TUMOR-DIAGNOSIS BY PCR-BASED DETECTION OF TUMOR-CELLS

Tumour cells shed from solid primary tumours can be detected by the po lymerase chain reaction (PCR) based on the selective amplification of mutated tumour genes or of genes expressed in a tissue specific manner . When tumour specific alterations are amplified, few tumour cells can be detected in excess of normal cells derived from the same tissue. T hus, malignant cells can be detected specifically in pancreatic juice, stool, urine, and sputum. Here we describe the adaptation of the muta nt enriched PCR in conjunction with the introduction of artificial pri mer mediated restriction sites to the selective amplification of mutan t K-ras genes in stool samples from patients with colorectal carcinoma s. In reconstitution experiments, down to 10 colorectal carcinoma cell s could be detected in 100 mg of stool. For the diagnosis of micrometa static disease, a sensitive and specific technique was established bas ed on the reverse transcription of mRNA specific for the carcinoembryo nic antigen followed by the amplification of the cDNA (RT-PCR). Attemp ts to establish a specific RT-PCR for cytokeratin-18 failed because of the existence of at least one pcocessed pseudogene.