Sinem Firtina

Deregulated WNT signaling in childhood T-cell acute lymphoblastic leukemia

WNT signaling has been implicated in the regulation of hematopoietic stem cells and plays an important role during T-cell development in thymus. Here we investigated WNT pathway activation in childhood T-cell acute lymphoblastic leukemia (T-ALL) patients. To evaluate the potential role of WNT signaling in T-cell leukomogenesis, we performed expression analysis of key components of WNT pathway. More than 85% of the childhood T-ALL patients showed upregulated β-catenin expression at the protein level compared with normal human thymocytes. The impact of this upregulation was reflected in high expression of known target genes (AXIN2, c-MYC, TCF1 and LEF). Especially AXIN2, the universal target gene of WNT pathway, was upregulated at both mRNA and protein levels in ∼40% of the patients. When β-CATENIN gene was silenced by small interfering RNA, the cancer cells showed higher rates of apoptosis. These results demonstrate that abnormal WNT signaling activation occurs in a significant fraction of human T-ALL cases independent of known T-ALL risk factors. We conclude that deregulated WNT signaling is a novel oncogenic event in childhood T-ALL.

Inflammatory hypothesis as a link between Alzheimer's disease and diabetes mellitus

The aim of the present study was to evaluate whether there was an inflammation‐mediated link between Alzheimers disease (AD) and type 2 diabetes mellitus (DM) status.

Comparison of plasma viscosity as a marker of endothelial dysfunction with nitric oxide and asymmetric dimethylarginine in subjects with dyslipidemia.

OBJECTIVE In this study, we aimed to investigate the alterations in plasma viscosity and whether there was a relationship between plasma viscosity and endothelial dysfunction markers such as nitric oxide (NOx), asymmetric dimethylarginine (ADMA) and oxidized Low Density Lipoprotein (oxLDL) in dyslipidemic subjects. MATERIAL AND METHOD 35 subjects with normolipidemia and 30 subjects with hyperlipidemia were involved in this study. Dyslipidemia was defined as total cholesterol levels ≥200 mg/dL and/or triglyceride level ≥150 mg/dL. Plasma total cholesterol, high density lipoprotein cholesterol, low density lipoprotein cholesterol, triglycerides, total protein, albumin levels and NOx were determined by enzymatic methods using commercial kits. Plasma ADMA concentrations and serum levels of total oxLDL were determined by ELISA. Plasma viscosity was measured by Harkness capillary viscometer. RESULT Plasma viscosity, ADMA and oxLDL values were significantly higher in subjects with dyslipidemia than in subjects with normolipidemia. Plasma NOx concentration was decreased in dyslipidemic subjects compared to the normo-lipidemic subjects. We found that fibrinogen had no effect upon plasma viscosity in selected patients with dyslipidemia. CONCLUSION Our results demonstrated that the rheological impairment of dyslipidemic patients was related with endothelial dysfunction and this was a possible cause of both micro and macrovascular complications. Therefore, as plasma viscosity is also a sensitive parameter, it can add useful information about the diagnosis and treatment of various disorders, and it should be utilized more frequently in clinical medicine.

A Possible Role for WNT5A Hypermethylation in Pediatric Acute Lymphoblastic Leukemia.

Objective: WNT5A is one of the most studied noncanonical WNT ligands and is shown to be deregulated in different tumor types. Our aim was to clarify whether hypermethylation might be the cause of low WNT5A mRNA levels and whether we could restore this downregulation by reversing the event. Materials and Methods: The expression of WNT5A mRNA was studied in a large acute lymphoblastic leukemia (ALL) patient group (n=86) by quantitative real-time PCR. The methylation status was detected by methylation-specific PCR (MSPCR) and bisulphate sequencing. In order to determine whether methylation has a direct effect on WNT5A expression, disease-representative cell lines were treated by 5’-aza-20-deoxycytidine. Results: Here we designed a validation experiment of the WNT5A gene, which was previously examined and found to be differentially expressed by microarray study in 31 T-cell ALL patients. The expression levels were confirmed by quantitative real-time PCR and the expression levels were significantly lower in T-cell ALL patients than in control thymic subsets (p=0.007). MSPCR revealed that 86% of the patients were hypermethylated in the WNT5A promoter region. Jurkat and RPMI cell lines were treated with 5’-aza-20-deoxycytidine and WNT5A mRNA expression was restored after treatment. Conclusion: According to our results, WNT5A hypermethylation does occur in ALL patients and it has a direct effect on mRNA expression. Our findings show that epigenetic changes of WNT signaling can play a role in ALL pathogenesis and reversing methylation might be useful as a possible treatment of leukemia.

