Muge Sayitoglu | Istanbul University (original) (raw)

Papers by Muge Sayitoglu

DergiPark (Istanbul University), Jul 4, 2022

Objective: Deregulated WNT signaling was reported in TALL and other cancers. AXIN2 is a negative ... more Objective: Deregulated WNT signaling was reported in TALL and other cancers. AXIN2 is a negative regulator of the active WNT signaling and AXIN2 gene variants were associated with increased cancer risk. In this study, we aimed to determine AXIN2 variations and compare with clinic features in TALL. Methods: Thirty-two diagnostic TALL patients were retrospectively enrolled in the study. Coding sites of the AXIN2 were amplified by PCR and then screened by denaturing highperformance liquid chromatography (dHPLC). Patients with differential chromatograms were evaluated by Sanger sequencing. Results: None of the patients had pathogenic AXIN2 variants. Besides that, AXIN2 polymorphisms, rs2240308/ rs1133683/ rs9915936 were detected in 14 (43.7%) TALL patients. Genotype distributions of the rs2240308 and rs1133683 variants in TALL group were significantly different from controls (rs2240308, GG/GA p=0.029; rs1133683, GG/GA p<0.0001) and G allele increased the overall risk of TALL compared to A allele in both polymorphisms. We did not observe any clinical differences between AXIN2 variant carriers or non-carriers. Conclusion: AXIN2 rs2240308 and rs1133683 variants revealed significant positive associations between susceptibility to TALL .

Mediterranean Journal of Hematology and Infectious Diseases, Jun 29, 2023

Acute lymphoblastic leukemia (ALL) is a malignant disease of hematopoietic stem cells. B cell ALL... more Acute lymphoblastic leukemia (ALL) is a malignant disease of hematopoietic stem cells. B cell ALL (B-ALL) is characterized by highly proliferative and poorly differentiated progenitor B cells in the bone marrow. Chromosomal rearrangements, aberrant cell signaling, and mutations lead to dysregulated cell cycle and clonal proliferation of abnormal B cell progenitors. In this study, we aimed to examine hot spot genetic variations in the RUNX1, IDH2, and IL2RA genes in a group of (n=52) pediatric BALL. Sanger sequencing results revealed a rare RUNX1 variant p.Leu148Gln in one BALL patient with disease recurrence. Additionally, common intronic variations rs12358961 and rs11256369 of IL2RA were determined in two patients. None of the patients had the IDH2 variant. RUNX1, IDH2, and IL2RA variations were rare events in ALL. This study detected a novel pathogenic RUNX1 variation in a patient with a poor prognosis. Examining prognostically important genetic anomalies of childhood lymphoblastic leukemia patients and the signaling pathway components will pilot more accurate prognosis estimations.

Expert Review of Respiratory Medicine, Aug 3, 2022

Blood, Nov 15, 2013

Introduction Amplicon deep-sequencing using second-generation sequencing technology is an innovat... more Introduction Amplicon deep-sequencing using second-generation sequencing technology is an innovative molecular diagnostic technique and enables a highly-sensitive detection of mutations. As an international consortium we had investigated previously the robustness, precision, and reproducibility of 454 amplicon next-generation sequencing (NGS) across 10 laboratories from 8 countries (Leukemia, 2011;25:1840-8). Aims In Phase II of the study, we established distinct working groups for various hematological malignancies, i.e. acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), and multiple myeloma. Currently, 27 laboratories from 13 countries are part of this research consortium. In total, 74 gene targets were selected by the working groups and amplicons were developed for a NGS deep-sequencing assay (454 Life Sciences, Branford, CT). A data analysis pipeline was developed to standardize mutation interpretation both for accessing raw data (Roche Amplicon Variant Analyzer, 454 Life Sciences) and variant interpretation (Sequence Pilot, JSI Medical Systems, Kippenheim, Germany). Results We will report on the design, standardization, quality control aspects, landscape of mutations, as well as the prognostic and predictive utility of this assay in a cohort of 8,867 cases. Overall, 1,146 primer sequences were designed and tested. In detail, for example in AML, 924 cases had been screened for CEBPA mutations. RUNX1 mutations were analyzed in 1,888 cases applying the deep-sequencing read counts to study the stability of such mutations at relapse and their utility as a biomarker to detect residual disease. Analyses of DNMT3A (n=1,041) were focused to perform landscape investigations and to address the prognostic relevance. Additionally, this working group is focusing on TET2, ASXL1, and TP53 analyses. A novel prognostic model is being developed allowing stratification of AML into prognostic subgroups based on molecular markers only. In ALL, 1,124 pediatric and adult cases have been screened, including 763 assays for TP53 mutations both at diagnosis and relapse of ALL. Pediatric and adult leukemia expert labs developed additional content to study the mutation incidence of other B and T lineage markers such as IKZF1, JAK2, IL7R, PAX5, EP300, LEF1, CRLF2, PHF6, WT1, JAK1, PTEN, AKT1, IL7R, NOTCH1, CREBBP, or FBXW7. Further, the molecular landscape of CLL is changing rapidly. As such, a separate working group focused on analyses including NOTCH1, SF3B1, MYD88, XPO1, FBXW7 and BIRC3. Currently, 922 cases were screened to investigate the range of mutational burden of NOTCH1 mutations for their prognostic relevance. In MDS, RUNX1 mutation analyses were performed in 977 cases. The prognostic relevance of TP53 mutations in MDS was assessed in additional 327 cases, including isolated deletions of chromosome 5q. Next, content was developed targeting genes of the cellular splicing component, e.g. SF3B1, SRSF2, U2AF1, and ZRSR2. In BCR-ABL1-negative MPN, nine genes of interest (JAK2, MPL, TET2, CBL, KRAS, EZH2, IDH1, IDH2, ASXL1) have been analyzed in a cohort of 155 primary myelofibrosis cases searching for novel somatic mutations and addressing their relevance for disease progression and leukemia transformation. Moreover, an assay was developed and applied to CMML cases allowing the simultaneous analysis of 25 leukemia-associated target genes in a single sequencing run using just 20 ng of starting DNA. Finally, nine laboratories are studying CML, applying ultra-deep sequencing of the BCR-ABL1 tyrosine kinase domain. Analyses were performed on 615 cases investigating the dynamics of expansion of mutated clones under various tyrosine kinase inhibitor therapies. Conclusion Molecular characterization of hematological malignancies today requires high diagnostic sensitivity and specificity. As part of the IRON-II study, a network of laboratories analyzed a variety of disease entities applying amplicon-based NGS assays. Importantly, the consortium not only standardized assay design for disease-specific panels, but also achieved consensus on a common data analysis pipeline for mutation interpretation. Distinct working groups have been forged to address scientific tasks and in total 8,867 cases had been analyzed thus far. Disclosures: Kohlmann: Roche Diagnostics: Honoraria; MLL Munich Leukemia Laboratory: Employment. Martinelli:Roche Diagnostics: Research Support Other. Alikian:Roche Diagnostics: Research Support Other. Auber:Roche Diagnostics: Research Support Other. Belickova:Roche Diagnostics: Research Support Other. Bronzini:Roche Diagnostics: Research Support Other. Cazzaniga:Roche Diagnostics: Research Support Other. Chiaretti:Roche Diagnostics: Research Support Other. Ernst:Roche Diagnostics: Research Support Other. Fuellgrabe:Roche Diagnostics: Research Support Other.…

