Variability of contribution of 1,25 (OH)2D3 (vitamin D) level to hematopoietic stem cell transplantation outcome.

BACKGROUND: The impact of vitamin D status on the outcome of allogeneic hematopoietic stem cell transplantation (HSCT) has recently been the focus of interest with a lot of controversy. In this study we aimed to evaluate the impact of pre-transplant vit. D level on the outcome of HSCT. METHODS: In this study, we evaluated the impact of vitamin D level on the risk of development of graft versus host disease (GVHD) and survival after HSCT. The study included 97 patients who received allogeneic HSCT from an identical sibling. Serum vitamin D level was measured before conditioning using ELIZA. Student t-test, Mann-Whitney U test, ANOVA F-test and Kruskal-Wallis H tests were used to determine significance of difference for quantitative data. Pearson correlation, Spearman correlation and Chi-square test were used to determine correlations and associations. Kaplan-Meier and Log rank (Mantel-Cox) tests were used for analysis of survival. P value ≤ 0.05 was considered significant. RESULTS: Vitamin D level showed a range of 18.24-84.6 with a mean of 38.14 ± 9.73 and a median of 36.26 ng/ml. Two patients had vitamin D level <20 and 17 had a level <30 ng/ml. Acute GVHD occurred in 33 (34 %) and chronic GVHD in 29 (29.9 %) patients. Vitamin D level had no impact on frequency or severity of GVHD; either did it impact survival. This might be attributable to the relatively normal level in the majority of our patients on account of the sunny weather of Egypt. This might also be a potential explanation for the inconsistency of the different studies with variable levels of vitamin D. CONCLUSIONS: The current study failed to demonstrate an impact of pre-transplant vitamin D level on the outcome of HSCT. This might be attributed to the low prevalence of vitamin D deficiency in our population on account of our almost always sunny weather. The marked variability in the level of vitamin D that is considered sufficient interferes with objective comparison between studies; a consensus on what is considered sufficient, insufficient, or deficient is essential.

Leukemia Stem Cell Frequency at Diagnosis Correlates With Measurable/Minimal Residual Disease and Impacts Survival in Adult Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a heterogenous disease in which the initiation and maintenance of the malignant clone is blamed on a rare population of leukemia stem cells (LSCs). The persistence of such a malignant population is referred to as measurable/minimal residual disease (MRD). Evaluation of MRD is the gold standard for follow-up of therapy and constitutes an independent prognostic parameter. As LSCs are the main contributor to the persistence of MRD, then MRD should correlate with the bulk of LSCs at the individual case level. MRD is measured at defined time points during therapy. However, LSCs can be evaluated at diagnosis, which ensures the advantage of early prediction of high-risk patients and allows for early therapeutic decisions. Using two simple four-color monoclonal antibody combinations (CD38/CD123/CD34/CD45 and CD90/CD133/CD45/CD33) and the prism function of the Coulter Navios flow cytometer, the frequency of LSC subsets was evaluated in 84 newly diagnosed adult AML patients. For each panel, 16 possible combinations were detected. Our results showed that there was extreme variability in the percentage of the LSC fraction between different cases, as well as at the individual case level. For each LSC subset, the median value was used to divide cases into low and high expressors. LSC subsets that showed an impact on overall survival (OS) and disease-free survival (DFS) included CD123+, CD 123+/CD34-, CD34-/CD38+/CD123+, CD34+/CD38-/CD123+, CD133+, and CD133+/CD33-. On multivariate analysis, only CD123 (p ≤ 0.001, SE = 0.266, HR = 2.8, 95% CI = 1.74.7) and CD133+/CD33- (p = 0.017, SE = 0.263, HR = 1.9, 95% CI = 1.1-3.1) retained their significance for OS. Likewise, only CD34+/CD38-/CD123+ (p ≤ 0.001, HR 2.3, SE: 0.499, 95% CI: 2.4-17.4) and CD133 (p = 0.015, HR 2.3, SE 0.34, 95% CI: 1.2-4.4) retained their statistical significance for DFS. The LSC frequency at diagnosis showed a moderate to strong correlation with MRD status at day 14 and day 28. In conclusion, the level of LSCs at diagnosis correlated with MRD status at day 14 and day 28 in AML patients and had a deleterious impact on OS and DFS. It may be used as an early marker for high-risk patients allowing for early therapeutic decisions.

