Effect of Blocking PYR Complex Binding to DNA Site by Peptide Nucleic Acid on γ-Globin Gene Expression / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of blocking polypyrimidine complex binding to DNA site by using peptide nucleic acid (PNA) on γ-globin gene expression.</p><p><b>METHODS</b>PYR-PNA, β-PNA and RS-PNA (random sequence-PNA) were designed and synthesized, then were transfected into K562 cells with the cationic liposome lipofectamine 2000 used as vector. The expression of γ-globin gene at both the transcriptional and translational level was detected by RT-PCR and the Western blot respectively at 24 h, 48 h and 72 h after transfection with PNAs.</p><p><b>RESULTS</b>Compared with RS-PNA and control groups, the expression of γ-globin gene at mRNA and protein levels in PYR-PNA group was significantly up-regulated(P<0.05), especially at 48 h after tranfection, the levels of mRNA and protein in PYR-PNA group were increased by 2.0 and 2.5 times than those in control group, respectively.</p><p><b>CONCLUSION</b>PYR-PNA can significantly up-regulate the expression of γ-globin gene in K562 cells, this study may provide a new research idea for gene therapy of β-thalassemia.</p>

Peptide Nucleic Acid Probe-Based Analysis as a New Detection Method for Clarithromycin Resistance in Helicobacter pylori

BACKGROUND/AIMS: Helicobacter pylori eradication rates are decreasing because of increases in clarithromycin resistance. Thus, finding an easy and accurate method of detecting clarithromycin resistance is important. METHODS: We evaluated 70 H. pylori isolates from Korean patients. Dual-labeled peptide nucleic acid (PNA) probes were designed to detect resistance associated with point mutations in 23S ribosomal ribonucleic acid gene domain V (A2142G, A2143G, and T2182C). Data were analyzed by probe-based fluorescence melting curve analysis based on probe-target dissociation temperatures and compared with Sanger sequencing. RESULTS: Among 70 H. pylori isolates, 0, 16, and 58 isolates contained A2142G, A2143G, and T2182C mutations, respectively. PNA probe-based analysis exhibited 100.0% positive predictive values for A2142G and A2143G and a 98.3% positive predictive value for T2182C. PNA probe-based analysis results correlated with 98.6% of Sanger sequencing results (κ-value=0.990; standard error, 0.010). CONCLUSIONS: H. pylori clarithromycin resistance can be easily and accurately assessed by dual-labeled PNA probe-based melting curve analysis if probes are used based on the appropriate resistance-related mutations. This method is fast, simple, accurate, and adaptable for clinical samples. It may help clinicians choose a precise eradication regimen.

Peptide Nucleic Acid Clamping and Direct Sequencing in the Detection of Oncogenic Alterations in Lung Cancer: Systematic Review and Meta-Analysis

PURPOSE: Molecular testing in non-small cell lung cancer (NSCLC) aids in identifying oncogenic alterations. The aim of this study was to compare the rates of detection of oncogenic alterations and responsiveness to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) according to EGFR mutation status as determined by peptide nucleic acid (PNA) clamping or direct sequencing (DS). MATERIALS AND METHODS: We performed a systematic literature search using MEDLINE, EMBASE, and the Cochrane Central Register. Data from included studies were pooled to yield summary sensitivity, specificity, positive and negative likelihood ratios, diagnostic odds ratio, and receiver operating characteristic curves. A meta-regression analysis was conducted to identify potential sources of heterogeneity between selected studies. RESULTS: We identified 10 studies comprising 924 patients. Oncogenic alterations were detected in 340 of 924 cases (36.8%) with PNA clamping and in 250 of 924 (27.1%) with DS. The pooled sensitivities of PNA clamping and DS were 0.93 [95% confidence interval (CI): 0.90−0.95] and 0.69 (95% CI: 0.64−0.73), respectively. According to meta-regression analysis, none of the covariates were found to be significant sources of heterogeneity. With respect to treatment responses to EGFR-TKIs, there was no significant difference therein between EGFR mutations detected by PNA clamping and DS (53.4% vs. 50.8%; risk ratio, 0.99; 95% CI 0.83−1.19; p=0.874). CONCLUSION: We demonstrated that PNA clamping has a higher sensitivity than DS for detecting oncogenic alterations in NSCLC. Our findings suggest that PNA clamping is a more useful method for clinical practice.

