acute myeloid leukemia ( aml ) is the most common type of leukemia among hematologic malignancies . this disease is a clonal hematopoietic disease that is caused by either inherited or acquired genetic mutations . aml is known to be a molecularly and clinically heterogeneous disease . in order to cure this disease , cytosine - arabinoside ( two thirds of aml patients who receive ara - c based chemotherapy regimen , will achieve complete remission ( cr ) . eventually , a great majority of aml patients die from progressive disease after relapse by various genetic changes that will endow tumor cells with resistance against most commonly used anticancer agents . therefore , it is prerequisite to identify prognostic markers that will help in deciding the suitable treatment modalities . a lot of studies have focused on finding somatic mutations in cancer cells by whole - exome sequencing ( wes ) . in the initial development of cancer in humans , knudson 's two - hit hypothesis has provided important insights into the pathogenesis of tumors in autosomal dominant tumor predisposition syndromes . with an analogy , we focused on the additional genetic changes that will predispose susceptible individuals with a pre - existing genetic abnormality to develop leukemia . to address this issue , germline single nucleotide polymorphism ( snp ) analysis was performed using the ucsc hg19 program in 10 aml patients ( samples , g1 to g10 ) , and somatic mutations were identified in the same tumor sample using somaticsniper and varscan2 . our results suggest that snps with additional somatic mutations may directly affect the prognosis of aml and emphasize the need to validate in a larger patient cohort . genomic dna samples were prepared for wes . they were freshly obtained from aml patients ' bone marrow at initial diagnosis . subsequent bone marrow samples were aspirated if necessary according the disease status of the patients . aml blast tumor cells were isolated from bone marrow of each patient using ficoll gradient separation . matching patients ' saliva samples we prepared and sequenced exomes using the solexa sequencing technology platform ( hiseq2000 ; illumina , san diego , ca , usa ) following the manufacturer 's instructions . we randomly sheared 3 g of genomic dna using the covaris system to generate 150-bp inserts . the fragmented dna was end - repaired using t4 dna polymerase and klenow polymerase , and illumina paired - end adaptor oligonucleotides were ligated to the sticky ends . we analyzed the ligation mixture by electrophoresis on an agarose gel and sliced and purified fragments of 200.250 bp . the purified dna library was hybridized with the sureselect human all exons probes set ( agilent , santa clara , ca , usa ) to capture 50 mb of targeted exons following the manufacturer 's instruction . we prepared the hiseq2000 paired - end flowcell with the manufacturer 's protocol using the captured exome library . clusters of pcr colonies were then sequenced on the hiseq2000 platform using recommended protocols from the manufacturer . this pipeline essentially includes 1 ) mapping and alignment , 2 ) discovery of sequence nucleotide variations ( snvs ) and insertions and deletions ( indels ) , and 3 ) filtering and annotation of snvs and indels . briefly , once the raw sequence data were created , the output short reads were aligned to a reference genome ( ncbi human genome assembly build 36 ) using ucsc hg19 . each alignment was assigned a mapping quality score by ucsc hg19 , which is the phred - scaled probability that the alignment is incorrect . we defined somatic mutation snvs as those detected by both somaticsniper and varscan2 with a somatic p - value < 0.05 . we also set a minimum coverage of 5 in both normal and tumor samples . genomic dna samples were prepared for wes . they were freshly obtained from aml patients ' bone marrow at initial diagnosis . subsequent bone marrow samples were aspirated if necessary according the disease status of the patients . aml blast tumor cells were isolated from bone marrow of each patient using ficoll gradient separation . matching patients ' saliva samples we prepared and sequenced exomes using the solexa sequencing technology platform ( hiseq2000 ; illumina , san diego , ca , usa ) following the manufacturer 's instructions . we randomly sheared 3 g of genomic dna using the covaris system to generate 150-bp inserts . the fragmented dna was end - repaired using t4 dna polymerase and klenow polymerase , and illumina paired - end adaptor oligonucleotides were ligated to the sticky ends . we analyzed the ligation mixture by electrophoresis on an agarose gel and sliced and purified fragments of 200.250 bp . the purified dna library was hybridized with the sureselect human all exons probes set ( agilent , santa clara , ca , usa ) to capture 