IL12 and IFNγ secretion by donor mononuclear cells in response to host antigens may predict acute GVHD after HSCT.

One persistent problem of allogeneic hematopoietic stem cell transplantation (HSCT) is acute graft versus host disease (GVHD). The role of cytokines in the pathogenesis of GVHD has been acknowledged. We aimed, in the current study, to investigate the possibility of prediction of acute GVHD through investigating the pattern of interleukin 12 (IL12) and interferon gamma (IFNγ) production of both patients' origin and donors' origin. A total of 45 patients, receiving allogeneic peripheral blood (PB) stem cells from an identical sibling, were included in the study. Patients' plasma was collected after conditioning, during aplastic phase (representing patients' origin) and after engraftment (representing donors' origin). In addition an aliquot from the graft was used as responders in mixed lymphocyte culture (MLC) for 3 days with patients' mitomycin-treated mononuclear cells as stimulators. Culture supernatant was used for detection of IL12 and IFNγ of donors' origin. Fourteen patients developed acute GVHD. In culture supernatant, IL12 was detectable in 7/14 cases with and in none of 31 cases without acute GVHD (p= <0.001). The corresponding figures for IFNγ were 10/14 and 3/31 with significantly higher IFNγ level in cases with than in cases without acute GVHD (p = 0.001). At engraftment the corresponding figures were 7/14 and 5/31 for IL12 and 11/14 and 7/31 for IFNγ with significantly higher cytokine levels in cases with acute GVHD (p = 0.008 and p = 0.001 respectively). At a cutoff of 0.89 pg/ml, IL12 in culture supernatant may predict acute GVHD with absolute specificity of 100% and a sensitivity of 50%. In conclusion, IL12 and IFNγ of donors' origin not of patients' origin may predict the occurrence of acute GVHD. The MLC model may allow prediction of acute GVHD upfront before conditioning of the patient or mobilization of the donor.

Variability of contribution of vitamin D receptor gene polymorphisms to outcome of HLA-matched sibling allogeneic bone marrow transplantation.

Graft-versus-host disease (GVHD) remains one of the major complications of hematopoietic stem cell transplantation (HSCT). Several etiological factors were investigated. Among these, vitamin D and hence its receptor (VDR) gene polymorphisms have gained much interest; however, the results are still controversial. Using PCR-RFLP, we genotyped VDR polymorphisms FokI (rs10735810), ApaI (rs7975232), and Taq1 (rs731236) in 80 patient/donor pairs according to DNA availability. No association was encountered between VDR polymorphisms and GVHD. Neither was there any impact on survival. Only grade II-IV acute GVHD was associated with inferior overall (p = .01), but not disease-free survival. The controversy between our results and the literature may be attributed to marked variability in the relative distribution of VDR genotypes in different populations. Also different environmental factors, including exposure to sun, may ensure vitamin D sufficiency nullifying the impact of VDR polymorphisms.

The impact of cytokine gene polymorphisms on the outcome of HLA matched sibling hematopoietic stem cell transplantation.