Cellular delivery of modified peptide nucleic acids: a review / 生物工程学报

Peptide nucleic acid (PNA) is a DNA surrogate in which the phosphate deoxyribose backbone of DNA is replaced by repeating N-(2-aminoethyl)glycine units. PNA can hybridize to the complementary DNA and RNA with higher affinity than their oligonucleotide counterparts. This character of PNA not only makes it a new tool for the studies of molecular biology but also the potential candidate for gene-targeting drugs. The non-ionic backbone of PNA leads to stable hybrids with the nucleic acids, but at the same time, the neutral backbone results in poor cellular uptake. To address this problem, studies on modified PNA progress rapidly in recent years. We reviewed literature reports combined with our study about the delivery methods, including backbone modified PNA and PNA-ligand conjugates, and the cellular uptake of modified PNA. In addition, we summarized the problems and future prospect of the cellular delivery of modified PNA.

Evaluation of Dual-Color Fluorescence In Situ Hybridization With Peptide Nucleic Acid Probes for the Detection of Mycobacterium tuberculosis and Non-Tuberculous Mycobacteria in Clinical Specimens

BACKGROUND: Peptide nucleic acid (PNA) probes are artificial DNA analogues with a hydrophobic nature that can penetrate the mycobacterial cell wall. We evaluated a FISH method for simultaneous detection and identification of Mycobacterium tuberculosis (MTB) and non-tuberculous mycobacteria (NTM) in clinical respiratory specimens using differentially labeled PNA probes. METHODS: PNA probes targeting the mycobacterial 16S ribosomal RNA were synthesized. The cross-reactivity of MTB- and NTM-specific probes was examined with reference strains and 10 other frequently isolated bacterial species. A total of 140 sputum specimens were analyzed, comprising 100 MTB-positive specimens, 21 NTM-positive specimens, and 19 MTB/NTM-negative specimens; all of them were previously confirmed by PCR and culture. The PNA FISH test results were graded by using the United States Centers for Disease Control and Prevention-recommended scale and compared with the results from the fluorochrome acid-fast bacterial stain. RESULTS: The MTB- and NTM-specific PNA probes showed no cross-reactivity with other tested bacterial species. The test results demonstrated 82.9% agreement with the culture results with diagnostic sensitivity of 80.2% and diagnostic specificity of 100.0% (kappa=0.52, 95% confidence interval: 0.370-0.676). CONCLUSIONS: Dual-color PNA FISH showed high specificity for detecting and identifying mycobacteria in clinical specimens. However, because of its relatively low sensitivity, this method could be more applicable to culture confirmation. In application to direct specimens, the possibility of false-negative results needs to be considered.

Detection of Plasma BRAF(V600E) Mutation Is Associated with Lung Metastasis in Papillary Thyroid Carcinomas

PURPOSE: The BRAF(V600E) mutation represents a novel indicator of the progression and aggressiveness of papillary thyroid carcinoma (PTC). The purpose of this study was to determine the clinical significance of free circulating mutant BRAF(V600E) in predicting the advanced disease of PTC. MATERIALS AND METHODS: Seventy seven matched tumor and plasma samples obtained from patients with both benign and PTC were analyzed for BRAF(V600E) mutation using a peptide nucleic acid (PNA) clamp real-time polymerase chain reaction (PCR). RESULTS: The BRAF(V600E) mutation was absent in tumor DNA samples obtained from patients with benign follicular adenomas or adenomatous goiter. In contrast, 49 of 72 (68.1%) PTC tumors were positive for the BRAF(V600E) mutation. Among them, 3 (6.1%) patients with PTC were positive for BRAF(V600E) mutation in plasma and tumor. However, all 3 patients (100%) had lateral lymph node and lung metastasis. CONCLUSION: These findings suggest that the BRAF(V600E) mutation can be detected using a PNA clamp real-time PCR in the blood of PTC patients with lung metastasis. Future studies are warranted to determine clinical significance of serum BRAF(V600E) mutation in large prospective studies.

