Barbaros YİĞİT, Pınar ASLAN KOŞAR, Emine Güçhan ALANOĞLU, Muhammet Yusuf TEPEBAŞI

HEMATOLOJİK MALİGNİTELERDE KONVANSİYONEL SİTOGENETİK, MOLEKÜLER SİTOGENETİK VE MOLEKÜLER GENETİK SONUÇLARININ DEĞERLENDİRİLMESİ

Amaç Hematolojik maligniteler, kemik iliği kaynaklı hücrele rin neoplazmlarıdır. Epidemiyolojik ve klinik çalışma lar sonucunda somatik hücrelerde meydana gelenmutasyonların, bu malignitelerin çoğunda sayısal veyapısal kromozomal anomaliler ile spesifik gen düze yindeki değişiklikler içerdiğini göstermektedir. Oluşanbu değişikliklerin hematolojik malignensilerin tanı vetakibinde aynı zamanda tedavi seçimi ve progno zu belirlemede kritik role sahip olduğu belirlenmiştir.Çalışmamız, hematolojik maligniteye sahip hasta gu ruplarında kromozomal ve moleküler olarak meydanagelen bu genetik değişikliklerin neler olduğunu tespitetmeyi amaçlamaktadır. Gereç ve Yöntem Bu çalışmaya Süleyman Demirel Üniversitesi Tıp Fa kültesi Hematoloji Anabilim Dalı'na hematolojik malig nite ön tanısı veya tanısı ile başvuran 110 hasta dahiledildi. Akut Miyeloid Lösemi (AML), Kronik MiyeloidLösemi (KML), Kronik Lenfoblastik Lösemi (KLL), Mi yelodisplastik Sendrom (MDS), Kronik Miyeloprolife ratif Neoplazm ( KMPN), Multipl Myelom (MM)/DiğerPlazma Hücreli Neoplazmlar (DPHN) ve Lenfomahastalıklarına sahip yedi grubun kemik iliği kültürleriyapılarak üç farklı analiz yöntemi ile incelendi. Sayı sal ve yapısal kromozomal değişiklikler sitogenetikkromozom analizi ve Floresan In Situ Hibridizasyon(FISH) yöntemleri ile ve JAK-2 V617F mutasyonu Re al-Time PCR (RT-PCR) ile analiz edildi. Bulgular Sitogenetik ve FISH analizileri sonucunda, KML içint(9;22) mutasyonu %30,8 ve %22,6 oranlarında, AMLiçin t(15;17) %7,7 oranında pozitif bulundu. Ayrıca,MDS için del (5q) ve del (7q) değerlendirildi ve deles yon saptanmadı. Hastalar del(13q14) ve del(17p13)açısından değerlendirildiğinde, lenfoma (%28,6 ve%71,4) KLL (%50 ve %62,5) ve MPHN (%50 ve%85,7) hastalarında değişen oranlarda bulundu. Ay rıca RT-PCR sonuçlarına göre JAK-2 V617F mutas yonu, KML hasta gurubunda (18.8%) KMPN hastagrubunda ise (81.2%) heterozigot positive olarak bu lunmuştur. Sonuç Laboratuvarımızda elde ettiğimiz bulgular ışığında,hematolojik maligniteye sahip hastaların rutinde ça lışılan mevcut genetik analizlerine ek olarak belirle diğimiz mutasyonlarında incelenmesi ve hasta grup larının da genişletilerek çalışılmasının hastalığın tanıve prognozunun değerlendirilmesine katkıda buluna cağını düşünmekteyiz.