Sabiad, Jun 29, 2020

Kodlama yapmayan küçük RNA'lar hücre farklılaşması, büyümesi, gelişmesi, immün reaksiyonlar, stre... more Kodlama yapmayan küçük RNA'lar hücre farklılaşması, büyümesi, gelişmesi, immün reaksiyonlar, stres adaptasyonu gibi fizyolojik süreçlerin yanı sıra, kanser, kalp hastalıkları gibi kompleks hastalıklarla da ilişkilendirilmiştir. MIR223, hematopoetik sisteme özgü bir miRNA' dır. T-hücreli Akut Lenfoblastik Lösemi (T-ALL) patogenezine katkıda bulunan miRNA'lar arasında yüksek anlatıma sahip olduğu ve onkomir olarak aktivite gösterdiği tespit edilmiştir. Bu çalışmada MIR223 geninin TALL hücre serilerinde alternatif bir yaklaşım olarak özgün kilitli nükleik asit (KNA) kullanılarak baskılanması ve gen sessizleştirme etkinliğinin gösterilmesi amaçlanmıştır. Gereç ve Yöntem: Kültüre edilen TALL hücre serilerine (Jurkat ve Molt4), 24 ve 48 saatlik sürelerde, 100 ve 150pmol konsantrasyonda MIR223' e özgü KNA uygulanmıştır. Her iki zaman aralığında RNA izolasyonu sonrası, stem loop polimeraz zincir reaksiyonu (PZR) ile cDNA sentezlenerek, kantitatif gerçek zamanlı PZR (QRT-PZR) ile miRNA anlatım düzeyleri belirlenmiştir. Bulgular: Her iki hücre serisinde de 24. saatte, 150pmol KNA, sadece transfeksiyon ajanı uygulanmış (Mock) hücrelerle karşılaştırıldığında MIR223 düzeyinin yüksek oranda baskılandığı gözlenmiştir (Jurkat %73, p=0,001 ve Molt4%80 p=0,04). Molt 4 hücre serisinde anlamlı düzeyde baskılanma 48. saatte devam etse de (p=0,005), Jurkat hücre serisinde 48. saatteki baskılama istatistiksel olarak anlamlı bulunmamıştır. Sonuç: MIR223 onkogenik etki gösteren bir miRNA olarak tanımlanmıştır ve antisens oligolar ile MIR223 geninin sessizleştirilmesi TALL gibi artmış MIR223 anlatımı gösteren kanserlerde hastalığın seyrini ve tedavi alternatiflerini araştırma imkanı sağlamaktadır. Bu çalışmada TALL hücrelerinde alternatif bir RNA interferans (sessizleştirme) uygulaması olarak KNA kullanılmıştır ve bu moleküllerin çok etkin ancak kısa süreli olarak kullanılabileceği görülmüştür. Anahtar Kelimeler: TALL , MIR223, kilitli nükleik asitler (KNA), gen sessizleştirmesi ABSTRACT Objective: Noncoding small RNAs play roles in physiological processes such as cell differentiation, growth, development, immune reactions, stress adaptation as well as complex diseases such as cancer and heart diseases. MIR223 is a specific miRNA to the hematopoietic system. Among the miRNAs associated with the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL), MIR223 was shown to have the highest expression level and functions as an oncomir. In this study we aimed to suppress MIR223 gene expression by using locked nucleic acid (LNA) in TALL cell lines as an alternative gene silencing technique and to show the silencing efficacy. Material and Methods: TALL cell lines (Jurkat and Molt4) were cultured and MIR223 specific LNA was applied in 100pmol and 150pmol concentrations for 24 hours and 48 hours. RNA was isolated from cells at both time points, followed by a stem loop polymerase chain reaction (PCR) for cDNA synthesis and the miRNA expression levels were evaluated by quantitative real time PCR. Results: In both cell lines a 150pmol application of LNA was compared to mock cells and the MIR223 expression was already suppressed at 24hours (73% in Jurkat (p=0,001) and 80%in Molt4 (p=0,04)). The suppression was continued at 48 hours in Molt4 cell line (p=0,005) where as there was no statistical significant difference in Jurkat at 48 hours. Conclusion: MIR223 has been identified as an oncomir and silencing the MIR223 with synthetic antisense oligos provide the opportunity to investigate the course and alternative therapy options of cancers with increased MIR223 expression, such as TALL. In this study LNA was used as an alternative RNA interference application in TALL cells and showed that LNA can effectively suppress gene expression when used in the short term.

