Predicting outcomes of EGFR-targeted therapy in non-small cell lung cancer patients using pleural effusions samples and peptide nucleic acid probe assay.

BACKGROUND: Mutation of epidermal growth factor receptor (EGFR) is a prediction marker of the response to tyrosine kinase inhibitor (TKI) drugs in non-small cell lung cancer (NSCLC) patients. As late stage lung cancer patients rarely undergo surgery, samples for EGFR mutation identification usually come from computed tomography (CT)-guided or endoscopic biopsies, which is invasive and costly. Pleural effusion may serve as a less invasive sample for EGFR mutation detection. METHODS: We designed a fluorophore-labeled peptide nucleic acid (PNA) probe assay for three types of EGFR mutations, including exon 19 deletions, L858R point mutations and T790M point mutations. The assay was applied in 39 pleural effusion samples from NSCLC patients. The correlation between detected EGFR status and clinical outcome were analyzed. RESULTS: In 15 paired samples, PNA probe assay in pleural effusion samples could detect all the mutations that were identified by conventional PCR plus Sanger sequencing in tissue biopsies. In addition, PNA probe assay detected three more T790M mutations. In all 39 pleural effusions, the PNA probe assay detected 27 having at least one of the three EGFR mutations. Among the patients before TKI treatment, those with a sensitizing mutation (either exon 19 deletion or L858R) but without T790M, had 94.1% response rate and longer progression-free survival (mean 10.8 months) than patients without detected mutation (mean 4.2 months) and patients with T790M (mean 1.7 months). CONCLUSIONS: Mutations detected in pleural effusions using PNA probe assay are highly associated with clinical outcome. This method appears to be a reliable way for the prediction of the efficacy of EGFR-targeted therapy.

Detection of Rare Somatic GNAS Mutation in McCune-Albright Syndrome Using a Novel Peptide Nucleic Acid Probe in a Single Tube.

McCune-Albright syndrome (MAS) is characterized by the triad of precocious puberty, café au lait pigmentation, and polyostotic fibrous dysplasia (FD) of bone, and is caused by post-zygotic somatic mutations-R201H or R201C-in the guanine nucleotide binding protein, alpha stimulating (GNAS) gene. In the present study, a novel peptide nucleic acid (PNA) probe with fluorescent labeling was designed to detect trace amounts of somatic mutant GNAS in a single tube reaction. The method was applied to screen GNAS mutations in six patients with MAS/FD. The results showed that the PNA probe assay could detect low abundant mutants in 200-fold excess of wild-type alleles. The GNAS mutation was found in three patients with severe disease (MAS) by using the assay. The other three patients with mild disease (having only FD) showed a wild-type result. This study has provided a simple method to detect trace amounts of GNAS mutants with high sensitivity in large amounts of wild-type DNA.

A primer and probe set for detecting multiple types of EGFR exon 19 deletions.

EGFR exon 19 deletion is an important indicator for tyrosine kinase inhibitor treatment in non-small cell lung cancer. However, detection of exon 19 deletions faces a challenge: there are more than 30 types of mutations reported at the hotspot. Moreover, considering the application in body fluid samples, assays with high sensitivity and specificity are necessary for the detection of rare mutant alleles. Here, we describe a single tube reaction which could detect at least 29 types of exon 19 deletions with only an unlabeled peptide nucleic acid (PNA) clamp and a pair of DNA probes. The PNA clamp was used to inhibit amplification of wild-type templates; and the DNA probes were used to generate melting peaks for multiple types of mutations. Under optimal condition, the assay was able to detect as low as 0.01% mutant DNA in wild-type background, and had a limit of detection of 10 pg genomic DNA. Feasibility of the assay was tested in body fluid samples from lung cancer patients. The assay detected 100% and 60% of deletions in pleural effusions and plasma, respectively. We believe the present assay can be used in the clinical laboratories and has potential to be adapted for a microfluidic device.

Detection of rare mutant K-ras DNA in a single-tube reaction using peptide nucleic acid as both PCR clamp and sensor probe.

The major problem of using somatic mutations as markers of malignancy is that the clinical samples are frequently containing a trace amounts of mutant allele in a large excess of wild-type DNA. Most methods developed thus far for the purpose of tickling this difficult problem require multiple procedural steps that are laborious. We report herein the development of a rapid and simple protocol for detecting a trace amounts of mutant K-ras in a single tube, one-step format. In a capillary PCR, a 17mer peptide nucleic acid (PNA) complementary to the wild-type sequence and spanning codons 12 and 13 of the K-ras oncogene was used to clamp-PCR for wild-type, but not mutant alleles. The designated PNA was labeled with a fluorescent dye for use as a sensor probe, which differentiated all 12 possible mutations from the wild-type by a melting temperature (T(m)) shift in a range of 9 to 16 degrees C. An extension temperature of 60 degrees C and an opposite primer 97 nt away from the PNA were required to obtain full suppression of wild-type PCR. After optimization, the reaction detected mutant templates in a ratio of 1:10,000 wild-type alleles. Using this newly devised protocol, we have been able to detect 19 mutants in a group of 24 serum samples obtained from patients with pancreatic cancer. Taken together, our data suggest that this newly devised protocol can serve as an useful tool for cancer screening as well as in the detection of rare mutation in many diseases.

A Sensitive Peptide Nucleic Acid Probe Assay for Detection of BRAF V600 Mutations in Melanoma.

