Berkay Saraymen, Behzat Çimen, İhsan Çetin, Mustafa Köker, Aysun Çetin, Bülent Eser

Hematolojik Malignansilerde P-Glikoprotein Ekspresyonunun Akım Sitometri İle Belirlenmesi

Amaç: Duyarlılık açısından sınırlı olan immünolojik yöntemler kullanılarak özellikle normal dokulardaki P-glikoprotein ekpresyonunun belirlenmesi sorun oluşturmaktadır. Bu çalışmada hematolojik malignansi tanısı almış ve tedaviye yanıt vermeyen hastalarda P-glikoprotein ve CD34 ekpresyonunun değerlendirilmesi için, P-glikoprotein and CD34 ekpresyonunun akım sitometri ile belirlenmesini amaçladık.Yöntemler: Erciyes Üniversitesi Mehmet-Kemal Dedeman Hematoloji-Onkoloji Hastanesine başvuran akut miyeloblastik lösemi ile akut lenfoblastik lösemi tanısı almış 50 hasta ve yirmi kontrol çalışmaya dâhil edildi. Hastaların kemik iliği örnekleri ile kontrol grubundan alınan periferik kan örneklerinden elde adilen süspanse hücreler, P-glikoprotein fikoeritrin ile ve CD34 FITC ya da PerCP Cy 5.5 antikorları ile işaretlendi ve daha sonra yüzey ekspresyonları akım sitometri ile ölçüldü.Bulgular: Çalışmamızda 30 akut miyeloblastik lösemi hastasının 6’sında (%20) P-glikoprotein ve CD34 ekspresyonu tespit edilirken, 20 akut lenfoblastik lösemi hastasının 6’sında (%30) P-glikoprotein, 5’inde (%25) CD34 ekspresyonu tespit edildi. Akut miyeloblastik lösemi ve akut lenfoblastik lösemi kemik iliği örneklerinde P-glikoprotein ve CD34 ekspresyonları arasında anlamlı bir ilişki olduğu görüldü. Sonuç: Bulgular, akım sitometri yöntemi ile P-glikoprotein ekspresyonunun akut miyeloblastik lösemi ve akut lenfoblastik lösemi kemik iliği örneklerinde moleküler yöntemlerden daha hızlı, güvenilir ve doğru bir şekilde ölçülebildiğini, P-glikoprotein ile CD34 ekspresyonları arasında anlamlı bir ilişki olabileceğini göstermiştir. Yüzeyinde CD34 eksprese eden blast hücrelerinin aynı zamanda P-glikoprotein eksprese etmeleri hücrelerdeki çoklu ilaç direnci geninin daha çok olgunlaşmamış hücrelerde aktif olduğunu göstermektedir

Anahtar Kelimeler:

Çoklu ilaç direnci-1 geni, AML, ALL, P-glikoprotein, akım sitometri

Determination of P-Glycoprotein Expression by Flow Cytometry in Hematological Malignancies

Objective: Determination the expression of P-glycoprotein is especially problematic for normal tissues because immunological methods are limited in terms of sensitivity. We aimed to determine the expression of P-glycoprotein and CD34 by flow cytometry, and to evaluate the level of expression of P-glycoprotein and CD34 with unresponsive to treatment in patients diagnosed with hematologic malignancy.Methods: Our study included fifty patients diagnosed with acute myeloblastic leukemia and acute lymphoblastic leukemia, and twenty healthy controls who were admitted to Erciyes University Hematology-Oncology Hospital. The suspended cells from bone marrow samples of patients and the peripheral blood samples of healthy people were marked with P-glycoprotein phycoerythrin and CD34 FITC or PerCP Cy 5.5; and then surface expression was measured by means of flow cytometry.Results: In 6 of 30 acute myeloblastic leukemia patients P-glycoprotein and CD34 expression, in 6 of 20 acute lymphoblastic leukemia patients P-glycoprotein, in 5 of them CD34 expression were determined. A significant relation between P-glycoprotein and CD34 expressions in acute myeloblastic leukemia and acute lymphoblastic leukemia bone marrow samples was reported.Conclusion: Our data indicate that flow cytometry is more reliable, precise and faster than molecular methods for measuring P-glycoprotein expression and suggests the possibility of a significant relationship between P-glycoprotein and CD34 expressions in acute myeloblastic leukemia and acute lymphoblastic leukemia bone marrow samples. The blast cells expressing CD34 on their surface along with P-glycoprotein simultaneously show that multi drug resistance 1 gene is mostly active in immature cells.Key words: MDR-1 gene, AML, ALL, P-glycoprotein, flow cytometry

Keywords:

