Acid-base and lipophilic properties of peptide nucleic acid derivatives / 药物分析学报

The first combined experimental and theoretical study on the ionization and lipophilic properties of peptide nucleic acid(PNA)derivatives,including eleven PNA monomers and two PNA decamers,is described.The acidity constants(pKa)of individual acidic and basic centers of PNA monomers were measured by automated potentiometric pH titrations in water/methanol solution,and these values were found to be in agreement with those obtained by MoKa software.These results indicate that single nucleobases do not change their pKa values when included in PNA monomers and oligomers.In addition,immobilized artificial membrane chromatography was employed to evaluate the lipophilic properties of PNA monomers and oligomers,which showed the PNA derivatives had poor affinity towards membrane phospholipids,and confirmed their scarce cell penetrating ability.Overall,our study not only is of po-tential relevance to evaluate the pharmacokinetic properties of PNA,but also constitutes a reliable basis to properly modify PNA to obtain mimics with enhanced cell penetration properties.

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.

Identification of cut off value and lower limit of detection for K-ras mutation by PNA-PCR clamping method / 中华胰腺病杂志

Objective To determine the lower limit of detection (LLOD) and cut off values of K-ras mutation detection by peptide nucleic acid (PNA) clamping-PCR.Methods The genomic DNA of pancreatic cancer cell lines (PANC1 and SW1990) with codonl2,13 mutation and the genomic DNA of placenta with K-ras wild type were mixed and diluted serially into samples with different mutation rate (0,0.1％,0.2％,0.4％,0.8％,1.6％,3.1％,6.25％,12.5％,25％,50％),PANC1 cells with 1％ mutation rate and SW1990 cells with 30％ mutation rate and 4 samples with the quantity of DNA was 50,20,5,1 ng and 50,10,5,1 ng was prepared.Codon 12,13 mutation of K-ras was determined by PNA-PCR,and the mutation Ct values,overall Ct values were collected,and the △Ct values (mutation Ct values-overall Ct values) were calculated,and the tests were repeated for 10 times.ROC curve was used to analyze the △Ct values and determine the best cut off values for K-ras mutation,and the positive diagnostic rate,LLOD was evaluated.Results The mutation Ct,△Ct values of codon 12 mutation of PANC1 and codon 13 mutation of SW1990 of all the different mutation rates were statistically significantly different (P ＜ 0.05) when compared with negative control group,but the overall Ct values were not statistically significantly different from that of negative control group.For detection of K-ras codon 12 mutation by ROC curve,the relevant area of ROC curve (AUC) was 0.926,the optimum cut off value of △CT was 11,the sensitivity and specificity were 84％ and 100％,respectively,and the LLOD was 0.4 ng.For detection of K-ras codon 13 mutation by ROC curve,the relevant AUC was 0.906,the optimum cut off value of △CT was 9.5,the sensitivity and specificity were 71％ and 100％,respectively,and the LLOD was 1.5 ng.The mutation detection results of fixed rate further confirmed the LLOD.Conclusions This study successfully defines LLOD and cut off value of PNA clamping-PCR/K-ras method in detection of K-ras 12 and 13 codon mutations.This method meets the requirement of clinical application.

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.

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.

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.

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.

Detection of K-ras mutation by PNA-PCR/K-ras method in diagnosis of colorectal cancer tissues / 第二军医大学学报

