Peptide Nucleic Acid Clamp PCR Research Articles

Efficient detection of somatic UBA1 variants and clinical scoring system predicting patients with variants in VEXAS syndrome.

To efficiently detect somatic UBA1 variants and establish a clinical scoring system predicting patients with pathogenic variants in VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome. Eighty-nine Japanese patients with clinically suspected VEXAS syndrome were recruited [81 males and 8 females; median age of onset 69.3 years (interquartile range 62.1-77.6)]. Peptide nucleic acid-clamping PCR (PNA-PCR), regular PCR targeting exon 3 clustering UBA1 variants and subsequent Sanger sequencing were conducted for variant screening. Partitioning digital PCR or targeted amplicon deep sequencing was also performed to evaluate the variant allele frequency (VAF). We developed our clinical scoring system to predict UBA1 variant-positive and -negative patients and assessed the diagnostic value of our system using receiver operating characteristics (ROC) curve analysis. Forty patients (44.9%) with reported pathogenic UBA1 variants were identified, including a case having a variant with VAF of 1.7%, using a highly sensitive method. Our clinical scoring system considering age >50 years, cutaneous lesions, lung involvement, chondritis and macrocytic anaemia efficiently predicted patients with UBA1 variants (the area under the curve for the scoring total was 0.908). Genetic screening with the combination of regular PCR and PNA-PCR detected somatic UBA1 variants with high sensitivity and specificity. Our scoring system could efficiently predict patients with UBA1 variants.

Monitoring of circulating tumour DNA in advanced pancreatic ductal adenocarcinoma predicts clinical outcome and reveals disease progression earlier than radiological imaging.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with a need for better tools to guide treatment selection and follow-up. The aim of this prospective study was to investigate the prognostic value and treatment monitoring potential of longitudinal circulating tumour DNA (ctDNA) measurements in patients with advanced PDAC undergoing palliative chemotherapy. Using KRAS peptide nucleic acid clamp-PCR, we measured ctDNA levels in plasma samples obtained at baseline and every 4 weeks during chemotherapy from 81 patients with locally advanced and metastatic PDAC. Cox proportional hazard regression showed that ctDNA detection at baseline was an independent predictor of progression-free and overall survival. Joint modelling demonstrated that the dynamic ctDNA level was a strong predictor of time to first disease progression. Longitudinal ctDNA measurements during chemotherapy successfully revealed disease progression in 20 (67%) of 30 patients with ctDNA detected at baseline, with a median lead time of 23 days (P = 0.01) over radiological imaging. Here, we confirmed the clinical relevance of ctDNA in advanced PDAC with regard to both the prediction of clinical outcome and disease monitoring during treatment.

Optimization of a Low-Cost, Sensitive PNA Clamping PCR Method for JAK2 V617F VariantDetection.

The JAK2 V617F variantis diagnostic for myeloproliferative neoplasms, a group of clonal disorders of hematopoietic stem and progenitor cells. Although several approaches have been developed to detect the variant, a gold standard diagnostic method has not yet been defined. We describe a simple, fast, and cost-effective PCR-based approach that enhances test specificity and sensitivity by blocking the amplification of the large excess of wild-type DNA. The method involves using an oligo peptide nucleic acid (PNA) perfectly matching its corresponding DNA sequence. The PCR protocol was optimized by collecting a detailed thermodynamic data set on PNA-DNA wild-type duplexes by circular dichroism melting experiments. The specificity and sensitivity of PNA clamping PCR were assessed by genotyping 50 patients with myeloproliferative neoplasm who carried the JAK2 V617F variantand 50 healthy donors. The optimized protocol enabled selective amplification of the variant alleles, achieving maximum sensitivity (100%) and specificity (100%). Analytical sensitivity was 0.05% of variant alleles as assessed by serial dilutions of DNA from the HEL cell line (which carries the JAK2 V617F variant) mixed to wild-type DNA from healthy donors. The JAK2 V617F variant test performed according to this method has better diagnostic performance than its 2 main PCR-based competitors, at much lower cost. High sensitivity and specificity and cost-effectiveness make PNA clamping PCR a useful testing platform for the detection of minor allele variants in small-scale diagnostic laboratories. It promises to improve patient care while enabling significant healthcare savings.