Evaluation of Peptide Nucleic Acid Probe-Based Fluorescence In Situ Hybridization for the Detection of Mycobacterium tuberculosis Complex and Nontuberculous Mycobacteria in Clinical Respiratory Specimens / 대한임상미생물학회지

BACKGROUND: Tuberculosis is globally the most important cause of death from single pathogen. Rapid and accurate identification of mycobacteria is essential for the control of tuberculosis. We evaluated a fluorescence in situ hybridization (FISH) method using peptide nucleic acid (PNA) probes for the differentiation of Mycobacterium tuberculosis complex (MTB) and nontuberculous mycobacteria (NTM) in direct smears of sputum specimens. METHODS: The cross-reactivity of MTB- and NTM-specific PNA probes was examined with reference strains of M. tuberculosis ATCC 13950, Mycobacterium kansasii ATCC 12479, Mycobacterium fortuitum ATCC 6841, several clinical isolates of mycobacteria (Mycobacterium abscessus, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium gordonae and Mycobacterium chelonae), and 11 frequently isolated respiratory bacterial species other than mycobacteria. A series of 128 sputa (89 MTB culture positive, 29 NTM culture positive, and 10 under treatment culture negative) with grades of trace to 4+ were used to evaluate the performance of the method. RESULTS: The MTB- and NTM-specific PNA probes showed specific reactions with the reference strains of MTB and M. kansasii and clinical isolates of mycobacteria except M. fortuitum ATCC 6841, and no cross-reactivity with other tested bacteria. The PNA probe-based FISH assay for detection of MTB had a sensitivity and specificity of 100%, respectively. The sensitivity and specificity of the NTM-specific PNA probe was 100%. The smear grades of the PNA FISH test were same as with those of the fluorescence AFB stain in 2+ or higher grade. CONCLUSION: Detection and differentiation based on PNA FISH is sensitive and accurate for detecting mycobacteria and for differentiating MTB from NTM in clinical sputum smears.

Peptide Nucleic Acid Clamping Versus Direct Sequencing for the Detection of EGFR Gene Mutation in Patients with Non-small Cell Lung Cancer / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Direct sequencing (DS) is the standard method for detection of epidermal growth factor receptor (EGFR) gene mutation in non-small cell lung cancer (NSCLC); however, low detection sensitivity is a problem. The aim of this study is to demonstrate higher detection rate of EGFR gene mutation with peptide nucleic acid (PNA) clamping compared with DS. MATERIALS AND METHODS: This is a single arm, prospective study for patients with stage IIIB/IV or relapsed NSCLC. Using tumor DNA from 138 patients, both DS and PNA clamping for EGFR gene in exon 18, 19, 20, and 21 were performed. Discrepant results between the two methods were verified using Cobas and a mutant enrichment based next generation sequencing (NGS). Patients with activating mutations were treated with EGFR tyrosine kinase inhibitor (EGFR-TKI, gefitinib, or erlotinib) as first line treatment. RESULTS: Of 138 paired test sets, 24 (17.4%) and 45 (32.6%) cases with activating mutations were detected by DS and PNA clamping, respectively. The difference of detection rate between the two methods was 15.2% (95% confidence interval, 8.7% to 17.8%; p < 0.001). Between the two methods, 25 cases showed discrepant results (n=23, PNA+/DS-; n=2, PNA-/DS+). Mutations were confirmed by Cobas or NGS in 22 of 23 PNA+/DS- cases. The response rates to EGFR-TKI were 72.2% in the PNA+/DS+ group and 85.0% in the PNA+/DS- group. CONCLUSION: PNA clamping showed a significantly higher detection rate of EGFR gene mutation compared with DS. Higher sensitivity of PNA clamping was not compromised by the loss of predictive power of response to EGFR-TKI.

