McCall Chad M, Mosier Stacy, Thiess Michele, Debeljak Marija, Pallavajjala Aparna, Beierl Katie, Deak Kristen L, Datto Michael B, Gocke Christopher D, Lin Ming-Tseh, Eshleman James R
Department of Pathology, The Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins University School of Medicine, Baltimore, Maryland.
Department of Pathology, The Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins University School of Medicine, Baltimore, Maryland; Department of Oncology, The Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins University School of Medicine, Baltimore, Maryland.
J Mol Diagn. 2014 Sep;16(5):541-549. doi: 10.1016/j.jmoldx.2014.06.001. Epub 2014 Jul 11.
Next-generation sequencing shows great promise by allowing rapid mutational analysis of multiple genes in human cancers. Recently, we implemented the multiplex PCR-based Ion AmpliSeq Cancer Hotspot Panel (>200 amplicons in 50 genes) to evaluate EGFR, KRAS, and BRAF in lung and colorectal adenocarcinomas. In 10% of samples, automated analysis identified a novel G873R substitution mutation in EGFR. By examining reads individually, we found this mutation in >5% of reads in 50 of 291 samples and also found similar events in 18 additional amplicons. These apparent mutations are present only in short reads and within 10 bases of either end of the read. We therefore hypothesized that these were from panel primers promiscuously binding to nearly complementary sequences of nontargeted amplicons. Sequences around the mutations matched primer binding sites in the panel in 18 of 19 cases, thus likely corresponding to panel primers. Furthermore, because most primers did not show this effect, we demonstrated that next-generation sequencing may be used to better design multiplex PCR primers through iterative elimination of offending primers to minimize mispriming. Our results indicate the need for careful sequence analysis to avoid false-positive mutations that can arise in multiplex PCR panels. The AmpliSeq Cancer panel is a valuable tool for clinical diagnostics, provided awareness of potential artifacts.
新一代测序技术通过对人类癌症中的多个基因进行快速突变分析展现出巨大潜力。最近，我们应用基于多重PCR的Ion AmpliSeq癌症热点区域检测试剂盒（50个基因中>200个扩增子）来评估肺腺癌和结直肠癌中的表皮生长因子受体（EGFR）、 Kirsten大鼠肉瘤病毒癌基因（KRAS）和B-Raf原癌基因（BRAF）。在10%的样本中，自动化分析在EGFR中鉴定出一种新的G873R替代突变。通过逐个检查读数，我们在291个样本中的50个样本中发现该突变存在于>5%的读数中，并且在另外18个扩增子中也发现了类似情况。这些明显的突变仅存在于短读数中，且位于读数两端的10个碱基范围内。因此，我们推测这些突变来自检测试剂盒引物与非靶向扩增子的近乎互补序列的杂乱结合。在19个病例中的18个病例中，突变周围的序列与检测试剂盒中的引物结合位点匹配，因此很可能对应于检测试剂盒引物。此外，由于大多数引物未显示出这种效应，我们证明可以通过迭代消除有问题的引物以尽量减少错配，利用新一代测序技术更好地设计多重PCR引物。我们的结果表明需要进行仔细的序列分析，以避免多重PCR检测试剂盒中可能出现的假阳性突变。AmpliSeq癌症检测试剂盒是一种有价值的临床诊断工具，但要意识到潜在的假象。
