Turkish Journal of Hematology Volume: 35 - Issue: 1

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Genitsari S, et al: MSCs in Childhood ALL Turk J Hematol 2018;35:19-2618. Blau O, Hofmann WK, Baldus CD, Thiel G, Serbent V, Schümann E, Thiel E, Blau IW.Chromosomal aberrations in bone marrow mesenchymal stroma cells from patientswith myelodysplastic syndrome and acute myeloblastic leukemia. Exp Hematol2007;35:221-229.19. Menendez P, Catalina P, Rodriguez R, Melen GJ, Bueno C, Arriero M, García-SánchezF, Lassaletta A, García-Sanz R, García-Castro J. Bone marrow mesenchymal stemcells from infants with MLL-AF4+ acute leukemia harbor and express the MLL-AF4fusion gene. J Exp Med 2009;206:3131-3141.20. Zhao Z, Tang X, You Y, Li W, Liu F, Zou P. Assessment of bone marrow mesenchymalstem cell biological characteristics and support hematopoiesis function in patientswith chronic myeloid leukemia. Leuk Res 2006;30:993-1003.21. Karaöz E, Çetinalp Demircan P, Erman G, Güngörürler E, Eker Sarıboyacı A.Comparative analyses of immune-suppressive characteristics of bone-marrow,Wharton’s jelly, and adipose tissue-derived human MSCs. Turk J Hematol2017;34:213-225.22. Choumerianou DM, Martimianaki G, Stiakaki E, Kalmanti L, Kalmanti M, DimitriouH. Comparative study of stemness characteristics of mesenchymal cells from bonemarrow of children and adults. Cytotherapy 2010;12:881-887.23. Dimitriou H, Linardakis E, Martimianaki G, Stiakaki E, Perdikogianni CH, Charbord P,Kalmanti M. Properties and potential of bone marrow mesenchymal stromal cellsfrom children with hematologic diseases. Cytotherapy 2008;10:125-133.24. Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C. A rapid and simplemethod for measuring thymocyte apoptosis by propidium iodide staining and flowcytometry. J Immunol Methods 1991;139:271-279.25. Lecoeur H, Ledru E, Prevost MC, Gougeon ML. Strategies for phenotypingapoptotic peripheral human lymphocytes comparing ISNT, annexin V and 7-AADcytofluorometric staining methods. J Immunol Methods 1997;209:111-123.26. Wagner W, Horn P, Castoldi M, Diehlmann A, Bork S, Saffrich R, Benes V, Blake J,Pfister S, Eckstein V, Ho AD. Replicative senescence of mesenchymal stem cells: acontinuous and organized process. PLoS One 2008;3:e2213.27. Stolzing A, Jones E, McGonagle D, Scutt A. Age-related changes in human bonemarrow-derived mesenchymal stem cells: consequences for cell therapies. MechAgeing Dev 2008;129:163-173.28. Horwitz EM, Le Blanc K, Dominici M, Mueller I, Slaper-Cortenbach I, Marini FC, DeansRJ, Krause DS, Keating A; International Society for Cellular Therapy. Clarification ofthe nomenclature for MSC: the International Society for Cellular Therapy positionstatement. Cytotherapy 2005;7:393-395.29. Lv FJ, Tuan RS, Cheung KM, Leung VY. Concise review: the surface markers andidentity of human mesenchymal stem cells. Stem Cells 2014;32:1408-1419.30. Nagasawa T. Microenvironmental niches in the bone marrow required for B-celldevelopment. Nat Rev Immunol 2006;6:107-116.31. Gomes AC, Gomes MS. Hematopoietic niches, erythropoiesis and anemia of chronicinfection. Exp Hematol 2016;44:85-91.32. Nie Y, Han YC, Zou YR. CXCR4 is required for the quiescence of primitivehematopoietic cells. J Exp Med 2008;205:777-783.33. Ge J, Hou R, Liu Q, Zhu R, Liu K. Stromal-derived