Eman Z. Kandeel

Human myeloid inhibitory C‐lectin: a highly specific and stable acute myeloid leukemia marker

The prognosis of acute myeloid leukemia (AML) is poor because of relapses occurring on conventional chemotherapy. The distinction between leukemic and normal stem cells relies on the expression of antigen combinations defining leukemia‐associated immunophenotypes (LAIPs), which are absent or extremely infrequent in normal bone marrow. However, LAIPs are very different from patient to patient and are not necessarily stable over the course of the disease. Accordingly, we addressed the applicability of human myeloid inhibitory C‐lectin (hMICL) by flow cytometry as a specific leukemic myeloid stem cell marker for the diagnosis of AML in CD34+ and CD34− cases and evaluated the stability of hMICL during the course of the disease. hMICL expression was assessed in 78 bone marrow aspirate specimens obtained from AML patients at diagnosis (n = 40), complete remission (CR) (n = 28), and relapse (n = 10). AML patients at diagnosis were compared to 20 newly diagnosed acute lymphoblastic leukemia (ALL) patients and 20 healthy controls. hMICL was reevaluated in CR and relapse specimens. hMICL was expressed in 100% AML patients at diagnosis (mean ± standard deviation [SD], 60.3 ± 19.9%), both CD34+ and CD34−, but not in ALL (mean ± SD, 3.3 ± 1.9%) or healthy controls (mean ± SD, 3.4 ± 2.6%) (P < .001). hMICL median fluorescence intensity ratio was higher in AML (mean ± SD, 15.9 ± 11.7) compared to ALL (mean ± SD, 4.5 ± 1.4) and healthy controls (mean ± SD, 4.4 ± 1.6) (P < .001). hMICL was expressed in all studied AML morphologic subtypes. Preserved stable expression of hMICL was found in CR and relapse specimens with no antigen loss. hMICL is a robust pan‐AML‐associated antigen with excellent diagnostic impact, extreme specificity to AML blasts, and stability throughout the course of the disease. hMICL could be incorporated into the routine flow cytometry setting within the initial diagnostic work‐up and follow‐up of AML.

Adhesion molecules expression in CLL: Potential impact on clinical and hematological parameters.

BACKGROUND B-cell chronic lymphocytic leukemia (CLL) is marked by the accumulation of CD5+ B lymphocytes within the blood, bone marrow (BM), and secondary lymphoid tissues. Abnormalities in the expression and function of cell adhesion molecules may account for the patterns of intra-nodal growth and hematogenous spread of the malignant cells. Chemokines and integrin-mediated adhesion and trans-endothelial migration (TEM) are central aspects in trafficking and retention of hematopoietic cells in the BM and lymphoid organs. AIM OF THE WORK This work was conducted to study adhesion molecules status in CLL and its potential impact on both hematological and clinical parameters. PATIENTS AND METHODS The study included 78 newly diagnosed CLL patients. Immunophenotyping was performed on peripheral blood using the chronic lymphoid panel. Adhesion molecules (CD11a, CD11b, CD49d, CD49C, CD29 and CD38) were tested using monoclonal antibodies and analyzed by Flow Cytometry. RESULTS Positive correlation was encountered between adhesion molecules: CD38 with CD49d (r=0.25, p=0.028), CD11a with CD11b, CD49d and CD29 (r=0.394, p=0.001; r=0.441, p=<0.01 and r=0.446, p<0.01 respectively) and CD29 with CD49c and CD49d (r=0.437, p<0.01; r=0.674, p<0.01 respectively). CD49c showed negative correlation with Rai staging (r=-0.269, p=0.033). CD11a and CD29 showed a significant relation with splenomegaly (p=0.04 and 0.03 respectively) and CD49d showed a significant relation with lymphadenopathy (p=0.02). CONCLUSION The level of different adhesion molecules expression in CLL is apparently reflected on the potential migratory behavior of the leukemic cells to different organs.