Pediatric Blood & Cancer

The European Union‐funded COST Action (LEukaemia GENe Discovery by data sharing, mining, and coll... more The European Union‐funded COST Action (LEukaemia GENe Discovery by data sharing, mining, and collaboration) LEGEND was an international and multidisciplinary collaboration between clinicians and researchers that covered a range of aspects of genetic predisposition in childhood leukemia. Within this framework, we explored the perception and handling of genetic predisposition in the daily practice of European treatment centers. Herein, we present the results of our questionnaire‐based survey. We found that the overall awareness is quite high, and respondents remarked that identification and treatment of the most common predisposition syndromes were present. Nevertheless, high demand for continuous education and routinely updated resources remains.

DergiPark (Istanbul University), Jul 4, 2022

Objective: Deregulated WNT signaling was reported in TALL and other cancers. AXIN2 is a negative ... more Objective: Deregulated WNT signaling was reported in TALL and other cancers. AXIN2 is a negative regulator of the active WNT signaling and AXIN2 gene variants were associated with increased cancer risk. In this study, we aimed to determine AXIN2 variations and compare with clinic features in TALL. Methods: Thirty-two diagnostic TALL patients were retrospectively enrolled in the study. Coding sites of the AXIN2 were amplified by PCR and then screened by denaturing highperformance liquid chromatography (dHPLC). Patients with differential chromatograms were evaluated by Sanger sequencing. Results: None of the patients had pathogenic AXIN2 variants. Besides that, AXIN2 polymorphisms, rs2240308/ rs1133683/ rs9915936 were detected in 14 (43.7%) TALL patients. Genotype distributions of the rs2240308 and rs1133683 variants in TALL group were significantly different from controls (rs2240308, GG/GA p=0.029; rs1133683, GG/GA p<0.0001) and G allele increased the overall risk of TALL compared to A allele in both polymorphisms. We did not observe any clinical differences between AXIN2 variant carriers or non-carriers. Conclusion: AXIN2 rs2240308 and rs1133683 variants revealed significant positive associations between susceptibility to TALL .

Turkish Journal of Hematology, 2019

Angiotensin II promotes growth and angiogenesis via type 1 receptors (AGTR1) in certain tumors. I... more Angiotensin II promotes growth and angiogenesis via type 1 receptors (AGTR1) in certain tumors. In this study, we examine the bone marrow AGTR1 expression in multiple myeloma (MM) and its relationship with the regulation of angiogenesis and prognostic factors. Materials and Methods: Bone marrow AGTR1 mRNA levels of 39 MM patients and 15 healthy controls were analyzed with quantitative RT-PCR. Immunohistochemical staining of the tissue vascular endothelial growth factor (VEGF), CD34, and factor VIIIrAg (fVIIIrAg) was used to assess bone marrow angiogenesis. Results: Bone marrow samples of the patients showed increased VEGF, fVIIIrAg, and CD34 staining and higher AGTR1 expression levels when compared to controls. Patients with severe-diffuse bone marrow infiltration showed higher bone marrow VEGF, fVIIIrAg, CD34, and AGTR1 mRNA levels when compared to other patients. Conclusion: AGTR1 expression was found positively correlated with plasma β2-microglobulin level and patients with increased AGTR1 expression showed increased bone marrow CD34 levels.

Journal of Cancer Research and Clinical Oncology, 2003

Scandinavian Journal of Clinical and Laboratory Investigation

Case reports in hematology, 2011

The current treatment of chronic phase chronic myeloid leukemia (CML) consists of oral tyrosine k... more The current treatment of chronic phase chronic myeloid leukemia (CML) consists of oral tyrosine kinase inhibitors (TKIs). However, high-risk CML may present with an aggressive course which may result in blastic crisis or a "difficult-to-manage" state with available treatments. The aim of this paper is to report a patient with complicated CML resistant to treatment and progressed despite the administration of bosutinib, imatinib mesylate, nilotinib, dasatinib, interferon alpha 2a, cytotoxic chemotherapy, and allogeneic hematopoietic stem cell transplantation. The striking point of this case story is that no Abl kinase domain mutation against TKIs has been detected during this very complicated disease course of CML. Meanwhile, challenging cases will always be present despite the hope and progress in CML in the TKI era.