50 mb of targeted exons following the manufacturer 's instruction . we prepared the hiseq2000 paired - end flowcell with the manufacturer 's protocol using the captured exome library . clusters of pcr colonies were then sequenced on the hiseq2000 platform using recommended protocols from the manufacturer . this pipeline essentially includes 1 ) mapping and alignment , 2 ) discovery of sequence nucleotide variations ( snvs ) and insertions and deletions ( indels ) , and 3 ) filtering and annotation of snvs and indels . briefly , once the raw sequence data were created , the output short reads were aligned to a reference genome ( ncbi human genome assembly build 36 ) using ucsc hg19 . each alignment was assigned a mapping quality score by ucsc hg19 , which is the phred - scaled probability that the alignment is incorrect . we defined somatic mutation snvs as those detected by both somaticsniper and varscan2 with a somatic p - value < 0.05 . we also set a minimum coverage of 5 in both normal and tumor samples . conclusively germline snp analysis was done to detect germline heterozygous mutations that are carried by aml patients . to address this issue , germline snp analysis was performed using the ucsc hg19 program in 10 aml patients ( samples g1 to g10 ) ( table 1 ) . we chose 18 major genes ( tet2 , asxl1 , dnmt3a , cebpa , phf6 , wt1 , tp53 , ezh2 , runx1 , pten , flt3 , npm1 , hras , kras , nras , kit , idh1 , and idh2 ) to be analyzed in 10 samples ( table 2 ) . among these 18 genes , kras and dnmt3a seemed to play a critical role in the progression of aml , associated with the chemotherapy response . kras is a protein product of the normal kras gene , which performs an essential function in normal tissue signaling , and the mutation of a kras gene is an essential step in the development of many cancers . dnmt3a is a key component of the epigenetic regulation of genes , as it catalyzes the addition of methyl groups to the cytosine residue of cpg dinucleotides . however , variations only in kras and dnmt3a are not enough to explain the mechanism of aml progression , since additional somatic changes exist in aml patients . snps of kras were detected in 4 different individuals ( g2 , g6 , g7 , and g8 ) , as shown in table 1 , while those of dnmt3a were detected in 5 individuals ( g3 , g4 , g6 , g8 , and g10 ) among 10 aml patients ( table 1 ) . we then searched for somatic mutations of each individual in order to prove the ' two - hit ' theory . snps of kras and dnmt3a were detected in both samples g6 and g8 ( table 3 ) . they both attained a cr after the first induction ; however , g6 relapsed within 390 days after attaining remission , while g8 did not relapse for 402 days . a somatic mutation in idh2 was identified in both g6 and g8 , while that of flt3 and npm1 was detected in g6 and g8 , respectively . npm1 mutation , on the other hand , is usually known as a good prognostic factor in aml . therefore , we could speculate that snps ( kras and dnmt3a ) with additional somatic mutations ( flt3 and npm1 ) affect the prognosis of aml differentially . in addition , g2 and g7 shared the same snps in kras but their clinical courses showed a striking difference ( table 4 ) . as we searched further for somatic mutations in g7 , a somatic mutation in tet2 was detected as a second hit . on the other hand , an npm1 mutation was observed in g2 , showing a divergent outcome compared with g7 . we observed a divergent outcome in patients having the same snps but with different somatic mutations as a second hit . somatic mutations that were differentially detected at the time of diagnosis strongly suggest clinical implications and warrant future study in a larger cohort to enhance our understanding of the mechanisms of disease progression in aml .
this study evaluated the effects of somatic mutations and single nucleotide polymorphisms ( snps ) on disease progression and tried to verify the two - hit theory in cancer pathogenesis . to address this issue , 
 snp analysis was performed using the ucsc hg19 program in 10 acute myeloid leukemia patients ( samples , g1 to g10 ) , and somatic mutations were identified in the same tumor sample using somaticsniper and varscan2 . 
 snps in kras were detected in 4 out of 10 different individuals , and those of dnmt3a were detected in 5 of the same patient cohort . in 2 patients , both kras and dnmt3a were detected simultaneously . 
 a somatic mutation in idh2 was detected in these 2 patients . 
 one of the patients had an additional mutation in flt3 , while the other patient had an npm1 mutation . 
 the patient with an flt3 mutation relapsed shortly after attaining remission , while the other patient with the npm1 mutation did not suffer a relapse . 
 our results indicate that snps with additional somatic mutations affect the prognosis of aml .