Department of Tumor Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Clin Chem. 2013 Jan;59(1):252-60. doi: 10.1373/clinchem.2012.188557. Epub 2012 Nov 7.
Molecular characterization of circulating tumor cells (CTCs) is pivotal to increasing the diagnostic specificity of CTC assays and investigating therapeutic targets and their downstream pathways on CTCs. We focused on epidermal growth factor receptor (EGFR) and genes relevant for its inhibition in patients with colorectal cancer (CRC).
We used the CellSearch® system for CTC detection in peripheral blood samples from 49 patients with metastatic CRC (mCRC) and 32 patients with nonmetastatic CRC (nmCRC). We assessed EGFR expression in 741 CTCs from 27 patients with mCRC and 6 patients with nmCRC using a fluorescein-conjugated antibody with the CellSearch Epithelial Cell Kit. DNA of a single CTC isolated by micromanipulation was propagated by whole-genome amplification and analyzed by quantitative PCR for EGFR gene amplification and sequencing for KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog), BRAF (v-raf murine sarcoma viral oncogene homolog B1), and PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit α) mutations.
At least 2 CTCs were detected in 24 of 49 patients with mCRC and 7 of 32 patients with nmCRC. In 7 of 33 patients, CTCs with increased EGFR expression were identified. Heterogeneity in EGFR expression was observed between CTCs from the same patient. EGFR gene amplification was found in 7 of 26 CTCs from 3 patients. The investigated BRAF gene locus was not mutated in 44 analyzed CTCs, whereas KRAS mutations were detected in 5 of 15 CTCs from 1 patient and PIK3CA mutations in 14 of 36 CTCs from 4 patients.
Molecular characterization of single CTCs demonstrated considerable intra- and interpatient heterogeneity of EGFR expression and genetic alterations in EGFR, KRAS, and PIK3CA, possibly explaining the variable response rates to EGFR inhibition in patients with CRC.
循环肿瘤细胞（CTCs）的分子特征对于提高 CTC 检测的诊断特异性以及研究 CTC 上的治疗靶点及其下游途径至关重要。我们专注于表皮生长因子受体（EGFR）和与结直肠癌（CRC）患者相关的基因。
我们使用 CellSearch®系统检测 49 例转移性 CRC（mCRC）患者和 32 例非转移性 CRC（nmCRC）患者外周血样本中的 CTC。我们使用 CellSearch Epithelial Cell Kit 中的荧光标记抗体评估 27 例 mCRC 患者和 6 例 nmCRC 患者的 741 个 CTC 中的 EGFR 表达。通过微操作分离的单个 CTC 的 DNA 通过全基因组扩增进行扩增，并通过定量 PCR 分析 EGFR 基因扩增，通过测序分析 KRAS（v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog）、BRAF（v-raf murine sarcoma viral oncogene homolog B1）和 PIK3CA（phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit α）突变。
在 49 例 mCRC 患者中有 24 例和 32 例 nmCRC 患者中有 7 例至少检测到 2 个 CTC。在 7 例 33 例患者中，鉴定出 EGFR 表达增加的 CTC。同一患者的 CTC 之间观察到 EGFR 表达的异质性。在 3 例患者的 26 个 CTC 中有 7 个发现 EGFR 基因扩增。在 44 个分析的 CTC 中未发现研究的 BRAF 基因座突变，而在 1 例患者的 15 个 CTC 中有 5 个检测到 KRAS 突变，在 4 例患者的 36 个 CTC 中有 14 个检测到 PIK3CA 突变。
单个 CTC 的分子特征表明 EGFR 表达和 EGFR、KRAS 和 PIK3CA 中的遗传改变存在相当大的个体内和个体间异质性，这可能解释了 CRC 患者对 EGFR 抑制的反应率不同。
