Potential CD34 signaling through phosphorylated-BAD in chemotherapy-resistant acute myeloid leukemia.

Acute myeloid leukemia (AML) constitutively express growth factors and cytokines for survival. Chemotherapy alters these signals to induce cell death. However, drug resistance in AML remains a major hindrance to successful treatment and early warning is unavailable. Modulation of signaling pathways during chemotherapy may provide a window to detect response and predict treatment outcome. Blood samples collected from AML patients before and at day-3 of induction therapy were compared for changes in expression of CD117, CD34, pro-inflammatory cytokines and mediators of Akt and MAPK pathways, using multi-color flow cytometry. Nine patients were diagnosed as drug-resistant and seven sensitive to chemotherapy. Twelve were paired. Average percentages of CD34 (66.8 ± 11.7% vs. 26.2 ± 5.8%, p = 0.033) and pBAD (66.9 ± 8.2% vs. 28.9 ± 8.2%, p = 0.016) were significantly increased in chemo-resistant (N = 9) compared to chemo-sensitive (N = 5) samples. Percentages of CD34 were strongly correlated with pBAD (R = 0.785; p = 0.001; N = 14) and pFKHR (R = 0.755; p = 0.002; N = 14) at day-3 induction. Chemo-sensitive cases expressed significantly higher percentages of IL-18Rα (71.9 ± 9.6% vs. 29.8 ± 5.8%, p = 0.016). Though not significantly different in the outcome, IL-1ß was strongly associated with activated Akt-S473, IL-6 with phosphorylated JNK and FKHR while TNF-α appeared to trigger Bim, in treated samples. These preliminary results suggested AML cells resistant to chemotherapy increased expression of CD34 and may signal through pBAD while cells sensitive to chemotherapy-induced IL18Rα expression. These were observed early during induction therapy. Identifying CD34 is interesting as it is a convenient marker to monitor drug-resistance in AML patients. Inhibition of CD34 and pBAD signaling may be important in treating drug-resistant AML.

Selection of reference genes for quantitative studies in acute myeloid leukaemia

@#Introduction: Quantitative polymerase chain reaction (qPCR) is commonly used in the investigation of acute myeloid leukaemias (AML). Stable reference genes (RG) are essential for accurate and reliable reporting but no standard method for selection has been endorsed. Materials and Methods: We evaluated simple statistics and published model-based approaches. Multiplex-qPCR was conducted to determine the expression of 24 candidate RG in AMLs (N=9). Singleplex-qPCR was carried out on selected RG (SRP14, B2M and ATP5B) and genes of interest in AML (N=15) and healthy controls, HC (N=12). Results: RG expression levels in AML samples were highly variable and coefficient of variance (CV) ranged from 0.37% to 10.17%. Analysis using GeNorm and Normfinder listed different orders of most stable genes but the top seven (ACTB, UBE2D2, B2M, NF45, RPL37A, GK, QARS) were the same. In singleplex-qPCR, SRP14 maintained the lowest CV in AML samples. B2M, one of most stable reference genes in AML, was expressed near significantly different in AML and HC. GeNorm selected ATP5B+SRP14 while Normfinder chose SRP14+B2M as the best two RG in combination. The median expressions of combined RG genes in AML compared to HC were less significantly different than individually implying smaller expression variation after combination. Genes of interest normalised with RG in combination or individually, displayed significantly different expression patterns. Conclusions: The selection of best reference gene in qPCR must consider all sample sets. Model-based approaches are important in large candidate gene analysis. This study showed combination of RG SRP14+B2M was the most suitable normalisation factor for qPCR analysis of AML and healthy individuals.