Turkiye Klinikleri Dahili Tıp Bilimleri Dergisi, 2007

Turkiye Klinikleri Hematoloji Ozel Dergisi, 2009

Turkish Journal of Medical Sciences, Feb 27, 2006

Polymorphisms in the genes encoding cytochrome p450 (CYP) and thiopurine S-methyl transferase (TP... more Polymorphisms in the genes encoding cytochrome p450 (CYP) and thiopurine S-methyl transferase (TPMT) enzymes catalyze the metabolic reactions of several drugs. These polymorphisms might be responsible for adverse drug reactions. Turkish population data for these genes still needs to be elucidated. We aimed to detect the allele frequencies of thiopurine S-methyl transferase (TPMT), cytochrome p4503A4*1B (CYP3A4*1B) and cytochrome p4503A4*3 (CYP3A5*3) gene variants in the Turkish population. We examined the TPMT (*1, *2, *3A, *3C), CYP3A4*1B and CYP3A5*3 variant allele frequencies in a group of healthy Turkish Caucasian blood donors by using PCR-RFLP, allele-specific PCR and direct sequencing techniques. The frequencies of four allelelic variants of TPMT gene, are *2 (238G>C)(2.0%), *3A (460G>A and 719A>G)(1.0%), *3B (460G>A)(0.0%) and *3C (719A>G) (1.4%). We observed CYP3A4*1B allele frequency in 1.4% and CYP3A5*3 allele frequency in 7.5% of our population. This study provides the first analysis of TPMT, CYP3A4*1B and CYP3A5*3 mutant allele frequencies in the Turkish population.

Experimed, 2021

Objective: Lymphoid enhancer-binding factor-1 (LEF1) is one of the key regulators of lymphocyte p... more Objective: Lymphoid enhancer-binding factor-1 (LEF1) is one of the key regulators of lymphocyte proliferation and its aberrant expression is a prognostic factor for lymphoid or myeloid malignancies. In this study, we focused on the expression of LEF1 isoforms in several hematological malignancies and found tissue-specific differential expression for the full-length (FL)-LEF1 gene and its tumor suppressor (∆LEF1) variant. Material and Method: Fifty-three leukemia/lymphoma patients were included in this study. Diagnostic samples of "lymphoid group" patients: Chronic Lymphoblastic Leukemia (CLL) (n=10), B-cell Acute Lymphoblastic Leukemia (B-ALL) (n=9) and "myeloid group" patients: Chronic Myeloblastic Leukemia (CML) (n=12), Acute Myeloid Leukemia (AML) (n=13), and Multiple Myeloma (MM) (n=9) were studied. Healthy bone marrow, peripheral blood cells, and CD34 positive cells were used as controls. Total (T) and FL-LEF1 transcript levels were examined by using quantitative real-time polymerase chain reaction (qRT-PCR). T and FL-LEF1 mRNA ratios were also evaluated for calculation of ∆LEF1. Results: LEF1 levels were significantly high in lymphoid malignancies, but MM and AML patients have decreased LEF1 levels. Although CLL patients have high FL-LEF1 levels, the ratio of the T/FL levels was significantly decreased. Conclusion: LEF1 is a proliferation factor for lymphocytes and not only its differential overexpression but also the ratio of T/FL isoforms seem to accompany leukemia progress.

Istanbul University Institute of Health Sciences Journal of Advanced Research in Health Sciences, 2020

PI3K/AKT sinyal yolağının bozulmuş aktivasyonu tümör gelişimi ile ilişkilendirilmiştir. PTEN/AKT ... more PI3K/AKT sinyal yolağının bozulmuş aktivasyonu tümör gelişimi ile ilişkilendirilmiştir. PTEN/AKT yolağı, hücre büyümesi ve sağ kalımında kritik role sahip olup, lösemilerde kemoterapi direnci ile ilişkilendirilmiştir. Bu çalışma kapsamında, çocukluk çağı TALL hastalarındaki PTEN ve AKT1 gen anlatımları belirlendi ve hastalığın prognozuna olan etkisi araştırıldı. Gereç ve Yöntem: Otuz dokuz çocukluk çağı TALL olgusunun, tanı zamanı kemik iliği örnekleri kantitatif gerçek zamanlı PZR ile PTEN ve AKT1 genleri için tarandı. Gen anlatım düzeylerinin hastaların yaş, cinsiyet, immünfenotip, tedavi yanıtı, nüks ve sağ kalım gibi klinik verileri ile ilişkisi incelendi. Bulgular: Çalışmamızda, TALL hastalarında PTEN gen anlatımında kontrollere göre bir farklılık gözlenmezken, AKT1 gen anlatımının anlamlı düzeyde arttığı belirlendi (p<0,0001). Hastaların klinik özellikleri, genel ve hastalıksız sağ kalım oranları ve gen anlatım düzeyleri arasında anlamlı bir ilişki saptanmazken, indüksiyon tedavi ilişkili sağ kalım analizleri PTEN ve AKT1 gen anlatımları ile genel sağ kalım oranları arasında (sırasıyla, p=0,026 ve p=0,034 Cox regresyon analizi) anlamlı bir ilişki ortaya koyuldu. Diğer klinik parametreler ile gen anlatım düzeyleri arasında anlamlı ilişki saptanmadı. Sonuç: Çalışmamızda, TALL örneklerinde PTEN gen anlatımından bağımsız olarak artmış AKT1 gen anlatımının varlığı ve sağ kalım sürelerini etkilediğine dair bulgular elde edildi. Gelecekteki çalışmalar, AKT1'in hedefe yönelik tedaviler için uygun bir aday olup olmayacağını ortaya çıkaracaktır.

