Ghada I. Mossallam

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.

Can Glypican3 be Diagnostic for Early Hepatocellular Carcinoma among Egyptian Patients

BACKGROUNDnBecause of the high prevalence of hepatocellular carcinoma (HCC) in Egypt, new markers with better diagnostic performance than alpha-feto protein (AFP) are needed to help in early diagnosis. The aim of this work was to compare the clinical utility of both serum and mRNA glypican3 (GPC3) as probable diagnostic markers for HCC among Egyptian patients.nnnMATERIALS AND METHODSnA total of 60 subjects, including 40 with HCC, 10 with cirrhosis and 10 normal controls were analyzed for serum GPC3 (sGPC3) by ELISA. GPC-3 mRNA from circulating peripheral blood mononuclear cells was amplified by RT-PCR. Both markers were compared to some prognostic factors of HCC, and sensitivity of both techniques was compared.nnnRESULTSnSerum glypican-3 and AFP were significantly higher in the HCC group compared to cirrhotic and normal controls (p<0.001). Sensitivity and specificity were (95% each) for sGlypican-3, (82.5% and 85%) for AFP, and (100% and 90%) for Glypican3 mRNA , and (80% and 95%) for double combination between sGPC3 and AFP respectively.nnnCONCLUSIONnBoth serum GPC-3 and GPC-3mRNA are promising diagnostic markers for early detection of HCC in Egyptian patients. RT- PCR proved to be more sensitive (100%) than ELISA (95%) in detecting glypican3.

Comparing prothrombin induced by vitamin K absence-II (PIVKA-II) with the oncofetal proteins glypican-3, Alpha feto protein and carcinoembryonic antigen in diagnosing hepatocellular carcinoma among Egyptian patients.

BACKGROUNDnHepatocellular carcinoma (HCC) is usually asymptomatic in the early stage and does not show elevated alpha-feto protein (AFP). AFP shows 60-80% sensitivity in diagnosing HCC. Glypican3 (GPC-3) is an oncofetal protein that is only detected in HCC cells but not in benign liver tissues, while Carcinoembryonic antigen (CEA) is expressed in various neoplasms including HCC. Although, it is not specific for HCC. Prothrombin induced by vitamin K absence-II (PIVKA-II) is an abnormal prothrombin protein that is increased in the serum of HCC patients. It has higher sensitivity and specificity compared to AFP. The aim of this study is to compare the clinical utility of PIVKA-II with GPC-3, AFP and CEA in diagnosing HCC.nnnPATIENTS AND METHODSnThis study included 40 patients with HCC, 10 patients with cirrhosis as a benign control group, and 10 apparently healthy volunteers as normal controls. Serum samples were subjected to routine laboratory investigations, measurement of CEA, AFP using MEIA technique (Axsym), glypican3, and PIVKA-II using ELISA technique in the sera of all patients and controls.nnnRESULTSnAll markers showed the highest results in the HCC group. Higher concentrations of PIVKA-II were detected in patients with splenomegaly, and in tumors with size (>3cm). Combination of Glypican-3 and PIVKA-II showed the highest sensitivity, while GPC-3 alone and combination of GPC-3 and AFP showed the highest specificity to differentiate HCC from liver cirrhosis and normal controls. GPC-3, PIVKAII, and combination of both showed the highest sensitivity, while GPC-3 alone showed the highest specificity to differentiate HCC from liver cirrhosis.nnnCONCLUSIONnGlypican-3 is the only oncofetal antigen that showed comparable high diagnostic accuracy as PIVKA-II in diagnosing HCC among Egyptian patients.

CTLA-4 polymorphism and clinical outcome post allogeneic hematopoietic stem cell transplantation.

CTLA-4 inhibitory molecule plays an important role in regulating T cell activation. It is considered a crucial element in keeping the immune balance and has been implicated in cancer, autoimmunity and transplantation immunology. Inconsistent observations are reported regarding its association with hematopoietic stem cell transplantation (HSCT). Genotyping of CTLA-4 was performed in recipients and their HLA-matched donors for +49A/G and CT60 polymorphisms (80 and 94 pairs, respectively) using PCR-RFLP. No association was encountered between both polymorphisms in patients and donors and acute or chronic graft versus host disease. Significant association was observed between recipient +49A/G G allele and lower disease-free survival and overall survival compared to AA genotype (HR: 2.17, p = 0.03, 95% CI: 1.05-4.48 and HR: 2.54, p = 0.01, 95% CI: 1.16-5.54), respectively. Our results suggest that CTLA-4 genotyping may predict outcome in patients post HSCT. To validate our results, further studies on a larger cohort are needed.