KRAS Mutation Detection in Non-small Cell Lung Cancer Using a Peptide Nucleic Acid-Mediated Polymerase Chain Reaction Clamping Method and Comparative Validation with Next-Generation Sequencing

BACKGROUND: KRAS is one of commonly mutated genetic "drivers" in non-small cell lung cancers (NSCLCs). Recent studies indicate that patients with KRAS-mutated tumors do not benefit from adjuvant chemotherapy, so there is now a focus on targeting KRAS-mutated NSCLCs. A feasible mutation detection method is required in order to accurately test for KRAS status. METHODS: We compared direct Sanger sequencing and the peptide nucleic acid (PNA)-mediated polymerase chain reaction (PCR) clamping method in 134 NSCLCs and explored associations with clinicopathological factors. Next-generation sequencing (NGS) was used to validate the results of discordant cases. To increase the resolution of low-level somatic mutant molecules, PNA-mediated PCR clamping was used for mutant enrichment prior to NGS. RESULTS: Twenty-one (15.7%) cases were found to have the KRAS mutations using direct sequencing, with two additional cases by the PNA-mediated PCR clamping method. The frequencies of KRAS mutant alleles were 2% and 4%, respectively, using conventional NGS, increasing up to 90% and 89%, using mutant-enriched NGS. The KRAS mutation occurs more frequently in the tumors of smokers (p=.012) and in stage IV tumors (p=.032). CONCLUSIONS: Direct sequencing can accurately detect mutations, but, it is not always possible to obtain a tumor sample with sufficient volume. The PNA-mediated PCR clamping can rapidly provide results with sufficient sensitivity.

Correlation analysis between abundance of K-ras mutation in plasma free DNA and its correlation with clinical outcome and prognosis in patients with metastatic colorectal cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To detect K-ras gene mutations in plasma free DNA by peptide nucleic acid clamp PCR assay (PNA-PCR) and nested primer PCR, and to analyze the correlation between K-ras mutations and prognosis in patients with metastatic colorectal cancer (mCRC).</p><p><b>METHODS</b>Peripheral blood was collected and free DNA was extracted from plasma in 106 patients with mCRC. Nested primer PCR and PNA-PCR were used to detect K-ras gene mutation in the plasma free DNA. The patients were divided into three groups by K-ras status: wild-type group (wild-type determined by both methods), low mutation group (mutation by PNA-PCR method, wild-type by nested primer PCR method) and high mutation group (mutation by two methods). The correlation between K-ras mutations and prognosis was analyzed.</p><p><b>RESULTS</b>The mutation rate of K-ras in tumor tissues of the 106 patients was 40.6%. The Mutation rate of K-ras in plasma free DNA detected by PNA-PCR was 31.1%, significantly higher than that of 15.1% detected by nested primer PCR (P = 0.006). The consistent rate of the K-ras status in plasma free DNA detected by PNA-PCR and that in tumor tissue detected by traditional method was up to 83.0%. The median overall survival (OS) of patients of the wild type, low mutation and high mutation groups was 23.5 months, 17.3 months and 13.9 months, respectively (P = 0.002). The median progression-free survival (PFS) of the K-ras wild-type, low mutation and high mutation groups with first-line chemotherapy was 6.8 months, 6.1 months and 3.2 months, respectively (P = 0.002), and the median OS of them were 23.0 months, 15.5 months and 13.9 months, respectively (P = 0.036). The overall response rate (ORR) was improved in the K-ras wide-type patients who received cetuximab combined with chemotherapy as first-line therapy (75.0% vs. 23.4%, P = 0.058). Cetuximab combined with in second-line therapy chemotherapy led to a significant improvement in disease control rate (DCR) ( 100% vs. 35.7%, P < 0.001) as compared with those of chemotherapy alone. COX regression model showed that K-ras status detected by PNA-PCR, ECOG PS, number of surgery and initially metastatic site were independent factors for prognosis.</p><p><b>CONCLUSIONS</b>PNA-PCR for the detection of K-ras mutation in plasma free DNA can be used to substitute the traditional method for detection of K-ras mutation in tumor tissues. The abundance of K-ras mutation in plasma free DNA is an independent prognostic factor for patients with metastatic colorectal cancer.</p>

Preparation and performance assessment of Gamma-peptide nucleic acid gene chip detection system based on surface plasmon resonance / 生物医学工程学杂志

