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• W96244539 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. 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. CME Accreditation Statement: This activity (“JMD 2013 CME Program in Molecular Diagnostics”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity (“JMD 2013 CME Program in Molecular Diagnostics”) for a maximum of 48 AMA PRA Category 1 Credit(s)TM. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. CME Accreditation Statement: This activity (“JMD 2013 CME Program in Molecular Diagnostics”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity (“JMD 2013 CME Program in Molecular Diagnostics”) for a maximum of 48 AMA PRA Category 1 Credit(s)TM. Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. The phosphatidylinositol-3-kinases (PI3Ks) are heterodimeric lipid kinases involved in regulation of cellular growth, transformation, adhesion, apoptosis, survival, and motility.1Samuels Y. Velculescu V.E. Oncogenic mutations of PIK3CA in human cancers.Cell Cycle. 2004; 3: 1221-1224Crossref PubMed Scopus (389) Google Scholar, 2Karakas B. Bachman K.E. Park B.H. Mutation of the PIK3CA oncogene in human cancers.Br J Cancer. 2006; 94: 455-459Crossref PubMed Scopus (375) Google Scholar PIK3CA, encoding the PI3K p110-α catalytic subunit, is mutated frequently in invasive breast cancer as well as gastric, colon, lung, brain, endometrial, and other carcinomas. There are three mutational hotspots: codons 542 and 545 of exon 9 (helical domain) and in codon 1047 of exon 20 (kinase domain).1Samuels Y. Velculescu V.E. Oncogenic mutations of PIK3CA in human cancers.Cell Cycle. 2004; 3: 1221-1224Crossref PubMed Scopus (389) Google Scholar, 2Karakas B. Bachman K.E. Park B.H. Mutation of the PIK3CA oncogene in human cancers.Br J Cancer. 2006; 94: 455-459Crossref PubMed Scopus (375) Google Scholar Based on data from the Catalogue of Somatic Mutations in Cancer (COSMIC) database, point mutations in these three codons account for more than 80% of PIK3CA mutations in human cancers.3Bamford S. Dawson E. Forbes S. Clements J. Pettett R. Dogan A. Flanagan A. Teague J. Futreal P.A. Stratton M.R. Wooster R. The COSMIC (Catalogue of Somatic Mutations in Cancer) database and website.Br J Cancer. 2004; 91: 355-358Crossref PubMed Scopus (889) Google Scholar Studies have shown that PIK3CA mutations are associated with the activation of the downstream PI3K/Akt/mTOR signaling, are transforming in cell culture, and are tumorigenic when overexpressed in the mammary gland in mouse models. In addition, they have prognostic and therapeutic implications.4Meric-Bernstam F. Akcakanat A. Chen H. Do K.A. Sangai T. Adkins F. Gonzalez-Angulo A.M. Rashid A. Crosby K. Dong M. Phan A.T. Wolff R.A. Gupta S. Mills G.B. Yao J. PIK3CA/PTEN mutations and akt activation as markers of sensitivity to allosteric mTOR inhibitors.Clin Cancer Res. 2012; 18: 1777-1789Crossref PubMed Scopus (178) Google Scholar, 5Yamaguchi H. Kuboki Y. Hatori T. Yamamoto M. Shiratori K. Kawamura S. Kobayashi M. Shimizu M. Ban S. Koyama I. Higashi M. Shin N. Ishida K. Morikawa T. Motoi F. Unno M. Kanno A. Satoh K. Shimosegawa T. Orikasa H. Watanabe T. Nishimura K. Harada Y. Furukawa T. Somatic mutations in PIK3CA and activation of AKT in intraductal tubulopapillary neoplasms of the pancreas.Am J Surg Pathol. 2011; 35: 1812-1817Crossref PubMed Scopus (72) Google Scholar, 6Wu G. Xing M. Mambo E. Huang X. Liu J. Guo Z. Chatterjee A. Goldenberg D. Gollin S.M. Sukumar S. Trink B. Sidransky D. Somatic mutation and gain of copy number of PIK3CA in human breast cancer.Breast Cancer Res. 2005; 7: R609-R616Crossref PubMed Scopus (186) Google Scholar, 7Troxell M.L. PIK3CA/AKT1 mutations in breast carcinoma: a comprehensive review of experimental and clinical studies.J Clin Exp Pathol. 