Abstract
Numerous studies have analyzed the expression and prognostic importance of various proteins in acute myelogenous leukemia (AML). We sought to determine whether the sample source and methodology used to measure protein expression affect the results obtained. To determine the importance of sample source, we used Western blotting to compare the expression of eight proteins and phosphoproteins in the leukemia blast-enriched fraction of 118 blood- and 108 marrow-derived samples, including 37 paired samples. To determine the importance of methodology, the expression of five proteins was measured in 20 paired samples by Western blotting, laser scanning cytometry (LSC), and flow cytometry. The mean expression and range of expression in blood- and marrow-derived samples were statistically identical for all eight proteins. Expression measurements for the 37 paired blood and marrow samples also had very high statistical correlation. The LSC and flow cytometry data had the highest concordance when compared using Kolmogorov–Smirnoff D-stats (range of R values, 0.8–1.0). High concordance was also observed between the LSC and flow cytometry results when the percentage of cells positive for expression was dichotomized into positive or negative expression. However, there was less correlation between LSC and flow cytometry when the actual percentages of positive cells were compared. The majority of discordant situations involved samples that were positive by flow cytometry but negative by LSC. The correlation between Western blotting signal intensity and the percentage of expression-positive cells measured by LSC or flow cytometry varied by protein but was limited when there was little heterogeneity in expression by either method. In conclusion, provided that leukemia blast-enriched fractions were analyzed, the blood- and marrow-derived samples had identical protein expression. There was good concordance of results between flow cytometry and LSC, which share similar technology, but more limited correlation between these methods and Western blotting.
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References
Kornblau SM, Vu HT, Ruvolo P, Estrov Z, O'Brien S, Cortes J et al. BAX and PKCalpha modulate the prognostic impact of BCL2 expression in acute myelogenous leukemia. Clin Cancer Res 2000; 6: 1401–1409.
Darzynkiewicz Z, Bedner E . Analysis of apoptotic cells by flow and laser scanning cytometry. Methods Enzymol 2000; 322: 18–39.
Kamentsky LA, Burger DE, Gershman RJ, Kamentsky LD, Luther E . Slide-based laser scanning cytometry. Acta Cytol 1997; 41: 123–143.
Bedner E, Li X, Gorczyca W, Melamed MR, Darzynkiewicz Z . Analysis of apoptosis by laser scanning cytometry. Cytometry 1999; 35: 181–195.
Nuessler V, Stotzer O, Gullis E, Pelka-Fleischer R, Pogrebniak A, Gieseler F et al. Bcl-2, bax and bcl-xL expression in human sensitive and resistant leukemia cell lines. Leukemia 1999; 13: 1864–1872.
He L, Fox MH . Comparison of flow cytometry and Western blotting to measure Hsp70. Cytometry 1996; 25: 280–286.
Revelen R, D'Arbonneau F, Guillevin L, Bordron A, Youinou P, Dueymes M . Comparison of cell-ELISA, flow cytometry and Western blotting for the detection of antiendothelial cell antibodies. Clin Exp Rheumatol 2002; 20: 19–26.
Kornblau SM, Womble M, Jackson CE, Chen W, Estey E, Andreeff M . Simultaneous activation of multiple signal transduction pathways confers poor prognosis in acute myelogenous leukemia [abstract]. Blood 2004; 104: 2992.
Banker DE, Groudine M, Norwood T, Appelbaum FR . Measurement of spontaneous and therapeutic agent-induced apoptosis with bcl-2 protein expression in acute myeloid leukemia. Blood 1997; 89: 243–255.
Grandage VL, Gale RE, Linch DC, Khwaja A . PI3-kinase/Akt is constitutively active in primary acute myeloid leukaemia cells and regulates survival and chemoresistance via NF-κB, MAPkinase and p53 pathways. Leukemia 2005; 19: 586–594.
Shen Y, Iqbal J, Hyang JZ, Zhou G, Chan WC . BCL2 protein expression parallels its mRNA level in normal and malignant B cells. Blood 2004; 205: 2936–2939.
Darzynkiewicz Z, Bedner E, Smolewski P . Flow cytometry in analysis of cell cycle and apoptosis. Semin Hematol 2001; 38: 179–193.
Darzynkiewicz Z, Smolewski P, Bedner E . Use of flow and laser scanning cytometry to study mechanisms regulating cell cycle and controlling cell death. Clin Lab Med 2001; 21: 857–873.
Deptala A, Bedner E, Darzynkiewicz Z . Unique analytical capabilities of laser scanning cytometry (LSC) that complement flow cytometry. Folia Histochem Cytobiol 2001; 39: 87–89.
Kornblau SM, Thall PF, Estrov Z, Walterscheid M, Patel S, Theriault A et al. The prognostic impact of BCL2 protein expression in acute myelogenous leukemia varies with cytogenetics. Clin Cancer Res 1999; 5: 1758–1766.
Kohler T, Leiblein S, Borchert S, Eller J, Rost AK, Lassner D et al. Absolute levels of MDR-1, MRP, and BCL-2 MRNA and tumor remission in acute leukemia. Adv Exp Med Biol 1999; 457: 177–185.
Invernizzi R, Pecci A, Bellotti L, Ascari E . Expression of p53, bcl-2 and ras oncoproteins and apoptosis levels in acute leukaemias and myelodysplastic syndromes. Leuk Lymphoma 2001; 42: 481–489.
Chen Y, Zhou J, Yue B, Xiang Z, Song X, Li C et al. Bcl-2 gene and its family genes Bax, Bcl-Xl as well as Fas/Apo-1 and their clinical significance in acute leukemia. Chin Med J 1998; 111: 682–685.
Zhou J, Chen Y, Li C, Liu W . The protein expression of Bcl-2, Bax, Fas/Apo-1 in acute myeloid leukemia. J Tongji Med Univ 1998; 18: 42–45.
Bosanquet AG, Bell PB, Burlton AR, Amos TA . Correlation of bcl-2 with P-glycoprotein expression in chronic lymphocytic leukaemia and other haematological neoplasms but of neither marker with ex vivo chemosensitivity or patient survival. Leuk Lymphoma 1996; 24: 141–147.
Acknowledgements
This work was supported by Leukemia and Lymphoma Society Grant 6448 and 6172 and National Institutes of Health Grants PO1 CA-55164 and CA-16672.
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Kornblau, S., Womble, M., Cade, J. et al. Comparative analysis of the effects of sample source and test methodology on the assessment of protein expression in acute myelogenous leukemia. Leukemia 19, 1550–1557 (2005). https://doi.org/10.1038/sj.leu.2403845
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DOI: https://doi.org/10.1038/sj.leu.2403845
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