Objective To determine the positive judgement standard of K-ras mutation detection method peptide nucleic acid (PNA)-PCR/K-ras (previously established in our laboratory) and to assess its diagnostic value for colorectal cancer tissues. Methods Plasmidswith K-ras codon 12 mutation and plasmids with K-ras wild-type plasmids were mixed and serially diluted into standard samples (mutation/total: 0, 1/3 200, 1/1 600, 1/800, 1/400, 1/200, 1/100) for six independent tests. The mutation CT, total CT and ΔCT (mutation CT-total CT) values were obtained by PNA-PCR/K-ras method. After the cut-off values of the mutation CT and ΔCT for K-ras diagnosis were identified by ROC analysis, the diagnostic criteria for K-ras mutation was defined by combining both the cut-off values of the mutation CT and ΔCT. A comparison was made between K-ras diagnostic rate by PNA-PCR/K-ns method and direct sequencing for 35 colorectal cancer tissues and their corresponding adjacent noncancerous tissues. Results The mutation CT and ΔCT values for 1/800 and the above standard samples were significantly different from those of the negative samples(P<0. 05), with the optimumcut-off values of mutation CT and ΔCT being 41. 7 and 15. 4, respectively. The diagnostic criteria (mutation CT≤41. 7 or ΔCT≤15. 4) for K-ras mutation was set up asAUC-ROC 0. 955 (P=0. 001). According this diagnostic criteria, the K-ras mutation diagnostic rates in each concentration gradient of the standard samples (0, 1/3 200, 1/1 600, 1/800, 1/400, 1/200, and 1/100) were 0%,66.7%,83.3%,100%, 100%, 100%,and 100%, receptivity, with the upper diagnostic limit being 1/800. The diagnostic rates of K-ras mutation by our method and by direct sequence method for colorectal cancer tissues were 45. 7% (32/70) and 18. 6% (13/70), respectively, showing significant difference (P = 0. 000). Conclusion PNA-PCR/K- ras method has higher sensitivity and positive detection rate than direct sequencing method for colorectal cancer tissues.

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.

The role of mucin gene 1 mediated anti-MKN-45 cell invasion / 中华消化杂志

Objective To create Mucin gene 1 (MUC1) antisense peptide nucleic acid (PNA),and to observe its effects on MKN-45 cell invasion and explore the mechanism. Methods The sequence of antisense PNA was designed according to MUC1 gene sequence and transfected into human gastric cancer cells (MKN-45) by liposome,and the empty vector group (randomized control group)and blank control group (negative control group) were involved. The expression of MUC1 was detected by real time quantitative PCR and the changes of E-cadherin expression were also observed.The effects on gastric cancer cell invasion were tested with transwell chamber assays.Results The expression of MUC1 gene was effectively suppressed by the 3 created antisense PNA,and their expression level (0.62±0.18,0.49±0.12 and 0.60±0.21) was significantly lower than that of negative control group (1.18 ± 0.03,P ＜ 0.01). There was no significant difference between radomized control group and negative control group (1.00±0.04,P=0.657).After MUC1 PNA transfected,the capability of gastric cancer cell invasion decreased significantly (P=0.005).And the expression of E-cadherin at mRNA and protein level was up-regulated.Conclusions There is negative correlation between MUC1 and E-cadherin expression in gastric cancer cell MKN-45.The capability of tumor cell invasion is significantly inhibited by suppressing MUC1 gene expression.

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 study on imaging of 99 Tcm-survivin mRNA antisense peptide nucleic acid in tumor and inflammation animal models / 中华核医学杂志

ObjectiveTo investigate the value of 99Tcm labeled survivin mRNA antisense peptide nucleic acid (PNA) as an imaging agent in the specific diagnosis for carcinoma.MethodsSurvivin mRNA antisense PNA was labeled directly with 99Tcm by the ligand-exchange method.Twenty nude mice with lung carcinoma A549 xenografts were randomly divided into 4 groups.Three groups were used for biodistribution study and one group was used for imaging study.Other twenty mice infected by staphylococcus aureus underwent the same procedure.The biodistribution and imaging of 99Tcm-survivin mRNA antisense PNA was studied at 1,2 and 4 h respectively after the intravenous injection in nude mice bearing lung carcinoma A549 xenografts or inflammation models.SPSS 13.0 was used in the study and all data were analyzed by t test.ResultsBiodistribution results showed that the highest radioactivity was found in the liver,and then in the kidney.Four hours after the administration of the imaging agent,the radioactivity ratios of target-tonon target (T/NT,tumor or inflamumatory lesions to the contralateral regions) in tumor model group were significantly higher than those in inflammation model group ( 3.69 ± 1.13 vs 2.03 ± 0.47,t =3.01,P =0.02 ).Tumors were clearly visible in the tumor model groups at 0.5 h and still clearly seen at 4 h after the injection of antisense PNA.On the contrary,inflammatory lesions could not be seen clearly.Conclusion 99Tcm labeled survivin mRNA antisense PNA can be used to distinguish tumor from inflammation and it may provide a new feasible method for specific tumor diagnosis.