The aim of this study was to build a gene chip system with surface plasmon resonance (SPR) technique, for which Gamma-peptide nucleic acid (Gamma-PNA) functioned as a probe, in order to improve sensitivity and its specificity. With the use of self-assembled monolayer (SAM) technology, surface chemistry of two-dimensional structure was used. Gamma-PNA was designed according to the bioinformatics, and was plated on the SPR chip modified by SAM. Subsequently, relevant parameters of the experiment were ensured and optimized. The results showed that the performances of Gamma-PNA probe was little affected by the ion concentration of buffer, and it had a strong light signal in a stable state. As the ion concentration was 0, there were still good hybrid reactions; pH value had less influence upon Gamma-PNA probe, and acid environment of buffer could be better. Gamma-PNA probe combined with sensor technologies achieved made the probe with dispensable labels and real-time detection. It also improved the efficiency of the hybridization and the stability, providing the foundation for clinical application.

Improvement of detection of paternally inherited fetal mutant genes for β-globin in maternal plasma by PNA clamp / 中华血液学杂志

<p><b>OBJECTIVE</b>To study improvement of detection of paternally herited fetal mutant genes for β-globin in maternal plasma by PNA clamp to seek a noninvasive prenatal diagnostic method for β-thalassemia.</p><p><b>METHODS</b>A total of 38 maternal blood samples were collected at 7 to 20 weeks of gestation, samples in which the father carried CD41-42 mutation and mother carried normal gene or the other point mutation for β-thalassemia were examined. The results of fetal DNA in amniotic fluid, cord blood or peripheral blood of newborns were used as the gold standard for comparison. In the study group, the total cell-free DNA was extracted from maternal plasma using QIAamp DNA Blood Mini Kit. After extraction, the total cell-free DNA was separated by agarose gel (1%) electrophoresis, and the cell-free DNA with a size of 100-300 bp was retrieved from the gel slice. Then, the retrieved DNA-free cell underwent PCR amplified with a PNA clamp. The genotype was confirmed by the conventional method (reverse dot blot hybridization), and the results were compared to gold standard. Simultaneously, two control groups with different PCR procedures were set up. The PCR procedure of control group A was amplified with the extracted total cell-free DNA and PNA clamp, and the PCR procedure of control group B was amplified with the retrieved size-fractionated DNA-free cell without PNA clamp.</p><p><b>RESULTS</b>Plasma samples from 38 pregnant women were detected using PCR products for hybridization, the results were compared with the gold standard. Regarding the 21 samples confirmed by gold standard with fetal genotype 41-42M/N, 19, 8, 12 cases were detected as fetal genotype 41-42M in study group, control group A and control group B respectively, the sensitivity was 90.5% (19/21), 38.1% (8/21) and 57.1% (12/21) respectively;Concerning the 17 samples confirmed by gold standard with fetal normal genotype, the amount of false positive cases were 1, 2 and 1 respectively. The respective specificity was 94.1% (16/17), 94.1% (16/17) and 88.2% (15/17) respectively. The respective accuracies were 92.1% (35/38), 63.2% (24/38) and 71.1% (27/38) respectively. The difference in sensitivity and specificity was pairwise compared by means of McNemar's test. There was significant difference between new study group and control group A or control group B (all P﹤0.05).</p><p><b>CONCLUSION</b>The method of detection of paternally inherited fetal mutation genes for β-thalassemia using small size of fetal DNA-free cell in maternal plasma with PNA clamp had several advantages of reliable sensitivity, specificity and accuracy, indicating its potential of clinical practicality.</p>

Comparison of Direct Sequencing, PNA Clamping-Real Time Polymerase Chain Reaction, and Pyrosequencing Methods for the Detection of EGFR Mutations in Non-small Cell Lung Carcinoma and the Correlation with Clinical Responses to EGFR Tyrosine Kinase Inhibito