2013; https://doi.org/10.4172/2161-0681.S1-002Crossref Google Scholar With the ongoing development of pharmacological agents targeting the PI3K pathway, detection of PIK3CA mutations will become increasingly important. Several assays have been developed to identify PIK3CA mutations including Sanger sequencing, high-resolution DNA melting analysis (HRM), real-time PCR, pyrosequencing, improved and complete enrichment co-amplification of major and minor alleles at lower denaturation temperature (ice-COLD)–PCR technology, competitive amplification of differentially melting amplicons (CADMA), peptide nucleic acid–mediated PCR clamping, multiplex PCR-mass spectroscopy (PCR-MS), and SNaPshot genotyping (Applied Biosystems, Life Technologies, Grand Island, NY).8Baker C.L. Vaughn C.P. Samowitz W.S. A PIK3CA pyrosequencing-based assay that excludes pseudogene interference.J Mol Diagn. 2012; 14: 56-60Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 9Miyake T. Yoshino K. Enomoto T. Takata T. Ugaki H. Kim A. Fujiwara K. Miyatake T. Fujita M. Kimura T. PIK3CA gene mutations and amplifications in uterine cancers, identified by methods that avoid confounding by PIK3CA pseudogene sequences.Cancer Lett. 2008; 261: 120-126Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 10Nosho K. Kawasaki T. Ohnishi M. Suemoto Y. Kirkner G.J. Zepf D. Yan L. Longtine J.A. Fuchs C.S. Ogino S. PIK3CA mutation in colorectal cancer: relationship with genetic and epigenetic alterations.Neoplasia. 2008; 10: 534-541PubMed Google Scholar, 11Board R.E. Thelwell N.J. Ravetto P.F. Little S. Ranson M. Dive C. Hughes A. Whitcombe D. Multiplexed assays for detection of mutations in PIK3CA.Clin Chem. 2008; 54: 757-760Crossref PubMed Scopus (69) Google Scholar, 12Lin R. Woodford T. Wu G. Eastlake P. Candau R. Legendre B.J. Richardson K. High sensitivity detection and robust enrichment for PIK3CA somatic mutations using ice COLD-PCR technology.J Clin Oncol. 2011; 29: e13546Crossref Scopus (4) Google Scholar, 13Kristensen L.S. Andersen G.B. Hager H. Hansen L.L. Competitive amplification of differentially melting amplicons (CADMA) enables sensitive and direct detection of all mutation types by high-resolution melting analysis.Hum Mutat. 2012; 33: 264-271Crossref PubMed Scopus (19) Google Scholar, 14Gonzalez-Bosquet J. Calcei J. Wei J.S. Garcia-Closas M. Sherman M.E. Hewitt S. Vockley J. Lissowska J. Yang H.P. Khan J. Chanock S. Detection of somatic mutations by high-resolution DNA melting (HRM) analysis in multiple cancers.PLoS One. 2011; 6: e14522Crossref PubMed Scopus (42) Google Scholar, 15Dupont Jensen J. Laenkholm A.V. Knoop A. Ewertz M. Bandaru R. Liu W. Hackl W. Barrett J.C. Gardner H. PIK3CA mutations may be discordant between primary and corresponding metastatic disease in breast cancer.Clin Cancer Res. 2011; 17: 667-677Crossref PubMed Scopus (167) Google Scholar, 16Su Z. Dias-Santagata D. Duke M. Hutchinson K. Lin Y.L. Borger D.R. Chung C.H. Massion P.P. Vnencak-Jones C.L. Iafrate A.J. Pao W. A platform for rapid detection of multiple oncogenic mutations with relevance to targeted therapy in non-small-cell lung cancer.J Mol Diagn. 2011; 13: 74-84Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 17Kwon M.J. Lee S.E. Kang S.Y. Choi Y.L. Frequency of KRAS, BRAF, and PIK3CA mutations in advanced colorectal cancers: comparison of peptide nucleic acid-mediated PCR clamping and direct sequencing in formalin-fixed, paraffin-embedded tissue.Pathol Res Pract. 2011; 207: 762-768Crossref PubMed Scopus (56) Google Scholar Direct Sanger sequencing is one of the most commonly applied, but has a low mutation detection sensitivity of about 15% to 25%. Detecting low-level somatic mutations in cancer can be challenging in several settings: in small biopsies, post-treatment samples, cases with significant inflammation, and in samples with low tumor content (due to the overwhelming presence of wild-type DNA).18Arcila M. Lau C. Nafa K. Ladanyi M. Detection of KRAS and BRAF mutations in colorectal carcinoma roles for high-sensitivity locked nucleic acid-PCR sequencing and broad-spectrum mass spectrometry genotyping.J Mol Diagn. 