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Diagnostic Technologies and Molecular and Cellular Profiling: Novel Assay Technologies

Novel mutation detection method for tumor samples grossly contaminated with normal tissue

SAIC-Frederick, Frederick, MD

Abstract

A109

For diagnostic purposes, mutation detection in cancer is difficult due to the presence of contaminating normal tissue infiltrated throughout the tumor tissue. Traditional PCR and sequencing methods are labor intensive and time consuming. Therefore, we have developed an assay using a nanochip array platform to find specific mutations in cancer-related genes. An amplicon-down method was applied. That is, the DNA sample was amplified with a biotin labeled primer and the resulting target amplicon was electronically addressed to a streptavidin membrane. This method is based on differences in melting temperatures. The data is collected either at a specific Tm, or during a series of temperature increments to create a melting curve. As proof of principle, we screened codon 12 of the K-ras gene, which is commonly mutated in several tumor types. The analysis was done on formalin fixed, paraffin-embedded tumor samples that showed various degrees of contaminating normal tissue. We screened 233 patient samples corresponding to various cancer types. These included colon, rectum, pancreas, lymph node, lung, spleen, liver, stomach, and thyroid cancers. We were able to detect mutations in samples that contained as little as 20% tumor cells. In addition, the sensitivity was 100% and the specificity was nearly 100%. These data agreed 100% with PCR enrichment and sequencing results that were performed in parallel. Moreover, the method presented here was even more sensitive in some instances. For example, we were able to detect very low amounts of mutant allele that was not detected by traditional sequencing due to background noise. In conclusion, we found this test to be extremely accurate, reproducible, fast, and due to its versatility, generally applicable to any mutation screening. Furthermore, in a therapeutic setting, it could provide an effective selection criterion for a patient treatment regimen.