FRI-287 - Quantification of hepatitis B virus covalently closed circular DNA by a peptide nucleic acid-clamping PCR method

FRI-287 - Quantification of hepatitis B virus covalently closed circular DNA by a peptide nucleic acid-clamping PCR method

DETECTION OF BRAF MUTATION IN MELANOMA FOUND IN TISSUE AND CFDNA BY USING A HIGHLY SENSITIVE PNA-CLAMP PCR.

Malignant melanoma results from ongoing activation of the mitogen activated protein kinases(MAPK ) pathway, commonly driven by mutations in BRAF. Several selective inhibitors of this pathway are used clinically, most notably the Vemurafenib and Dabrafenib. Different methods for BRAF mutation detection exist in the United Kingdom, including pyrosequencing, COBAS test and Sanger sequencing. However, there is significant variability in the analytical sensitivity of these tests. A highly sensitive PCR assay was developed to detect low tumor cell percentage of 0.1% in formalin fixed paraffin embedded (FFPE) tissue and plasma. The assay was developed on stable cell lines containing BRAF (codon 600) mutations. Peptide nucleic acid (PNA) Clamp PCR was used for the selective amplification of DNA target sequences. Total DNA was extracted from 48 FFPE tissues and 20 blood plasma (14 matched) stage II-IV melanoma patients and screened for BRAF mutations. Results were correlated with COBAS test data and clinical parameters. PNA Clamp PCR on FFPE tissue showed 46% (n=22) BRAF mutants. Four more BRAF mutant cases were identified using PNA clamp methodology when compared to COBAS. Sample cases were independently tested. Matched samples showed 85% (n=12) correlation for BRAF mutation status (6 + ve, 4 – ve, 2 with no residual tumor burden). There were two cases which were positive for COBAS test and PNA Clamp PCR but negative for cfDNA. This demonstrates an innovative and highly sensitive technique for the detection of the common driver mutations in melanoma using exceptionally low tumor burden samples, representing a useful tool for future research and clinical application.

The usefulness of a novel fully automated PCR-based Idylla test for detection of the BRAF V600E mutation in thyroid tissue: comparison with PNA-clamping PCR, real-time PCR and pyrosequencing

IntroductionThe BRAF V600E mutation is the most common genetic event in papillary thyroid carcinoma (PTC). The BRAF V600E mutational status has a significant diagnostic and prognostic role in PTC since...

Clinical relevance of circulating KRAS mutated DNA in plasma from patients with advanced pancreatic cancer

We used KRAS mutations to investigate the clinical relevance of circulating tumor DNA (ctDNA) measurements in patients with advanced pancreatic cancer. Fifty-three blood samples were collected from 14 prospectively recruited patients prior to chemotherapy (gemcitabine or FOLFIRINOX) and subsequently every month during treatment. Samples were processed by density centrifugation and plasma DNA isolation. A Peptide–nucleic acid–clamp PCR was then used to detect KRAS mutations (present in >90% of pancreatic cancers) as a surrogate marker for ctDNA. Plasma samples from 29 healthy individuals were analyzed as a reference group. Results were compared to conventional monitoring measures and survival data. Median follow-up time was 3.7 months (range 0.6–12.9 months).Ten (71%) patients had a positive KRAS status in the plasma samples obtained prior to chemotherapy, indicating the presence of ctDNA. Among the patients who were ctDNA-positive before chemotherapy, nine (90%) experienced disease progression during follow-up, compared to one (25%) of four ctDNA-negative patients (P = 0.01). The pre-therapy ctDNA level was a statistically significant predictor of both progression-free and overall survival (P = 0.014 and 0.010, respectively). Of the 14 patients, ten had ≥2 follow-up samples; in several of these patients, the ctDNA level changed substantially during the course of chemotherapy. Changes in ctDNA levels corresponded both with radiological follow-up data and CA19-9 levels for several patients.This pilot study supports the hypothesis that ctDNA may be used as a marker for monitoring treatment efficacy and disease progression in pancreatic cancer patients. Recruitment of more patients is ongoing to corroborate these findings.