factor-1 deficiency in the bonemarrow of acute myeloid leukemia. Int J Hematol 2011;93:750-759.34. Khandany BK, Hassanshahi G, Khorramdelazad H, Balali Z, Shamsizadeh A, ArababadiMK, Ostadebrahimi H, Fatehi A, Rezazadeh M, Ahmadi Z, Karimabad MN. Evaluationof circulating concentrations of CXCL1 (Gro-α), CXCL10 (IP-10) and CXCL12 (SDF-1) in ALL patients prior and post bone marrow transplantation. Pathol Res Pract2012;208:615-619.35. Karakurt N, Aksu T, Koksal Y, Yarali N, Tunc B, Uckan-Cetinkaya D, Ozguner M.Angiopoietins in the bone marrow microenvironment of acute lymphoblasticleukemia. Hematology 2016;21:325-331.36. Pelagiadis I, Stiakaki E, Choulaki C, Kalmanti M, Dimitriou H. The role of children’sbone marrow mesenchymal stromal cells in the ex vivo expansion of autologousand allogeneic hematopoietic stem cells. Cell Biol Int 2015;39:1099-1110.Appendix: Supplementary Materials and MethodsBM MNC Isolation and MSC Culture and ExpansionBM MNCs, following separation with Ficoll-Hypaque (1077 g/mL; Lymphoprep,Nycomed, Oslo, Norway), were cultured in a-MEM without nucleotides in the presenceof 10% lot-selected fetal calf serum (Invitrogen Ltd., Paisley, UK) as described previously[21]. They were seeded at a concentration of 5x10 4 cells/cm 2 in the presence of 1 ng/mL FGF-2 (FGF-2; Abcys SA, Paris, France). A complete medium change was performedtwice a week. When layers became confluent at ~90%, cells were detached using 0.25%trypsin/1 mM EDTA (Invitrogen Ltd.) and then replated at a concentration of 1x10 3 cells/cm 2 (passage 1, P1). MSCs were maintained in culture for up to five passages. Assayswere performed at any of P1 to P4 depending on the cell availability.Immunophenotyping EvaluationPhenotypic characterization of MSCs was performed by flow cytometry at variouspassages using the following monoclonal antibodies: CD105-phycoerythrin (PE)CD146-PE, CD73-PE CD29-fluorescein isothiocyanate (FITC), CD44-FITC, CD90-FITC,CD14-FITC, CD45-FITC, CD34-PE, and CD95-FITC (BD Biosciences, San Jose, CA, USA).One hundred thousand cells were stained with the markers as described previously [21].At least 10,000 events were acquired for each analysis.Cell-Cycle Analysis - ApoptosisMSCs, at either P2 or P3, after detachment by trypsinization (trypsin/EDTA 0.25%) werecentrifuged at 150 x g for 10 min at 4 °C and washed with PBS. In order to estimate thepercentage of cells in each phase of the cell cycle, 1x10 6 MSCs were stained with 1 mLof propidium iodide staining solution (50 µg/mL propidium iodide, 1 mg/mL RNAse inPBS without Ca ++ /Mg ++ , pH 7.4) for 30 min at room temperature. After the acquisitionof at least 10,000 events for each sample, cells were gated according to forward vs.side scatter (FSC/SSC) characteristics. Cell-cycle analysis was performed using WinMDIsoftware, version 2.8 [22].Apoptotic MSCs at passages P2 and P4 were detected by flow cytometry and 7-aminoactinomycinD (7-AAD; Sigma, St. Louis, MO, USA) staining [23]. They were initiallygated according to their morphology (FSC/SSC). Then a scattergram was generated bycombining FSC with 7-AAD fluorescence to quantitate 7-AAD negative (alive), 7-AAD low(early apoptotic), and 7-AAD high (late apoptotic/dead) cells.Cell DTDT was calculated according to the formula DT=t/n=t×log(2)/log (cells harvested/cellsinoculated), where