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Arch Pathol Lab Med. 2012 Sep;136(9):983-92. doi: 10.5858/arpa.2011-0176-OA.

High-resolution melting analysis as a sensitive prescreening diagnostic tool to detect KRAS , BRAF , PIK3CA , and AKT1 mutations in formalin-fixed, paraffin-embedded tissues.

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1
Institute of Pathology, University of Bonn, Germany.

Abstract

CONTEXT:

As the availability of targeted therapies for several tumor types increases, the need for rapid and sensitive mutation screening is growing. KRAS mutations constitutively activate the RAS/RAF/mitogen-activated protein kinase (MAPK) pathway and therefore play an important role in anti-epidermal growth factor receptor therapy for patients with colorectal cancers. Mutationally activated PIK3CA and AKT1 genes are promising therapeutic targets in breast cancer. In 60% to 70% of malignant melanomas, a mutation in BRAF can be found. Thus, the blocking of the oncogenic signaling induced by this mutation is now used as treatment approach.

OBJECTIVE:

To establish high-resolution melting assays for routinely used predictive analyses of KRAS , AKT1 , PIK3CA , and BRAF mutations.

DESIGN:

High-resolution melting assays were developed by using specifically designed primers and genomic DNA isolated either from cell lines or formalin-fixed paraffin-embedded tissues, oligonucleotides, or plasmids. Melting curve analyses were performed on the LightCyler platform and mutation analyses were additionally confirmed by Sanger sequencing.

RESULTS:

We developed high-resolution melting assays by using genomic DNA containing the desired mutation, which enabled us to detect percentages of mutated DNA (3.1% to 12.5%) mixed in a wild-type background. Assays were evaluated by hybridization probes and/or Sanger sequencing to exclude pseudogene amplification. The high-resolution melting assays were validated with genomic DNA from different tumor entities. The concordance between Sanger sequencing and high-resolution melting was 99% for KRAS exon 2 and PIK3CA exon 20 and 100% for the remaining assays.

CONCLUSIONS:

High-resolution melting provides a valid and powerful tool for detecting genomic mutations efficiently.

PMID:
22938585
DOI:
10.5858/arpa.2011-0176-OA
[Indexed for MEDLINE]
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