Graft-versus-host disease (GVHD) is the major complication of allogeneic hematopoietic stem cell transplantation (HSCT); cytokines are recognized as important mediators in its pathogenesis. In this study we investigated the role of cytokine gene polymorphisms on HSCT outcome. A total of 106 patient and 98 donors were genotyped by polymerase chain reaction sequence specific primers (PCR-SSP) based assay for tumor necrosis factor-α-308 (TNFα -308), interleukin (IL)-6-174, IL-10-1082, -819, -592, Interferon-γ+874 (IFN-γ+874), and transforming growth factor-ß1 (TGF-ß1) codon10 and 25 polymorphisms. Except one in each category, all patients and donors were TNFα -308 high producers and the majority were IL-6-174 high producers (93.3% and 90.8% respectively); a pattern that would alleviate any potential biological impact. Patient's IFN-γ+874 showed significant association with the development of chronic GVHD. Patients with IFN-γ +874 high producer showed an 8 folds likelihood to develop chronic GVHD as compared to those with IFN-γ+874 low producer predicted phenotype (95% CI: 1.59-40.2, p = 0.01). Patient's TGFß1-codon 10 and 25 high/intermediate producers showed a lower incidence of acute GVHD though it did not achieve statistical significance (p = 0.065) on account of the low frequency of this genotype in our patients and donors (11.4 and 8.2% respectively). Other factors contributing to risk of GVHD included older age for both acute and chronic (p = 0.01 and 0.02 respectively) with age 24 as the best discriminating cutoff; CD34+ cell dose for chronic GVHD (p = 0.045) with a dose of 8 × 106/kg as the best discriminating cutoff; and conditioning regimen with Flu/Bu associated with the lowest incidence of acute GVHD (p = 0.003) and no impact on chronic GVHD. In conclusion the current study further indicates a potential role of some cytokine gene polymorphisms in the development of GVHD. The relative distribution of high and low producer genotypes in different ethnic groups contributes to their biological impact in different populations.

The effect of killer cell immunoglobulin-like receptor genotype on outcome of hematopoietic stem cell transplantation from matched sibling.

The alloreactivity of natural killer (NK) cell after allogeneic hematopoietic stem cell transplantation (AHSCT) is regulated by the interaction between donor killer immunoglobulin-like receptors (KIRs) and recipient human leukocyte antigen (HLA)-class I molecules. The aim was to identify KIR genes, haplotypes and their HLA-class I ligands and to investigate their association with transplantation outcome. The study included 65 patient/donor pairs who received AHSCT from HLA-matched identical siblings. KIR genotyping was done for donors using reverse sequence specific oligonucleotide probes (rSSO) coupled with luminex technology, while HLA-C genotyping was performed in patients using rSSO strip assay. In multivariate analysis, KIR2DS4 was associated with significant reduced incidence of relapse (p = .002). A trend towards reduced incidence of relapse was also observed with more than two KIR B motifs (p = .09), whereas a significant increased relapse was associated with homozygous HLA-C2 ligand compared to combined C1/C2 and C1/C1 (p = .04). Activating KIR2DS3 was associated with rapid leukocyte engraftment (p = .02). While, KIR 2DL5 was associated with decreased CMV infection (p = .03) and better platelets engraftment (p = .05). KIR genes, haplotypes and HLA-C alleles have an impact on HSCT outcome. Better selection of donors with favorable KIR genotype can improve HLA-matched sibling HSCT outcome especially for AML patients.

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

BACKGROUND: 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. METHODS: The study included 85 females; 52 breast cancer, 13 benign breast lesions and 20 age-matched healthy controls. BORIS expression in the neutrophils was detected by Flow Cytometry. RESULTS: High level of BORIS was detected in all malignant (64.4 ± 16.6%) and benign cases (67 ± 12.3), mean florescent intensity ratio (MFIR) of 7.2 ± 4.1 and 7 ± 3.5, median 5.8 and 6.6%; and staining index (SI) 8.3 ± 3.9 and 8.2 ± 3.4, median 7.6 and 7.9 respectively vs.13.4 ± 11.5% MFI 1.8 ± 0.7, median1.6 and SI 2.6 ± 0.69, median 2.5 for the control. BORIS level was comparable in the malignant and benign group (P = 0.934) and significantly higher than control (P = 0.0001). There was no correlation between neutrophil BORIS expression and ER/PR status, HER-2/neu expression or tumor stage or size. CONCLUSIONS: Increased BORIS expression in peripheral blood neutrophils is associated with both benign and malignant breast lesions; apparently, increased proliferation of breast tissue is the determining factor. This excludes BORIS as a tumor marker but it does not jeopardize its value as a potential therapeutic target. © 2016 International Clinical Cytometry Society.

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.

Plasma DNA integrity index as a potential molecular diagnostic marker for breast cancer.

