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

Kim Leitzel, Binh Lieu, Elizabeth Curley, Jill Smith, Vernon Chinchilli, Wojciech Rychlik, Allan Lipton

Research output: Contribution to journalArticle

41 Scopus citations

Abstract

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 β2-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 RTPCR 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.

Original languageEnglish (US)
Pages (from-to)3037-3043
Number of pages7
JournalClinical Cancer Research
Volume4
Issue number12
StatePublished - Dec 1 1998

All Science Journal Classification (ASJC) codes

  • Oncology
  • Cancer Research

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