DergiPark (Istanbul University), Jul 4, 2022

Objective: Deregulated WNT signaling was reported in TALL and other cancers. AXIN2 is a negative ... more Objective: Deregulated WNT signaling was reported in TALL and other cancers. AXIN2 is a negative regulator of the active WNT signaling and AXIN2 gene variants were associated with increased cancer risk. In this study, we aimed to determine AXIN2 variations and compare with clinic features in TALL. Methods: Thirty-two diagnostic TALL patients were retrospectively enrolled in the study. Coding sites of the AXIN2 were amplified by PCR and then screened by denaturing highperformance liquid chromatography (dHPLC). Patients with differential chromatograms were evaluated by Sanger sequencing. Results: None of the patients had pathogenic AXIN2 variants. Besides that, AXIN2 polymorphisms, rs2240308/ rs1133683/ rs9915936 were detected in 14 (43.7%) TALL patients. Genotype distributions of the rs2240308 and rs1133683 variants in TALL group were significantly different from controls (rs2240308, GG/GA p=0.029; rs1133683, GG/GA p<0.0001) and G allele increased the overall risk of TALL compared to A allele in both polymorphisms. We did not observe any clinical differences between AXIN2 variant carriers or non-carriers. Conclusion: AXIN2 rs2240308 and rs1133683 variants revealed significant positive associations between susceptibility to TALL .

Mediterranean Journal of Hematology and Infectious Diseases, Jun 29, 2023

Acute lymphoblastic leukemia (ALL) is a malignant disease of hematopoietic stem cells. B cell ALL... more Acute lymphoblastic leukemia (ALL) is a malignant disease of hematopoietic stem cells. B cell ALL (B-ALL) is characterized by highly proliferative and poorly differentiated progenitor B cells in the bone marrow. Chromosomal rearrangements, aberrant cell signaling, and mutations lead to dysregulated cell cycle and clonal proliferation of abnormal B cell progenitors. In this study, we aimed to examine hot spot genetic variations in the RUNX1, IDH2, and IL2RA genes in a group of (n=52) pediatric BALL. Sanger sequencing results revealed a rare RUNX1 variant p.Leu148Gln in one BALL patient with disease recurrence. Additionally, common intronic variations rs12358961 and rs11256369 of IL2RA were determined in two patients. None of the patients had the IDH2 variant. RUNX1, IDH2, and IL2RA variations were rare events in ALL. This study detected a novel pathogenic RUNX1 variation in a patient with a poor prognosis. Examining prognostically important genetic anomalies of childhood lymphoblastic leukemia patients and the signaling pathway components will pilot more accurate prognosis estimations.

Expert Review of Respiratory Medicine, Aug 3, 2022

Blood, Nov 15, 2013

Introduction Amplicon deep-sequencing using second-generation sequencing technology is an innovat... more Introduction Amplicon deep-sequencing using second-generation sequencing technology is an innovative molecular diagnostic technique and enables a highly-sensitive detection of mutations. As an international consortium we had investigated previously the robustness, precision, and reproducibility of 454 amplicon next-generation sequencing (NGS) across 10 laboratories from 8 countries (Leukemia, 2011;25:1840-8). Aims In Phase II of the study, we established distinct working groups for various hematological malignancies, i.e. acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), and multiple myeloma. Currently, 27 laboratories from 13 countries are part of this research consortium. In total, 74 gene targets were selected by the working groups and amplicons were developed for a NGS deep-sequencing assay (454 Life Sciences, Branford, CT). A data analysis pipeline was developed to standardize mutation interpretation both for accessing raw data (Roche Amplicon Variant Analyzer, 454 Life Sciences) and variant interpretation (Sequence Pilot, JSI Medical Systems, Kippenheim, Germany). Results We will report on the design, standardization, quality control aspects, landscape of mutations, as well as the prognostic and predictive utility of this assay in a cohort of 8,867 cases. Overall, 1,146 primer sequences were designed and tested. In detail, for example in AML, 924 cases had been screened for CEBPA mutations. RUNX1 mutations were analyzed in 1,888 cases applying the deep-sequencing read counts to study the stability of such mutations at relapse and their utility as a biomarker to detect residual disease. Analyses of DNMT3A (n=1,041) were focused to perform landscape investigations and to address the prognostic relevance. Additionally, this working group is focusing on TET2, ASXL1, and TP53 analyses. A novel prognostic model is being developed allowing stratification of AML into prognostic subgroups based on molecular markers only. In ALL, 1,124 pediatric and adult cases have been screened, including 763 assays for TP53 mutations both at diagnosis and relapse of ALL. Pediatric and adult leukemia expert labs developed additional content to study the mutation incidence of other B and T lineage markers such as IKZF1, JAK2, IL7R, PAX5, EP300, LEF1, CRLF2, PHF6, WT1, JAK1, PTEN, AKT1, IL7R, NOTCH1, CREBBP, or FBXW7. Further, the molecular landscape of CLL is changing rapidly. As such, a separate working group focused on analyses including NOTCH1, SF3B1, MYD88, XPO1, FBXW7 and BIRC3. Currently, 922 cases were screened to investigate the range of mutational burden of NOTCH1 mutations for their prognostic relevance. In MDS, RUNX1 mutation analyses were performed in 977 cases. The prognostic relevance of TP53 mutations in MDS was assessed in additional 327 cases, including isolated deletions of chromosome 5q. Next, content was developed targeting genes of the cellular splicing component, e.g. SF3B1, SRSF2, U2AF1, and ZRSR2. In BCR-ABL1-negative MPN, nine genes of interest (JAK2, MPL, TET2, CBL, KRAS, EZH2, IDH1, IDH2, ASXL1) have been analyzed in a cohort of 155 primary myelofibrosis cases searching for novel somatic mutations and addressing their relevance for disease progression and leukemia transformation. Moreover, an assay was developed and applied to CMML cases allowing the simultaneous analysis of 25 leukemia-associated target genes in a single sequencing run using just 20 ng of starting DNA. Finally, nine laboratories are studying CML, applying ultra-deep sequencing of the BCR-ABL1 tyrosine kinase domain. Analyses were performed on 615 cases investigating the dynamics of expansion of mutated clones under various tyrosine kinase inhibitor therapies. Conclusion Molecular characterization of hematological malignancies today requires high diagnostic sensitivity and specificity. As part of the IRON-II study, a network of laboratories analyzed a variety of disease entities applying amplicon-based NGS assays. Importantly, the consortium not only standardized assay design for disease-specific panels, but also achieved consensus on a common data analysis pipeline for mutation interpretation. Distinct working groups have been forged to address scientific tasks and in total 8,867 cases had been analyzed thus far. Disclosures: Kohlmann: Roche Diagnostics: Honoraria; MLL Munich Leukemia Laboratory: Employment. Martinelli:Roche Diagnostics: Research Support Other. Alikian:Roche Diagnostics: Research Support Other. Auber:Roche Diagnostics: Research Support Other. Belickova:Roche Diagnostics: Research Support Other. Bronzini:Roche Diagnostics: Research Support Other. Cazzaniga:Roche Diagnostics: Research Support Other. Chiaretti:Roche Diagnostics: Research Support Other. Ernst:Roche Diagnostics: Research Support Other. Fuellgrabe:Roche Diagnostics: Research Support Other.…

