| This thesis will comprise two parts：Part I: Molecular genetics research on genemutations in acute promyelocytic leukemia; Part II: Clinical and laboratory characteristicsof two cases of atypical chronic myeloid leukemia.PART I Molecular genetics research on gene mutations in acutepromyelocytic leukemiaObjective:To analyze the mutation incidence of multiple common leukemia-related genes inacute promyelocytic leukemia (APL), to explore whether these mutations synergesticallyinvolved in the leukemogenesis of APL and their relevance with clinical features,cytogenetics and molecular risk stratification.Methods:A total of84specimens of admitted de novo APL patients in the First AffiliatedHospital of Soochow University from February2005to October2010was recruited in thisresearch to assess the genetic mutation patterns. The genomic DNA is extracted from bonemarrow mononuclear cells and amplified by PCR(polymerase chain reaction). Theanalysis of TET2, DNMT3A, IDH1, IDH2, EZH2, CBL, ASXL1, MLL-PTD, NPM1, WT1,RUNX1, c-KIT, FLT3-ITD, FLT3-TKD, N-RAS and JAK2V617F gene mutations andclinical features of mutation-positive patients were analyzed by massively DNAsequencing and statistical methods.Results:1. We demonstrate the presence of66cryptic genomic aberrations in84patients(78.6%) consisting of mutations (51patients), in which the mutations with the highestincidence were found as FLT3-ITD, reaching27.4%(23/84). All the duplications of FLT3 were heterozygosis and sustained in open reading frame, which differ in length andinsertion sites. Next, there were12cases WT1mutations,9for FLT3-TKD,7for TET2,5for N-RAS,4for ASXL1,2for EZH2mutation and1positive in MLL-PTD, IDH1and CBLrespectively. No mutation was found in other JAK1, DNMT3A, c-Kit, NPM1, IDH2,RUNX1and JAK2(V617F) common leukemia-related genes.2. Median age of our patients was34years (range,8to79years) and44patients(52.4%) were male and40(47.6%) were female. Combined analysis with clinical datademonstrated that the patients with FLT3-ITD mutation displayed higher white blood cellcounts (WBC=24.3×10~9/L VS WBC=7.2×10~9/L, P=0.004), which displayed nosignificant difference compared with wild-type in median age, platelets counts,hemoglobin and blast percentage. The patients with N-RAS mutation showed lower plateletcounts (PLT=70.0×10~9/L VS PLT=21.5×10~9/L, P=0.012), which displayed no significantdifference compared with wild-type in median age, white blood cell counts, hemoglobinand blast percentage. The median age of NRAS-positive patients is significantly larger, butthere was no significant difference (P>0.05). Overall survival of these patients wasobviously shorten as compared with wild-type (for FLT3-ITD,39.1months vs71.7months;p=0.010; for NRAS,24.79months vs68.02months; p=0.028). The difference of OSbetween mutant and wild-type of all above mentioned cases was statistically significance(P＜0.05). There were no significant correlations between age, sex, WBC and mutations inWT1, FLT3-TKD, TET2, ASXL1, EZH2, MLL-PTD, IDH1or CBL.3. In the cytogenetic analysis of84APL patients, recurrent chromosomalabnormalities that are the cytogenetic hallmarks of genetically except simple t(15;17)entities were identified in13cases, namely+8(4/84),17abnormal (2/84),9abnormal(2/84); while t (7;12)(1/84),+21,+15,-Y,+6,-6,7q+and11abnormal were in1caserespectively. The difference between APL with simple t(15;17) and additional abnormalkaryotype did not show a significant statistically variant OS (28.94months vs67.10months; p=0.576) or a mutation incidence of leukemia-related genes (6/13vs45/71;p=0.219) as other clinical features. Conclusion:The FLT3-ITD mutation is recurring genetic change in APL, together with N-RASmutation indicates poor prognosis. Additional abnormal karyotype does not associate withprognosis and mutation incidence of