Analysis Of The Relationship Between The FLT3 Gene Mutation And The Prognosis Of Leukemia Using DNA In Stored Bone Marrow Slides

Background and Objectives:Fms-like tyrosine kinase-3 (FLT3) is a member of the class III receptor tyrosine kinase (RTK)family, and plays an important role in regulating the proliferation of the hematopoietic cells by the activation of the tyrosine residues and subsequent signal transduction with its ligand called FLT3 ligand(FL).Recently, most studies have indicated that FLT3 gene has a class of activation mutation:internal tandem duplication (ITD) in the juxtamembrane (JM) domain, which also can be called FLT3 length mutation, and the patients with FLT3-ITD have some clinical features. Mutant FLT3 exhibit constitutive activity and autophosphorylation in the absence of FL binding, and then cause an aberrant signal transduction pathway, furthermore having an important pathological effect on the hematopoietic malignancy process. Some of acute leukemia (AL) patients displayed both receptor and ligand suggesting a possible autocrine or paracrine stimulation. High level of FLT3 expression may play an important role in the pathogenesis of acute leukemia by strengthening the common signal transduction of Wt-FLT3.The study was to extract micro amount DNA from bone marrow slides and to investigate FLT3-ITD in patients with ANLL and the clinical features of the patients with FLT3-ITD.Method:Micro amount DNA was extracted from 79 bone marrow slides which had been kept in storage for 1 to 5 years without Wright's staining or formalin fixation. Analysis of FLT3-ITD was performed on these bone marrow slides samples from 48 patients with de novo acute nonlymphocytic leukemia (ANLL) (1 case for M1,18case for M2,13 case for M3,0 case for M4, 15 case for M5,1 case for M6,0 case for M7),28patients with de novo acute lymphocytic leukemia (ALL),1 patients with acute mixed lineage leukemia,1 patient from myelodysplastic syndrome (MDS) to M2,1 patient from multiple myeloma (MM) to M2, and 10 controls without hematologic malignancies by polymerase chain reaction (PCR). Logistic analysis was used to reveal whether age, gender, bone marrow blast cells, peripheral white blood cells, hemoglobin (Hb), platelet(PLT), creatinine(Cr), alanine aminotransferase (ALT), splenohepatomegalia, CNS infiltration and FLT3 mutation have any relationship with complete remission (CR) after the first chemotherapy. Survival analysis was carried out by COX regression about age, gender, FAB type, peripheral white blood cells, Hb, PLT, peripheral blood blast cells, bone marrow blast cells, serum lactate dehydrogenase (LDH), Cr or ALT, splenohepatomegalia, lymphadenectasis, CNS infiltration and FLT3 mutation.Result:FLT3-ITD was detected in 5 patients of these 79 patients.5 of 48(10.42%) ANLL patients (1 with M2,1 with M3,2 with M5, and 1 with M6). There were no noticeable deviation among the FAB subtypes (P<0.05). No FLT3-ITD was found in 28 ALL,1 acute mixed lineage leukemia,1 from MDS to M2,1 from MM to M2 or 10 healthy controls. No correlation can be found between FLT3-ITD and age, gender, peripheral white blood cells, Hb, PLT, peripheral blood blast cells, bone marrow blast cells, serum LDH, Cr or ALT(P>0.05).FLT3-ITD ANLL patients have lower CR rate tendency and shorter survival time (P<0.05) comparing with FLT3-ITD negative ANLL patients. Logistic analysis reveals that age, gender, bone marrow blast cells, peripheral white blood cells, Hb, PLT, Cr, ALT, splenohepatomegalia, CNS infiltration or FLT3 mutation have no relationship with CR after the first chemotherapy (P>0.05).COX regression survival analysis demonstrates that FLT3-ITD and CNS infiltration are risk factors which affect the survival of AL patients (respectively, RR=5.910, RR=13.884).Conclusion:Micro amount DNA can be extracted from bone marrow slides by phenol/ chloroform/isoamyl alcohol protocol, which is an important empirical method for retrospective study of molecular biology. FLT3-ITD represent a common genetic abnormality in ANLL patients especially M5 and M3. ANLL patients with FLT3-ITD were associated with a lower CR rate tendency and shorter survival time comparing with FLT3-ITD negative patients. FLT3-ITD, age, gender, bone marrow blast cells, peripheral white blood cells, Hb, PLT, splenohepatomegalia, CNS infiltration, Cr, or ALT have no relationship with CR after the first chemotherapy for AL patients. FLT3-ITD and CNS infiltration are risk factors of poor prognosis in patients with newly diagnosed AL.