Sabiad, Jun 29, 2020

Kodlama yapmayan küçük RNA'lar hücre farklılaşması, büyümesi, gelişmesi, immün reaksiyonlar, stre... more Kodlama yapmayan küçük RNA'lar hücre farklılaşması, büyümesi, gelişmesi, immün reaksiyonlar, stres adaptasyonu gibi fizyolojik süreçlerin yanı sıra, kanser, kalp hastalıkları gibi kompleks hastalıklarla da ilişkilendirilmiştir. MIR223, hematopoetik sisteme özgü bir miRNA' dır. T-hücreli Akut Lenfoblastik Lösemi (T-ALL) patogenezine katkıda bulunan miRNA'lar arasında yüksek anlatıma sahip olduğu ve onkomir olarak aktivite gösterdiği tespit edilmiştir. Bu çalışmada MIR223 geninin TALL hücre serilerinde alternatif bir yaklaşım olarak özgün kilitli nükleik asit (KNA) kullanılarak baskılanması ve gen sessizleştirme etkinliğinin gösterilmesi amaçlanmıştır. Gereç ve Yöntem: Kültüre edilen TALL hücre serilerine (Jurkat ve Molt4), 24 ve 48 saatlik sürelerde, 100 ve 150pmol konsantrasyonda MIR223' e özgü KNA uygulanmıştır. Her iki zaman aralığında RNA izolasyonu sonrası, stem loop polimeraz zincir reaksiyonu (PZR) ile cDNA sentezlenerek, kantitatif gerçek zamanlı PZR (QRT-PZR) ile miRNA anlatım düzeyleri belirlenmiştir. Bulgular: Her iki hücre serisinde de 24. saatte, 150pmol KNA, sadece transfeksiyon ajanı uygulanmış (Mock) hücrelerle karşılaştırıldığında MIR223 düzeyinin yüksek oranda baskılandığı gözlenmiştir (Jurkat %73, p=0,001 ve Molt4%80 p=0,04). Molt 4 hücre serisinde anlamlı düzeyde baskılanma 48. saatte devam etse de (p=0,005), Jurkat hücre serisinde 48. saatteki baskılama istatistiksel olarak anlamlı bulunmamıştır. Sonuç: MIR223 onkogenik etki gösteren bir miRNA olarak tanımlanmıştır ve antisens oligolar ile MIR223 geninin sessizleştirilmesi TALL gibi artmış MIR223 anlatımı gösteren kanserlerde hastalığın seyrini ve tedavi alternatiflerini araştırma imkanı sağlamaktadır. Bu çalışmada TALL hücrelerinde alternatif bir RNA interferans (sessizleştirme) uygulaması olarak KNA kullanılmıştır ve bu moleküllerin çok etkin ancak kısa süreli olarak kullanılabileceği görülmüştür. Anahtar Kelimeler: TALL , MIR223, kilitli nükleik asitler (KNA), gen sessizleştirmesi ABSTRACT Objective: Noncoding small RNAs play roles in physiological processes such as cell differentiation, growth, development, immune reactions, stress adaptation as well as complex diseases such as cancer and heart diseases. MIR223 is a specific miRNA to the hematopoietic system. Among the miRNAs associated with the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL), MIR223 was shown to have the highest expression level and functions as an oncomir. In this study we aimed to suppress MIR223 gene expression by using locked nucleic acid (LNA) in TALL cell lines as an alternative gene silencing technique and to show the silencing efficacy. Material and Methods: TALL cell lines (Jurkat and Molt4) were cultured and MIR223 specific LNA was applied in 100pmol and 150pmol concentrations for 24 hours and 48 hours. RNA was isolated from cells at both time points, followed by a stem loop polymerase chain reaction (PCR) for cDNA synthesis and the miRNA expression levels were evaluated by quantitative real time PCR. Results: In both cell lines a 150pmol application of LNA was compared to mock cells and the MIR223 expression was already suppressed at 24hours (73% in Jurkat (p=0,001) and 80%in Molt4 (p=0,04)). The suppression was continued at 48 hours in Molt4 cell line (p=0,005) where as there was no statistical significant difference in Jurkat at 48 hours. Conclusion: MIR223 has been identified as an oncomir and silencing the MIR223 with synthetic antisense oligos provide the opportunity to investigate the course and alternative therapy options of cancers with increased MIR223 expression, such as TALL. In this study LNA was used as an alternative RNA interference application in TALL cells and showed that LNA can effectively suppress gene expression when used in the short term.

