Detection of cancer cells in peripheral blood of breast cancer patients using reverse transcription polymerase chain reaction for epidermal growth factor receptor

The epidermal growth factor receptor (EGFR) has been reported to be express ed in high levels in primary breast cancer by immunohistochemistry. In the present study, a reverse transcription (RT)-PCR assay using EGFR primers wa s 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 brea st cancer patients. After reverse transcription, outer and nested primers f or EGFR were used for cDNA amplification. RNA integrity was confirmed with parallel RT-PCR amplification using beta(2)-microglobulin primers. PCR prod ucts were electrophoresed on agarose gels containing ethidium bromide and v isualized by UV photography. Southern blotting was used to confirm EGFR spe cificity. The nested EGFR RT-PCR assay was capable of detecting a lower lim it 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 l ocally recurrent breast cancer patients, 0 of 13 adjuvant breast cancer pat ients, and 0 of 23 controls (P = 0.03, metastatic versus control). The 18 m etastatic 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 pati ents were not receiving treatment at the time of blood collection. RT-PCR f or EGFR is a sensitive and specific method for the detection of circulating micrometastases in a proportion of patients with metastatic breast cancer.