Presence of nucleotide substitutions in the ABO promoter in individuals with phenotypes A3 and B3

Recently, the involvement of mutation and deletion of transcription regulatory elements in the Bm , Am , A3 and B3 phenotypes has been reported. In the present study, we carried out genetic analysis of individuals with A3 and B3 using peptide nucleic acid-clamping PCR to exclude amplification of O alleles. Two single-point mutations, -76G>C and -68G>T, were found in the ABO promoter on the A-allele in three A3 individuals and on the B allele in a B3 individual, respectively. Transient transfection of luciferase reporter plasmids carrying the same mutations into K562 cells revealed decreased luciferase activity in comparison with that carrying the wild-type promoter. These observations suggest that the mutations downregulate the promoter activity, leading to reduction in A- or B-antigen expression on red blood cells in individuals with the A3 and B3 phenotypes.

The Impact of EGFR T790M Mutations and BIM mRNA Expression on Outcome in Patients with EGFR-Mutant NSCLC Treated with Erlotinib or Chemotherapy in the Randomized Phase III EURTAC Trial

Concomitant genetic alterations could account for transient clinical responses to tyrosine kinase inhibitors of the EGF receptor (EGFR) in patients harboring activating EGFR mutations. We have evaluated the impact of pretreatment somatic EGFR T790M mutations, TP53 mutations, and Bcl-2 interacting mediator of cell death (BCL2L11, also known as BIM) mRNA expression in 95 patients with EGFR-mutant non-small-cell lung cancer (NSCLC) included in the EURTAC trial (trial registration: NCT00446225). T790M mutations were detected in 65.26% of patients using our highly sensitive method based on laser microdissection and peptide-nucleic acid-clamping PCR, which can detect the mutation at an allelic dilution of 1 in 5,000. Progression-free survival (PFS) to erlotinib was 9.7 months for those with T790M mutations and 15.8 months for those without, whereas among patients receiving chemotherapy, it was 6 and 5.1 months, respectively (P < 0.0001). PFS to erlotinib was 12.9 months for those with high and 7.2 months for those with low/intermediate BCL2L11 expression levels, whereas among chemotherapy-treated patients, it was 5.8 and 5.5 months, respectively (P = 0.0003). Overall survival was 28.6 months for patients with high BCL2L11 expression and 22.1 months for those with low/intermediate BCL2L11 expression (P = 0.0364). Multivariate analyses showed that erlotinib was a marker of longer PFS (HR = 0.35; P = 0.0003), whereas high BCL2L11 expression was a marker of longer PFS (HR = 0.49; P = 0.0122) and overall survival (HR = 0.53; P = 0.0323). Low-level pretreatment T790M mutations can frequently be detected and can be used for customizing treatment with T790M-specific inhibitors. BCL2L11 mRNA expression is a biomarker of survival in EGFR-mutant NSCLC and can potentially be used for synthetic lethality therapies.

Detecting KRAS mutations in peripheral blood of colorectal cancer patients by peptide nucleic acid clamp PCR

We investigated the effectiveness of peptide nucleic acid (PNA) clamp PCR for detecting KRAS mutations in peripheral blood samples of colorectal cancer (CRC) patients. We compared KRAS point mutations between tumour tissue and blood samples. Forty-two patients were included in this study. We observed KRAS mutations in formalin-fixed, paraffin-embedded tissues by PCR direct sequencing and in blood samples by PNA clamp PCR. KRAS point mutations were detected in primary tumour tissue samples of 13 patients (31.0%) and in peripheral blood samples of 10 patients (23.8%). KRAS point mutations were detected in both samples for 8 patients (19.0%). The sensitivity, specificity and accuracy for detecting KRAS mutations in peripheral blood and tumour tissue samples were 61.5, 93.1 and 83.3%, respectively. The positive and negative predictive values were 80.0 and 84.4%, respectively. Five patients with mutant KRAS in their plasma preoperatively, did not exhibit KRAS mutations postoperatively. Our method detected KRAS point mutations in peripheral blood samples of CRC patients, which contained extremely small amounts of mutant cells. This method is helpful for identifying metastatic CRC patients in whom metastases will respond to EGFR-targeted monoclonal antibody therapy.

