Detection of germ-cell-tumor-specific gene products in peripheral blood by immunomagnetic tumor cell enrichment followed by RT-PCR.

Abstract

Therapeutic procedures in patients with testicular germ cell tumors (GCT) are determined by the histopathology of the primary tumor and the tumor extension. The aim of our study was to determine whether conventional staging could be supplemented by combining enrichment of disseminated testicular GCT cells from peripheral blood with subsequent detection of germ-cell-specific gene products. Blood samples from 46 patients with GCT of different clinical stages (CS) were examined by RT-PCR before therapy and >/=8 weeks thereafter for alpha-fetoprotein, beta-human chorionic gonadotropin and germ-cell-specific alkaline phosphatase mRNA. In addition, we performed titration experiments to evaluate whether the sensitivity can be improved by previous immunomagnetic tumor cell enrichment with anti-epithelial HEA-125 microbeads. No positive results were found in controls (n=15; specificity 100%). The overall ratio of positive PCR results in the group of patients with GCTs was 28.26%. The ratio was 35.7% for CS >IIb (n=5/14 patients), 20.0% for CS IIa-b (n=4/20) and 33.3% for CS I (n=4/12). FACS analysis in titration experiments with GCT cell lines showed that previous immunomagnetic tumor cell enrichment achieved a significant increase ranging up to 185.6 times the initial ratio and thus improved the measuring conditions for detection of tumor-specific transcripts. The sole qualitative RT-PCR of tumor-specific gene products in peripheral blood is not sensitive enough to improve staging in GCT patients. Immunomagnetic enrichment of GCT cells in peripheral blood seems a promising approach for increasing the sensitivity of RT-PCR.