Plasma DNA integrity index is increased in various malignancies including breast cancer, the most common cancer in women worldwide; early detection is crucial for successful treatment. Current screening methods fail to detect many cases of breast cancer at an early stage. In this study, we evaluated the level of plasma DNA integrity index in 260 females (95 with breast cancer, 95 with benign breast lesions, and 70 healthy controls) to verify its potential value in discriminating malignant from benign breast lesions. The criteria of the American Joint Committee on Cancer were used for staging of breast cancer patients. DNA integrity index was measured by real-time PCR. DNA integrity index was significantly higher in breast cancer than in benign breast patients and healthy subjects (P = <0.001). DNA integrity index is correlated with TNM stage. Given 100 % specificity, the highest sensitivity achieved in detecting cancer group was 85.3 % at 0.55 DNA integrity index cutoff. In conclusion, the plasma DNA integrity index may be a promising molecular diagnostic marker of malignancy in breast lesions.

N-Acetyltransferase 2 (NAT2) polymorphism as a risk modifier of susceptibility to pediatric acute lymphoblastic leukemia.

N-Acetyltransferases (NAT) have been known to modify the risk to a variety of solid tumors. However, the role of NAT2 polymorphism in risk susceptibility to childhood acute lymphoblastic leukemia (ALL) is still not well known. We performed a case-control study to determine if the common NAT2 polymorphisms play a role in altering susceptibility to pediatric ALL. DNA of 92 pediatric ALL patients and 312 healthy controls was analyzed for the NAT2 polymorphisms using the PCR-RFLP method. The wild-type NAT2*4 was encountered in 8.6 % of patients versus 11.8 % of controls (P = 0.23). The rapid acetylators NAT2*12 803A>G, AG, GG, and AG/GG were overrepresented in controls (P = 0.0001; odds ratio (OR) 0.22, 0.19, and 0.21 respectively). NAT2*5D 341T>C and NAT2*11A 481C>T were of comparable frequencies. For their combination, NAT2*5A, a slow acetylator, both TCTT and CCCT were overrepresented in patients (P < 0.001; OR 15.8 and 17.9 respectively). NAT2*5B (803A>G, 341T>C, 481C>T) was overrepresented in controls (P < 0.001; OR 0.12). Apparently, 803A>G ameliorated the combined effect of 341T>C and 481C>T. A similar effect was obtained with NAT2*5C (341T>A, 803A>G) (P < 0.0001; OR 0.11). For slow acetylator NAT2*7A 857G>A, GA and GA/AA were overrepresented in patients (P = 0.009 and 0.01; OR 2.74 and 2.72 respectively). NAT2*13 282C>T, NAT2*6B 590G>A, and NAT2*14A 191G>A were of comparable frequencies. NAT2 282C>A in combination with NAT2 857G>A (NAT2*7B) showed a synergistic effect in patients versus controls (P < 0.0001; OR 3.51). In conclusion, NAT2 gene polymorphism(s) with slow acetylator phenotype is generally associated with the risk of development of ALL in children.

The prognostic significance of minimal residual disease in adult Egyptian patients with precursor acute lymphoblastic leukemia.

BACKGROUND: Minimal residual disease (MRD) studies in adult acute lymphoblastic leukemia (ALL) give highly significant prognostic information superior to other standard criteria as age, gender and total leucocytic count (TLC) in distinguishing patients at high and low risk of relapse. OBJECTIVES: We aimed to determine the value of MRD monitoring by flowcytometry (FCM) in predicting outcome in adult Precursor ALL patients. PATIENTS AND METHODS: Bone marrow (BM) samples were analyzed by 4-color FCM collected at diagnosis and after induction therapy (MRD1) to correlate MRD positivity with disease free survival (DFS) and overall survival (OS). RESULTS: Study included 57 adult ALL patients (44 males and 13 females) with a median age of 22 years (18-49). DFS showed no significant difference with age, gender and initial TLC (p=0.838, 0.888 and 0.743, respectively). Cumulative DFS at 2 years was 34% for B-lineage ALL (n: 35) and 57% for T-lineage ALL (n: 18) (p = 0.057). Cumulative DFS at 2 years was 7% for MRD1 positive (high risk, HR) versus 57% for MRD1 negative patients (Low risk, LR) (p < 0.001). Cumulative DFS at 2 years was 29% for HR patients (n: 26) versus 55% for LR (n: 27) according to GMALL classification (p = 0.064). Cumulative OS did not differ according to age, gender and TLC (p = 0.526, 0.594 and 0.513, respectively). Cumulative OS at 2 years was 36% for B ALL (n: 39) versus 77% for TALL (n: 18) (p = 0.016) and was 49% for Philadelphia chromosome (Ph) negative patients versus 0% for Ph-positive patients (p < 0.001). Regarding MRD1, OS at 2 years was 18% for MRD1 HR (n: 17) versus 65% for MRD1 LR (n: 38) (p < 0.001). OS was 35% for high-risk patients (n: 30) and 62% for low-risk patients (n: 27) classified according to GMALL risk stratification (p = 0.017). CONCLUSION: MRD by FCM is a strong independent predictor of outcome in terms of DFS and OS and is a powerful informative parameter in guiding individual treatment in ALL patients.