2011; 13: 64-73Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar There are several enrichment methods to enhance mutant detection.19Milbury C.A. Li J. Makrigiorgos G.M. PCR-based methods for the enrichment of minority alleles and mutations.Clin Chem. 2009; 55: 632-640Crossref PubMed Scopus (147) Google Scholar One of these methods involves the use of locked nucleic acid (LNA)-PCR. In this study, we designed a 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. This study was approved by Oregon Health & Science University Institutional Review Board. H&E-stained slides from breast resections were carefully reviewed to identify lesional tissue, and the corresponding area of the paraffin tissue block was then punched with a 1-mm or 2-mm core device (range, 2 to 8 punches pooled). The tissue cores were aligned on edge and re-embedded in a paraffin block using the smallest available mold, and an H&E slide was prepared at shallow depth for diagnostic confirmation of the lesion and assessment of the percentage of lesional tissue (reviewed by M.T., D.A., and R.W.). For proliferative breast lesions, the resulting cores contained lesional epithelium, myoepithelium, and stromal cells, with epithelial content ranging from <10% up to 30% in the core samples. Carcinoma samples had greater tumor cellularity. The samples were then deparaffinized in xylene for 5 minutes. After centrifugation, the samples were washed in 100% ethanol. The pellet was air dried and DNA was extracted using DNeasy QIAamp Mini Kit (Qiagen, Valencia, CA) following the manufacturer's protocol. DNA yield was assessed by absorbance on a NanoDrop 1000 spectrophotometer (ND-1000; Thermo Scientific, Wilmington, DE). We tested 60 breast DNA samples that were previously screened for PIK3CA exon 9 and exon 20 hotspot point mutations using a PCR-MS assay as previously described.20Beadling C. Heinrich M.C. Warrick A. Forbes E.M. Nelson D. Justusson E. Levine J. Neff T.L. Patterson J. Presnell A. McKinley A. Winter L.J. Dewey C. Harlow A. Barney O. Druker B.J. Schuff K.G. Corless C.L. Multiplex mutation screening by mass spectrometry evaluation of 820 cases from a personalized cancer medicine registry.J Mol Diagn. 2011; 13: 504-513Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 21Troxell M.L. Brunner A.L. Neff T. Warrick A. Beadling C. Montgomery K. Zhu S. Corless C.L. West R.B. Phosphatidylinositol-3-kinase pathway mutations are common in breast columnar cell lesions.Mod Pathol. 2012; 25: 930-937Crossref PubMed Scopus (32) Google Scholar PIK3CA codons 542, 545, and 1047 and flanking sequences were amplified using primers described in Table 1. Due to the presence of a pseudogene located in chromosome 22 that has 95% sequence homology with exons 9 through 13 of the PIK3CA gene,22Muller C.I. Miller C.W. Hofmann W.K. Gross M.E. Walsh C.S. Kawamata N. Luong Q.T. Koeffler H.P. Rare mutations of the PIK3CA gene in malignancies of the hematopoietic system as well as endometrium, ovary, prostate and osteosarcomas, and discovery of a PIK3CA pseudogene.Leuk Res. 2007; 31: 27-32Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar the exon 9 reverse sequencing primer was also designed to have one LNA at a base pair mismatched to the pseudogene sequence to minimize amplification of the pseudogene. The PCR reaction was performed in a 20-μL volume mixture containing 200 ng of DNA, 10 μL of LC480 Probe Master Mix (Roche Applied Science, Indianapolis, IN), and 0.3 μmol/L of each forward and reverse primer. The PCR amplification was performed under the following cycling conditions: initial denaturation at 95°C for 2 minutes, followed by 40 cycles of denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, and elongation at 72°C for 30 seconds, and final elongation at 72°C for 7 minutes.Table 1PIK3CA Primers Used for PCR Amplification and Direct SequencingExonPrimerSequence9Forward5′-ACAGAGTAACAGACTAGCTAGAG-3′Reverse5′-CATGTAAATTCTGCTT+TATTTATTCC-3′20Forward5′-ATGATGCTTGGCTCTGGAAT-3′Reverse5′-GGTCTTTGCCTGCTGAGAGT-3′The nucleotide followed by the plus (+) sign indicates the locked nucleotide. Open table in a new tab The nucleotide followed by the plus (+) sign indicates the locked nucleotide. Five microliters of amplified products were then purified with 2 μL of ExoSAP-IT (Affymetrix, Santa Clara, CA). The mixture was treated at 37°C for 15 minutes, followed by enzyme deactivation at 80°C for 15 minutes. Cycle sequencing reaction was performed using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) according to the manufacturer's protocol. Centri-Sep columns (Princeton Separations, Adelphia, NJ) were used for postsequencing purification according to the manufacturer's protocol. The sequence data were collected on an ABI 3130 BioAnalyzer (Applied Biosystems) using POP 7 as polymer (Applied Biosystems, Life Technologies), and sequence analysis was performed with the DNA Sequence Analysis Software version 3.1.1 (Applied Biosystems). LNA is a nucleic acid analog that contains a 2′-O,4′-C-methylene bridge in the ribose moiety.18Arcila M. Lau C. Nafa K. Ladanyi M. Detection of KRAS and BRAF mutations in colorectal carcinoma roles for high-sensitivity locked nucleic acid-PCR sequencing and broad-spectrum mass spectrometry genotyping.J Mol Diagn. 2011; 13: 64-73Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 23Pfundheller H.M. Sorensen A.M. Lomholt C. Johansen A.M. Koch T. Wengel J. Locked nucleic acid synthesis.Methods Mol Biol. 2005; 288: 127-146PubMed Google Scholar, 24Vester B. Wengel J. LNA (locked nucleic acid): high-affinity targeting of complementary RNA and DNA.Biochemistry. 2004; 43: 13233-13241Crossref PubMed Scopus (572) Google Scholar When incorporated into a DNA oligonucleotide, it raises thermal stability with complementary DNA. Non-extendable oligonucleotides containing wild-type–specific LNAs anneal tightly to wild-type sequences, effectively suppressing wild-type allele amplification. The LNA oligonucleotide sequences for exons 9 and 20 are shown in Table 2. Multiple concentrations of LNA oligonucleotide were tested to optimize the assay. We sought a concentration of LNA oligonucleotide such that amplification of wild-type alleles was not completely suppressed, yet mutant alleles were preferentially amplified. Higher concentrations of LNA oligonucleotide resulted in increased suppression of wild-type sequences. Conversely, decreasing the concentration of LNA oligonucleotide decreased the resultant mutant:wild-type peak height ratio. PCR cycling conditions were similar to those in the standard Sanger sequencing assay except for the addition of 0.075 μmol/L PIK3CA exon 9 LNA oligonucleotide or 0.03 μmol/L PIK3CA exon 20 LNA oligonucleotide to the PCR reaction. Otherwise, all samples were sequenced following the same procedure as the standard Sanger sequencing assay. The assay was validated using known exon 9 and exon 20 mutant cases, as well as wild-type cases, including replicate assays.Table 2PIK3CA LNA Probes Used for LNA-PCR SequencingExonCodonLNA probe sequence9542, 5455′-T+G+A+A+A+TCA+C+T+G+A+GCAGGAGA/3InvdT/-3′2010475′-T+G+C+A+C+A+T+C+A+T+GGTGGCTGGA/3InvdT/-3′The nucleotides followed by the plus (+) signs indicate the locked nucleotides. Open table in a new tab The nucleotides followed by the plus (+) signs indicate the locked nucleotides. Serial dilutions of PIK3CA exons 9 and 20 mutant formalin-fixed paraffin-embedded (FFPE) samples were performed to assess the sensitivity of mutation detection in the presence of the LNA-oligonucleotide probe. Mutant and wild-type FFPE DNA samples were mixed to generate the following estimated mutant concentrations (relative peak heights of sequencing traces20Beadling C. Heinrich M.C. Warrick A. Forbes E.M. Nelson D. Justusson E. Levine J. Neff T.L. Patterson J. Presnell A. McKinley A. Winter L.J. Dewey C. Harlow A. Barney O. Druker B.J. Schuff K.G. Corless C.L. Multiplex mutation screening by mass spectrometry evaluation of 820 cases from a personalized cancer medicine registry.J Mol Diagn. 2011; 13: 504-513Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar): 30%, 15%, 7%, 3%, 1.5%, and 0.75% of exon 9 mutant DNA; 40%, 20%, 10%, 5%, 2.5%, 1.3%, and 0.6% of exon 20 mutant DNA. Statistical differences between the results obtained from direct sequencing as compared to LNA-PCR and PCR-MS were analyzed by Fisher's exact test. A P value of <0.05 was considered statistically significant. We evaluated the point mutation status of PIK3CA hotspot codons E542, E545, and H1047 in a group of 60 breast lesions by PCR-MS, LNA-PCR sequencing, and standard Sanger sequencing. The 60 cases evaluated included preneoplastic breast lesions (31), breast carcinoma (21, including in situ, invasive, and metastatic), and normal