Sensitive detection of rare cancer cells in sputum and peripheral blood samples of patients with lung cancer by preproGRP-specific RT-PCR

Abstract

RT-PCR-based amplification of transcripts expressed in cancer but not in normal non-neoplastic cells is increasingly used for the sensitive detection of rare disseminated or exfoliated cancer cells to improve cancer staging and early detection protocols. However, these assays are frequently hampered by false-positive test results due to low-level transcription of the marker genes in normal cells. To overcome these limitations, target transcripts have to be identified that are tightly suppressed in normal non-neoplastic tissues, whereas they should be actively transcribed in the respective cancer cells. Here, we tested RT-PCR assays for 7 neuroendocrine marker transcripts including NCAM, PGP 9.5, gastrin, gastrin receptor, synaptophysin, preprogastrin-releasing peptide (preproGRP) and GRP-receptor to detect rare exfoliated tumor cells in peripheral venous blood and sputum samples from patients with lung cancer. Among these preproGRP RT-PCR was the only assay with which illegitimate transcription in blood or sputum samples from healthy donors or patients with unrelated diseases did not interfere. However, it reproducibly detected up to 10 small-cell lung cancer cells diluted in either 10 ml blood or 5 ml sputum samples. Single blood and sputum samples were collected directly before diagnostic bronchoscopy from 175 patients suspected to have lung cancer. Twenty-six of these had small-cell lung cancer (SCLC). Thereof, 13 patients (50%) tested positive in the blood sample and 5 of 23 patients (22%) tested positive in the sputum sample. Moreover, among 92 patients with non-small-cell lung cancer (NSCLC) 25 patients (27%) had disseminated cancer cells in peripheral blood. Amplification of preproGRP transcripts from clinical samples is a sensitive and specific assay to detect disseminated or exfoliated lung cancer cells either in peripheral blood or sputum samples.