CAJ | 학술논문

目的探讨焦磷酸测序法检测结直肠癌患者肿瘤组织K-ras基因外显子2第12和13密码子点突变方法的临床应用价值.方法以已知K-ras基因突变的结直肠癌细胞株SW480、DLD-1和野生型细胞株HT-29 DNA作为测序模板检验焦磷酸测序法的准确性.对含不同比例(2％、3％、5％、10％、20％、30％和50％)结直肠癌细胞株K-ras基因的DNA混合样本采用焦磷酸测序法进行基因突变率检测,并与Sanger测序结果平行进行Fisher精确检验比较,评价其灵敏度.同时用焦磷酸测序法检测分析30份临床结直肠癌患者石蜡包埋组织中K-ras基因第12和13密码子突变.结果当混合已知突变类型的结直肠癌细胞株K-ras基因的DNA样本突变DNA比例在5％和10％浓度时,Sanger测序法检出K-ras基因突变率分别为33.3％ (4/12)和58.3％ (7/12),焦磷酸测序法分别为91.7％(11/12)和100％( 12/12),且2种方法检出K-ras基因突变率的差异有统计学意义(P＜0.05).此外,用焦磷酸测序法从30例结直肠癌患者石蜡包埋组织标本中检出K-ras基因外显子2第12和13密码子突变10例,均为杂合型突变,突变率为33.3％ (10/30).最常见的突变类型为G＞A转换[50％(5/1O)]和G＞T颠换[(30％(3/10)].结论焦磷酸测序法检测结直肠癌K-ras基因外显子2第12和13密码子突变具有敏感、准确的优点,可用于临床个体化治疗中肿瘤基因突变检测.
목적 탐토초린산측서법검측결직장암환자종류조직K-ras기인외현자2제12화13밀마자점돌변방법적림상응용개치.방법 이이지K-ras기인돌변적결직장암세포주SW480、DLD-1화야생형세포주HT-29 DNA작위측서모판검험초린산측서법적준학성.대함불동비례(2％、3％、5％、10％、20％、30％화50％)결직장암세포주K-ras기인적DNA혼합양본채용초린산측서법진행기인돌변솔검측,병여Sanger측서결과평행진행Fisher정학검험비교,평개기령민도.동시용초린산측서법검측분석30빈림상결직장암환자석사포매조직중K-ras기인제12화13밀마자돌변.결과 당혼합이지돌변류형적결직장암세포주K-ras기인적DNA양본돌변DNA비례재5％화10％농도시,Sanger측서법검출K-ras기인돌변솔분별위33.3％ (4/12)화58.3％ (7/12),초린산측서법분별위91.7％(11/12)화100％( 12/12),차2충방법검출K-ras기인돌변솔적차이유통계학의의(P＜0.05).차외,용초린산측서법종30례결직장암환자석사포매조직표본중검출K-ras기인외현자2제12화13밀마자돌변10례,균위잡합형돌변,돌변솔위33.3％ (10/30).최상견적돌변류형위G＞A전환[50％(5/1O)]화G＞T전환[(30％(3/10)].결론 초린산측서법검측결직장암K-ras기인외현자2제12화13밀마자돌변구유민감、준학적우점,가용우림상개체화치료중종류기인돌변검측.
Objective To investigate the clinical significance of pyrosequencing assay for determining K-ras mutations in exon 2 codons 12 and 13 in clinical colorectal cancer tissues.Methods Genomic DNA,extracted from K-ras mutant cell lines SW480 (homozygous,c.35G ＞ T), DLD-1 (heterozygous,c.38G ＞ A) and wild-type HT-29,was first used as the sequencing template respectively to test the accuracy of pyrosequencing methodology.The SW480 and DLD-1 DNA was separately mixed with wild-type HT-29 DNA in proportions of 2％,3％,5％,10％,20％,30％ and 50％,the sensitivity for mutation detection was measured separately by pyrosequencing assay and directed Sanger DNA sequencing in the serial DNA mixture samples.The pyrosequencing assay results were compared with the corresponding Sanger sequencing and the datas were analysized by Fisher exact test.Pyrosequencing analysis was then performed for screening K-ras exon 2 mutations at codons 12 and 13 on DNA isolated from a panel of 30 colorectal cancer samples derived from clinical formalin-fixed and paraffin embedded (FFPE)tissues.Results Cancer cell lines with known K-ras mutations ( SW480 and DLD-1 ) were readily detectable by pyrosequencing-based analysis.When the proportions of mutant colorectal cancer cell line DNA were 5％ and 10％ content,the mutation rates of K-ras gene detected by conventional Sanger DNA sequencing were 33.3％ (4/12) and 58.3％ (7/12) respectively,whereas the mutation rates detected by pyrosequencingbased assay were 91.7％ (11/12) and 100％ (12/12) respectively,there were significant differences between those two sequencing methodology ( P ＜0.05).Furthermore,we found 10 patients with K-ras exon 2 point mutations at codons 12 and 13 by pyrosequencing-based assay from 30 colorectal cancer FFPE tissues,the point mutation rate was 33.3％ (10/30) and all of the mutations determined were heterozygous.The codon 12 was most frequently affected [30％ (9/30)].Mutations with the highest frequency were G ＞ A transitions [ 50％ ( 5/10 ) ],followed by G ＞ T transversions [ 30％ ( 3/10 ) ].Conclusion The pyrosequencing assay provides an accurate and sensitive method for mutation screening of K-ras exon 2 codons 12 and 13 in routine diagnostic specimens,thereby allowing the selection of the cancer treatment in clinical individualized practice.