Experimental And Clinical Research Of FLT3, NPM1 And C-KIT Gene In Acute Myeloid Leukemia By Bone Marrow Slides

Background and ObjectivesAcute myeloid leukemia (AML) is a group of malignant hematologic diseases with different biological characteristics. In recent years, with the progress in molecular genetics, most of AML can be detected in abnormal gene levels. FLT3 gene plays an important role in proliferation and differentiation of hematopoietic stem /progenitor cells, precursor B cells and so on. FLT3 gene mutations may disrupt the proliferation, differentiation and apoptosis of the normal hematopoietic cells leading to the occurrence of leukemia. Nucleophosmin(NPM, NO38, B23 or NPM1 protein) is expressed in the nucleolus, which can shuttle between the nucleolus and the cytoplasm. It takes part in the transport and synthesis of ribosomal precursor, the replication of centrosome, genomic stability, the activity of DNA polymerase, and make it regulate cell cycle progression and proliferation. Besides, the interaction among NPM1 protein, P53 and P19 protein plays an important role in tumor inhibition. NPM1 mutations can make NPM1 protein transfer to the cytoplasm and the tumor suppressor protein in the nucleolus named Arf(alternate-reading-frame protein) inactivates in this way. Then it promotes proliferation of leukemia cells by p53 or non-dependent p53 pathway. C-KIT gene encodes transmembrane tyrosine kinase receptor of molecular weight 145kD. Its ligand is stem cell factor (SCF). SCF is an important hematopoietic growth factor, which promotes the proliferation and differentiation of hematopoietic stem cells together with other cytokines. C-KIT gene mutations make the C-KIT spontaneous receptor dimerization which are not dependent on the ligand and lead to the C-KIT receptor consititutive activative. These result in hematopoietic cells excessive proliferation or apoptosis inhibiting. All of these cause leukemia. National Comprehensive Cancer Network (NCCN) in 2011 has regarded FLT3 gene, NPM1 gene and C-KIT gene mutations as an important risk stratification of Molecular Genetics logo in AML. It suggests that gene mutations have closely relationship with the occurrence, development, prognosis and efficacy of AML. The research studys the FLT3 gene, NPM1 gene and C-KIT gene mutations in AML by extracting DNA from the storage of bone marrow slides and investigates the relationship of the three gene mutations and clinical features in AML. The research can provide more scientific experiments and theoretical basis for the AML clinical stratification, prognosis and molecular targeted therapy.Methods1. Extract DNA from the storage of bone marrow slides by a improved phenol: chloroform:isoamyl alcohol method.2. Use polymerase chain reaction (PCR) technology and agarose gel electrophoresis to detect the 55 AML patients with FLT3 (internal tandem duplication, ITD) gene mutations.3. Use PCR, DNA sequencing and molecular cloning to detect and analyse the 55 AML patients with NPM1 gene mutations.4. Use PCR, DNA sequencing and molecular cloning to detect and analyse the 55 AML patients with C-KIT gene mutations.ResultsWe can extract DNA from the bone marrow slides of 55 cases with-20℃frozen storage without Wright stained and 10 cases with room temperature storage Wright stained by a improved phenol:chloroform:isoamyl alcohol method and DNA can be used in PCR, direct sequencing and molecular cloning sequencing analysis.10 of the 55 cases are FLT3-ITD positive.9 cases are heterozygous mutations and 1 case is homozygous mutation. The positive rate was 18.2%. M5 is more frequent in the FLT3-ITD positive patients, but there are no noticeable deviation among the FAB subtypes (P> 0.05). FLT3-ITD-positive group has lower complete remission (CR) rate after the initial remission induction therapy, shorter event-free survival (EFS) time and overall survival (OS) time than the negative group(P<0.05).9 of the 55 cases(16.4%) are NPM1 heterozygous gene mutations by PCR technology,DNA reverse direct sequencing and molecular cloning sequencing.9 cases are type A, which inserted the TCTG (reverse complementary is CAGA) between the 960-961 nucleotides. NPM1 gene mutations only be found in M2 and M5 in this study. NPM1 gene mutation group has higher white blood cell counts and bone marrow blast percentages than the wild group (P<0.05), but the sex, age, hemoglobin, platelets counts, lactate dehydrogenase, and the CR rate of the two groups show no significant differences (P> 0.05). The EFS rate of the mutation is higher in 10 months and the OS rate is higher in 19 months (P<0.05).3 cases of NPM1 mutations patients are FLT3-ITD positive. There are NPM1+/FLT3-ITD+, NPM1+/FLT3-ITD-, NPM1-/FLT3-ITD+, NPM1-/FLT3-ITD-four groups.We compare the four groups and the result of white blood cell counts is:NPM1+/FLT3-ITD+> NPM1+/FLT3-ITD-> NPM1-/FLT3-ITD+> NPM1-/FLT3-ITD-(P<0.05). The result of the CR rate of the four groups after initial remission induction therapy is:NPM1+/FLT3-ITD-> NPM1-/FLT3-ITD-> NPM1-/FLT3-ITD+>NPM1+/FLT3-ITD+(P<0.05). Blast percentages and lactate dehydrogenase are no significant differences among four groups (P>0.05). We use Cox regression to evaluate the six prognostic factors including age, bone marrow blast percentages, NPM1+/FLT3-ITD+, NPM1+/FLT3-ITD-, NPM1-/FLT3-ITD+and NPM1-/FLT3-ITD-which affect survival time of AML. The results suggest that NPM1-/FLT3-ITD+(P=0.005, RR=1.250) is a risk factor affecting the OS. We only find 2 cases(3.6%) have C-KIT gene mutations using PCR amplification of exon 17. The two case are the mutant D816H and mutant D816V respectively in M2a and M3. We use the Logistic Regression to analysize the relationgships between the factors and the CR rate after initial remission induction therapy. It shows that FLT3-ITD mutations are a kind of risk factor(OR= 66.940). C-KIT gene mutations are not found in patients with FLT3 and NPM1 mutations.Conclusions1. Improved phenol:chloroform:isoamyl alcohol method is a higher successful way to extract DNA from bone marrow slides. It is suitable for frozen storage without Wright stained and room temperature storage with Wright stained, and the DNA can be used for PCR, direct sequencing and molecular cloning sequencing analysis.2. FLT3-ITD mutations appear in AML patients frequently. FLT3-ITD positive patients have lower CR rate, shorter EFS and OS time. It suggests that FLT3-ITD mutations is a poor prognosis molecular marker in AML.3. The mutation rate of NPM1 gene and FLT3-ITD gene are similar. Type A of NPM1 gene mutations is frequent. NPM1 mutation group has some features with a high white cell counts and high bone marrow blast percentages. The EFS rate of the mutation is higher in 10 months and the OS rate is higher in 19 months (P<0.05). It suggests that NPM1 mutations are the better factors influencing the prognosis.4. 3 patients can be found with NPM1 and FLT3-ITD gene mutations in this study.4 groups of NPM1 gene and FLT3-ITD gene combinations affect CR rate. The highest CR rate after the initial remission induction therapy is the NPM1+/FLT3-ITD- group and the lowest is the NPM1+/FLT3-ITD+ group. Cox regression analysis shows that the NPM1-/FLT3-ITD+ group is a risk factor affecting OS time.5. The rate of C-KIT gene mutations is low in AML. C-KIT gene mutations are not found in patients with FLT3 and NPM1 mutations.6. Logistic regression analysis shows that FLT3-ITD mutations are risk factors which affect CR rate after initial remission induction therapy. NPM1 gene and C-KIT gene mutations do not affect the CR rate.