ABSTRACT
This study was aimed to investigate the clinical value of quantification of AML1/ETO fusion transcripts using real-time reverse transcription PCR. Fourteen AML1/ETO positive children out of 52 AML children were selected. A serial dilution of AML1/ETO plasmid was used as a template for the AML1/ETO real-time PCR. AML1/ETO was quantified according to the expression of the GAPDH housekeeping gene at new diagnosis and during/after chemotherapy and transplantation. SPSS statistics was used to analyze the data. The results showed that the ratio of AML1/ETO: GAPDH expression level at new diagnosis varied in the range 0.219-2.080 (median 0.648) among the patients, without relevance with percentage of blasts. The detection sensitivity was up to the dilution of 1:10(5). Six patients showed a slight decline of AML1/ETO (higher than 5 x 10(-2)) at 1 month, three of whom relapsed in the early stage and one later. Five patients had a higher level than 5 x 10(-3) at 3 months, three of whom relapsed. Four patients with always a higher level than 5 x 10(-3) all relapsed in early stage. After six months, four out of them with constant low-level expression (10(-4) - 10(-6)) were in continuous complete hematological remission (CCR). In another patient, a rapid rise of AML1/ETO transcripts could be detected at CR stage and he relapsed 5 months later. The AML1/ETO gene expression leveling off by 10(-5) - 10(-6) could be detected in 3 patients at their complete remission after 9 months. It is concluded that real-time RT-PCR is a suitable approach for quantifying AML1/ETO transcripts in monitoring of AML patients with t(8;21) during/after chemotherapy and provides data of diagnostic relevance.
Subject(s)
Adolescent , Child , Child, Preschool , Female , Humans , Male , Acute Disease , Core Binding Factor Alpha 2 Subunit , Genetics , Gene Expression Regulation, Leukemic , Leukemia, Myeloid , Diagnosis , Genetics , Therapeutics , Neoplasm, Residual , Diagnosis , Genetics , Oncogene Proteins, Fusion , Genetics , RUNX1 Translocation Partner 1 Protein , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , Methods , Transcription, GeneticABSTRACT
<p><b>OBJECTIVE</b>Hematologic relapse remains the greatest obstacle to the cure of acute lymphoblastic leukemia (ALL), especially T-lineage acute lymphoblastic leukemia (T-ALL) in children. Recent studies have shown that patients with increased risk of relapse can be identified by measuring residual leukemic cells, called minimal residual disease (MRD), during clinical remission. Current polymerase chain reaction (PCR) methods, however, for measuring MRD are cumbersome and time-consuming. To improve and simplify MRD assessment, the author developed a real-time quantitative PCR (RQ-PCR) assay for the detection of leukemic cells that harbor the tal-1 deletion. In addition, the author discussed the significance of MRD levels at different stages in treatment and prognosis of children with T-ALL.</p><p><b>METHODS</b>A total of 50 consecutively enrolled patients with T-ALL were analysed for detection of leukemic cells harboring the most common tal-1 deletion. Serial dilutions of leukemic DNA were studied to find the sensitivity of detection with RQ-PCR assay. The MRD of 28 samples in clinical remission from 10 patients were quantified by RQ-PCR assay and limiting dilution assay. The results detected by both methods were compared statistically with correlation analysis.</p><p><b>RESULTS</b>(1) A total of 10 patients presented tal-1 deletion involving the sildb1 breakpoint rearranged to tal1db1 in 50 cases with T-ALL. The breakpoints of relapsed samples are the same as those of the corresponding diagnostic samples; (2) The RQ-PCR assay had a sensitivity of detection of one leukemic cell among 100,000 normal cells. In 24 samples, MRD levels > 10(-5) could be detected with both methods. The percentages of leukemic cells measured by the two methods correlated well (r = 0.898, P < 0.001); (3) The MRD levels of 3 patients out of the 8 cases undergoing disciplinary regimen were over 10(-4) at the end of induction chemotherapy. They all relapsed in bone marrow during chemotherapy. The higher the MRD levels, the earlier the relapse. The other 5 patients with MRD levels < 10(-4) had been relapse-free survival (RFS) for 4-59 months, one of whom with increased MRD levels > 10(-4) for twice at the continuation stage had been RFS for 27 months till now.</p><p><b>CONCLUSIONS</b>The sildb1-taldb1 deletion presents in 20% of T-ALL, and is an ideal PCR marker for its specificity, uniform and stability; The tal-1 RQ-PCR can be used for the rapidly, sensitively and accurately quantitative assessment of MRD in T-ALL with the tal-1 deletion. MRD levels at different stages of chemotherapy have different significance in prognosis and treatment.</p>