Hempelmann Jennifer A, Scroggins Sheena M, Pritchard Colin C, Salipante Stephen J
Department of Laboratory Medicine, University of Washington, Seattle, Washington.
Department of Laboratory Medicine, University of Washington, Seattle, Washington.
J Mol Diagn. 2015 Nov;17(6):705-14. doi: 10.1016/j.jmoldx.2015.05.008. Epub 2015 Aug 29.
Molecular analysis of colon cancers currently requires multiphasic testing that uses various assays with different performance characteristics, adding cost and time to patient care. We have developed a single, next-generation sequencing assay to simultaneously evaluate colorectal cancers for mutations in relevant cancer genes (KRAS, NRAS, and BRAF) and for tumor microsatellite instability (MSI). In a sample set of 61 cases, the assay demonstrated overall sensitivity of 100% and specificity of 100% for identifying cancer-associated mutations, with a practical limit of detection at 2% mutant allele fraction. MSIplus was 97% sensitive (34 of 35 MSI-positive cases) and 100% specific (42 of 42 MSI-negative cases) for ascertaining MSI phenotype in a cohort of 78 tumor specimens. These performance characteristics were slightly better than for conventional multiplex PCR MSI testing (97% sensitivity and 95% specificity), which is based on comparison of microsatellite loci amplified from tumor and matched normal material, applied to the same specimen cohort. Because the assay uses an amplicon sequencing approach, it is rapid and appropriate for specimens with limited available material or fragmented DNA. This integrated testing strategy offers several advantages over existing methods, including a lack of need for matched normal material, sensitive and unbiased detection of variants in target genes, and an automated analysis pipeline enabling principled and reproducible identification of cancer-associated mutations and MSI status simultaneously.
目前，结肠癌的分子分析需要多阶段检测，即使用具有不同性能特征的各种检测方法，这增加了患者护理的成本和时间。我们开发了一种单一的下一代测序检测方法，可同时评估结直肠癌相关癌基因（KRAS、NRAS和BRAF）的突变以及肿瘤微卫星不稳定性（MSI）。在一组61例样本中，该检测方法在识别癌症相关突变方面的总体灵敏度为100%，特异性为100%，实际检测限为2%的突变等位基因比例。在一组78个肿瘤标本中，MSIplus在确定MSI表型方面的灵敏度为97%（35例MSI阳性病例中的34例），特异性为100%（42例MSI阴性病例中的42例）。这些性能特征略优于传统的多重PCR MSI检测（灵敏度97%，特异性95%），后者基于对从肿瘤和匹配的正常材料中扩增的微卫星位点进行比较，并应用于同一标本队列。由于该检测方法采用扩增子测序方法，因此速度快，适用于可用材料有限或DNA片段化的标本。这种综合检测策略比现有方法具有多个优势，包括无需匹配的正常材料、对靶基因变异进行灵敏且无偏倚的检测，以及一个自动化分析流程，能够同时原则性且可重复地识别癌症相关突变和MSI状态。