Prognostic significance of WT1 expression at diagnosis and end of induction in Egyptian adult acute myeloid leukemia patients

Abstract Background Wilms tumor (WT1) gene overexpression has been reported in the majority of acute myeloid leukemia (AML) patients at diagnosis and has been evaluated as prognostic and minimal residual disease (MRD) marker. Patients and methods WT1 overexpression was evaluated in 68 adult AML patients at diagnosis and at the end of induction using quantitative real-time polymerase chain reaction (PCR). Results No significant associations were encountered between WT1 overexpression at diagnosis and other prognostic factors. Complete remission (CR) was achieved in 74% of the patients with WT1 overexpresssion compared to 80% of patients with normal levels (P = 0.5). No significant associations were encountered between WT1 overexpression at diagnosis and disease-free survival (DFS) or overall survival (OS) (P = 0.6 and 0.3, respectively). At the end of induction, the median duration of DFS in patients achieving ≥2 log reduction was not reached compared to only 5 months (range: 2.1–7.9 months) in those attaining <2 log reduction (P = 0.2). The median duration of OS in patients achieving ≥2 log reduction was 13 months (range: 0–33.3 months) compared to 7.5 months (5.4–9.6 months) in those attaining <2 log reduction (P = 0.2). The survival at 1 year in patients achieving ≥2 log was double the group with <2 log reduction (67% compared to 33%). Conclusion Our results, although not reaching the level of significance, probably due to the small sample size, still suggest that the early assessment of WT1 transcript level at the end of induction in patients in CR may have a prognostic significance on clinical outcome and may thus be a useful marker for risk stratification especially in patients lacking disease-specific marker.

HLA-E polymorphism and clinical outcome after allogeneic hematopoietic stem cell transplantation in Egyptian patients.

UNLABELLEDnHuman leukocyte antigen-E (HLA)-E in a non-classical major histocompatibility complex (MHC) class I (Ib) molecule. HLA-E-peptide complex acts as a ligand for natural killer (NK) cells and CD8+ T lymphocytes playing a dual role in natural and acquired immune responses. The difference in expression levels between HLA-E alleles was suggested to have impact on transplantation outcome. The aim of the study is to evaluate the clinical effect of HLA-E alleles on transplantation in a group of Egyptian patients. HLA-E genotyping was analyzed in eighty-eight recipients of stem cell transplantation using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). HLA-E*01:03 allele showed a trend towards lower cumulative incidence of relapse at 2 years compared to homozygous HLA-E*01:01 genotype (8% versus 21.5%, p=0.09, HR: 0.30, 95% CI: 0.91-1.69). HLA-E was the only factor showing near significant association with relapse incidence. HLA-E polymorphism did not affect the cumulative incidence of acute GVHD grades II-IV at 100 days, the 2-year cumulative incidence of extensive chronic GVHD, transplant related mortality (TRM) or overall survival (OS).nnnCONCLUSIONnthe suggested association of HLA-E polymorphism with reduced risk of relapse needs verification in a larger cohort. However, its proposed role in GVL helps better understanding of alloreactivity of T cells and NK cells and their implication in immunotherapy post allogeneic hematopoietic stem cell transplantation.

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 (pu202f=u202f.002). A trend towards reduced incidence of relapse was also observed with more than two KIR B motifs (pu202f=u202f.09), whereas a significant increased relapse was associated with homozygous HLA-C2 ligand compared to combined C1/C2 and C1/C1 (pu202f=u202f.04). Activating KIR2DS3 was associated with rapid leukocyte engraftment (pu202f=u202f.02). While, KIR 2DL5 was associated with decreased CMV infection (pu202f=u202f.03) and better platelets engraftment (pu202f=u202f.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.

HLA Class I in Egyptian patients with Behçet’s disease: new association with susceptibility, protection, presentation and severity of manifestations

ABSTRACT Introduction: Behçet’s disease is an autoimmune disease with diverse clinical manifestations with vasculitis being the hallmark of the disease. The aim of this work is to study the genetic association between human leukocyte antigen (HLA) class-I molecules of Egyptians with Behçet’s disease and the disease susceptibility and clinical patterns. Methods: Fifty-seven patients diagnosed with Behçet’s disease according to the 1990 International Study Group (ISG) criteria for Behçet’s disease coming from Egyptian origin up to the third grandfather were included in the study. Healthy controls were taken from HLA Class-I case control studies in Egyptian population yielding a pool of 221 healthy controls. HLA Class-I typing for patients was done using Reverse Sequence specific oligonucleotide probes (rSSO). Results: Male patients represented 89% of the sample. Mean age of onset was 25.81 (± 6.7) years and mean disease duration was 9.47 (± 7.4) years. Behçet’s disease was associated with HLA-A*24 and HLA-B*42 (p = 0.001) and highly associated with HLA-A*68 and B*15 and B*51 (p < 0.001). While HLA A*03 and B*52 were protective for Behçet’s (p = 0.002 and 0.007). Interestingly, HLA-B*51 and HLA-A*68 (p = 0.005 and 0.023) were associated with the blinding eye disease. HLA-B*51 was protective from Neurological and vascular involvement (p = 0.005 and 0.032, respectively). Conclusion: Behçet’s disease is associated with HLA Class-I A*24, A*68 and B*15, B*42 and B*51 in Egyptian patients while A*03 and B*52 were found to be protective. Interestingly, HLA B*51 and A*68 could be considered as poor prognostic factor for eye involvement.