breast samples (8). On the basis of prior PCR-MS screening, 48 cases had one or more PIK3CA mutations (14 E545K, 1 E545G, 4 E542K, 25 H1047R, one case with both E545K and E542K, one case with both E542K and H1047R, one case with both E545K and H1047R, and one case with E545K and Q546E), and 12 were wild type (Table 3).Table 3PIK3CA Mutation Testing Results by Three Different PlatformsMutationMS-PCRSanger sequencingLNA-PCR sequencingAdditional LNA mutant cases compared to SangerH1047R2720307 of 30 (23%)E542K6264 of 6 (67%)E545K, G17, 113, 018, 15 of 18 (28%)Wild type12258One additional Q546E mutation, detected with all assays, was not included in the totals. Open table in a new tab One additional Q546E mutation, detected with all assays, was not included in the totals. We developed a protocol using competitive LNA oligonucleotides to partially suppress amplification of wild-type PIK3CA exon 9 or exon 20 sequences (Tables 1 and 2) allowing detection of PIK3CA hotspot point mutations (E542, E545, H1047) in small or mixed samples. LNA oligonucleotide concentrations of 0.075 μmol/L and 0.03 μmol/L for PIK3CA exon 9 and exon 20, respectively, were found to be optimal for mutation detection. These LNA oligonucleotide concentrations were tested against 12 PIK3CA wild-type DNAs, and amplification was not inhibited. After optimization of assay conditions and assay validation, the LNA sequencing method was used to test the cohort of previously screened breast lesions. In total, 38 breast lesions were tested for exon 9 hotspot mutations and 35 for exon 20 hotspot mutations. LNA-PCR sequencing confirmed all 52 of the known PIK3CA mutations (Table 3). Of the 12 cases screened as PIK3CA wild type by PCR-MS, LNA-PCR sequencing detected additional mutations in 4 of 12 (33%) cases, including three H1047R mutations in normal breast samples and one E545K mutation in usual ductal hyperplasia (Table 3 and Figure 1). Interestingly, histopathological re-review of these three normal breast specimens showed contaminating columnar cell change in two. In both of these patients, columnar cell change had been separately isolated and tested, and demonstrated H1047R mutations. The third case had focal apocrine metaplasia. All 35 PIK3CA exon 9 LNA-PCR sequencing assays showed no amplification of the pseudogene. Although PIK3CA codon 546 was not covered with a LNA oligonucleotide, our PIK3CA exon 9 assay allowed detection of a Q546E PIK3CA mutation in one patient, as described in detail below. All 60 breast lesions were also assayed by standard Sanger sequencing; there was one assay failure with insufficient material remaining for repeat studies. In comparison to PCR-MS, Sanger sequencing detected only 69% (35 of 51) of the mutations. In comparison to LNA-PCR, the Sanger sequencing identified only 64% (36 of 55) mutations (Table 3). The differences between mutation detection by Sanger sequencing as compared to LNA-PCR and PCR-MS were statistically significant (P = 0.0001 and 0.0038, respectively). Among the 15 mutant cases that showed discordant genotype by Sanger sequencing as compared to LNA-PCR sequencing and PCR-MS, 27% (4 of 15) cases had double mutations. Two cases showed two PIK3CA exon 9 mutations: one case had both E542K and E545K mutations by both LNA-PCR and PCR-MS, but Sanger sequencing only detected the E545K mutation; the second case had E545K and Q546E mutations by both LNA-PCR and PCR-MS, but only Q546E by Sanger sequencing. The other two cases had exon 9 and exon 20 PIK3CA mutations: one case had E545K and H1047R mutations by LNA-PCR and PCR-MS, yet only showed H1047R by Sanger; the second case had E542K and H1047R mutations by LNA-PCR and PCR-MS, but was wild type for both PIK3CA exons 9 and 20 by Sanger sequencing (Table 4 and Figure 1). Histopathological review of these four double-mutant cases show 80% to 100% lesional tissue.Table 4Discordant Cases across Three MethodologiesCasePCR-MSSangerLNA-PCR1E545K, H1047RH1047RE545K, H1047R2E542K, H1047RWTE542K, H1047R3E545K, Q546EQ546EE545K, Q546E4E542K, E545KE545KE542K, E545K5H1047RWTH1047R6H1047RWTH1047R7H1047RWTH1047R8H1047RWTH1047R9H1047RWTH1047R10H1047RWTH1047R11E545GWTE545G12E545KWTE545K13E545KWTE545K14E542KWTE542K15E542KWTE542K16WTWTE545K∗Case 16: usual ductal hyperplasia.17WTWTH1047R†Cases 17 and 18: normal breast specimens with three to four glands with columnar cell lesion (columnar cell lesion with known H1047R mutation).18WTWTH1047R†Cases 17 and 18: normal breast specimens with three to four glands with columnar cell lesion (columnar cell lesion with known H1047R mutation).19WTWTH1047R‡Case 19: normal breast specimen with six glands with apocrine metaplasia.WT, wild type.