Peptide Nucleic Acid-locked Nucleic Acid Research Articles

Rapid detection of CALR type 1 and type 2 mutations using PNA-LNA clamping loop-mediated isothermal amplification on a CD-like microfluidic chip

Bleeding and thrombosis represent common complications in myeloproliferative neoplasms (MPN) and significantly contribute to morbidity and mortality. Molecular markers, including CALR mutations, were considered not only as diagnostic markers, but also as risk factors for bleeding and thrombosis associated with MPN, especially for patients in remote primary hospitals. We sought to develop an easy-to-use assay for the rapid detection of CALR type 1 (CALR-1) and type 2 (CALR-2) mutations in Philadelphia chromosome-negative MPN patients. Peptide nucleic acid-locked nucleic acid (PNA-LNA) clamping loop-mediated isothermal amplification (LAMP) assays were established, which were integrated into a centrifugal compact disc (CD) microfluidic platform. A total of 158 clinical blood samples were tested simultaneously by this microfluidic platform and an in-house real time PCR assay. The detection performance of the LAMP arrays was validated and conflicting results were identified by Sanger sequencing. The results suggested that the LAMP methods we developed exhibited good sensitivity, specificity, and precision. By real time fluorescence assay the detection limit for CALR-1 and CALR-2 mutations could reach as low as 1% and 0.5% respectively, and 10% and 5% respectively by visual method. There were no nonspecific background amplifications among different detection systems. For the CALR-1 and CALR-2 LAMP detection systems, intra-batch CV values of 1% mutated plasmid were 10.56% and 10.51% respectively, and the inter-batch CV values were 19.55% and 18.39%, respectively. The products were all analyzed by melting curve analysis and electrophoresis followed by Sanger sequencing analysis, which were consistent with the database sequences. The microfluidic platform could complete rapid detection of CALR-1/2 mutations within 60 min. The results of clinical samples detected by our CD-like microfluidic chipLAMP assay and rtPCR assay suggested that 133 samples were CALR wild type, 15 were CALR-1 mutation type, and 9 were CALR-2 mutation type. The correlation coefficient value (Kendall's tau_b) of the two assays was 0.99. Interestingly, by the newly established detection platform, we were surprised to find that one patient of Chinese origin harbored both CALR-1 and CALR-2 mutations. This result was verified by Sanger sequencing analysis. The LAMP detection systems developed herein displayed good sensitivity, specificity, and stability. Additionally, the detection results could be directly judged by color changes of the reaction systems without any auxiliary equipment. Thus, the platform we developed has the potential of being widely used in remote and economically undeveloped areas in the future.

Droplet digital polymerase chain reaction assay and peptide nucleic acid-locked nucleic acid clamp method for RHOA mutation detection in angioimmunoblastic T-cell lymphoma.

Angioimmunoblastic T‐cell lymphoma (AITL) is a subtype of nodal peripheral T‐cell lymphoma (PTCL). Somatic RHOA mutations, most frequently found at the hotspot site c.50G > T, p.Gly17Val (G17V RHOA mutation) are a genetic hallmark of AITL. Detection of the G17V RHOA mutations assists prompt and appropriate diagnosis of AITL. However, an optimal detection method for the G17V RHOA mutation remains to be elucidated. We compared the sensitivity and concordance of next‐generation sequencing (NGS), droplet digital PCR (ddPCR) and peptide nucleic acid‐locked nucleic acid (PNA‐LNA) clamp method for detecting the G17V RHOA mutation. G17V RHOA mutations were identified in 27 of 67 (40.3%) PTCL samples using NGS. ddPCR and PNA‐LNA clamp method both detected G17V mutations in 4 samples in addition to those detected with NGS (31 of 67, 46.3%). Additionally, variant allele frequencies with ddPCR and those with NGS showed high concordance (P < .001). Three other RHOA mutations involving the p.Gly17 position (c.[49G > T;50G > T], p.Gly17Leu in PTCL198; c.[50G > T;51A > C], p.Gly17Val in PTCL216; and c.50G > A, p.Gly17Glu in PTCL223) were detected using NGS. These sequence changes could not appropriately be detected using the ddPCR assay and the PNA‐LNA clamp method although both indicated that the samples might have mutations. In total, 34 out of 67 PTCL samples (50.7%) had RHOA mutations at the p.Gly17 position. In conclusion, our results suggested that a combination of ddPCR/PNA‐LNA clamp methods and NGS are best method to assist the diagnosis of AITL by detecting RHOA mutations at the p.Gly17 position.

