Quantitation Of LYL1 Expresion In Patients With Myeliod Leukemia Using Real Time Quantitative Polymerase Chain Reaction

Background and Objectives Leukemia is the most common haematologic malignancy. Although the pathogenesis of leukemia is not known clearly, many leukemia-related oncogenes have been found. The LYL1 gene was first identified as a partner in the chromosome translocation t(7;19) found in some cases of T-cell acute lymphoblastic leukemia (T-ALL). It encodes a basic helix-loop-helix transcription factor. Many clinic studies have shown the ectopic expression of LYL1 gene in patients with T-ALL. And it has been considered as an oncogenic transcription factor related to T-ALL. However, some scholars recently noticed that LYL1 gene was also ectopicly expressed in some patients with AML, high-risk MDS, and some cell lines of myeloid leukemia. Based on those investigations, we used reverse transcription polymerase chain reaction (RT-PCR) and fluorescent quantitative reverse transcription polymerase chain reaction (Q-RT-PCR) to detect the expression level of LYL1 mRNA in patients with acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML) and myelodysplastic syndrome (MDS) in order to determine the role of LYL1 gene in the pathogenesis of leukemia and the potential use in gene diagnosis and gene therapy of leukemia.Methods1. Patients: We seleted 50 patients admitted to the second hospital of Shandong University from March, 2003 to February, 2005, which include 25 cases of AML, 15 cases of CML, and 10 cases of MDS.2.We established methods of RT-PCR and Q-RT-PCR to detect the expression level of LYL1 mRNA in periphal blood leukocytes or Bone marrow cells of patients with AML, CML or MDS. And 10 healthy donors were studied as control group. We used the plasmid containing LYL1 sequence as positive control, and GAPDH was detected as internal control. Ten fold serial dilutions of LYL1 plasmid yielded standard curve that allowed the quantification of LYL1 copies.3. We performed analysis on the patients with the high expression level of LYLl gene, and wanted to know whether the high expression of LYLl had an effect on the response of patients with leukemia to chemotherapy.Results1. The results obtained in RT-PCR: The LYLl was expressed in 72% (18/25) of patients with AML. The expression of LYLl was negative in 4 patients with CML in chronic phase, but it's positive in 72.7%(8/ll) of patients with CML in acceleration phase or blast crisis. The positive rate of LYLl was 70%(7/10) in patients with MDS. The expression of LYLl was negative in all 10 healthy donors.2. The results obtained in Q-RT-PCR: LYLl mRNA was expressed at very low levels in normal bone marrow cells, but was found at high levels in the majority of patients with AML or MDS or CML. The expression level of LYLl was at least 2 times greater than normal bone marrow in 80% of AML cases (20/25) and 80% of MDS cases (8/10). In some cases the level of LYLl was very high. LYLl was also expressed at very low levels in 4 patients of CML in chronic phase. The expression level of LYLl was at least 2 times greater than normal bone marrow in 72.7% of CML cases (8/11) in acceleration phase or blast crisis.3. The consistency of results from RT-PCR and Q-RT-PCR: The expression level of LYLl was at least 2 times greater than normal bone marrow in Q-RT-PCR in the cases whose expression of LYLl was positive in RT-PCR. The results obtained from two methods were basicly consistent.4. The high expression level of LYLl was observed in all subsets of AML, CML in acceleration phase or blast crisis, and high-risk of MDS. The rate of complete remission (CR) was lower, and the time of attaining complete remission was postponed in cases with very high level of LYL.Conclusions1 .The specificity, sensitivity and reproducibility were good for the fluorescent quantitative reverse transcription polymerase chain reaction (Q-RT-PCR) to detect LYLl expression. It can be considered as a promising tool for quantitative assessment of LYLl gene in experimental as well as clinical studies.2.The results show that the expression level of LYLl was at least 2 times greater than normal bone marrow in 70% of leukemia cases.3. The level of LYL1 expression was related in some degree to the response to chemotherapy in cases of leukemia.