t is the time between initial plating and harvest for the respectivepassage.CFU-F FormationAt day 0.1x10 5 MNCs were seeded in each well of a 24-well plate (in triplicate) in theabsence of FGF-2. At subsequent passages, MSCs were plated in 20-cm 2 petri platesat a concentration of 10 cells/cm 2 (in duplicate). Following 14 days of culture at 37 °Cand 5% CO 2, CFU-F was quantified after staining with Giemsa stain and categorizedaccording to size as small CFU-F (S: <50 cells), medium CFU-F (M: 50-500 cells), andlarge CFU-F (highly proliferating; L: >500 cells). The sum of CFU-F of all sizes is denotedas CFU-F.Detection of SDF-1α and Ang-1 (ELISA)A quantitative sandwich ELISA was employed for the determination of both SDF-1αand Ang-1 in the supernatant of MSCs at any of P1 to P3 cultures (and of MNCs atd0) within the leukemia group only. All subgroups were examined for the evaluationof these factors through the whole course of the disease, diagnosis, and treatment. TheELISA kits were purchased from R&D Systems, and the instructions of the manufacturerwere followed. More specifically, 100 µL for SDF-1α (50 µL for Ang-1) of standard orsample per well was added and incubated for 2 h at room temperature on a shaker.After well aspiration and washing, 200 µL of the corresponding conjugate was added.Incubation was continued for 2 h further under the same conditions. After washing,200 µL of substrate solution was added to each well for 30 min at room temperatureand then 50 µL of stop solution terminated the reaction. The optical density of eachwell was determined at 450 nm with wavelength correction at 570 nm.26
RESEARCH ARTICLEDOI: 10.4274/tjh.2017.0021Turk J Hematol 2018;35:27-34Juvenile Myelomonocytic Leukemia in Turkey: A RetrospectiveAnalysis of Sixty-five PatientsTürkiye’de Juvenil Miyelomonositik Lösemi: Altmış Beş Hastanın Retrospektif AnaliziÖzlem Tüfekçi 1 , Ülker Koçak 2 , Zühre Kaya 2 , İdil Yenicesu 2 , Canan Albayrak 3 , Davut Albayrak 3 , Şebnem Yılmaz Bengoa 1 ,Türkan Patıroğlu 4 , Musa Karakükçü 4 , Ekrem Ünal 4 , Elif Ünal İnce 5 , Talia İleri 5 , Mehmet Ertem 5 , Tiraje Celkan 6 , Gül Nihal Özdemir 6 ,Nazan Sarper 7 , Dilek Kaçar 8 , Neşe Yaralı 8 , Namık Yaşar Özbek 8 , Alphan Küpesiz 9 , Tuba Karapınar 10 , Canan Vergin 10 , Ümran Çalışkan 11 ,Hüseyin Tokgöz 11 , Melike Sezgin Evim 12 , Birol Baytan 12 , Adalet Meral Güneş 12 , Deniz Yılmaz Karapınar 13 , Serap Karaman 14 , VedatUygun 15 , Gülsun Karasu 15 , Mehmet Akif Yeşilipek 15 , Ahmet Koç 16 , Erol Erduran 17 , Berna Atabay 18 , Haldun Öniz 18 , Hale Ören 11Dokuz Eylül University Faculty of Medicine, Department of Pediatric Hematology, İzmir, Turkey2Gazi University Faculty of Medicine, Department of Pediatric Hematology, Ankara, Turkey3Ondokuz Mayıs University Faculty of Medicine, Department of Pediatric Hematology, Samsun, Turkey4Erciyes University Faculty of Medicine, Department of Pediatric Hematology and Oncology, Kayseri, Turkey5Ankara University Faculty of Medicine, Department of Pediatric Hematology and Oncology, Ankara, Turkey6İstanbul University Cerrahpaşa Faculty of Medicine, Department of Pediatric Hematology and Oncology, İstanbul, Turkey7Kocaeli University Faculty of Medicine, Department of Pediatric Hematology, Kocaeli, Turkey8Ankara Children’s Hematology and