Molecular detection of cancer cells by competitive reverse transcription-polymerase chain reaction analysis of specific CD44 variant RNAs

Background: CD44 is a cell surface glycoprotein implicated in such diverse biologic processes as lymphocyte activation and homing, extracellular matrix adhesion, and cellular migration. Primary transcripts of the CD44 gene can be alternatively spliced to produce a variety of messenger RNA (mRNA) species. The standard form of CD44 mRNA contains sequences from at least 20 genomic exons; variant mRNAs contain sequences from one or more additional exons (v1-10). Predominant expression of a specific CD44 variant, i.e., CD44v8-10, in several human carcinomas has been described previously. In this study, we developed a novel molecular approach for detecting cancer cells that overexpress CD44v8-10 mRNA. Methods: After finding that CD44v8-10 was predominantly expressed in non-small-cell lung and bladder carcinomas and that CD44v10 was predominantly expressed in leukocytes, we developed a competitive reverse transcription-polymerase chain reaction assay (CC-RT- PCR) that allows quantification of the relative expression of these two mRNA species in clinical specimens (i.e., determination of a v8-10/v10 ratio). CC- RT-PCR analysis was applied to pleural effusion specimens from patients with benign or malignant lung diseases as well as to spontaneously voided urine samples from patients with benign or malignant urologic diseases. Results: Fifty two of 54 samples from patients with benign diseases expressed CD44v10 predominantly (v8-10/v10 ratio ≤0.65), whereas 46 of 61 samples from patients with malignant diseases expressed CD44v8-10 predominantly (v8- 10/v10 ratio >1.00) (two-sided P<.001). CC-RT-PCR detected predominant expression of CD44v8-10 in cytologically negative samples from 11 patients who were later diagnosed with malignant disease. Conclusions: CC-RT-PCR analysis of CD44v8-10 expression could be an important adjunct to cytologic examination in cancer diagnosis, especially in detecting exfoliated cancer cells in pleural effusions and urine.