Novel method for PIK3CA mutation analysis: Locked nucleic acid-PCR sequencing

Daphne Ang, Rebecca O'Gara, Amy Schilling, Carol Beadling, Andrea Warrick, Megan Troxell, Christopher Corless

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)312-318
Number of pages7
JournalJournal of Molecular Diagnostics
Volume15
Issue number3
DOIs
StatePublished - May 2013

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Polymerase Chain Reaction
Mutation
Exons
Codon
Pseudogenes
Breast
Chromosomes, Human, Pair 22
DNA
Metaplasia
Mutation Rate
Oligonucleotides
Hyperplasia
locked nucleic acid
Mass Spectrometry
Breast Neoplasms
Carcinoma

ASJC Scopus subject areas

  • Molecular Medicine
  • Pathology and Forensic Medicine

Cite this

Novel method for PIK3CA mutation analysis : Locked nucleic acid-PCR sequencing. / Ang, Daphne; O'Gara, Rebecca; Schilling, Amy; Beadling, Carol; Warrick, Andrea; Troxell, Megan; Corless, Christopher.

In: Journal of Molecular Diagnostics, Vol. 15, No. 3, 05.2013, p. 312-318.

Research output: Contribution to journalArticle

Ang, Daphne ; O'Gara, Rebecca ; Schilling, Amy ; Beadling, Carol ; Warrick, Andrea ; Troxell, Megan ; Corless, Christopher. / Novel method for PIK3CA mutation analysis : Locked nucleic acid-PCR sequencing. In: Journal of Molecular Diagnostics. 2013 ; Vol. 15, No. 3. pp. 312-318.
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