BACKGROUND: The aims of this study were to evaluate the abilities of direct sequencing (DS), peptide nucleic acid (PNA) clamping, and pyrosequencing methods to detect epidermal growth factor receptor (EGFR) mutations in formalin-fixed paraffin-embedded (FFPE) non-small cell lung carcinoma (NSCLC) samples and to correlate EGFR mutational status as determined by each method with the clinical response to EGFR tyrosine kinase inhibitors (TKIs). METHODS: Sixty-one NSCLC patients treated with EGFR TKIs were identified to investigate somatic mutations in the EGFR gene (exons 18-21). RESULTS: Mutations in the EGFR gene were detected in 38 of the 61 patients (62%) by DS, 35 (57%) by PNA clamping and 37 (61%) by pyrosequencing. A total of 44 mutations (72%) were found by at least one of the three methods, and the concordances among the results were relatively high (82-85%; kappa coefficient, 0.713 to 0.736). There were 15 discordant cases (25%) among the three different methods. CONCLUSIONS: All three EGFR mutation tests had good concordance rates (over 82%) for FFPE samples. These results suggest that if the DNA quality and enrichment of tumor cells are assured, then DS, PNA clamping, and pyrosequencing are appropriate methods for the detection of EGFR mutations.

Detection of BRAFV600E Mutations in Papillary Thyroid Carcinomas by Peptide Nucleic Acid Clamp Real-Time PCR: A Comparison with Direct Sequencing

BACKGROUND: Papillary thyroid carcinoma (PTC) of the thyroid is the most common endocrine malignancy. High prevalence of an activating point mutation of BRAF gene, BRAFV600E, has been reported in PTC. We assessed the efficiency of peptide nucleic acid clamp real-time polymerase chain reaction (PNAcqPCR) for the detection of BRAFV600E mutation in PTC in comparison with direct sequencing (DS). METHODS: A total of 265 thyroid lesions including 200 PTCs, 5 follicular carcinomas, 60 benign lesions and 10 normal thyroid tissues were tested for BRAFV600E mutation by PNAcqPCR and DS. RESULTS: The sensitivity and accuracy of the PNAcqPCR method were both higher than those of DS for the detection of the BRAFV600E mutation. In clinical samples, 89% of PTCs harbored the BRAFV600E mutation, whereas 5 follicular carcinomas, 50 benign lesions and 10 normal thyroid tissues lacked the mutation. The mutation was associated with aggressive clinical behaviors as extrathyroid invasion (p=0.015), lymph node metastasis (p=0.002) and multiple tumor numbers (p=0.016) with statistical significance. CONCLUSIONS: The PNAcqPCR method is efficiently applicable for the detection of the BRAFV600E mutation in PTCs in a clinical setting.

Evaluation of Peptide Nucleic Acid Probe-based Real-time PCR for Detection of Mycobacterium tuberculosis Complex and Nontuberculous Mycobacteria in Respiratory Specimens

BACKGROUND: A peptide nucleic acid (PNA) probe-based real-time PCR (PNAqPCR(TM) TB/NTM detection kit; PANAGENE, Korea) assay has been recently developed for the simultaneous detection of Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM) in clinical specimens. The study was aimed at evaluation of the performance of PNA probe-based real-time PCR in respiratory specimens. METHODS: To evaluate potential cross-reactivity, the extracted DNA specimens from Mycobacterium species and non-mycobacterial species were tested using PNA probe-based real-time PCR assay. A total of 531 respiratory specimens (482 sputum specimens and 49 bronchoalveolar washing fluid specimens) were collected from 230 patients in July and August, 2011. All specimens were analyzed for the detection of mycobacteria by direct smear examination, mycobacterial culture, and PNA probe-based real-time PCR assay. RESULTS: In cross-reactivity tests, no false-positive or false-negative results were evident. When the culture method was used as the gold standard test for comparison, PNA probe-based real-time PCR assay for detection of MTBC had a sensitivity and specificity of 96.7% (58/60) and 99.6% (469/471), respectively. Assuming the combination of culture and clinical diagnosis as the standard, the sensitivity and specificity of the new real-time PCR assay for detection of MTBC were 90.6% (58/64) and 99.6% (465/467), respectively. The new real-time PCR for the detection of NTM had a sensitivity and specificity of 69.0% (29/42) and 100% (489/489), respectively. CONCLUSIONS: The new real-time PCR assay may be useful for the detection of MTBC in respiratory specimens and for discrimination of NTM from MTBC.