∗ Case 16: usual ductal hyperplasia.† Cases 17 and 18: normal breast specimens with three to four glands with columnar cell lesion (columnar cell lesion with known H1047R mutation).‡ Case 19: normal breast specimen with six glands with apocrine metaplasia. Open table in a new tab WT, wild type. The PIK3CA exon 9 LNA oligonucleotide was tested at concentrations of 0.05 μmol/L, 0.075 μmol/L, and 0.10 μmol/L, whereas the exon 20 LNA oligonucleotide was run at concentrations of 0.03 μmol/L, 0.0375 μmol/L, and 0.075 μmol/L. LNA oligonucleotide concentrations of 0.075 μmol/L and 0.0375 μmol/L for PIK3CA exon 9 and exon 20, respectively, were found to be optimal for mutation detection in our hands. The addition of higher LNA concentration resulted in complete suppression of wild-type amplification. The optimal LNA oligonucleotide concentrations may vary in individual laboratories and will have to be tested in assay development/validation. Serial dilutions of six different mutations from FFPE-derived DNA showed technical sensitivities of between 0.6% and 2.5% mutant allele (Table 5 and Figure 2). Thus, our LNA-PCR assay will theoretically be able to detect PIK3CA mutations in these three codons of the PIK3CA gene if mutation is present in <5% of the sampled population (accounting for heterozygosity).Table 5LNA-PCR Assay Sensitivity for PIK3CA Exons 9 and 20PIK3CA ExonMutationNucleic acid changeSensitivity with LNA probe9E545KGAG to AAG1.0%E545DGAG to GAT2.5%E542KGAA to AAA2.5%20H1047RCAT to CGT1.3%H1047LCAT to CTT0.6%H1047YCAT to TAT1.3% Open table in a new tab A number of pharmacological agents targeting PIK3CA or its downstream pathway are under development, with some agents already in clinical trials, including PI3K inhibitors XL147, PX866, GDC0941, BKM120, CH5132799, and dual PI3K and mTOR inhibitors BEZ235, BGT226, XL765, SF1126, GSK1059615, among others.25Engelman J.A. Targeting PI3K signalling in cancer: opportunities, challenges and limitations.Nat Rev Cancer. 2009; 9: 550-562Crossref PubMed Scopus (1968) Google Scholar, 26Myers A.P. Cantley L.C. 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Sakata K. Tachibana Y. Ohwada J. Ebiike H. Kuramoto S. Morita K. Yoshimura Y. Yamazaki T. Ishii N. Kondoh O. Aoki Y. The selective class I PI3K inhibitor CH5132799 targets human cancers harboring oncogenic PIK3CA mutations.Clin Cancer Res. 2011; 17: 3272-3281Crossref PubMed Scopus (79) Google Scholar Further, tumors harboring activating PIK3CA mutations may be resistant to agents targeting upstream elements of the pathway. For instance, in a retrospective study of patients with metastatic colorectal cancer who had received epidermal growth factor receptor–directed antibody therapy, Sood et al32Sood A. McClain D. Maitra R. Basu-Mallick A. Seetharam R. Kaubisch A. Rajdev L. Mariadason J.M. Tanaka K. Goel S. PTEN gene expression and mutations in the PIK3CA gene as predictors of clinical benefit to anti-epidermal growth factor receptor antibody therapy in patients with KRAS wild-type metastatic colorectal cancer.Clin Colorectal Cancer. 2012; 11: 143-150Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar found that among patients with KRAS wild-type col" @default.
• W96244539 created "2016-06-24" @default.
• W96244539 creator A5020561263 @default.
• W96244539 creator A5026593581 @default.
• W96244539 creator A5053306009 @default.
• W96244539 creator A5061310592 @default.
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• W96244539 date "2013-05-01" @default.
• W96244539 modified "2023-10-16" @default.
• W96244539 title "Novel Method for PIK3CA Mutation Analysis" @default.
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