Sensitive detection of small cell lung carcinoma cells by reverse transcriptase–polymerase chain reaction for prepro–gastrin‐releasing peptide mRNA

Abstract

BACKGROUND. Gastrin-releasing peptide (GRP) is an autocrine growth factor in patients with small cell lung carcinoma (SCLC). The authors developed a reverse transcriptase-polymerase chain reaction (RT-PCR) assay for the detection of SCLC cells in the peripheral blood and the pleural effusion using preproGRP mRNA as a target. METHODS. The current study was conducted to determine the utility of preproGRPspecific nested RT-PCR on the peripheral blood, bone marrow, and pleural effusion samples from 32 patients with SCLC, 39 patients with non-small cell lung carcinoma (NSCLC), 28 patients with nonmalignant pulmonary disease, and 20 healthy volunteers. The internal primers were designed to amplify a 244-base pair PCR product, a sequence encompassing exon 1 and exon 2 by the nested RT-PCR assay. RESULTS. Amplification of the preproGRP message was detected in SCLC cell lines (LU165, SBC1, SBC2, and SBC3) but not in other NSCLC cell lines (A549, ABC1, EBC1, and Oka-1). The SCLC cells (LU165) were detected in dilutions of tumor cells of up to 10 Ϫ7 in hematopoietic cells from healthy donors. The preproGRP mRNA was detected in 16 of 32 (50%) blood samples, 2 of 11 (18%) marrow samples, and in all 6 (100%) pleural effusion samples. Blood samples gave positive results in 11 of 19 (58%) patients with extensive disease compared with 5 of 13 (38%) patients with limited disease. In contrast, only 1 blood sample (2.6%) from a patient with lung adenocarcinoma gave a positive result among patients with NSCLC. No other samples of blood, bone marrow, and pleural effusion from patients with NSCLC and none of the blood samples from patients with nonmalignant diseases and healthy volunteers were positive. CONCLUSIONS. The current RT-PCR approach may be a sensitive and specific assay to detect SCLC cells in circulating blood as well as in pleural effusions from SCLC patients.

Review: Biological relevance of disseminated tumor cells in cancer patients

“…While no transcripts could be detected among NSCLC cases, in 50% of the blood and 18% of BM samples from SCLC patients GRP mRNA was detected. 174 In conclusion, around half of the published studies on the prognostic role of DTC in BM in nonsmall cell lung cancer have found an association between the presence of DTC and worse overall survival or recurrence rate. However, most studies conducted so far have only investigated a small number of patients, and thus larger standardized studies are needed to confirm the prognostic value in lung cancer.…”

Reciprocal CD4+ T-Cell Balance of Effector CD62Llow CD4+ and CD62LhighCD25+ CD4+ Regulatory T Cells in Small Cell Lung Cancer Reflects Disease Stage

“…However, although no distant metastases are detected in LD-SCLC patients, regional treatments such as surgical resection or thoracic radiation therapy alone seldom result in a cure suggesting systemic micrometastases. Thus, repeated invasion by SCLC cells into the peripheral blood, some of which remain in the blood as circulating tumor cells, is considered to occur not only in extended-stage disease (ED) patients possessing prominent distant metastases but also in LD patients (1,2). The reason why circulating tumor cells are unable to establish visible distant metastases in LD patients is unclear because no biological differences have been detected between tumor cells of LD-SCLC and ED-SCLC.…”

Pre-analytical issues in effusion cytology

“…Freezing of cell pellets at -80°C or in liquid nitrogen prior to PCR analysis has been used by several groups, i. e. in analysis of effusions for prepro-gastrin releasing peptide (prepro-GRP) in small cell lung carcinoma [70], hMAM and hMAMB, coding for the breast carcinoma markers mammaglobin and mammaglobin B [71], and in analysis of different epithelial markers, including CLDN1, CLDN4, CLDN18, CEA, EPCAM, CK19, CK20, MUC1 and MUC16 using quantitative PCR (qPCR) [72].…”