Comparative Analysis of Peptide Nucleic Acid (PNA)-Mediated Real-Time PCR Clamping and DNA Direct Sequencing for EGFR Mutation Detection / 결핵및호흡기질환

BACKGROUND: Although the gold standard method for research trials on epidermal growth factor receptor (EGFR) mutations has been direct sequencing, this approach has the limitations of low sensitivity and of being time-consuming. Peptide nucleic acid (PNA)-mediated polymerase chain reaction (PCR) clamping is known to be a more sensitive detection tool. The aim of this study was to compare the detection rate of EGFR mutation and EGFR-tyrosine kinase inhibitor (TKI) responsiveness according to EGFR mutation status using both methodologies. METHODS: Clinical specimens from 112 NSCLC patients were analyzed for EGFR mutations in exons 18, 19, 20, and 21. All clinical data and tumor specimens were obtained from 3 university hospitals in Korea. After genomic DNA was extracted from paraffin-embedded tissue specimens, both PNA-mediated PCR clamping and direct-sequencing were performed. The results and clinical response to EGFR-TKIs were compared. RESULTS: Sequencing revealed a total of 35 (22.9%) mutations: 8 missense mutations in exon 21 and 26 deletion mutations in exon 19. PNA-mediated PCR clamping showed the presence of genomic alterations in 45 (28.3%) samples, including the 32 identified by sequencing plus 13 additional samples (6 in exon 19 and 7 in exon 21). CONCLUSION: PNA-mediated PCR clamping is simple and rapid, as well as a more sensitive method for screening of genomic alterations in EGFR gene compared to direct sequencing. This data suggests that PNA-mediated PCR clamping should be implemented as a useful screening tool for detection of EGFR mutations in clinical setting.

Comparative Analysis of Peptide Nucleic Acid (PNA)-Mediated Real-Time PCR Clamping and DNA Direct Sequencing for EGFR Mutation Detection / 결핵및호흡기질환

BACKGROUND: Although the gold standard method for research trials on epidermal growth factor receptor (EGFR) mutations has been direct sequencing, this approach has the limitations of low sensitivity and of being time-consuming. Peptide nucleic acid (PNA)-mediated polymerase chain reaction (PCR) clamping is known to be a more sensitive detection tool. The aim of this study was to compare the detection rate of EGFR mutation and EGFR-tyrosine kinase inhibitor (TKI) responsiveness according to EGFR mutation status using both methodologies. METHODS: Clinical specimens from 112 NSCLC patients were analyzed for EGFR mutations in exons 18, 19, 20, and 21. All clinical data and tumor specimens were obtained from 3 university hospitals in Korea. After genomic DNA was extracted from paraffin-embedded tissue specimens, both PNA-mediated PCR clamping and direct-sequencing were performed. The results and clinical response to EGFR-TKIs were compared. RESULTS: Sequencing revealed a total of 35 (22.9%) mutations: 8 missense mutations in exon 21 and 26 deletion mutations in exon 19. PNA-mediated PCR clamping showed the presence of genomic alterations in 45 (28.3%) samples, including the 32 identified by sequencing plus 13 additional samples (6 in exon 19 and 7 in exon 21). CONCLUSION: PNA-mediated PCR clamping is simple and rapid, as well as a more sensitive method for screening of genomic alterations in EGFR gene compared to direct sequencing. This data suggests that PNA-mediated PCR clamping should be implemented as a useful screening tool for detection of EGFR mutations in clinical setting.

Rapid and Sensitive Detection of KRAS Mutation by Peptide Nucleic Acid-based Real-time PCR Clamping: A Comparison with Direct Sequencing between Fresh Tissue and Formalin-fixed and Paraffin Embedded Tissue of Colorectal Cancer

