Study On Detection Of Minimal Residual Disease In Acute Leukemia With Immunolgical Method And Polymerase Chain Reaction

The detection of minimal residual leukemic cells(MRLC) represents a major goal in acute leukemia with great applications: identification of patients at high risk of relapse, earlier detection of relapse, possible improvement of autologous bone marrow transplantation (determination of the best time for bone marrow harvest and assessment of its quality) etc.Of the numerous methods (cytology, immunology, cytogenetics, clonogenic assays, molecular biology, etc.), both immunologic (double color immunofluorescence staining, DCIF) and molecular biologic method (polymerase chain recation, PCR) offer sensitive tools to study MRLC. In this study we developed two practical techniques to detect MRLC. One is the combination of DCIF and discontinuos Percoll gradient(density 1.070g/ml). This approach was capable of detecting one leukemic cell out of approximately 1,000 to 10,000 peripheral blood mo-nonuclear cells (PB HNC) with reliable specificity. We have applied this method to monitor 10 patients with CALLA-positive acute lymphoblastic leukemia(ALL). Residual leukemic cells at frequency of 0.08%~8% were demon-strated in 4 PB samples obtained from the 4 patients during complete remission, and the recurrent leukemia was predicted in one case six weeks priorto clinical morphologic examination.Another is PCR and oligotyping assay (PCR) for detection of MRLC in acute myelogenous leukemia (AML) exhibiting a H-ras mutation. The amplified N-ras sequence was detectable when as little as 10 pg of PB MNC genomic DNA was tested by this techneque. With this method leukemic cells harboring the N-ras point nutation were specifically detected at a minimum concentration of 0.1%~0.5%. Presentation DNAs from ten patients with AML were screened for mutations in codons 12, 13, and 61 of N-ras using PCR method. Three of then (30%) contained mutation--all in codon 13 of N-ras. We have conducted a follow-up study of two of the three N-ras-mutated patients. The initial results of follow-up showed that N-ras point mutation detectable by suitable oligonucleotide probes, could be taken as a clonal marker to trace the MRLC carring the N-ras mutation through the course of the disease. We concluded that both methods proposed here are sensitive, specific tools to deteet MRLC, and can be of value in clinical or experimental studying on MRLC.