Clinical Significance Of The IDH1 And IDH2 Mutations In Patients With Acute Myeloid Leukemia

Background and ObjectThe acquired genetic alterations and epigenetic changes in hematopoietic progenitor cells is one of the pathogenesis of acute myeloid leukemia (AML) and these changes alter the normal mechanisms of cell growth, proliferation, and differentiation. At diagnosis, most patients with AML harbor at least one chromosome aberration in their marrow blasts. Some of the cytogenetic aberrations are not only diagnostic markers for specific AML subtypes, but also constitute independent prognostic factors for attainment of complete remission (CR), relapse risk(RR), and overall survival (OS). However, studies indicate that the rate of the cytogenetic aberrations of AML is 60～80%. Therefore, there are some patients who do not have cytogenetic aberrations and are called cytogenetically normal (CN) patients. Patients with normal karyotype have usually been classified in an intermediate-risk prognostic category according to cytogenetical analysis.From clinical observation, CN-AML patients with the same AML subtypes are treated with the same protocols, but the differences among complete remission, clinical effects and prognosis are remarkable. The reasons for the differences are the different molecular biology features. Recently, the markers associated with prognosis are being explored. For example, the PML-RARa and AML1-ETO genes had been studied thoroughly with the pathogenesis of AML; The overexpression of the EVI-1, ERG, BAALC, MN1, NF-κB are adverse for prognosis;some mutations are advantageous, for example, NPM1 and C/EBPα; however, some genes mutation are adverse, such as FLT3, WT1, c-KIT, MLL-PTD.The family of isocitrate dehydrogenases (IDHs) comprises some enzymes which catalyze oxidative decarboxylation of isocitrate intoα-ketoglutarate by using nicotinamide adenine dinucleotide (NAD) or NAD phosphate (NADP) as a cofactor to yield NADH or NADPH, respectively. In mammalian cells, there are three classes of IDH isoenzymes:mitochondrial NAD-dependent IDH, mitochondrial NADP-dependent IDH, and cytosolic NADP-dependent IDH. The human NADP-dependent IDH1 enzyme is encoded by the IDH1 gene at chromosome band 2q33.36 and is localized in cytoplasm and peroxisomes, and the mitochondrial NADP-dependent IDH2 enzyme is encoded by the IDH2 gene at chromosome band 15q26.1[6]. Although both IDH1 and IDH2 are thought to be involved in the cellular defense of oxidative damage, IDH1 has also an important role in lipid metabolism.Recently, mutations in IDH1 and IDH2 gene encoding isocitrate dehydrogenase have been evidenced in AML, ALL, MPD by whole-genome sequencing, but there are no mutations in CML. Now, the rate of the IDH1 and IDH2 gene in CN-AML is 5.5～9.6% and 3～11%, respectively. The patients with 1DH1 and IDH2 mutations have lower CR, higher RR and shorter OS. There have been no studies about IDH 1 and IDH2 mutations in China so far.Reverse transcriptase-polymerase chain reaction (RT-PCR) combined with sequencing technology were used in this study. The aim was to assess the frequencies, evaluate the prognostic significance of IDH1 and IDH2 mutations in 96 patients, and to explore their associations with clinical, cytogenetic and molecular characteristics as well as with other known gene mutations.Patients and Methods1. The 96 primary patients, including inpatient and outpatient of our hospital, were classified according to the FAB and MIC schema from Sep.2009 to Jan.2011. R-banding method was used in the karyotype analysis, At least 20 metaphases were assessable in all cases.2. The bone marrow (BM) or peripheral-blood (PB) specimens 2ml of untreated patients were used to analyze Immunophenotyping. A panel of monoclonal antibodies, including myeloid-associated antigens (CD13, CD33, CD11b, CD117, CD64, cMPO, CD15, CD14, and CD41a), lymphoid-associated antigens (CD2, CD5, CD7, CD4, CD3, CD8, cCD3, CD19, CD10, CD20, CD22 and CD79a), and the