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RESEARCH ARTICLEDOI: 10.4274/tjh.galenos.2019.2019.0282Turk J Hematol 2020;37:98-103PTEN and AKT1 Variations in Childhood T-Cell AcuteLymphoblastic LeukemiaÇocukluk Çağı T-hücreli Akut Lenfoblastik Lösemi Hastalarında PTEN ve AKT1 VaryasyonlarFulya Küçükcankurt 1,2,a , Yücel Erbilgin 1,a , Sinem Fırtına 1,3 , Özden Hatırnaz Ng 1,4 , Zeynep Karakaş 5 , Tiraje Celkan 6 ,Ayşegül Ünüvar 5 , Uğur Özbek 1,7 , Müge Sayitoğlu 11İstanbul University, Aziz Sancar Institute of Experimental Medicine, Department of Genetics, İstanbul, Turkey2Altınbaş University Faculty of Medicine, İstanbul, Turkey3İstinye University Faculty of Art and Science, Department of Molecular Biology and Genetics, İstanbul, Turkey4Acıbadem Mehmet Ali Aydınlar University Faculty of Medicine, Department of Medical Biology, İstanbul, Turkey5İstanbul University Faculty of Medicine, Department of Pediatrics Hematology, İstanbul, Turkey6İstanbul University-Cerrahpaşa Cerrahpaşa Faculty of Medicine, Department of Pediatric Hematology, İstanbul, Turkey7Acıbadem Mehmet Ali Aydınlar University Faculty of Medicine, Department of Medical Genetics, İstanbul, TurkeyaF.K. and Y.E. contributed equally to this work.AbstractObjective: PTEN/AKT pathway deregulations have been reported tobe associated with treatment response in acute leukemia. This studyexamined pediatric T-cell acute lymphoblastic leukemia (T-ALL)samples for PTEN and AKT1 gene variations and evaluated the clinicalfindings.Materials and Methods: Fifty diagnostic bone marrow samples ofchildhood T-ALL cases were investigated for the hotspot regions ofthe PTEN and AKT1 genes by targeted next-generation sequencing.Results: A total of five PTEN variations were found in three of the 50T-ALL cases (6%). Three of the PTEN variations were first reported inthis study. Furthermore, one patient clearly had two different mutantclones for PTEN. Two intronic single-nucleotide variations were foundin AKT1 and none of the patients carried pathogenic AKT1 variations.Conclusion: Targeted deep sequencing allowed us to detect both lowlevelvariations and clonal diversity. Low-level PTEN/AKT1 variationfrequency makes it harder to investigate the clinical associationsof the variants. On the other hand, characterization of the PTEN/AKT signaling members is important for improving case-specifictherapeutic strategies.Keywords: T-ALL, PTEN, AKT1, Next-generation sequencingÖzAmaç: PTEN/AKT yolak düzensizliklerinin akut lösemide tedavi yanıtıile ilişkili olduğu bildirilmiştir. Çalışmanın kapsamı, pediatrik T-ALLhastalarının PTEN ve AKT1 genlerinin sıcak bölge varyasyonları içinincelenmesi ve klinik bulgularla değerlendirilmesidir.Gereç ve Yöntemler: Elli pediatrik T-ALL olgusunun tanı zamanıkemik iliği örnekleri, PTEN ve AKT1 genlerinin sıcak bölgeleri içinhedefe yönelik yeni nesil dizileme ile dizilenmiştir.Bulgular: Elli T-ALL olgusunun %6’sında PTEN varyasyonusaptanmıştır. Tespit edilen varyasyonlardan üçü ilk defa bu çalışmadagösterilmiştir. Ayrıca bir hastanın PTEN açısından iki farklı mutantklon taşıdığı belirlenmiştir. AKT1 geninde iki intronik tek nükleotidpolimorfizmi tespit edilirken hiçbir olguda patojenik AKT1 varyasyonusaptanmamıştır.Sonuç: Derin dizileme, hem düşük düzeydeki varyasyonların hemde klonal çeşitliliğin belirlenmesine olanak sağlamıştır. T-ALLhastalarındaki düşük düzey PTEN/AKT1 varyasyon sıklığı, varyantlarınklinikle ilişkisinin ortaya çıkarılmasını zorlaştırmaktadır. Diğer yandan,PTEN/AKT sinyal yolağının karakterizasyonu hasta spesifik terapötikstratejilerin uygulanabilirliği için önemlidir.Anahtar Sözcükler: T-ALL, PTEN, AKT1, Yeni nesil dizilemeIntroductionOne of the key signal transduction pathways involved inmalignant transformation is the PTEN/PI3K/AKT pathway,which regulates cellular metabolism, cell growth, translation,chromosome stability, and cell survival [1]. Phosphatase andtensin homolog deleted on chromosome ten (PTEN) is a lipidand dual function phosphatase that antagonizes the PI3K/AKT pathway; by dephosphorylating phosphoinositide 3-kinase©Copyright 2020 by Turkish Society of HematologyTurkish Journal of Hematology, Published by Galenos Publishing HouseAddress for Correspondence/Yazışma Adresi: Müge Sayitoğlu, Ph.D., İstanbul University, Aziz Sancar Institute ofExperimental Medicine, Department of Genetics, İstanbul, TurkeyPhone : +90 212 414 20 00 -33314E-mail : mugeay@istanbul.edu.tr ORCID: orcid.org/0000-0002-8648-213XReceived/Geliş tarihi: July 24, 2019Accepted/Kabul tarihi: November 19, 201998