EVALUATION OF CONVENTIONAL CYTOGENETIC,MOLECULAR CYTOGENETICS AND MOLECULARGENETICS RESULTS IN HEMATOLOGICALMALIGNANCES

Objective Hematological malignancies are neoplasms of bonemarrow-derived cells. Epidemiological and clinicalstudies show that the mutations occurring in soma tic cells include numerical and structural chromoso mal abnormalities and specific gene-level changes inmost of these malignancies. It has been determinedthat these changes have a critical role in the diagno sis and follow-up of hematological malignancies, aswell as in the choice of treatment and determining theprognosis. Our study aims to determine what thesegenetic changes occur as chromosomal and molecu lar in patient groups with hematologic malignancy. Materials and Methods In this study, 110 patients who were admitted to theDepartment of Hematology of the Süleyman DemirelUniversity Faculty of Medicine with a pre-diagnosis ordiagnosis of Hematologic Malignancy were included.Three different analyses applied to the cultured bonemarrow tissue samples of seven groups of hemato logy patients who suffered from Acute Myeloid Leuke mia (AML), Chronic Myeloid Leukemia (CML), Chro nic Lymphoblastic Leukemia (CLL), MyelodysplasticSyndrome (MDS), Chronic Myeloproliferative Ne oplasm (CMPN), Malignant Plasma Cell Neoplasm(MPCN) and Lymphoma diseases. Numerical andstructural chromosomal changes were examined bycytogenetic chromosome analysis and Fluorescent InSitu Hybridization (FISH) methods and JAK-2 V617Fmutation was analyzed by Real-Time PCR (RT-PCR). Results As a result of cytogenetic and FISH analyzes, thet(9;22) mutation was found to be 30.8% and 22.6%positive for CML, and t(15;17) was found to be positiveat 7.7% for AML. Also, del(5q) and del(7q) were eva luated for MDS and no deletion was detected. Whenthe patients were evaluated in terms of del (13q14)and del (17p13), varying rates were found in lymp homa (28.6% and 71.4%) CLL (50% and 62.5%) andMPHN (50% and 85.7%) patients. Also, according tothe results of RT-PCR, the JAK-2 V617F mutationwas found as heterozygous positive in the CML pa tient group (18.8%) and CMPN patient group (81.2%).Conclusion In the light of the findings we have obtained in our la boratory, we think that examining patients with hema tological malignancies in addition to the existing ge netic analyzes that are routinely studied and studyingthe patient groups by expanding them will contributeto the evaluation of the diagnosis and prognosis ofthe disease.

