increased early apoptotic cells (especially T cells) in DS children may emphasize the fact that the function of cells- and not their number- is main mechanism responsible for the impairment of the immune system in DS children and may further add to the known fact that cellular immunity is more severely affected than humoral immunity in these children. Further studies on apoptotic cellular phenotype in larger number of DS are needed.