A novel potential effective strategy for enhancing the antitumor immune response in breast cancer patients using a viable cancer cell‑dendritic cell‑based vaccine

Dendritic cells (DCs) have been used in a number of clinical trials for cancer immunotherapy; however, they have achieved limited success in solid tumors. Consequently the aim of the present study was to identify a novel potential immunotherapeutic target for breast cancer patients through in vitro optimization of a viable DC-based vaccine. Immature DCs were primed by viable MCF-7 breast cancer cells and the activity and maturation of DCs were assessed through measuring CD83, CD86 and major histocompatibility complex (MHC)-II expression, in addition to different T cell subpopulations, namely CD4+ T cells, CD8+ T cells, and CD4+CD25+ forkhead box protein 3 (Foxp3)+ regulatory T cells (Tregs), by flow cytometric analysis. Foxp3 level was also measured by enzyme-linked immunosorbent assay (ELISA) in addition to reverse-transcription quantitative polymerase chain reaction. The levels of interleukin-12 (IL-12) and interferon-γ (IFN-γ) were determined by ELISA. Finally, the cytotoxicity of cytotoxic T lymphocytes (CTLs) was evaluated through measuring lactate dehydrogenase (LDH) release by ELISA. The results demonstrated that CD83+, CD86+ and MHC-II+ DCs were significantly elevated (P<0.001) following priming with breast cancer cells. In addition, there was increased activation of CD4+ and CD8+ T-cells, with a significant decrease of CD4+CD25+Foxp3+ Tregs (P<0.001). Furthermore, a significant downregulation of FOXP3 gene expression (P<0.001) was identified, and a significant decrease in the level of its protein following activation (P<0.001) was demonstrated by ELISA. Additionally, significant increases in the secretion of IL-12 and IFN-γ (P=0.001) were observed. LDH release was significantly increased (P<0.001), indicating a marked cytotoxicity of CTLs against cancer cells. Therefore viable breast cancer cell-DC-based vaccines could expose an innovative avenue for a novel breast cancer immunotherapy.

Impact of FLT3 Receptor (CD135) Detection by Flow Cytometry on Clinical Outcome of Adult Acute Myeloid Leukemia Patients

Micro‐Abstract In a large study focusing on the impact of CD135 on clincal presentation and its relation to other prognostic parameters, we addressed whether CD135 could be predictor of FMS‐like tyrosine kinase 3 (FLT3) mutation. We found an association between CD135 expression and persistence of positive minimal residual disease postinduction status. CD135 is a crucial indicator of unfavorable outcome that influences complete remission status, disease‐free survival, and overall survival in adult Egyptian acute myeloid leukeia patients. Background The significance of FMS‐like tyrosine kinase 3 (FLT3)‐ITD mutation in acute myeloid leukemia (AML) prognosis has been well established. The aims of this study were to investigate the prognostic impact of the FLT3 protein (CD135) expression and its association with FLT3‐ITD mutation, and to identify its role in minimal residual disease. Patients and Methods CD135 was measured by flow cytometry on leukemic blasts of 257 adults with de novo AML. High expression of CD135 ≥ 20% was correlated with clinical, laboratory, and other prognostic factors that influenced treatment outcome. FLT3‐ITD mutation was tested by PCR. Results The frequency of CD135 expression was 138 (53.7%) of 257. FLT3‐ITD was detected in (21.4%). Positive CD135 expression was associated with high total leukocyte count (P = .006), platelet count (P = .003), monocytic leukemia (P < .001), and CD34 (P = .008) and CD117 (P = .006) expression. CD135 expression ≥ 25% was a predictor of FLT3‐ITD mutation (P = .03). CD135 overexpression was a negative predictor of complete remission and of postinduction minimal residual disease at days 14 and 28 (P < .001). CD135 had an adverse impact on overall and disease‐free survival (68.5% vs. 15%, P = .002). Multivariate analysis indicated CD135 was the sole independent prognostic factor for overall survival (hazard ratio = 2.49; 95% confidence interval, 1.855‐3.345; P < .001). Conclusion CD135 is emerging as a prognostic factor, a new marker for minimal residual disease, and a potential novel therapeutic target of AML.