Evaluation of PAX5 gene in the early stages of leukemic B cells in the childhood B cell acute lymphoblastic leukemia.

B-lineage acute lymphoblastic leukemia (B-ALL) is a common subtype of acute leukemia in children. PAX5 plays a central role in B-cell development and differentiation. In this study, we analyzed PAX5 expression levels, transactivation domain mutations/deletions in B-ALL patients (n=115) and healthy controls (n=10). Relative PAX5 mRNA levels were significantly increased in B-ALL patients (p<0.0001). PAX5 expression was also evaluated in three different B-ALL subgroups (pro B, Common B and Pre B ALL) and showed stage specific expression levels. Pro B (p=0.04) and pre B (p=0.04) patients showed significantly high PAX5 mRNA levels compared to stage specific controls. At least one deletion of exons 7-8 or 9 has been identified in the 41% of the patients. CD34 positivity in patients and presence of large deletions (Δ7/8/9) showed a significant correlation (p=0.05). None of our patients showed PAX5 point mutations, but two previously identified SNPs (rs3780135 and rs35469494) were detected. Our results support that PAX5 is a critical factor in B-ALL development and aberrant PAX5 expression especially at early stages may leads to leukemic transformation.

Germline mutations of aryl hydrocarbon receptor-interacting protein (AIP) gene and somatostatin receptor 1–5 and AIP immunostaining in patients with sporadic acromegaly with poor versus good response to somatostatin analogues

ObjectiveTo determine aryl hydrocarbon interacting protein (AIP) gene variations and AIP and somatostatin receptor (SSTR) 1–5 immunostaining in patients with apparently sporadic acromegaly with poor versus good response to somatostatin analogues (SRLs).MethodsA total of 94 patients (66 with poor and 28 with good response to SRLs) were screened for the AIP gene variations using Sanger sequencing. Immunostaining was performed in 60 tumors.ResultsSeveral variations, albeit some with undetermined significance, were detected, especially in poor responder patients. The prevalence of AIP mutation was 2.1% in the whole group and 1.5% in patients with poor response to SRLs. AIP, SSTR2A, and SSTR2B immunostainings were decreased in patients with poor response (p < 0.05 for all), and other SSTRs did not differ between the groups (p > 0.05 for all). Patients with low AIP had decreased levels of SSTR2A and SSTR3 (p < 0.05 for all). AIP and SSTR2A immunostainings were positively correlated to the treatment response and age at diagnosis was negatively correlated (p < 0.05 for all). In poor responder patients with high SSTR2A immunostaining, SSTR2B immunostaining and preoperative tumor size were positively and negatively correlated, respectively, to SRL response (p < 0.05 for all).ConclusionsLack of response to SRLs does not necessarily increase the risk of harboring AIP mutations. The finding of decreased AIP, SSTR2A, and SSTR2B immunostaining in patients with poor response to SRLs and decreased SSTR2A and SSTR3 level in those with low AIP immunostaining suggests a possible interaction between AIP and some SSTR subtypes that might alter SRL sensitivity.

Deep sequencing of BCR-ABL1 kinase domain mutations in chronic myeloid leukemia patients with resistance to tyrosine kinase inhibitors

Abstract Tyrosine kinase inhibitor (TKI) therapy is the current treatment of choice for patients with chronic phase chronic myeloid leukemia (CML) leading to rapid and durable hematological as well as molecular responses. However, emergence of resistance to TKIs has been the major obstacle to treatment success on long term. In this regard kinase domain mutations are the most common mechanism of therapy failure. In this study, we analyzed peripheral blood samples from 17 CML patients who had developed resistance to various TKIs by using next-generation sequencing parallel to Sanger sequencing. BCR-ABL1 kinase domain mutations have been found in 59% of the cohort. Our results demonstrate that next-generation sequencing results in a higher mutational detection rate than reported with conventional sequencing methodology. Furthermore, it showed the clonal diversity more accurately.

