The Importance of Evaluation of DNA Amplificability in KRAS Mutation Testing with Dideoxy Sequencing using Formalin-fixed and Paraffin-embedded Colorectal Cancer Tissues
The Importance of Evaluation of DNA Amplificability in KRAS Mutation Testing with Dideoxy Sequencing using Formalin-fixed and Paraffin-embedded Colorectal Cancer Tissues
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DOI：
10.1093/jjco/hyq173
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发表时间：
2011-02-01
影响因子：
2.4
通讯作者：
Hinoda, Yuji
中科院分区：
文献类型：
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作者：
Okayama, Naoko;Nishioka, Mitsuaki;Hinoda, Yuji
我们使用最近临床试验中获得的福尔马林固定和石蜡包埋的结直肠癌组织样本，评估了 DNA 扩增能力，以实现双脱氧测序 KRAS 突变检测中 100% 的成功率。我们评估了脱蜡、福尔马林固定或储存时间以及扩增子大小对从 19 个福尔马林固定和石蜡包埋的结直肠癌组织样本中提取的 DNA 扩增能力的影响。我们对来自 31 家医院的转移性结直肠癌患者的 112 份样本进行了 KRAS 突变分析，这些患者参加了二线 FOLFIRI（5-氟尿嘧啶 + 亚叶酸 + 伊立替康）+ 西妥昔单抗方案的 II 期试验。脱石蜡、福尔马林固定和储存时间似乎不会影响 DNA 的恢复和扩增。然而，扩增子大小对 DNA 的扩增能力有显着影响。在所有测试的样品中，使用聚合酶链式反应成功扩增了大小 < 278 bp (96-278 bp) 的较小片段，而大小 >= 298 bp (298-565 bp) 的较大片段未扩增。我们临床试验的所有样本均使用扩增子大小分别为 201、221 和 240 bp 的三组引物进行了成功分析，在 112 例病例中的 49 例 (43.8%) 中检测到了外显子 2 和 3 中的 KRAS 突变。这些数据表明，DNA 扩增性和扩增子大小的评估对于突变检测测试的成功非常重要，例如使用福尔马林固定和双脱氧测序进行的 KRAS 测试。临床环境中的石蜡包埋组织样本。
We evaluated DNA amplificability to achieve a 100% success rate in KRAS mutation testing with dideoxy sequencing using formalin-fixed and paraffin-embedded colorectal cancer tissue samples obtained from a recent clinical trial.We evaluated the effects of deparaffinization, formalin fixation or storage time, and amplicon size on the amplificability of DNAs extracted from 19 formalin-fixed and paraffin-embedded colorectal cancer tissue samples. We subjected to KRAS mutation analysis 112 samples from metastatic colorectal cancer patients in 31 hospitals enrolled in a Phase II trial of a second-line FOLFIRI (5-fluorouracil+ leucovorin + irinotecan) + cetuximab regimen.Deparaffinization, formalin fixation and storage times did not appear to affect the recovery and amplificability of DNAs. However, amplicon size had a remarkable effect on the amplificability of DNAs. The smaller fragments with a size of < 278 bp (96-278 bp) were successfully amplified with polymerase chain reaction in all samples tested, whereas the larger fragments with a size of >= 298 bp (298-565 bp) were not amplified. All samples from our clinical trial were successfully analyzed using three sets of primers with the amplicon sizes of 201, 221 and 240 bp, and KRAS mutations in exons 2 and 3 were detected in 49 of the 112 cases (43.8%).These data suggest that the evaluation of DNA amplificability and amplicon size is important for the success of mutation detection tests such as the KRAS test with dideoxy sequencing using formalin-fixed and paraffin-embedded tissue samples in the clinical setting.