Pediatric Blood & Cancer

The European Union‐funded COST Action (LEukaemia GENe Discovery by data sharing, mining, and coll... more The European Union‐funded COST Action (LEukaemia GENe Discovery by data sharing, mining, and collaboration) LEGEND was an international and multidisciplinary collaboration between clinicians and researchers that covered a range of aspects of genetic predisposition in childhood leukemia. Within this framework, we explored the perception and handling of genetic predisposition in the daily practice of European treatment centers. Herein, we present the results of our questionnaire‐based survey. We found that the overall awareness is quite high, and respondents remarked that identification and treatment of the most common predisposition syndromes were present. Nevertheless, high demand for continuous education and routinely updated resources remains.

DergiPark (Istanbul University), Jul 4, 2022

Objective: Deregulated WNT signaling was reported in TALL and other cancers. AXIN2 is a negative ... more Objective: Deregulated WNT signaling was reported in TALL and other cancers. AXIN2 is a negative regulator of the active WNT signaling and AXIN2 gene variants were associated with increased cancer risk. In this study, we aimed to determine AXIN2 variations and compare with clinic features in TALL. Methods: Thirty-two diagnostic TALL patients were retrospectively enrolled in the study. Coding sites of the AXIN2 were amplified by PCR and then screened by denaturing highperformance liquid chromatography (dHPLC). Patients with differential chromatograms were evaluated by Sanger sequencing. Results: None of the patients had pathogenic AXIN2 variants. Besides that, AXIN2 polymorphisms, rs2240308/ rs1133683/ rs9915936 were detected in 14 (43.7%) TALL patients. Genotype distributions of the rs2240308 and rs1133683 variants in TALL group were significantly different from controls (rs2240308, GG/GA p=0.029; rs1133683, GG/GA p<0.0001) and G allele increased the overall risk of TALL compared to A allele in both polymorphisms. We did not observe any clinical differences between AXIN2 variant carriers or non-carriers. Conclusion: AXIN2 rs2240308 and rs1133683 variants revealed significant positive associations between susceptibility to TALL .

Turkish Journal of Hematology, 2019

Angiotensin II promotes growth and angiogenesis via type 1 receptors (AGTR1) in certain tumors. I... more Angiotensin II promotes growth and angiogenesis via type 1 receptors (AGTR1) in certain tumors. In this study, we examine the bone marrow AGTR1 expression in multiple myeloma (MM) and its relationship with the regulation of angiogenesis and prognostic factors. Materials and Methods: Bone marrow AGTR1 mRNA levels of 39 MM patients and 15 healthy controls were analyzed with quantitative RT-PCR. Immunohistochemical staining of the tissue vascular endothelial growth factor (VEGF), CD34, and factor VIIIrAg (fVIIIrAg) was used to assess bone marrow angiogenesis. Results: Bone marrow samples of the patients showed increased VEGF, fVIIIrAg, and CD34 staining and higher AGTR1 expression levels when compared to controls. Patients with severe-diffuse bone marrow infiltration showed higher bone marrow VEGF, fVIIIrAg, CD34, and AGTR1 mRNA levels when compared to other patients. Conclusion: AGTR1 expression was found positively correlated with plasma β2-microglobulin level and patients with increased AGTR1 expression showed increased bone marrow CD34 levels.

Journal of Cancer Research and Clinical Oncology, 2003

Scandinavian Journal of Clinical and Laboratory Investigation

Case reports in hematology, 2011

The current treatment of chronic phase chronic myeloid leukemia (CML) consists of oral tyrosine k... more The current treatment of chronic phase chronic myeloid leukemia (CML) consists of oral tyrosine kinase inhibitors (TKIs). However, high-risk CML may present with an aggressive course which may result in blastic crisis or a "difficult-to-manage" state with available treatments. The aim of this paper is to report a patient with complicated CML resistant to treatment and progressed despite the administration of bosutinib, imatinib mesylate, nilotinib, dasatinib, interferon alpha 2a, cytotoxic chemotherapy, and allogeneic hematopoietic stem cell transplantation. The striking point of this case story is that no Abl kinase domain mutation against TKIs has been detected during this very complicated disease course of CML. Meanwhile, challenging cases will always be present despite the hope and progress in CML in the TKI era.

