COLD-PCR Finds Hot Application in Mutation Analysis

Abstract

With the rapid development of targeted therapy and personalized medicine, gaining knowledge of the genetic and molecular characteristics of a patient’s tumor has become a crucial step in therapeutic and prognostic decision-making in oncology. A good example is the administration of therapy targeting the epidermal growth factor receptor (EGFR)1 in cancer patients. Several studies have shown that mutations that cause constitutive activation of the KRAS 2 oncogene (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) predict resistance to anti-EGFR therapy (1)(2)(3), whereas activating mutations in EGFR predict response to therapy and favorable clinical outcome (4)(5). Because of such findings, the clinical demand for mutation testing has exploded recently in cancer diagnostics. The specimens used for molecular testing range from large surgical resections to tiny fine-needle aspiration biopsies to cytology smears. Samples are submitted as either frozen or formalin-fixed, paraffin-embedded tissues, and the percentage of tumor cells varies substantially among different specimens. One of the major challenges that molecular diagnostics laboratories face is to detect mutations in samples with a low percentage of mutation-carrying tumor cells in a background of wild-type nonmalignant cells. The fact that the mutation-detection limit is approximately 20% for the gold standard of Sanger sequencing and approximately 5%–10% for pyrosequencing (6) necessitates implementation of tumor-enrichment strategies for samples with a small tumor component. When tissue sections or smears on …