Heterogeneous distribution of K-ras mutations in primary colon carcinomas: implications for EGFR-directed therapy

K-ras mutations predict resistance against epidermal growth factor receptor (EGFR)-directed therapy of metastatic colorectal cancer (CRC). The purpose of this study was to analyze the distribution of K-ras mutations in primary tumors and corresponding sentinel lymph nodes (SLNs) from colon cancer patients. Tumor biopsies and SLNs from 158 patients with non-metastatic colon cancer were analyzed for K-ras mutations in codons 12 and 13 by a sensitive and quantitative peptide nucleic acid clamp PCR assay. Analyses of single fresh-frozen tumor biopsies revealed K-ras mutations in 67 (42%) of the patients. Apparently low levels of K-ras mutations in 13 of the mutated primary tumors and the presence of K-ras mutations in SLNs from seven patients with a wild-type primary tumor biopsy suggested possible intratumoral heterogeneity for 20 of the patients. To confirm this hypothesis, we analyzed tissue sections from all available formalin-fixed, paraffin-embedded (FFPE) tumor blocks from these 20 patients. Ten of the patients had a mixture of tissue sections positive and tissue sections negative for K-ras mutations, two patients had K-ras mutations in all sections, and eight patients had no detectable K-ras mutations in tumor FFPE tissue blocks. Among these eight patients, five had K-ras mutations detected in SLNs. Thus, evidence supporting a heterogeneous distribution of K-ras mutations was obtained for 15 patients. Heterogeneous distribution of K-ras codon 12 and 13 mutations within primary tumor, or between primary tumor and lymph node metastases, was demonstrated for 15 (20%) of 74 colon cancer patients having K-ras mutations. This may have implications for tissue sampling routines with regard to EGFR-directed therapy of CRC, both in adjuvant and metastatic settings.

MRNA Expression and BRAF Mutation in Circulating Melanoma Cells Isolated from Peripheral Blood with High Molecular Weight Melanoma-Associated Antigen-Specific Monoclonal Antibody Beads

The detection of circulating tumor cells (CTCs) in the peripheral blood of melanoma patients by quantitative real-time reverse-transcription PCR (qRT-PCR) analysis correlates with a poor prognosis. The assessment of CTCs from blood has been difficult because of lack of a good monoclonal antibody (mAb) directed against surface cell antigens to capture melanoma cells. Blood was collected prospectively from 57 melanoma patients (43 test and 14 test-development cases) and 5 healthy donors. High molecular weight melanoma-associated antigen (HMW-MAA)-specific mAbs bound to immunomagnetic beads were used to isolate CTCs. mRNA and/or DNA were extracted from CTCs. Testing for the expression of a melanoma-associated gene panel (MLANA, MAGEA3, and MITF) with qRT-PCR and for the presence of BRAFmt (a BRAF gene variant encoding the V600E mutant protein) verified the beads-isolated CTCs to be melanoma cells. A peptide nucleic acid-clamping PCR assay was used for BRAFmt analysis. Spiking of peripheral blood cells (PBCs) with melanoma cells showed that the beads-based detection assay can detect approximately 1 melanoma cell in 5 x 10(6) PBCs. qRT-PCR analysis detected MLANA, MAGEA3, and MITF expression in 19 (44%), 29 (67%), and 19 (44%) of the patients, respectively. At least one biomarker of the panel was positive in 40 (93%) of the 43 melanoma patients. BRAFmt was detected in 17 (81%) of the 21 assessed stage IV melanoma patients. The assay of bead capture coupled with the PCR has utility for assessing CTCs in melanoma patients, which can then be characterized for both genomic and transcriptome expression.

