Routine expression profiling of microarray gene signatures in acute leukaemia by real‐time PCR of human bone marrow*

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Abstract
Summary: Cancer subtype diagnosis using microarray signatures has the potential to transform pathological diagnosis but the routine measurement of genes signatures remains difficult. Reverse transcription polymerase chain reaction (RT‐PCR) measurement of Indicator genes for acute myeloid leukaemia (AML) and acute lymphoblastic leukaemia (ALL) was used to determine gene signatures. Bone marrow (BM) mononuclear cells were sorted into total, CD34+ and CD34− fractions, and mRNAs globally amplified from each fraction using polyA PCR. The expression profile of the 17 top‐ranked genes distinguishing AML and ALL were measured by RT‐PCR in five ALL, 26 AML, 12 AML remission, four chronic myeloid leukaemia (CML) and nine morphologically normal BM samples. All but two of the genes measured showed similar expression in AML and ALL to that reported previously. Specifically, c‐MYB (P ≤ 0·04) was significantly increased in ALL in the total fraction, whilst HOXA9 (P ≤ 0·19) and cystatin c (P ≤ 0·01) were increased in AML in the CD34+ and CD34− fractions, respectively. c‐MYB, hSNF2, RBAP48, HKRT‐1, LYN, CD33, Adipsin and HOXA9 were increased in AML compared with remission AML, indicating an ability to determine disease activity. The method used is simple, sensitive and robust, enabling routine clinical use, and it can also be extended to other tumours types with gene signatures.