Report from the Killer-cell Immunoglobulin-like Receptors (KIR) component of the 17th International HLA and Immunogenetics Workshop

The goals of the KIR component of the 17th International HLA and Immunogenetics Workshop (IHIW) were to encourage and educate researchers to begin analyzing KIR at allelic resolution, and to survey the nature and extent of KIR allelic diversity across human populations. To represent worldwide diversity, we analyzed 1269 individuals from ten populations, focusing on the most polymorphic KIR genes, which express receptors having three immunoglobulin (Ig)-like domains (KIR3DL1/S1, KIR3DL2 and KIR3DL3). We identified 13 novel alleles of KIR3DL1/S1, 13 of KIR3DL2 and 18 of KIR3DL3. Previously identified alleles, corresponding to 33 alleles of KIR3DL1/S1, 38 of KIR3DL2, and 43 of KIR3DL3, represented over 90% of the observed allele frequencies for these genes. In total we observed 37 KIR3DL1/S1 allotypes, 40 for KIR3DL2 and 44 for KIR3DL3. As KIR allotype diversity can affect NK cell function, this demonstrates potential for high functional diversity worldwide. Allelic variation further diversifies KIR haplotypes. We determined KIR3DL3u202f∼u202fKIR3DL1/S1u202f∼u202fKIR3DL2 haplotypes from five of the studied populations, and observed multiple population-specific haplotypes in each. This included 234 distinct haplotypes in European Americans, 191 in Ugandans, 35 in Papuans, 95 in Egyptians and 86 in Spanish populations. For another 35 populations, encompassing 642,105 individuals we focused on KIR3DL2 and identified another 375 novel alleles, with approximately half of them observed in more than one individual. The KIR allelic level data gathered from this project represents the most comprehensive summary of global KIR allelic diversity to date, and continued analysis will improve understanding of KIR allelic polymorphism in global populations. Further, the wealth of new data gathered in the course of this workshop component highlights the value of collaborative, community-based efforts in immunogenetics research, exemplified by the IHIW.

Impact of CYP1A1, GSTP1 and XRCC1 genes polymorphisms on toxicity and response to chemotherapy in childhood acute lymphoblastic leukemia

BACKGROUNDnAcute lymphoblastic leukemia (ALL) is the most common childhood malignancy. The interindividual genetic variations in drug metabolizing enzymes and DNA repair genes influence the efficacy and toxicity of numerous chemotherapeutic drugs affecting the treatment outcome.nnnAIM OF THE WORKnThe aim of the study was to investigate the impact of drug metabolizing CYP1, GSTP1 and DNA repair (XRCC1) genes polymorphisms on the toxicity and response to chemotherapy in childhood ALL.nnnPATIENTS AND METHODOLOGYnNinety seven ALL pediatric patients were genotyped for CYP1A1, GSTP1 ILe105Val and XRCC1 Arg194Tryp single nucleotide polymorphisms (SNPs) using PCR-RFLP.nnnRESULTSnNo statistically significant differences were observed between the wild and variant (homozygous and heterozygous) genotypes of the polymorphisms studied in CYP1A1, GSTP1 or XRCC1 genes regarding age, total leukocyte count, immunophenotyping, cytogenetic or risk group. The SNPs in CYP1A1, GSTP1 and XRCC1 genes did not show significant association with complete remission (CR) rate, overall survival (OS) or event free survival (EFS). However, XRCC1 Arg194Trp SNP was associated with higher drug toxicity; carriers of variant genotypes (CT and TT) had a significantly higher frequency of myelosuppression compared to those with the wild CC genotype (21/43[48.8%]) compared to (14/54[25.9%]) (p=0.020). The analysis of the combined effect of studied SNPs did not show any significant association with patient outcome.nnnCONCLUSIONnOur study reported a significant association between the DNA repair gene polymorphism and myelosuppression in childhood ALL patients. Adjustment of the dose of chemotherapeutic agents according to XRCC1 Arg194Trp polymorphism may improve outcome in cases with risk of toxicity.