Rapid Screening Assay for KRAS Mutations by the Modified Smart Amplification Process

Previously, the smart amplification process version 2 (SMAP-2) was developed to detect mutations from tissue and in crude cell lysates and has been used for rapid diagnosis of specific somatic mutations with single-nucleotide precision. The purpose of this study was to develop a rapid and practical method to detect cancer and metastasis in specimens using the SMAP-2 assay. We developed modified SMAP-2 assays that enabled detection of any change in a single codon using a single assay. Rapid SMAP-2 screening assays are suitable for routine clinical identification of critical amino acid substitutions such as codon 12 mutations in KRAS. Primers bracketing the first two nucleotides of KRAS codon 12 were designed so that all possible alleles would be amplified by the SMAP-2 assay. In combination with the peptide nucleic acid (PNA) with exact homology to the wild-type allele, our assay amplified all mutant alleles except for the wild-type sequence. With this new assay design (termed PNA-clamp SMAP-2), we could detect KRAS mutations within 60 minutes, including sample preparation. We compared results from PNA-clamp SMAP-2 assay, polymerase chain reaction-restriction fragment length polymorphism, and direct sequencing of clinical samples from pancreatic cancer patients and demonstrated perfect concordance. The PNA-clamp SMAP-2 method is a rapid, simple, and highly sensitive detection assay for cancer mutations.

High-Fidelity DNA Polymerase Enhances the Sensitivity of a Peptide Nucleic Acid Clamp PCR Assay for K- ras Mutations

Sensitive detection of tumor-specific point mutations is of interest in both the early detection of cancer and the monitoring of treatment at a molecular level. Recently, peptide nucleic acid (PNA) clamp real-time PCR has provided a time-sparing and sensitive method for the detection of mutations in the presence of a large excess of wild-type DNA. We present the first report that the sensitivity of PNA clamp PCR is limited by the low fidelity of TaqDNA polymerase. Replication errors introduced by Taq polymerase in the PNA-binding site were amplified during PCR due to the resulting mismatches between PNA and DNA. To reduce the frequency of polymerase-induced errors, we developed a PNA clamp PCR assay for the detection of mutations in codons 12 and 13 of the K-ras gene based on a high-fidelity DNA polymerase. The sensitivity of our assay increased approximately 10-fold, significantly detecting mutant DNA diluted 20,000-fold in wild-type DNA (P = 0.025), compared with its detection at 2000-fold dilution (P = 0.039) when Taq polymerase was used. Our data suggest that the replication errors caused by Taq polymerase must be taken into consideration for PNA clamp PCR and for other methods based on selective PCR amplification, and that these assays can be enhanced by high-fidelity DNA polymerases.

Peptide nucleic acid clamp PCR: a novel K-ras mutation detection assay for colorectal cancer micrometastases in lymph nodes.

Inaccurate staging of colorectal cancer (CRC) has been attributed to the failure to detect lymph node metastases by conventional pathology. We have previously reported the use of lymphatic mapping to accurately identify those lymph nodes most likely to harbor micrometastatic disease and permit focused pathologic examination. Mutation of K-ras allele at codons 12 or 13 occurs frequently in early stages of CRC development. The purpose of our study was to assess sentinel lymph nodes (SLN) for occult CRC micrometastases using a unique peptide nucleic acid (PNA) clamp PCR assay specific for K-ras mutations. Seventy-two paraffin-embedded primary CRC and paired SLN were evaluated by PNA clamp PCR for K-ras mutations. Thirty primary tumors (42%) were positive for K-ras mutations, and in 5 of these cases the SLN were positive for metastases by Hematoxylin and Eosin staining. PNA clamp PCR identified occult metastases in an additional 6 patients, upstaging 24% of K-ras positive primary CRCs (p = 0.014). No K-ras mutations were detected among the 20 noncancer lymph nodes assessed. This study demonstrates the utility, specificity and sensitivity of PNA clamp PCR assay in identifying occult micrometastases in the SLN of CRC patients by single-base mutation analysis.