lineage-nonspecific antigens (HLA-DR, CD34, and CD56), were used to determine the immunophenotypes of leukemia cells.3. The bone marrow (BM) or peripheral-blood (PB) specimens 5ml of untreated patients were used to extract mononuclear cells by Ficoll-Hypaque gradient, then the cells were stored at -80℃.4. Mutational Analysis of 1DH gene:Total RNA was isolated following RNA extraction kit; Reverse transcriptase reaction 20ul was made up of:dNTP Mixture(10mMeach)1ul, Random 6 mers(20uM), Template RNA 100pg～1ug, RNase Free dH2O up to 10ul,65℃5min,4℃; 5 X PrimeScriptTM Buffer 4ul, RNase inhibitor(40U/ul)0.5ul, PrimeScriptTM RTase(for 2 step)0.5ul, RNase Free dH2O 5ul,30℃10min,42℃15min,95℃5min,4℃; Polymerase chain reaction was carried out in a final volume of 25μL, amplification was carried out at 94℃for 5 minutes, followed by 30 PCR cycles at 94℃for 1 minute,60℃for 1 minute, 72℃for 1 minute and with a final step of 72℃for 7 minutes. IDH primers as: Forward-IDH1-Ex4 5'-CTTCAGAGAAGCCATTATCTG-3' and Reverse-IDH1-Ex4 5'-TCACTTGGTGTGTAGGTTATC-3'(Amplification product 159bp); For-ward-IDH2-Ex4 5'-GTTCAAGCTGAAGAAGATGTG-3'and Reverse-IDH2-Ex5～6 5'-TGAGATGGACTCGTCGGTG-3'(Amplification product 313bp).5. At the same time, we analyzed the mutations of FLT3-ITD, FLT3-TKD, and c-kit. Total DNA was isolated following hematology genome DNA extraction kit. Polymerase chain reaction was carried out in a final volume of 25μL amplification was carried out at 95℃for 5 minutes, followed by 40 PCR cycles at 94℃for 30 seconds,55℃for 1 minute,72℃for 1 minute and with a final step of 72℃for 10 minutes. Primers as:Forward-FLT3-ITD-Exl4 5'-GCAATTTAGG TATGAAAGCCAGC-3 and Reverse-FLT3-ITD-Ex15 5'-CTTTCAGCATTTTG ACGGCAACC-3'(Amplification product 329bp);Forward-FLT3-TKD-Ex20 5'-C CGCCAGGAACGTGCTTG-3'and Reverse-FLT3-TKD-Ex20 5'-GCAGACGGG CATTGCCCC-3'(Amplification product 114bp); Forward-c-kit-Ex17 5'-ATGG AGGATGACTTG-3'and Reverse-c-kit-Ex17 5'-ATCCCATAGGACCAGACG-3'(Amplification product 302bp)6. PCR products were detected by agarose gel electrophoresis, then the positive samples were reclaimed by DNA product purification kit. Finally, the mutations were analyzed directly by sequencing technology.Results1. Karyotype analysis:Among the 96 cases, normal karyotype appeared in 67 cases; abnormal karyotype 24 cases, including t(8;21) 6 cases, t(15;17) 8 cases,-111 case,-7 1 case,+8 3 cases,+21 1 case, t(7;11) 1 case, complex karyotype 3 cases;failed analysis 5 cases.2. Mutational analysis of IDH gene:the mutation sites of IDH1 were at codon 127 and 130 in our study, while there was no mutation at codon 132;the mutation sites of IDH2 were at codon 140 in our study, while there was no mutation at codon 172. The prevalence of IDH 1 and IDH2 was 14.6%(14/96) and 2.17%(2/96).3. Mutational analysis of FLT3-ITD, FLT3-TKD and c-KIT:there were 15 cases with FLT3-ITD(+),2 cases FLT3-TKD(+),2 cases c-kit(+). The prevalence of FLT3 mutation in AML was 17.7%(17/96) and the prevalence of c-kit mutation in CBF-AML was 33.3%(2/6).4. The relationship between IDH and FLT3-ITD, FLT3-TKD, c-KIT:IDH mutations were mostly found in patients with cytogenetically normal, but because the rate of the latter two genes was low in our study, their relationship were not statistically analysed.5.The relationship between IDH and clinical features:the patients with IDH mutation had higher white blood cell counts and lower platelet counts; IDH2 mutations were found at older age at diagnosis.6. The relationship between IDH and Immunophenotyping:Immunophenotyping study showed that the IDH mutation was inversely associated with the expression of HLA-DR, CD34, CD33, and CD 13 on leukemia cells.7. The relationship between IDH and therapeutic effect:the patients with IDH mutation had lower CR, higher RR.Conciusion1. IDH mutation is one of the mutational genes in AML.2. IDH mutation in AML are possibly associated with poor prognosis.3. IDH mutation may provide basis for the layered treatment of the leukemia.