Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri.
Cancer Cytopathol. 2014 Feb;122(2):104-13. doi: 10.1002/cncy.21361. Epub 2013 Nov 12.
Molecular testing of cancer is increasingly critical to medicine. Next-generation sequencing (NGS) provides comprehensive, unbiased, and inexpensive mutation analysis of multiple genes with a single test. However, to the authors' knowledge, the usefulness of NGS in fine-needle aspiration (FNA) specimens, which may be the only specimens available, is unknown. Non-small cell lung cancer (NSCLC) is an ideal model in which to evaluate cytopathologic applications of NGS because FNA is used for diagnosis and staging and specific molecular therapeutic targets in NSCLC are known. Herein, the performance and quality of targeted NGS in FNA specimens from a small series of lung adenocarcinomas is evaluated.
Sequence data were generated from FNA specimens and paired formalin-fixed paraffin-embedded (FFPE) tissues from 5 patients with lung adenocarcinoma. DNA was isolated from FNA aspirate smears and cores of FFPE tissue. Multiplex sequencing of 27 cancer-related genes was performed after hybrid capture enrichment. Read-quality metrics and single-nucleotide variant calls were compared.
The overall concordance of total reads across specimens was > 99% and the average concordance of single-nucleotide variants was 99.5%. The total reads generated, as well as the percentages of mapped, on-target, and unique reads were statistically indistinguishable (P > 0.05) between FFPE and FNA preparations. There also was no difference in the depth of sequencing coverage, including exon-level coverage in known lung cancer mutation hotspots.
DNA isolated from FNA slides yields comprehensive, accurate, and statistically indistinguishable sequence information compared with that obtained from FFPE tissue. These results support the integration of NGS technologies into the standard cytopathology workflow. Cancer (Cancer Cytopathol) 2014;122:104-13. © 2013 American Cancer Society.
癌症的分子检测对医学越来越重要。下一代测序（NGS）提供了一种综合、无偏、廉价的方法，可在单次检测中分析多个基因的突变。然而，据作者所知，NGS 在细针穿刺（FNA）标本中的实用性尚不清楚，而 FNA 标本可能是唯一可用的标本。非小细胞肺癌（NSCLC）是评估 NGS 细胞病理学应用的理想模型，因为 FNA 用于诊断和分期，并且 NSCLC 中存在已知的特定分子治疗靶点。在此，评估了小系列肺腺癌 FNA 标本中靶向 NGS 的性能和质量。
从 5 例肺腺癌患者的 FNA 标本和配对的福尔马林固定石蜡包埋（FFPE）组织中生成序列数据。从 FNA 抽吸涂片和 FFPE 组织的核心中分离 DNA。在杂交捕获富集后，对 27 个与癌症相关的基因进行多重测序。比较了读质量指标和单核苷酸变异的调用。
标本之间的总读取量总体一致性>99%，单核苷酸变异的平均一致性为 99.5%。FFPE 和 FNA 制备物之间，总读取量、映射百分比、靶向百分比和唯一读取百分比的统计学差异无显著性（P>0.05）。测序覆盖率的深度也没有差异，包括已知肺癌突变热点的外显子水平覆盖率。
与从 FFPE 组织中获得的序列信息相比，从 FNA 载玻片上分离的 DNA 可提供全面、准确且统计学上无差异的序列信息。这些结果支持将 NGS 技术集成到标准细胞病理学工作流程中。癌症（癌症细胞病理学）2014；122：104-13。2013 年美国癌症协会。
