Evaluation of denaturing high‐performance liquid chromatography as a rapid detection method for identification of epidermal growth factor receptor mutations in nonsmall‐cell lung cancer

Abstract

Objectives: The polymorphic site rs4647905 of the FGFR1 gene was previously associated with a decrease in cephalic index (CI). Here, we evaluate the relationships between genotypes and cephalometric measurements and indices in one Mexican Native and two mestizo Mexican populations using two haplotype‐tag SNPs (rs4647905 and rs3213849) that represent >85% of the FGFR1 variability, plus three other SNPs (rs2293971, rs2304000, and rs930828) situated nearby. In addition, we genotyped five South American natives, two European, one African, and one Siberian populations to evaluate their intra and intercontinental population diversity. Methods: The five SNPs were tested and the craniofacial measurements and indices were collected using standardized procedures. Principal Component Analysis was used to verify individual/population comparisons. Associations were performed through the generalized linear model (GLM), coefficient of determination R2 and linear regression tests. Results: We found a tendency for a decrease in CI in individuals homozygous for allele rs4647905C, regardless of the population to which they belong, though the effect is more pronounced in mestizo. When the GLM analyses were performed using the absolute/linear cephalometric measurements, a statistically significant association was found between four SNPs and head length in the mestizo population. Conclusions: FGFR1 polymorphisms, especially rs4647905, can have an important role in the normal human skull variation, primarily due to their influence in head length, which would affect other cephalometric absolute/linear measures as well as indices like CI as a result of the pervasive nature of the morphological integration that characterizes the human skull. Am. J. Hum. Biol. 2013. © 2012 Wiley Periodicals, Inc.

Epidermal growth factor receptor mutations detected by denaturing high‐performance liquid chromatography in nonsmall cell lung cancer

“…Our own group previously reported on dHPLC (the same assay used in this study) for detection of EGFR mutations, showing that this method was an efficient and more sensitive method for screening compared with sequence analysis. 27 In that study, all amplicons with shifts detected by dHPLC were because of a DNA sequence variation; the false-positive rate of dHPLC in that analysis was 0%.…”

“…28,29 Recently, our group demonstrated that dHPLC is capable of extracting DNA from significantly smaller tumor sample volumes than required for sequencing. 27 This is particularly advantageous given that pathological and molecular analysis must often be performed on less than optimal tissue specimens. It is interesting to note that in our study population, 26% of evaluable tissue specimens were found to contain mutation, which is higher than the 10% to 15% incidence typically reported in the general North American and Western European populations.…”

Lung adenocarcinoma: guiding EGFR-targeted therapy and beyond

“…In some cases with EGFR mutations, this limit is somewhat lower because the mutant allele may be present in multiple copies per tumor cell (concurrent amplification). More recently, a number of studies of EGFR in lung cancer have employed more sensitive mutation detection techniques, including mutation-specific PCR assays, 37,38 PCR with hybridization in real-time with mutation-specific fluorescent probes, 39,40 single-strand conformational polymorphism, 35 denaturing high-performance liquid chromatography, 41,42 peptide nucleic acidlocked nucleic acid PCR clamp 11 and more experimental techniques such as mass spectrometry and single molecule sequencing. 43 These have been recently reviewed.…”

The Role of Molecular Pathology in Non-Small-Cell Lung Carcinoma—Now and in the Future

“…Direct sequencing is the first widely used method for the detection of EGFR mutation, but it lacks sensitivity and might miss 25% of positive cases 21 . Several other more sensitive techniques have been applied, including polymerase chain reaction (pcr) single-strand conformation polymorphism 22 , TaqMan (Roche Molecular Diagnostics, Pleasanton, CA, U.S.A.) pcr 23 , loop-hybrid mobility shift assay 24 , Cycleave (Clontech Laboratories, Mountain View, CA, U.S.A.) pcr 25 , pcr restriction fragment length polymorphism and length analysis 26 , matrixassisted laser desorption/ionization time-of-flight mass spectrometry genotyping 27 , peptide nucleic acid-locked nucleic acid pcr clamp 28 , Scorpions (DxS Limited, Manchester, U.K.) amplified refractory mutation system 29 , denaturing high-performance liquid chromatography 30 , single-molecule sequencing 31 , mutant-enriched pcr 32 , and Smart Amplification Process (DNAFORM, Yokohama City, Japan) 33 . These techniques not only vary in sensitivity, but also differ in terms of the detection of new and alreadyknown mutations and the comprehensive detection of deletions and insertions 21 .…”