-,

PDF

___

Faderl S, O’Brien S, Pui CH, et al. Adult acute lymphoblastic leukemia: concepts and strategies. Cancer 2010;116:1165-1176.
Ribera JM. Advances in acute lymphoblastic leukemia in adults. Curr Opin Oncol 2011;23:692-699.
Ayyıldız O, Işıkdoğan A, Çelik M, et al. Invasive pulmonary aspergillosis in a patient with acute lymphoblastic leukemia. Dicle Medical Journal 2004;31:57-61.
Döhner H, Estey EH, Amadori S, et al. European LeukemiaNet.
Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel, on behalf of the European Leukemia Net. Blood 2010;115:453-474.
Consoli U, Santonocito A, Stagno F, et al. Multidrug resistance
mechanisms in chronic lymphocytic leukaemia. Br J Haematol 2002;116:774-780.
Gross HJ, Verwer B, Houck D, et al. Detection of rare cells at a frequency of one per million by flow cytometry. Cytometry 1993;14:519-526.
Gross HJ, Verwer B, Houck D, et al. Model study detecting breast cancer cells in peripheral blood mononuclear cells at frequencies as low as 10(-7), Proc Natl Acad Sci USA 1995;92:537-541.
Rosenblatt JI, Hokanson JA, McLaughlin SR, et al. Theoretical
basis for sampling statistics useful for detecting and isolating rare cells using flow cytometry and cell sorting. Cytometry 1997;27:233-238.
Rehsem MA, Corpuzm S, Heimfeldm S, et al. Use of fluorescence
threshold triggering and high-speed flow cytometry for rare event detection. Cytometry 1995;22:317-322.
Bay A, Boşnak V, Coşkun E, et al. Severe Rotavirus astroenteritis
in a patient with infant leukemia. Dicle Medical Journal 2011;38:101-103.
Drach D, Zhao S, Drach J, et al. Subpopulations of normal peripheral blood and bone marrow cells express a functional multidrug resistant phenotype. Blood 1992;80:2729-2734.
Fackler MJ, Krause DS, Smith OM, et al. Full-length but not truncated CD34 inhibits hematopoietic cell differentiation of M1 cells. Blood 1995;85:3040-3047.
Healy L, May G, Gale K, et al. The stem cell antigen CD34 functions as a regulator of hemopoietic cell adhesion. Proc Natl Acad Sci USA 1995;92:1220-1224.
Cheng J, Baumhueter S, Cacalano G, et al. Hematopoietic defects in mice lacking the sialomucin CD34. Blood 1996;87:479-490.
Salati S, Zini R, Bianchi E, et al. Role of CD34 antigen in myeloid differentiation of human hematopoietic progenitor cells. Stem Cells 2008;26:950-959.
Haase D, Feuring-Buske M, Könemann S, et al. Evidence for malignant transformation in acute myeloid leukemia at the level of early hematopoietic stem cells by cytogenetic analysis of CD34+ subpopulations. Blood 1995;86:2906-2912.
Feller N, Schuurhuis GJ, van der Pol MA, et al. High percentage of CD34 positive cells in autologous AML peripheral blood stem cell products reflects inadequate in vivo purging and low chemotherapeutic toxicity in a subgroup of patients with poor clinical outcome. Leukemia 2003;17:68-75.
Yıldırım AT, Gülen H. A child AML-M1 with CD79a, CD56, and CD5 coexpressions and misdiagnosed as biphenotypic acute leukemia. Dicle Medical Journal 2015;42:89-92.
Samdani A, Vijapurkar U, Grimm MA, et al. Cytogenetics and P-glycoprotein (PGP) are independent predictors of treatment outcome in acute myeloid leukemia (AML). Leuk Res 1996;20:175-180.
Rosenau H. Legal prerequisites for clinical trials under the revised Declaration of Helsinki and the European Convention on Human Rights and Biomedicine. Eur J Health Law 2000;7:105-121.
Tiirikainen MI, Syrjälä MT, Jansson SE. Flow cytometric analysis of P-glycoprotein in normal and leukemic cell. Ann Hematol 1992;65:124-130.
Chin KV, Pastan I, Gottesman MM. Function and regulation of the human multidrug resistance gene. Advances in Cancer Research 1993;60:157-181.
Nooter K, Herweijer H. Multidrug resistance (mdr) genes in human cancer. Br J Cancer 1991;63:663-669.
Beck WT, Grogan TM, Willman CL, et al. Methods to detect P-glycoprotein associated multidrug resistance in patients’ tumors: Consensus recommendations. Cancer Res 1996;56:3010-3020.
Fenaux P, Preudhomme C, Laï JL, et al. Mutations of p53 gene in B-CLL report on 39 cases with cytogenetic analysis. Leukemia 1992;6:246-250.
Pall G, Spitaler M, Hofmann J, et al. Multidrug resistance in acute leukemia: a comparison of different diagnostic methods. Leukemia 1997;11:1067-1072.
Chevillard S, Vielh P, Validire P, et al. French multicentric evaluation of mdr1 gene expression by RT-PCR in leukemia and solid tumors. standardization of RT-PCR and preliminary comparisons between RT-PCR and immunohistochemistry in solid tumors. Leukemia 1997;11:1095-1106.
Pirker R, Wallner J, Geissler K. MDR 1 gene expression and treatment outcome in acut myeloid leukemia. J Natl Cancer Inst 1991;83:708-712.
Kuwazuru Y, Yoshimura A, Hanada S, et al. Expression of the multidrug transporter, P-glycoprotein, in acute leukemia cells and correlation to clinical drug resistance. Cancer 1990;66:868-873.
Senent L, Jarque I, Martín G, et al. P-glycoprotein expression and prognostic value in acute myeloid leukemia. Haematologica 1998;83:783-790.
Gruber A, Vitols S, Norgren S, et al. Quantitative determination
of mdr-1 gene expression in leukemic cells from patients with acute leukemia. Br J Cancer 1992;66:266-272.
te Boekhorst PA, de Leeuw K, Schoester M, et al. Predominance
of functional multidrug resistance (MDR-1) phenotype in D34+acute myeloid leukemia cells. Blood 1993;82:3157-3162.
Abd El-Ghaffar HA, Aladle DA, Farahat SE, et al. P-glycoprotein
(P-170) expression in acute leukemias. Hematology 2006;11:35-41.