Pang N K B, Nga M E, Chin S Y, Ismail T M, Lim G L, Soong R, Salto-Tellez M
Divisions of Cytopathology, Department of Pathology, National University Health System, Singapore.
Cytopathology. 2011 Dec;22(6):358-64. doi: 10.1111/j.1365-2303.2010.00812.x. Epub 2010 Oct 4.
Sanger sequencing is one of several reliable methods in use to detect KRAS and BRAF mutations to facilitate clinical patient selection for anti-epidermal growth factor receptor (EGFR) monoclonal antibody therapy in unresectable metastatic colorectal adenocarcinoma (CRC). Most analyses are made on pretreatment biopsy or resection specimens. There is a scarcity of published studies on the suitability of cytological samples for KRAS testing in this setting.
DNA extraction was attempted on 11 search-retrieved paired cases of histological resections or excisions of CRC and their corresponding cytological samples (representing metastases) and tested for KRAS mutations in exon 2 and 3, as well as BRAF exon 15 mutations by Sanger sequencing. Only KRAS wild-type cases were subjected to BRAF analysis because this is the setting with true diagnostic value, as these mutations are mutually exclusive.
Of the 11 paired cases analysed, only eight histology cases showed satisfactory DNA quality for sequencing. Thus, only eight of the corresponding cytology cases were analysed. Seven of the eight cases tested showed the same KRAS genotype on both the aspirated cytology specimen of metastatic carcinoma and the primary tumour (histological specimen), from which we derive an overall concordance rate of 87.5%. The single discordant case was likely to be a true difference as it was demonstrated again on repeat testing of both samples. No BRAF mutations were detected on the four KRAS wild-type cases.
A range of cytological samples are suitable for KRAS and BRAF mutation testing, be it from previously stained preparations or cell blocks. These samples would be highly valuable in cases where cytological samples are the only material available for mutation testing.
桑格测序是用于检测KRAS和BRAF突变的几种可靠方法之一，有助于临床选择不可切除转移性结直肠癌（CRC）患者进行抗表皮生长因子受体（EGFR）单克隆抗体治疗。大多数分析是基于治疗前的活检或切除标本。关于在这种情况下细胞学样本用于KRAS检测的适用性，已发表的研究较少。
对11例检索到的配对病例进行DNA提取，这些病例包括CRC的组织学切除或切除标本及其相应的细胞学样本（代表转移灶），通过桑格测序检测KRAS基因第2和3外显子的突变以及BRAF基因第15外显子的突变。仅对KRAS野生型病例进行BRAF分析，因为在这种情况下这些突变相互排斥，具有真正的诊断价值。
在分析的11对配对病例中，只有8例组织学病例显示出用于测序的满意DNA质量。因此，仅对相应的8例细胞学病例进行了分析。在8例检测的病例中，7例转移癌的抽吸细胞学标本和原发肿瘤（组织学标本）显示出相同的KRAS基因型，由此得出总体一致率为87.5%。唯一不一致的病例可能是真正的差异，因为在两个样本的重复检测中再次得到证实。在4例KRAS野生型病例中未检测到BRAF突变。
一系列细胞学样本适用于KRAS和BRAF突变检测，无论是来自先前染色的标本还是细胞块。在细胞学样本是唯一可用于突变检测的材料的情况下，这些样本将具有很高的价值。