Oncology Training and Research Hospital, Ankara, Turkey9Akdeniz University Faculty of Medicine, Department of Pediatric Hematology and Oncology, Antalya, Turkey10Dr. Behçet Uz Children Training and Research Hospital, Clinic of Pediatric Hematology and Oncology, İzmir, Turkey11Necmettin Erbakan University Meram Faculty of Medicine, Department of Pediatric Hematology, Konya, Turkey12Uludağ University Faculty of Medicine, Department of Pediatric Hematology, Bursa, Turkey13Ege University Faculty of Medicine, Department of Pediatric Hematology, İzmir, Turkey14Şişli Hamidiye Etfal Training and Research Hospital, Clinic of Pediatric Hematology and Oncology, İstanbul, Turkey15Bahçeşehir University Faculty of Medicine, Department of Pediatric Hematology and Oncology, İstanbul, Turkey16Marmara University Faculty of Medicine, Department of Pediatric Hematology and Oncology, İstanbul, Turkey17Karadeniz Technical University Faculty of Medicine, Department of Pediatric Hematology and Oncology, Trabzon, Turkey18Tepecik Training and Research Hospital, Clinic of Pediatric Hematology and Oncology, İzmir, TurkeyAbstractObjective: This study aimed to define the status of juvenilemyelomonocytic leukemia (JMML) patients in Turkey in terms oftime of diagnosis, clinical characteristics, mutational studies, clinicalcourse, and treatment strategies.Materials and Methods: Data including clinical and laboratorycharacteristics and treatment strategies of JMML patients werecollected retrospectively from pediatric hematology-oncology centersin Turkey.Results: Sixty-five children with JMML diagnosed between 2002and 2016 in 18 institutions throughout Turkey were enrolled in thestudy. The median age at diagnosis was 17 months (min-max: 2-117months). Splenomegaly was present in 92% of patients at the time ofdiagnosis. The median white blood cell, monocyte, and platelet countswere 32.9x10 9 /L, 5.4x10 9 /L, and 58.3x10 9 /L, respectively. MonosomyÖzAmaç: Türkiye’deki juvenil miyelomonositik lösemi (JMML) hastalarınındurumunu, tanı zamanı, klinik özellikler, mutasyon çalışmaları, klinikgidiş ve tedavi stratejileri açısından ortaya koymaktır.Gereç ve Yöntemler: Ülkemizdeki pediatrik hematoloji ve onkolojikliniklerinden veri istenerek, JMML tanısı ile takip ve tedavisi yapılanhastaların klinik ve laboratuvar bulguları geriye dönük olarakdeğerlendirildi.Bulgular: On sekiz merkezden, 2002-2016 tarihleri arasında JMMLtanısı alan toplam 65 hasta çalışmaya dahil edildi. Ortanca tanıyaşı 17 ay idi (2-117 ay). Splenomegali tanıda %92 hastada vardı.Ortanca lökosit, monosit ve trombosit sayıları sırasıyla 32,9x10 9 /L,5,4x10 9 /L ve 58,3x10 9 /L idi. Monozomi 7, %18 hastada saptanmıştı.JMML mutasyonları 32 hastada (%49) çalışılmış olup, en sık rastlananmutasyon PTPN11 idi. Hematopoetik kök hücre nakli (HKHN)©Copyright 2018 by Turkish Society of HematologyTurkish Journal of Hematology, Published by Galenos Publishing HouseAddress for Correspondence/Yazışma Adresi: Özlem TÜFEKÇİ, M.D.,Dokuz Eylül University Faculty of Medicine, Department of Pediatric Hematology, İzmir, TurkeyPhone : +90 232 412 61 50E-mail : ozlemtufekci@hotmail.com ORCID-ID: orcid.org/0000-0002-0721-1025Received/Geliş tarihi: January 19, 2017Accepted/Kabul tarihi: February 07, 201727
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