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1. Heerema NA. Cytogenetic analysis of hematologic malignant diseases. The AGT Cytogenetics Laboratory Manual. 2017:499-575.
2. Dewald W G. Cytogenetic and FISH studies in myelodysplasia, acute myeloid leukemia, chronic lymphocytic leukemia and lymphoma. International journal of hematology. 2002;76:65-74.
3. Sultana TA, Mottalib MA, Islam MS, Khan MA, Choudhury S. rtPCR method for diagnosis and follow-up of hematological malignancies: first approach in Bangladesh. Bangladesh Medical Research Council Bulletin. 2008;34(1):1-11.
4. Hokland P, Pallisgaard N, editors. Integration of molecular methods for detection of balanced translocations in the diagnosis and follow-up of patients with leukemia. Seminars in hematology; 2000: Elsevier.
5. Rowley J, Mitelman F. Principles of molecular cell biology of cancer: chromosome abnormalities in human cancer and leukemia. Cancer: Principles & Practice of Oncology DeVita, VT, Hellman, S, Rosenberg, SA,(Eds), Philadelphia: JB Lippincott Company. 1993:67-91.
6. Sreekantaiah C. FISH panels for hematologic malignancies. Cytogenetic and genome research. 2007;118(2-4):284-96.
7. Dewald GW, Wyatt WA, Juneau AL, Carlson RO, Zinsmeister AR, Jalal SM, et al. Highly sensitive fluorescence in situ hybridization method to detect double BCR/ABL fusion and monitor response to therapy in chronic myeloid leukemia. Blood. 1998;91(9):3357-65.
8. Pallisgaard N, Hokland P, Riishøj DC, Pedersen B, Jørgensen P. Multiplex reverse transcription-polymerase chain reaction for simultaneous screening of 29 translocations and chromosomal aberrations in acute leukemia. Blood. 1998;92(2):574-88.
9. Hochhaus A, Weisser A, La Rosee P, Emig M, Müller M, Saussele S, et al. Detection and quantification of residual disease in chronic myelogenous leukemia. Leukemia. 2000;14(6):998.
10. Hermans A, Heisterkamp N, von Lindern M, van Baal S, Meijer D, van der Plas D, et al. Unique fusion of bcr and c-abl genes in Philadelphia chromosome positive acute lymphoblastic leukemia. Cell. 1987;51(1):33-40.
11. Rabbitts T. Chromosomal translocations in human cancer. Nature. 1994;372(6502):143.
12. Willis T, Zalcberg I, Jadayel D, Coignet L, Stul M, Treleaven J, et al. Molecular cloning of translocation t (1; 14)(q21; q32) defines a novel gene (BCL9) at chromosome 1q21. Blood. 1997;90(10):1720-.
13. Barnes DJ, Melo JV. Cytogenetic and molecular genetic aspects of chronic myeloid leukaemia. Acta haematologica. 2002;108(4):180-202.
14. Sazawal S, Bajaj J, Chikkara S, Jain S, Bhargava R, Mahapatra M, et al. Prevalence of JAK2 V617F mutation in Indian patients with chronic myeloproliferative disorders. Indian Journal of Medical Research. 2010;132(4):423.
15. Murugesan G, Aboudola S, Szpurka H, Verbic MA, Maciejewski JP, Tubbs RR, et al. Identification of the JAK2 V617F mutation in chronic myeloproliferative disorders using FRET probes and melting curve analysis. American Journal of Clinical Pathology. 2006;125(4):625-33.
16. Shin M-G, Kim HJ, Kim H-R, Lee S-Y, Lee I-K, Shin J-H, et al. Frequency of the JAK2 mutation and its usefulness as a marker for treatment response and disease progression in Korean patients with chronic myeloproliferative disorders. Am Soc Hematology; 2006.
17. Vardiman JW. The World Health Organization (WHO) classification of tumors of the hematopoietic and lymphoid tissues: an overview with emphasis on the myeloid neoplasms. Chemico-biological interactions. 2010;184(1-2):16-20.
18. Başaran N. Tıbbi genetik: ders kitabı: Anadolu Üniversitesi; 1984.
19. Miranda RN, Mark H, Medeiros LJ. Fluorescent in situ hybridization in routinely processed bone marrow aspirate clot and core biopsy sections. The American journal of pathology. 1994;145(6):1309.
20. Ou J, Vergilio JA, Bagg A. Molecular diagnosis and monitoring in the clinical management of patients with chronic myelogenous leukemia treated with tyrosine kinase inhibitors. American journal of hematology. 2008;83(4):296-302.
21. Kantarjian H, Schiffer C, Jones D, Cortes J. Monitoring the response and course of chronic myeloid leukemia in the modern era of BCR-ABL tyrosine kinase inhibitors: practical advice on the use and interpretation of monitoring methods. Blood, The Journal of the American Society of Hematology. 2008;111(4):1774-80.
22. Bain BJ. Overview. Best Practice & Research Clinical Haematology. 2001;14(3):463-77.
23. Linenberger ML, Hong T, Flowers D, Sievers EL, Gooley TA, Bennett JM, et al. Multidrug-resistance phenotype and clinical responses to gemtuzumab ozogamicin. Blood. 2001;98(4):988- 94.
24. Mitelman F, Heim S. Quantitative acute leukemia cytogenetics. Genes, Chromosomes and Cancer. 1992;5(1):57-66.
25. Foon KA, Rai KR, Gale RP. Chronic lymphocytic leukemia: new insights into biology and therapy. Annals of Internal Medicine. 1990;113(7):525-39.
26. Choi W, Kim M, Lim J, Han K, Lee S, Lee JW, et al. Four cases of chronic myelogenous leukemia in mixed phenotype blast phase at initial presentation mimicking mixed phenotype acute leukemia with t (9; 22). Annals of laboratory medicine. 2014;34(1):60-3.
27. Maciejewski JP, Selleri C. Evolution of clonal cytogenetic abnormalities in aplastic anemia. Leukemia & lymphoma. 2004;45(3):433-40.
28. Komrokji RS, Bennett JM. What Is “WHO “? Myelodysplastic Syndromes Classification. Clinical Leukemia. 2008;2(1):20-7.
29. Lakatošová M, Holečková B. Fluorescence in situ hybridisation. Biologia. 2007;62(3):243-50.
30. Tefferi A, Vardiman J. Classification and diagnosis of myeloproliferative neoplasms: the 2008 World Health Organization criteria and point-of-care diagnostic algorithms. Leukemia. 2008;22(1):14.
31. Levine RL, Pardanani A, Tefferi A, Gilliland DG. Role of JAK2 in the pathogenesis and therapy of myeloproliferative disorders. Nature reviews cancer. 2007;7(9):673.
32. James C, Ugo V, Le Couédic J-P, Staerk J, Delhommeau F, Lacout C, et al. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature. 2005;434(7037):1144.
33. Hussein K, Bock O, Seegers A, Flasshove M, Henneke F, Buesche G, et al. Myelofibrosis evolving during imatinib treatment of a chronic myeloproliferative disease with coexisting BCR-ABL translocation and JAK2V617F mutation. Blood. 2007;109(9):4106-7.
34. Karkucak M, Yakut T, Ozkocaman V, Ozkalemkas F, Ali R, Bayram M, et al. Evaluation of the JAK2-V617F gene mutation in Turkish patients with essential thrombocythemia and polycythemia vera. Molecular biology reports. 2012;39(9):8663-7.
35. Çetinkaya S. Trakya Üniversitesi Tıp Fakültesi Hematoloji Bilim Dalına başvuran hastalarda JAK 2 geni nokta mutasyonu ve hastalık ilişkisinin değerlendirilerek fenotip-genotip ilişkisinin kurulması: Trakya Üniversitesi Sağlık Bilimleri Enstitüsü; 2012.