Peripheral blood mammaglobin gene expression for diagnosis and prediction of metastasis in breast cancer patients.

AIM: To evaluate the value of peripheral blood mammaglobin (MG) gene expression for diagnosis and prediction of metastasis in breast cancer patients. METHODS: MG expression was detected by nested reverse-transcription polymerase chain reaction in the peripheral blood of 46 females (32 breast cancer, 12 benign breast lesions, 2 no breast abnormalities). In total 28 breast cancer patients were followed up through a period of 34 months for the development of metastasis. RESULTS: MG expression was detected in 16/32 (50%) breast cancer patients but not in patients with benign lesions or healthy participants. Five patients had metastasis at diagnosis. During the 34 months of follow up, five more MG-positive patients showed metastatic lesions and none of the MG negative patients who were followed up developed metastasis. CONCLUSION: The study suggests blood MG expression is a specific molecular marker for detection of occult mammary carcinoma cells of patients with operable breast cancer. It might be of value as a predictor of subsequent metastasis. Large-scale studies and longer follow-up periods are needed.

Rationale for an international consortium to study inherited genetic susceptibility to childhood acute lymphoblastic leukemia.

Acute lymphoblastic leukemia is the major pediatric cancer in developed countries. To date most association studies of acute lymphoblastic leukemia have been based on the candidate gene approach and have evaluated a restricted number of polymorphisms. Such studies have served to highlight difficulties in conducting statistically and methodologically rigorous investigations into acute lymphoblastic leukemia risk. Recent genome-wide association studies of childhood acute lymphoblastic leukemia have provided robust evidence that common variation at four genetic loci confers a modest increase in risk. The accumulated experience to date and relative lack of success of initial efforts to identify novel acute lymphoblastic leukemia predisposition loci emphasize the need for alternative study designs and methods. The International Childhood Acute Lymphoblastic Leukaemia Genetics Consortium includes 12 research groups in Europe, Asia, the Middle East and the Americas engaged in studying the genetics of acute lymphoblastic leukemia. The initial goal of this consortium is to identify and characterize low-penetrance susceptibility variants for acute lymphoblastic leukemia through association-based analyses. Efforts to develop genome-wide association studies of acute lymphoblastic leukemia, in terms of both sample size and single nucleotide polymorphism coverage, and to increase the number of single nucleotide polymorphisms taken forward to large-scale replication should lead to the identification of additional novel risk variants for acute lymphoblastic leukemia. Ethnic differences in the risk of acute lymphoblastic leukemia are well recognized and thus in assessing the interplay between inherited and non-genetic risk factors, analyses using different population cohorts with different incidence rates are likely to be highly informative. Given that the frequency of many acute lymphoblastic leukemia subgroups is small, identifying differential effects will realistically only be possible through multi-center pooled analyses. Here, we review the rationale for identifying genetic risk variants for acute lymphoblastic leukemia and our proposed strategy for establishing the International Childhood Acute Lymphoblastic Leukaemia Genetics Consortium.

Prognostic utility of routine chimerism testing at 2 to 6 months after allogeneic hematopoietic cell transplantation.