Increased expression of brother of the regulator of imprinted sites in peripheral blood neutrophils is associated with both benign and malignant breast lesions

BORIS, a paralog of the multifunctional CCCTC‐binding factor (CTCF) gene is restricted to testis and normally not present in females. It is aberrantly activated in various human cancers including cancer breast. Using immunohistochemistry, western blot and/or RT‐PCR, significantly higher levels of BORIS expression were reported in the neutrophils of cancer breast patients. We hypothesized that Flow Cytometry might be a better technique for objective quantitative evaluation of BORIS in neutrophils and we wanted to investigate if BORIS would discriminate between benign and malignant breast lesions.

FLT3-ITD Mutations in Egyptian Patients of Acute Myeloid Leukemia: Correlation with Cytogenetic, FAB Subgroups and Prognosis

BACKGROUND FLT3-ITD mutations are common in AML subgroups, particularly in Acute Promyelocytic Leukemia (APL). It infers poor prognosis in AML patients; however, its prognostic value in APL is still controversial. We aimed to assess the distribution and prognostic value of FLT3-ITD mutation within AML subgroups, focusing on APL. METHODS In NCI, Cairo University, 346 newly diagnosed AML patients were included. Morphological, immunophenotypic and cytogenetic analysis were done at presentation and fixed follow-up points with monitoring in follow up visits of patients. FLT3-ITD mutations were detected using RT-PCR. RESULTS FLT3-ITD mutations were detected in 18.5% of AML patients with significant higher incidence in M3 patients (35.5%, p = 0.027) in comparison to other types. There was significant negative association between FLT3ITD mutations and CD34 expression (p = 0.026). FLT3 wild patients had a significantly better response on Day 35 than the mutant group (p = 0.046). Overall Survival (OS) of the wild group was significantly better than that of the mutant group (p = 0.003). In APL patients, OS of wild-FLT3 patients was significantly better than of those with mutant FLT3 (p = 0.018). In non-APL patients, favorable cytogenetic changes - t(8;21) and inv(16) - are more common in FLT3 wild group (12.8%) than in FLT3 mutant group (6.3%). In non-APL patients, FLT3-ITD mutation retained its adverse prognostic effect (p = 0.016). CD34 expression affected survival in both FLT3 mutant and wild groups. CONCLUSIONS We conclude that FLT3-ITD mutations infer poor prognostic effects both in AML patients generally and in the APL group specifically. CD34 may have a prognostic impact in FLT3-ITD mutant patients, which is to be further investigated.

Clinical Impact of Overexpression of FOXP3 and WT1 on Disease Outcome in Egyptian Acute Myeloid Leukemia Patients

Background: In the last decade, it has become clear that change of gene expression may alter the hematopoietic cell quiescent state and consequently play a major role in leukemogenesis. WT1 is known to be a player in acute myeloid leukemia (AML) and FOXP3 has a crucial role in regulating the immune response. Objectives: To evaluate the impact of overexpression of WT1and FOXP3 genes on clinical course in adult and pediatric AML patients in Egypt. Patients and methods: Bone marrow and peripheral blood samples were obtained from 97 de novo non M3 AML patients (63 adult and 34 pediatric). Real-time quantitative PCR was used to detect overexpression WT1 and FOXP3 genes. Patient follow up ranged from 0.2 to 39.0 months with a median of 5 months. Results: In the pediatric group; WT1 was significantly expressed with a high total leukocyte count median 50X109/L (p=0.018). In the adult group, WT1 had an adverse impact on complete remission induction, disease-free survival and overall survival (p=0.02, p=0.035, p=0.019 respectively). FOXP3 overexpression was associated with FAB subtypes AML M0 +M1 vs. M2, M4+M5 (p =0.039) and the presence of hepatomegaly (p=0.005). Conclusions: WT1 and FOXP3 overexpression has an adverse impact on clinical presentation, treatment response and survival of pediatric and adult Egyptian AML patients.