Turkiye Klinikleri Dahili Tıp Bilimleri Dergisi, 2007

Turkiye Klinikleri Hematoloji Ozel Dergisi, 2009

Turkish Journal of Medical Sciences, Feb 27, 2006

Polymorphisms in the genes encoding cytochrome p450 (CYP) and thiopurine S-methyl transferase (TP... more Polymorphisms in the genes encoding cytochrome p450 (CYP) and thiopurine S-methyl transferase (TPMT) enzymes catalyze the metabolic reactions of several drugs. These polymorphisms might be responsible for adverse drug reactions. Turkish population data for these genes still needs to be elucidated. We aimed to detect the allele frequencies of thiopurine S-methyl transferase (TPMT), cytochrome p4503A4*1B (CYP3A4*1B) and cytochrome p4503A4*3 (CYP3A5*3) gene variants in the Turkish population. We examined the TPMT (*1, *2, *3A, *3C), CYP3A4*1B and CYP3A5*3 variant allele frequencies in a group of healthy Turkish Caucasian blood donors by using PCR-RFLP, allele-specific PCR and direct sequencing techniques. The frequencies of four allelelic variants of TPMT gene, are *2 (238G>C)(2.0%), *3A (460G>A and 719A>G)(1.0%), *3B (460G>A)(0.0%) and *3C (719A>G) (1.4%). We observed CYP3A4*1B allele frequency in 1.4% and CYP3A5*3 allele frequency in 7.5% of our population. This study provides the first analysis of TPMT, CYP3A4*1B and CYP3A5*3 mutant allele frequencies in the Turkish population.

Experimed, 2021

Objective: Lymphoid enhancer-binding factor-1 (LEF1) is one of the key regulators of lymphocyte p... more Objective: Lymphoid enhancer-binding factor-1 (LEF1) is one of the key regulators of lymphocyte proliferation and its aberrant expression is a prognostic factor for lymphoid or myeloid malignancies. In this study, we focused on the expression of LEF1 isoforms in several hematological malignancies and found tissue-specific differential expression for the full-length (FL)-LEF1 gene and its tumor suppressor (∆LEF1) variant. Material and Method: Fifty-three leukemia/lymphoma patients were included in this study. Diagnostic samples of "lymphoid group" patients: Chronic Lymphoblastic Leukemia (CLL) (n=10), B-cell Acute Lymphoblastic Leukemia (B-ALL) (n=9) and "myeloid group" patients: Chronic Myeloblastic Leukemia (CML) (n=12), Acute Myeloid Leukemia (AML) (n=13), and Multiple Myeloma (MM) (n=9) were studied. Healthy bone marrow, peripheral blood cells, and CD34 positive cells were used as controls. Total (T) and FL-LEF1 transcript levels were examined by using quantitative real-time polymerase chain reaction (qRT-PCR). T and FL-LEF1 mRNA ratios were also evaluated for calculation of ∆LEF1. Results: LEF1 levels were significantly high in lymphoid malignancies, but MM and AML patients have decreased LEF1 levels. Although CLL patients have high FL-LEF1 levels, the ratio of the T/FL levels was significantly decreased. Conclusion: LEF1 is a proliferation factor for lymphocytes and not only its differential overexpression but also the ratio of T/FL isoforms seem to accompany leukemia progress.

Istanbul University Institute of Health Sciences Journal of Advanced Research in Health Sciences, 2020

PI3K/AKT sinyal yolağının bozulmuş aktivasyonu tümör gelişimi ile ilişkilendirilmiştir. PTEN/AKT ... more PI3K/AKT sinyal yolağının bozulmuş aktivasyonu tümör gelişimi ile ilişkilendirilmiştir. PTEN/AKT yolağı, hücre büyümesi ve sağ kalımında kritik role sahip olup, lösemilerde kemoterapi direnci ile ilişkilendirilmiştir. Bu çalışma kapsamında, çocukluk çağı TALL hastalarındaki PTEN ve AKT1 gen anlatımları belirlendi ve hastalığın prognozuna olan etkisi araştırıldı. Gereç ve Yöntem: Otuz dokuz çocukluk çağı TALL olgusunun, tanı zamanı kemik iliği örnekleri kantitatif gerçek zamanlı PZR ile PTEN ve AKT1 genleri için tarandı. Gen anlatım düzeylerinin hastaların yaş, cinsiyet, immünfenotip, tedavi yanıtı, nüks ve sağ kalım gibi klinik verileri ile ilişkisi incelendi. Bulgular: Çalışmamızda, TALL hastalarında PTEN gen anlatımında kontrollere göre bir farklılık gözlenmezken, AKT1 gen anlatımının anlamlı düzeyde arttığı belirlendi (p<0,0001). Hastaların klinik özellikleri, genel ve hastalıksız sağ kalım oranları ve gen anlatım düzeyleri arasında anlamlı bir ilişki saptanmazken, indüksiyon tedavi ilişkili sağ kalım analizleri PTEN ve AKT1 gen anlatımları ile genel sağ kalım oranları arasında (sırasıyla, p=0,026 ve p=0,034 Cox regresyon analizi) anlamlı bir ilişki ortaya koyuldu. Diğer klinik parametreler ile gen anlatım düzeyleri arasında anlamlı ilişki saptanmadı. Sonuç: Çalışmamızda, TALL örneklerinde PTEN gen anlatımından bağımsız olarak artmış AKT1 gen anlatımının varlığı ve sağ kalım sürelerini etkilediğine dair bulgular elde edildi. Gelecekteki çalışmalar, AKT1'in hedefe yönelik tedaviler için uygun bir aday olup olmayacağını ortaya çıkaracaktır.