The utility of routine chimerism analysis as a prognostic indicator of subsequent outcomes after allogeneic hematopoietic cell transplantation (HCT) with myeloablative conditioning regimens remains controversial. To address this controversy, routine chimerism test results at 2 to 6 months after HCT with myeloablative conditioning regimens were evaluated for association with subsequent risk of chronic graft-versus-host disease (GVHD), nonrelapse mortality (NRM), relapse, and overall mortality. Only 70 of 1304 patients (5%) had < 95% donor-derived cells in the marrow. Low donor chimerism in the marrow occurred more often in patients with low-risk diseases compared with those with higher-risk diseases and was significantly associated with a reduced risk of chronic GVHD. Among 673 patients evaluated, 164 (24%) had < 85% donor-derived T cells in the blood. Low donor T cell chimerism was more frequent in patients with low-risk diseases compared with those with higher-risk diseases, in those who received conditioning with busulfan compared with those who received conditioning with total body irradiation, and in those with lower-grade acute GVHD. Low donor T cell chimerism in the blood was significantly associated with a reduced risk of chronic GVHD but not with a reduced risk of relapse, NRM, or overall mortality. Routine testing of chimerism in the marrow and blood at 2 to 6 months after HCT with myeloablative conditioning regimens may be helpful in documenting engraftment in clinical trials, but provides only limited prognostic information in clinical practice.

Synergistic effect of methyltetrahydrofolate reductase (MTHFR) C677T and A1298C polymorphism as risk modifiers of pediatric acute lymphoblastic leukemia.

BACKGROUND AND PURPOSE: ALL is the most common pediatric cancer. The causes of the majority of pediatric acute leukemia are unknown and are likely to involve an interaction between genetic and environmental factors. Therefore, unfavourable gene-environmental interactions might be involved in the genesis of ALL. The aim of this work was to evaluate, in a case-control study, whether the common polymorphisms in 5, 10-methylenetetrahydrofolate reductase (MTHFR) namely (C677T and A1298C) and methionine synthase (MS) (A2756G) genes may play a role in altering susceptibility to pediatric ALL as individual genes and in combination. PATIENTS AND METHODS: DNA of 88 ALL patients (age < or = 18 years) and 311 healthy control subjects was analyzed for the polymorphisms of MTHFR and MS genes using PCR-RFLP method. RESULTS: The frequencies of the wild types of MTHFR 677CC, MTHFR 1298AA and MS 2756AA, the homozygous genotypes of MTHFR 677TT, MTHFR 1298CC and MS 2756GG and heterozygous genotypes of MTHFR 677CT and MS 2756AG showed no statistically significant differences between patients and controls. The frequency of the MTHFR 1298AC heterozygous genotype was 25% among patients compared to 45.0% among controls; the difference was found to be statistically significant (p value =0.001, O.R=0.382 & 95% C.I=0.222-0.658). The frequency of the MTHFR1298AC heterozygous genotype plus 1298CC homozygous genotype was 34% among patients compared to 54.3% among controls and the difference was statistically significant (p value =0.001). A synergistic effect of 677CT and1298AC (CTAC) was observed, (p value=0.002) with 3.65 fold protection (OR 0.273 & 95% C.I=0.155-0.9) compared to 2.6 folds for MTHFR 1298AC alone. This protective effect of CTAC polymorphism was abolished when combined with MS 2756AA or AG. CONCLUSION: The present study provided further evidence for the protective role of MTHFR 1298AC mutant alleles in acute lymphoblastic leukemia in children (2.6 fold protection). This suggests that folate and methionine metabolism play an important role in the pathogenesis of pediatric ALL. In contrast to the main bulk of literature, we did not find any protective role of either MTHFR C677T or MS A2756G polymorphisms. This may reflect the ethnic variation in both the polymorphism frequencies, variation in plasma level of folate, in addition to the possible role of gene-environment interaction mainly dietary availability of folate. The synergistic effect of MTHFR 1298AC and 677CT and its abolishment by MS 2756AA or AG further emphasizes that the interaction of genes, rather than the polymorphism in any single one, determines risk susceptibility to disease.

Prevalence of anti human herpes virus-6 IgG and its receptor in acute leukemia (membrane cofactor protein: MCP, CD46).

