TY - JOUR
T1 - Novel method for PIK3CA mutation analysis
T2 - Locked nucleic acid-PCR sequencing
AU - Ang, Daphne
AU - O'Gara, Rebecca
AU - Schilling, Amy
AU - Beadling, Carol
AU - Warrick, Andrea
AU - Troxell, Megan L.
AU - Corless, Christopher L.
N1 - Funding Information:
Supported by a Career Catalyst grant from Susan G. Komen for the Cure , KG100112 (M.L.T.).
PY - 2013/5
Y1 - 2013/5
N2 - Somatic mutations in PIK3CA are commonly seen in invasive breast cancer and several other carcinomas, occurring in three hotspots: codons 542 and 545 of exon 9 and in codon 1047 of exon 20. We designed a locked nucleic acid (LNA)-PCR sequencing assay to detect low levels of mutant PIK3CA DNA with attention to avoiding amplification of a pseudogene on chromosome 22 that has >95% homology to exon 9 of PIK3CA. We tested 60 FFPE breast DNA samples with known PIK3CA mutation status (48 cases had one or more PIK3CA mutations, and 12 were wild type) as identified by PCR-mass spectrometry. PIK3CA exons 9 and 20 were amplified in the presence or absence of LNA-oligonucleotides designed to bind to the wild-type sequences for codons 542, 545, and 1047, and partially suppress their amplification. LNA-PCR sequencing confirmed all 51 PIK3CA mutations; however, the mutation detection rate by standard Sanger sequencing was only 69% (35 of 51). Of the 12 PIK3CA wild-type cases, LNA-PCR sequencing detected three additional H1047R mutations in "normal" breast tissue and one E545K in usual ductal hyperplasia. Histopathological review of these three normal breast specimens showed columnar cell change in two (both with known H1047R mutations) and apocrine metaplasia in one. The novel LNA-PCR shows higher sensitivity than standard Sanger sequencing and did not amplify the known pseudogene.
AB - Somatic mutations in PIK3CA are commonly seen in invasive breast cancer and several other carcinomas, occurring in three hotspots: codons 542 and 545 of exon 9 and in codon 1047 of exon 20. We designed a locked nucleic acid (LNA)-PCR sequencing assay to detect low levels of mutant PIK3CA DNA with attention to avoiding amplification of a pseudogene on chromosome 22 that has >95% homology to exon 9 of PIK3CA. We tested 60 FFPE breast DNA samples with known PIK3CA mutation status (48 cases had one or more PIK3CA mutations, and 12 were wild type) as identified by PCR-mass spectrometry. PIK3CA exons 9 and 20 were amplified in the presence or absence of LNA-oligonucleotides designed to bind to the wild-type sequences for codons 542, 545, and 1047, and partially suppress their amplification. LNA-PCR sequencing confirmed all 51 PIK3CA mutations; however, the mutation detection rate by standard Sanger sequencing was only 69% (35 of 51). Of the 12 PIK3CA wild-type cases, LNA-PCR sequencing detected three additional H1047R mutations in "normal" breast tissue and one E545K in usual ductal hyperplasia. Histopathological review of these three normal breast specimens showed columnar cell change in two (both with known H1047R mutations) and apocrine metaplasia in one. The novel LNA-PCR shows higher sensitivity than standard Sanger sequencing and did not amplify the known pseudogene.
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U2 - 10.1016/j.jmoldx.2012.12.005
DO - 10.1016/j.jmoldx.2012.12.005
M3 - Article
C2 - 23541593
AN - SCOPUS:84876479270
SN - 1525-1578
VL - 15
SP - 312
EP - 318
JO - Journal of Molecular Diagnostics
JF - Journal of Molecular Diagnostics
IS - 3
ER -