Dysregulation of the DKK1 gene in pediatric B-cell acute lymphoblastic leukemia

BACKGROUND/AIM The canonical Wingless-type (WNT) pathway is involved in normal hematopoietic cell development and deregulated WNT signaling is implicated in the development of hematological malignancies. Dickkopf 1 (DKK1) acts as a modulator of the β-catenin regulated canonical pathway. Activation of DKK1 leads to apoptosis and growth suppression, whereas silencing by promoter hypermethylation results in abnormal WNT activation. The secreted inhibitor Dickkopf can antagonize WNT signaling by competitively binding to low density lipoprotein receptors (LRPs) 5 and 6. MATERIALS AND METHODS We studied DKK1 gene promoter methylation and investigated DKK1, β-catenin, LRP5, and LRP6 mRNA levels in B-cell acute lymphoblastic leukemia (B-ALL) patients (n = 90). Methylation-specific PCR and bisulfite sequencing were used for methylation profiling and quantitative real-time PCR was used for mRNA detection. RESULTS The DKK1 gene was examined for its promoter methylation and only 33% of patients were found methylated. On the other hand, B-ALL cases showed high expression of DKK1 (P = 0.01), LRP5 (P = 0.04), and LRP6 (P = 0.02) compared to normal bone marrow cells. CONCLUSION DKK1 methylation exists in some of cases but is not sufficient for WNT pathway activation alone in pediatric B-ALL.

A novel pathogenic frameshift variant of CD3E gene in two T-B+ NK+ SCID patients from Turkey

Severe combined immunodeficiency (SCID) is the most severe form of primary immunodeficiency, which is characterized by the dysfunction and/or absence of T lymphocytes. Early diagnosis of SCID is crucial for overall survival, and if it remains untreated, SCID is often fatal. Next-generation sequencing (NGS) has become a rapid, high-throughput technology, and has already been proven to be beneficial in medical diagnostics. In this study, a targeted NGS panel was developed to identify the genetic variations of SCID by using SmartChip-TE technology, and a novel pathogenic frameshift variant was found in the CD3E gene. Sanger sequencing has confirmed the segregation of the variant among patients. We found a novel deletion in the CD3E gene (NM000733.3:p.L58Hfs*9) in two T-B+ NK+ patients. The variant was not found in the databases of dbSNP, ExAC, and 1000G. One sibling in family I was homozygous and the rest of the family members were heterozygous for this variant. T cell receptor excision circle (TREC) and kappa-deleting recombination excision circle (KREC) analyses were performed for T and B cell maturation. TRECs were not detected in both patients and the KREC copy numbers were similar to the other family members. In addition, heterozygous family members showed decreased TREC levels when compared with the wild-type sibling, indicating that carrying this variant in one allele does not cause immunodeficiency, but does effect T cell proliferation. Here, we report a novel pathogenic frameshift variant in CD3E gene by using targeted NGS panel.

Comparing Oxidative Stress Markers and S100B, Aβ-40 Proteins as Independent Neurological Markers in Distinguishing the Relation of Alzheimer's Disease and Diabetes Mellitus

Objective: This study compares biomarkers of oxidative stress and neurological in patients of Alzheimers disease (AD) and Diabetes Mellitus (DM) to evaluate mechanisms of oxidative stress and inflammation in the pathogenesis of AD. Methods: Serum concentrations of 3-nitrotyrosine, Isoprostane, Protein Carbonyl, Malondialdehyde, Nitric Oxide, Asymmetrical Dimethyl-L-Arginine, Glutathione, and Homocysteine (as oxidative stress markers); adiponectin, fetuin A, insulin, soluble receptor of advanced glycation end products (sRAGE), IGF-I and Amylin (Insulin Resistance (IR) markers), serum amyloidβ (Aβ) and S100B protein (as neurological markers) were measured in controls, Type 2DM with or without AD, patients with AD with or without DM. Results: Even though levels of Aβ-40 was significantly higher in DM -OAD+ AD patients than DM-OAD patients (p<0.001) in AD-CEI and AD-CEI+DM groups no significant difference was observed; suggesting that higher Aβ-40 levels can be used in diagnosing the diabetes-related Alzhimer’s disease. S100B, on the other hand, was higher significantly in all AD groups (AD, AD-CEI, AD+DM-OAD) compared to Control and to solely DM group without AD (p<0.001 for control and DM respectively) which suggest that S100B can be used as a neurological biomarker independent from DM and can be used in monitoring the progression of AD. Also fetuin-A can be used in monitoring the progression and adiponectin for determining the onset of AD. Conclusions: By regulating the glycemia and IR, oxidative stress can be controlled and development of Alzheimers disease in diabetic patients can be slowed down.