BACKGROUND: CD46 is a membrane cofactor protein, which acts as a cofactor for factor I proteolytic cleavage of C3, so it protects the cells expressing it on their surface from autologous complement attack. It has been recently described as a receptor for HHV-6. Also, it has been shown to be highly expressed on malignant cells as compared to normal cells, thus playing a major role by which these cells, either cells of haematological malignancy or cells of other body cancers, can protect themselves against complement attack so they can survive and metastasize. PATIENTS AND METHODS: This study has been done to detect the seroprevalence of HHV-6 among 47 Egyptian adult cases of acute leukemia using the anti-HHV-6 IgG ELISA serological technique. CD46 receptor expression and immunophenotyping technique were performed using FCM. Twenty nine of the cases were ANLL, while 18 were ALL cases. Sixteen age- and sex-matched control cases were also studied for both anti-HHV-6 IgG and CD46 receptor expression. RESULTS: HHV-6 IgG antibodies were encountered in 29 (100%), 14 (77.8%) and 12 (75%) of the ANLL, ALL and the control group, cases, respectively. CD46 expression was encountered in 21 (72.4%) of the ANLL cases and in 10 (55.6%) of the ALL cases. Concordance between HHV- 6 seropositivity and CD46 expression was encountered in 31 cases (29 positive and 2 negative). Disconcordance was encountered in 16 cases with 14 showing HHV-6 IgG seropositivity with no CD46 expression and 2 showing the reverse. CONCLUSION: The lack of significant correlation between CD46 expression and seropositivity would exclude CD46 expression as a cause of contracting HHV-6 infection in leukemic patients.

P-gp expression and Rh 123 efflux assay have no impact on survival in Egyptian pediatric acute lymphoblastic leukemia patients.

PURPOSE: In a previous work we have studied MDR status in terms of P-glycoprotein (P-gp) expression and Rhodamine 123 efflux assay in Egyptian acute leukemia patients. We have reported results comparable to the literature as regards ANLL both in pediatric and adult cases. However, higher figures were encountered for the functional assay in ALL. As our ALL cases especially in pediatric age group show worse prognosis compared to literature, we hypothesized that the higher percentage of cases with positive Rh123 efflux assay might be a contributing factor. MATERIAL AND METHODS: A total of 108 cases were studied including 80 ALL and 28 ANLL. ALL cases included 48 male and 32 female with an age range of 6m to 18 yrs and a median of 7 yrs. ANLL cases included 18 male and 10 female with an age range of 6m to 18 yrs and a median of 8 yrs. P-gp expression was evaluated using 4E3 and UIC2 mAb, analyzed by Coulter XL flow cytometer and expressed as a ratio at a cut off of >or= 1.1 and/or >or= 5% positive cells. For the evaluation of MDR function Rh123 efflux assay using cyclosporine as a blocker and expressed as a ratio at a cutoff of >or= 1.1 and/or >or= 10% positive cells was performed. MDR expression and function were correlated to age, Hb, TLC, CD34 expression, immunophenotype and DNA index in ALL, FAB subtypes in ANLL as well as to CR, DFS and EFS in ALL. RESULTS: In ALL, P-gp expression was encountered in 26.4% of cases. Positive Rh efflux was reported in 61.5%. No correlation was encountered between neither expression nor functional assay with age, Hb, TLC, CD34 expression or immunophenotype. CR was achieved in 89.8%; neither P-gp expression nor Rh123 efflux had an impact on CR except for Rh123 efflux in T-ALL where a cutoff of 1.25 could predict CR at a total accuracy of 70.6%. DFS was 92.3% while EFS was 72.2% for the whole group. No significant difference was encountered neither between cases expressing or lacking P-gp nor between cases with negative or positive Rh123 efflux assay. In ANLL P-gp expression was encountered in 47.6% of cases, while positive Rh123 efflux assay was encountered in 75% of cases. No correlation as encountered between neither expression nor Rh123 efflux assay and neither age, Hb, TLC, CD34 expression nor FAB subtypes. CONCLUSION: Neither P-gp expression nor Rh123 efflux assay has any impact on survival in pediatric ALL. Rh123 ratio of 1.25 is predictive of CR in TALL.