APL. PART II Clinical and laboratory characteristics of two atypicalchronic myeloid leukemia cases.Object:Patients with clinical features of chronic myeloid leukemia (CML) who lack thePhiladelphia chromosome and/or the BCR-ABL fusion gene are generally considered ashaving atypical CML (aCML) or, more broadly, an unclassifiable myeloproliferative ormyelodysplastic/myeloproliferative disease. Here we report two aCML patient with raregenetic abnormality and research on clinical manifestations, laboratory features andindividual targeted therapy.Methods:Mononuclearcells of two patients with atypical chronic myeloid leukemia by bonemarrow aspiration were disposed with conventional chromosome suspension preparationof hypotension, fixation, harvest, RHG banding, Giemsa-staining and karyotype analysis.9p24probe (5’-D9S1969and3’-SHGC-149694, KREATECH) was investigated bydual-color Fluorescence in situ hybridization (FISH). BCR-JAK2and ETV6-PDGFRβprimers were designed and amplificated PCR product were sequenced.Results:Patient1: A28-years-old Chinese male was admitted to our hospital with the routineexamination finding WBC increased four months on July10th,2010. Physical examinationrevealed skin pallor, and splenomegaly were observed. Examination of peripheral blood(PB) indicated a white blood cell count of50.1×109/L (83%neutrophil,8%lymphocytes, 8%monocytes,1%metamyelocytes and2%eosinophil), a platelet count of159×10~9/L,and128g/L hemoglobin. Bone marrow (BM) aspirate was markedly with granulocytichyperplasia and characterized by increasing eosinocytes. Flow cytometricimmunophenotyping identified granuloid cells with dysmaturity and no evidence of anincrease in myeloid blasts. Cytogenetic analysis (RHG banding) demonstrated a karyotypeof ins(22;9)(q11;p13p24). Reversetranscription polymerase chain reaction (RT-PCR)analysis failed to detect BCR-ABL fusion transcripts as well as JAK2V617F mutationfrom the bone marrow cells. The BM biopsy tissue was considered as aCML. Based onabove datas, diagnosis of aCML was made. Since this patient had negative BCR/ABLfusion gene, low-dose interferon alpha and hydroxyurea therapy was given upon remaineduntil now.Patient2: The patient was a55-year-old male who presented with the routine examinationfinding white blood cells increased half months in June2012. A complete blood countrevealed leukocytosis (WBC=40.8×10~9/L), hemoglobin (145g/L) and thrombocyte(278×10~9/L). Examination of his peripheral blood film revealed nucleated red blood cells,and the differential consisted primarily of neutrophils and neutrophil precursors, rangingfrom metamyelocytes to bands. There was an increased number of metamyelocytes (10%),but relatively few monocytes (2%) and lymphocytes (3%). Eosinophils and basophils arealso visible. A bone marrow aspirate revealed hypercellularity, an increased M:E ratio andmegaloblastic degeneration of erythroid. Blasts were normal in number. The originalcytogenetics were reported as a t(5;12)(q33;p13) in20of20spreads. Testing by RT-PCRdemonstrated the ETV6-PDGFRB fusion gene. Treatment with hydrea and imatinibmesylate (formerly STI571; now Gleevec in the UnitedStates and Glivec in Europe) wasstarted at400mg per day. At this dose, a complete hematologic and cytogenetic responsewas attained within three month. Then low-dose100mg/d maintenance treatment wasadopted. Minimal residual disease analysis by nested RT-PCR was still negative at thisjuncture. Until March2013the patient is still in molecular genetics remission.Conclusion: Atypical chronic myeloid leukemia patients with BCR-JAK2and ETV6-PDGFRβfusion gene were resulted from the ins (22;9)(q11;p13p24) resulting in the BCR gene andthe JAK2gene juxtaposed and t(5;12)(q33;p13) causing the ETV6genes and PDGFRβgenes juxtaposition.. |
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