Enrichment of mutant KRAS alleles in pancreatic juice by subtractive iterative polymerase chain reaction

The detection of mutant tumor genes holds great promise for an early diagno sis of primary tumors and residual malignant disease. When few tumor cells are present with an excess of nonmalignant cells of the same lineage, the e xcess of wild-type alleles over mutant tumor alleles presents an analytical problem. The subtractive iterative PCR (siPCR) assay presents a new approa ch to solving this problem. To achieve an enrichment of mutant alleles, wil d-type alleles are removed by differential hybridization to complementary o ligonucleotides spanning the region of the gene in which point mutations ar e expected. The nonbound fraction is reamplified by PCR. By iterating this process, mutant alleles can be detected in the presence of an excess of wil d-type alleles with high sensitivity. To prove the feasibility of siPCR, pa ncreatic juice samples were analyzed for WAS mutations. Pancreatic juice ob tained from patients with pancreatic carcinoma or chronic pancreatitis duri ng endoscopic retrograde cholangiopancreatography was analyzed for point mu tations in codons 12 and 13 of the KRAS gene. In each of six samples from t umor patients, mutations in codon 12 were detected. One of nine samples fro m patients with chronic pancreatitis scored positive.