Turk J Hematol 2020;37:98-103Küçükcankurt F, et al: PTEN and AKT1 Variations in T-ALL(PI3K) it produces PIP2 (phosphatidylinositol 4,5-bisphosphate)and PIP3 (phosphatidylinositol (3,4,5)-triphosphate) and soterminates the transmission of the signal to AKT and otherPIP3-effector targets [2]. AKT1 is a serine threonine kinasethat modulates the cell cycle checkpoint [3]. AKT1 is activatedby platelet-derived growth factor and its activation isderegulated by mutations in the pleckstrin homology domain ofAKT1. Survival factors can suppress apoptosis in a transcriptionindependentmanner by activating the serine/threonine kinaseAKT1, which then phosphorylates and inactivates componentsof the apoptotic machinery [4].PTEN as a tumor suppressor is frequently mutated in cancersand its inactivation results in constitutive activation of thePI3K/AKT pathway. PTEN is a regulatory key to prevent themalignant transformation of T-cells [5]. The PTEN/AKT pathwayhas an important role in the β-selection checkpoint in T-celldevelopment and lymphocyte homeostasis [6]. PTEN-deficientT-cells are found to be highly proliferative as a cause of increasedphosphorylation of AKT [7]. AKT1 is highly expressed in thymustissue and knockout studies showed that terminal differentiationin CD8+ T-cells failed, with increased proliferation, cytokinesecretion, and prolonged survival [8,9]. PTEN/AKT abnormalitiesresulting in deletion, insertion, or missense mutations lead todifferential regulation in different hematologic malignancies[10,11,12,13,14]. Genomic resequencing results showed thatPI3K/AKT pathway genes are commonly mutated in pediatricand young adult T-cell acute lymphoblastic leukemia (T-ALL)cases [11,15]. In this study, PTEN and AKT1 variations and theirclinical associations were analyzed in a group of childhoodT-ALL cases.Materials and MethodsChildhood T-ALL cases (n=50) diagnosed at the İstanbulUniversity Faculty of Medicine and Cerrahpaşa UniversityFaculty of Medicine were included in this study. Patients weretreated according to the BFM-ALL protocol. Diagnostic bonemarrow samples with a blast count of >80% were included inthe study. The T-cell origin of ALL was defined by the expressionof immunophenotype markers that included CD1a, CD2,cytoplasmic CD3, surface CD3, CD4, CD5, CD7, and CD8. T-ALLcases were evaluated according to the European Group for theImmunological Characterization of Leukemia classification scaleas immature (n=20), cortical (n=17), or mature (n=4); however,nine cases were not able to be further classified due to limitedimmunological marker information [11]. Median age was 8(range=0.9-17) years and other clinical features of the T-ALLcases are summarized in Table 1. Written and oral informedconsent was obtained from the legal representatives of thepediatric patients.Identification of PTEN and AKT1 variationsThe mononuclear cells of the bone marrow samples were isolatedby the Ficoll density gradient procedure [16]. Genomic DNA wasisolated with the QIAamp DNA Mini Kit (QIAGEN GmbH, Hilden,Germany) according to the manufacturer’s protocol. DNAquality and quantity were checked with a spectrophotometer(NanoDrop 100, Thermo Scientific, USA). The hotspot regions ofPTEN (exons 7 and 8) and AKT1 (exon 2) were covered by primerpairs, which were designed and validated by the ALL packageof the IRON-II (Interlaboratory Robustness of Next-GenerationSequencing) study (Table 2). Exons were amplified using theFastStart High Fidelity PCR System and GC-RICH PCR Systemkits (Roche Applied Science, Penzberg, Germany). Ampliconswere purified with Ampure XP beads (Beckman Coulter, Krefeld,Germany) and libraries were quantified by Quant-iT PicoGreendsDNA Reagent (Invitrogen, Carlsbad, CA, USA). Deep sequencingwas performed on a Roche FLX GS Junior (454-Life Sciences,Branford, CT, USA) according to the manufacturer’s instructions.The minimum read depth threshold per amplicon per samplewas set to 500x. Sanger sequencing was used to confirm thevariations, and low-level variants (variant calling was <20%)were re-sequenced by using a different MID. After the dataquality assessment, variant detection analyses were done byAVA software (GS Amplicon Variant Analyzer software version2.5.3, Roche Applied Science). The in silico prediction toolsMutationTaster [17] and SIFT [18] were used to evaluate thefunctional effects of identified variants in PTEN (NM_000314.4)and AKT1 (NM_005163.2).ResultsA total of 50 childhood T-ALL patients were screened forhotspot regions of PTEN and AKT1 by targeted deep sequencing.All detected variations are listed in Table 3. A total of five PTENvariations were found in three of the 50 T-ALL cases (6%) andall the variations occurred in exon 7, truncating PTEN in theC2-domain.A nonsense c.781C>T, p.Q261* (rs730882131) pathogenic variantwas found in one patient (P#7) with a low frequency (2.1%),and this somatic variation was evaluated as a small backgroundclone without any clinical significance. P#7 is a 3.5-year-old boywho was classified in the medium-risk group (MRG), a responderto induction therapy who was followed for 27 months.Three novel variants including insertions and deletions weredetected in two T-ALL cases. One patient (P#48) had twodifferent mutant clones for PTEN; the first clone carriedc.700_701insCTGGAGCCGAC p.R234Pfs*26 with 40% frequencyand the second clone harbored c.707_720delACAAGTTCATGTACand c.724_740delGAGTTCCCTCAGCCGTT deletions that causep.D236Vfs*6 with 16% frequency, which are classified as“deleterious” by SIFT. The deletion area was able to be detectedby conventional sequencing; however, it was not possible to99
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