Preparation of 99Tcm labeled survivin mRNA antisense PNA and gene imaging in nude mice bearing lung carcinoma A549 xenografts / 中华核医学杂志

Objective To prepare the 99Tcm-survivin mRNA antisense peptide nucleic acid (PNA)and investigate its value as a gene imaging agent in tumor bearing mice and early diagnosis in tumor.Methods Survivin mRNA antisense PNA and mismatch PNA were synthesized.Four amino acids (Gly- (D)Ala-Gly-Gly) and Aba (4-aminobutyric acid) were linked to the 5' end of PNA.Gly- (D)Ala-Gly-Gly served as a chelating moiety for strong chelation of 99Tcm and Aba acted as a spacer to minimize the steric hindrance.PNAs were labeled with 99Tcm by the ligand-exchange method.The labeling efficiency and radiochemical purity were measured by HPLC and ITLC methods.There were five BALB/c nude mice bearing human lung carcinoma ( A549 ) in each of antisense PNA and mismatch PNA groups.Gene imaging of 99Tcm-survivin mRNA antisense and mismatch PNAs were performed at 1,2 and 4 h post the injection,respectively,and the T/NT ratio was measured by the method of ROI.The statistical comparisons of average values were performed with the two-group t-test for independent sample by SPSS 13.0.Results The product kept stable in vitro.The labeling efficiency of 99Tcm-survivin mRNA antisense PNA was (95.48 ±1.92)％ and more than 85％ after the incubation for24 h in serum.The radiochemical purity was ＞ 95％.The labeling efficiency of mismatch PNA was similar to the antisense PNA.99Tcm-survivin mRNA antisense PNA was especially uptaken by tumor lesion,and its accumulation reached the top at 4 h post the injection.T/NT ratios at 1,2,and 4 h were 2.70 ± 0.28,3.44 ± 0.35,4.21 ± 0.63,respectively.In the comparison,the T/NT ratio of 99Tcm-survivin mRNA mismatch PNA at 4 h (3.12 ±0.50) was significantly lower (t =2.918,P =0.019).Conclusions 99Tcm-survivin mRNA antisense PNA has high labeling efficiency,good stability and no need of purification.Its characteristic of especial uptake by tumor lesion provides the potential value in early diagnosis of tumor.

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.

The value of the K-ras mutations in FNA samples of pancreas on the diagnosis of pancreatic cancer / 中华胰腺病杂志

Objective To investigate the diagnostic value of the K-ras mutations in FNA samples for early detection of pancreatic cancer. Methods FNA samples of 27 patients with pancreatic cancers, 9 patients with other malignant tumors and 14 patients with non malignant pancreatic mass (NMPM) were collected. DNA was extracted, and K-ras gene was amplified through PNA-mediated PGR clamping, the products were sequenced to determine the mutation type. Results The positive rate of K-ras mutations in pancreatic cancers,other malignant tumors and NMPM were 88.9%, 44.4%, 35.7%. There was significant difference in K-ras gene mutations in FNA samples between pancreatic cancer and other malignant tumors ( P = 0. 013 ) and NMPM ( P = 0. 001 ). The sensitivity, specificity, positive predictive value, negative predictive value,accuracy of K-ras mutations in FNA samples of pancreatic cancers were 88.9%, 55.6%, 85.7%, 62.5%,80.6% when compared with other malignant tumors, and the difference between the two groups was significant (P =0. 013) ;Those were 88.9%, 64.3%, 82.8%, 75.0%, 80. 5% when compared with NMPM, and the difference between the two groups was significant ( P = 0. 001 ). When cytology of FNA samples and K-ras mutations was combined, the positive rate of pancreatic cancer was up to 96.3%. Conclusions The detection of K-ras mutations in EUS-FNA samples helped improve the positive diagnostic rate of pancreatic cancer.

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.