BACKGROUND: Rapid and sensitive detection of KRAS mutation is needed to maximize the benefits for patients who are being treated with monoclonal antibodies to target the epidermal growth factor receptor in colorectal cancer. The aim of this study is to evaluate the efficacy of the peptide nucleic acid clamp real-time PCR (PCqPCR) as compared to that of direct sequencing (DS) between using fresh colorectal cancer tissue and the matched formalin-fixed and paraffin-embedded (FFPE) colorectal cancer tissue. METHODS: The efficacy of PCqPCR was evaluated and compared with that of DS using fresh tissue and matched FFPE tissue from 30 cases of colorectal cancer. RESULTS: PCqPCR is more sensitive than DS for detecting KRAS mutation. PCqPCR detected 1% of mutants in 1 ng DNA. PCqPCR detected mutation in 1% of mutant cells, while DS barely detected, by manual reading, that in 20-50% of mutant cells. In the clinical samples, PCqPCR detected KRAS mutation in 60.0% while DS detected KRAS mutation in 53.3% of the colorectal cancers. The two methods showed a 100% concordance rate for detecting KRAS mutation between the fresh tissue and FFPE tissue. CONCLUSIONS: The PCqPCR method is efficiently applicable for the detection of KRAS mutation in a clinical setting.

PNA-Mediated PCR Clamping for the Detection of EGFR Mutations in Non-Small Cell Lung Cancer / 결핵및호흡기질환

BACKGROUND: Recent studies have demonstrated that the epidermal growth factor receptor (EGFR) genotype is the most important predictive marker to EGFR-tyrosine kinase inhibitors (TKIs) and first-line gefitinib treatment will be approved in the near future for use in non-small cell lung cancer (NSCLC) patients with the EGFR mutation. Direct sequencing is known to be the standard for detecting EGFR mutations; however, it has limited sensitivity. Peptide nucleic acids (PNA)-mediated PCR clamping method is a newly introduced method for analyzing EGFR mutations with increased sensitivity and stability. METHODS: A total of 71 NSCLC patients were analyzed for EGFR mutations using the PNA-mediated PCR clamping technique. Sixty-nine patients were analyzed for clinicopathologic correlation with EGFR genotype; 2 patients with indeterminate results were excluded. In order to determine EGFR-TKI drug response, 57 patients (42 gefitinib, 15 erlotinib) were included in the analysis. RESULTS: The EGFR mutation rate was 47.8%. Being female, a non-smoker, and having adenocarcinoma were favorable clinicopathologic factors, as expected. However, more than a few smokers (33.3%), male (28.1%), and patients with non-adenocarcinoma (28.6%) had the EGFR mutation. Having a combination of favorable clinicopathologic factors did not increase the EGFR mutation rate significantly. Drug response to EGFR-TKIs showed significant differences depending on the EGFR genotype; ORR was 14.3% for wild type vs 69.0% for mutant type; DCR is 28.6% for wild type vs 96.6% for mutant type. The median EGFR-TKI treatment duration is 7.6 months for mutant type group and 1.4 months for wild type group. CONCLUSION: EGFR genotype determined using the PNA-mediated PCR clamping method is significantly correlated with the clinical EGFR-TKI responses and PNA-mediated PCR.

Effects of blocking the CXC chemokine receptor 3 pathway on acute rejection of islet allograft / 中华外科杂志

<p><b>OBJECTIVE</b>To identify the effect of PNA CXCR3 on acute rejection of islet allograft.</p><p><b>METHODS</b>The mice islet transplant models were used. The mice were divided into three groups including saline group, PNA CXCR3 group and mismatch PNA group. In vitro the proliferation capability of T cell was assessed by proliferative responses. RT-PCR and western blot were used to detect the expression of mRNA and protein. Flow cytometry was applied to determine the expression level of CXCR3 in spleen CD3(+) T cells.</p><p><b>RESULTS</b>Compared with saline [(6.72 +/- 1.48) d] and PNA mismatch-treated recipients [(6.54 +/- 0.86) d], PNA CXCR3-treated recipients demonstrated statistically significant prolongation [(9.70 +/- 1.57) d] in functional allograft survival. The CXCR3 mRNA expression level of PNA CXCR3 group (1.06 +/- 0.07) was significantly down-regulated compared with saline (1.98 +/- 0.22) and PNA mismatch (1.87 +/- 0.10) group at the 7th day after transplant. The date showed that CXCR3 protein and lymphocytes proliferation capability was significantly down-regulated in PNA CXCR3 group compared with saline and PNA mismatch group (P<0.01).</p><p><b>CONCLUSIONS</b>The present study indicates that PNA CXCR3 can inhibit T cell activating and prolonging the survival time of islet allograft and has a substantial therapeutic effect on inhibiting acute allograft rejection.</p>