Mutated v-Raf murine sarcoma viral oncogene homolog B (BRAF) is an important biomarker for the prediction of therapeutic efficacy of several anticancer drugs. The detection of BRAF mutation faces two challenges: Firstly, there are multiple types of mutations, and secondly, tumor samples usually contain various amounts of wild-type, normal tissues. Here, we describe a newly established method for sensitive detection of multiple types of BRAF V600 mutations in excess wild-type background. The method introduced a fluorophore-tagged peptide nucleic acid (PNA) to serve as both polymerase chain reaction (PCR) clamp and sensor probe, which inhibited the amplification of wild-type templates during PCR and revealed multiple types of mutant signals during melting analysis. We demonstrated the design and optimization process of the method, and applied it in the detection of BRAF mutations in 49 melanoma samples. This PNA probe assay method detected three types of mutations in 17 samples, and was much more sensitive than conventional PCR plus Sanger sequencing.

Circulating Messenger RNA Profiling with Microarray and Next-generation Sequencing: Cross-platform Comparison.

BACKGROUND: Circulating mRNA is a less invasive and more easily accessed source of samples for biomedical research and clinical applications. However, it is of poor quality. We explored and compared the ability of two high-throughput platforms for the profiling of circulating mRNA regarding their ability to retrieve useful information out of this type of samples. MATERIALS AND METHODS: Circulating mRNAs from three non-small cell lung cancer patients and three healthy controls were analyzed by the cDNA-mediated annealing, selection, extension, and ligation (DASL) assay and high-throughput RNA sequencing (RSEQ). Twelve genes were selected for further confirmation by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). RESULTS: The overall expression profiles derived from the two platforms showed modest-to-moderate correlation. Genes with higher expression levels had higher cross-platform concordance than those of medium- and low-expression levels. In addition, the pathway signatures identified by gene set enrichment analysis from both platforms were in agreement. The RT-q PCR results for the selected genes correlated well with that of RSEQ. CONCLUSION: Genes with higher expression levels have cross-platform concordance and can be potential biomarkers. Furthermore, RSEQ is a better tool for profiling circulating mRNAs.

Detection of low copies of drug-resistant influenza viral gene by a single-tube reaction using peptide nucleic acid as both PCR clamp and sensor probe.

Influenza virus infection causes endemics almost yearly and pandemics occasionally. Although antivirals are available for the clinical treatment of influenza virus infection, the emergence of a drug-resistant virus has reduced the effectiveness of therapy and prophylaxis. Therefore, the timely detection of drug-resistant influenza viruses is important. A single-tube reaction using peptide nucleic acid (PNA) as both a polymerase chain reaction (PCR) clamp and a sensor probe was established to detect the low numbers of copies of viral genes that carry the resistant marker. Influenza A H1N1 viruses resistant to a clinically used antiviral, amantadine, are selected for the experimental design. The PNA-mediated reverse transcription-PCR detected 10 copies/µL of RNA from the resistant strain among 2 × 10(4) copies/µL of RNA from the sensitive strain. A rapid and sensitive method was established for detecting low numbers of drug-resistant genes of the influenza virus. The assay would help to monitorthe emergence of adrug-resistant influenza virus.

Detection of pandemic (H1N1) 2009 influenza virus by allele discrimination.

BACKGROUND: The pandemic (H1N1) 2009 has become a threat of public health. To manage rapidly increased infections and disease control, a simple and reliable first-line screening test for viral infection is on urgent demand. METHODS: Through comprehensive bioinformatics analysis, a single nucleotide polymorphism in nucleoprotein gene which differentiates swine lineage virus and human seasonal H1N1 virus was selected as target. A TaqMan probe-based allele discrimination analysis was designed to analyze clinical samples. In total, 93 clinical specimens and 39 viral isolates were used to test the assay efficacy. Traditional viral culture and molecular analysis were used as gold standard. RESULTS: The testing results showed that the established assay has high sensitivity and specificity (92% and 100%) for pandemic (H1N1) 2009. The assay could detect as low as 5 copies of NP gene of pandemic (H1N1) 2009 or 2 viral particles of human seasonal H1N1. CONCLUSION: This assay can be used as a first-line screening and confirmation test for pandemic (H1N1) 2009 virus and human seasonal flu in one-tube reaction. The assay can serve as a convenient method to reduce the burden of PCR manipulation for diagnostic laboratories when large amount of samples need to be analyzed in a short time.

Single-tube reaction using peptide nucleic acid as both PCR clamp and sensor probe for the detection of rare mutations.

The detection of rare mutant DNA from a background of wild-type alleles usually requires laborious manipulations, such as restriction enzyme digestion and gel electrophoresis. Here, we describe a protocol for homogeneous detection of rare mutant DNA in a single tube. The protocol uses a peptide nucleic acid (PNA) as both PCR clamp and sensor probe. The PNA probe binds tightly to perfectly matched wild-type DNA template but not to mismatched mutant DNA sequences, which specifically inhibits the PCR amplification of wild-type alleles without interfering with the amplification of mutant DNA. A fluorescein tag (which undergoes fluorescence resonance energy transfer with the adjacent fluorophore of an anchor probe when both are annealed to the template DNA) also allows the PNA probe to generate unambiguous melting curves to detect mutant DNA during real-time fluorescent monitoring. The whole assay takes about only 1 h. This protocol has been used for detecting mutant K-ras DNA and could be applied to the detection of other rare mutant DNAs.