Leitzel K, Lieu B, Curley E, Smith J, Chinchilli V, Rychlik W, Lipton A
Section of Hematology/Oncology, Pennsylvania State University College of Medicine, Hershey Medical Center, 17033, USA.
Clin Cancer Res. 1998 Dec;4(12):3037-43.
The epidermal growth factor receptor (EGFR) has been reported to be expressed in high levels in primary breast cancer by immunohistochemistry. In the present study, a reverse transcription (RT)-PCR assay using EGFR primers was developed and evaluated for the detection of circulating micrometastases in the blood of breast cancer patients. Total RNA was extracted from breast cancer cell lines and from the blood of 23 control individuals and 37 breast cancer patients. After reverse transcription, outer and nested primers for EGFR were used for cDNA amplification. RNA integrity was confirmed with parallel RT-PCR amplification using beta2-microglobulin primers. PCR products were electrophoresed on agarose gels containing ethidium bromide and visualized by UV photography. Southern blotting was used to confirm EGFR specificity. The nested EGFR RT-PCR assay was capable of detecting a lower limit of 100 fg of total RNA from the A431 cell line. EGFR RNA was identified from the blood of 4 of 18 (22%) metastatic breast cancer patients, 0 of 6 locally recurrent breast cancer patients, 0 of 13 adjuvant breast cancer patients, and 0 of 23 controls (P = 0.03, metastatic versus control). The 18 metastatic breast cancer patients all had progressive disease at the time of blood sampling. The identity of the four EGFR-positive bands was confirmed by Southern blotting. The presence of RT-PCR positivity for EGFR was not a treatment-related phenomenon, because three of the four EGFR-positive patients were not receiving treatment at the time of blood collection. RT-PCR for EGFR is a sensitive and specific method for the detection of circulating micrometastases in a proportion of patients with metastatic breast cancer.
据报道，通过免疫组织化学检测发现，原发性乳腺癌中表皮生长因子受体（EGFR）呈高水平表达。在本研究中，开发并评估了一种使用EGFR引物的逆转录（RT）-PCR检测方法，用于检测乳腺癌患者血液中的循环微转移灶。从乳腺癌细胞系以及23名对照个体和37名乳腺癌患者的血液中提取总RNA。逆转录后，使用EGFR的外部引物和巢式引物进行cDNA扩增。使用β2-微球蛋白引物进行平行RT-PCR扩增以确认RNA完整性。PCR产物在含溴化乙锭的琼脂糖凝胶上进行电泳，通过紫外摄影进行观察。使用Southern印迹法确认EGFR的特异性。巢式EGFR RT-PCR检测方法能够检测到来自A431细胞系的低至100 fg的总RNA。在18名转移性乳腺癌患者中的4名（22%）、6名局部复发性乳腺癌患者中的0名、13名辅助性乳腺癌患者中的0名以及23名对照中的0名患者的血液中鉴定出EGFR RNA（转移性患者与对照相比，P = 0.03）。这18名转移性乳腺癌患者在采血时均患有进展性疾病。通过Southern印迹法确认了四条EGFR阳性条带的身份。EGFR的RT-PCR阳性并非与治疗相关的现象，因为四名EGFR阳性患者中有三名在采血时未接受治疗。EGFR的RT-PCR是一种敏感且特异的方法，可用于检测一部分转移性乳腺癌患者血液中的循环微转移灶。