Utility of Liquid Biopsy by Improved PNA-LNA PCR Clamp Method for Detecting EGFR Mutation at Initial Diagnosis of Non–Small-Cell Lung Cancer: Observational Study of 190 Consecutive Cases in Clinical Practice

BackgroundThe clinical benefit of liquid biopsy for unselected patients at initial diagnosis has thus far been unclear. We aimed to evaluate the utility of liquid biopsy at initial diagnosis, as well as the efficacy of epidermal growth factor receptor–tyrosine kinase inhibitor (EGFR-TKI) based on liquid biopsy results in clinical practice, using the improved peptide nucleic acid–locked nucleic acid (PNA-LNA) PCR clamp method. Patients and MethodsWe routinely performed liquid biopsy using the improved PNA-LNA PCR clamp method for all patients diagnosed with non–small-cell lung cancer (NSCLC) between June 2015 and October 2016. We retrospectively evaluated the reliability of liquid biopsy based either on clinical stage or between sensitizing EGFR mutation and T790M mutation, and the clinical benefit of EGFR-TKI based on the liquid biopsy results in practice. ResultsA total of 244 patients underwent liquid biopsies, with 168 patients tested at diagnosis and 22 tested for T790M after pretreatment of EGFR-TKI. For detecting a sensitizing EGFR mutation, the sensitivity, specificity, positive predictive value, and negative predictive value were 72.7%, 100%, 100%, and 93.7% in the group with advanced-stage NSCLC and 0, 100%, not evaluable, and 70.5% in the group with early-stage NSCLC. The positive predictive value and negative predictive value for T790M were 33.3% and 55.6%, respectively. Fourteen patients in the liquid-positive group and 16 patients in the tissue-positive group received EGFR-TKI. The objective response rates of first- and second-generation EGFR-TKI for the liquid-positive and tissue-positive groups were 90.0% and 90.9%, respectively. There was no significant difference in median progression-free survival between the liquid-positive and tissue-positive groups (P = .839). ConclusionPatients with early-stage NSCLC should not be candidates for this liquid biopsy method. We recommend tissue biopsy as the preferred initial method of molecular analysis, with the exception of patients who are T790M positive or patients who are unable to tolerate invasive biopsy.

EGFR T790M Detection in Circulating Tumor DNA from Non-small Cell Lung Cancer Patients Using the PNA-LNA Clamp Method.

To evaluate the utility of plasma circulating tumor DNA (ctDNA) using the peptide nucleic acid-locked nucleic acid (PNA-LNA) clamp method to detect epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) patients who had progressed under treatment with EGFR-tyrosine kinase inhibitors (TKIs). Blood samples were collected from patients with EGFR mutation-positive NSCLC who had progressed on EGFR-TKIs between March 2016 and August 2016 at the Kyoto University Hospital. Extracted ctDNA was analyzed using the PNA-LNA clamp method. In eligible patients, tissue re-biopsy was also performed and EGFR mutation status was compared between tissue and plasma samples. Thirty-one patients were enrolled in this study. Known activating EGFR mutations and the T790M mutation were detected in 18 (58%) and 5 patients (16%), respectively. Twenty-five patients underwent tissue re-biopsy. Adequate samples for mutation analysis were obtained from 21 patients and 10 patients were found to be tissue T790M-positive. Among these 10 patients, 4 patients were positive for T790M in plasma ctDNA (sensitivity 40% and specificity 100%). All patients with T790M-positive plasma ctDNA responded to osimertinib. Sensitivity of the PNA-LNA clamp method in detecting the plasma EGFR T790M mutation was moderate with elevated, however, specificity. Plasma EGFR T790M testing may be adequate for the initial step; however, tissue re-biopsy should be considered for plasma T790M-negative patients because of its high false-negative rate.

Abstract 578: Molecular subgroup analysis of clinical outcomes in a phase 3 study of gemcitabine and oxaliplatin with or without erlotinib in advanced biliary tract cancer

Abstract BACKGROUND. We previously reported that the addition of erlotinib to gemcitabine and oxaliplatin (GEMOX) resulted in greater antitumor activity and might be a treatment option for patients with biliary tract cancers. Molecular subgroup analysis of treatment outcomes in patients who had specimens available for analysis was undertaken. METHODS. EGFR, KRAS, and PIK3CA mutations were evaluated using peptide nucleic acid-locked nucleic acid (PNA-LNA) PCR clamp reactions. Survival and response rates were analyzed according to the mutational status. 64 patients (48.1%) were available for mutational analysis in the chemotherapy alone group and 61 (45.1%) in the chemotherapy plus erlotinib group. RESULTS. 1.6% (2/116) harboured an EGFR mutation (2 patients; exon 20), 9.6% (12/121) harboured a KRAS mutation (12 patients; exon 2), and 9.6% (12/118) a PIK3CA mutation (10 patients; exon 9 and 2 patients; exon 20). The addition of erlotinib to GEMOX in patients with KRAS wild type disease (n = 109) resulted in significant improvements in overall response compared with GEMOX alone (30.2% vs. 12.5%, P = 0.024). In 95 patients with both wild type KRAS and PIK3CA, there was evidence of a benefit associated with the addition of erlotinib to GEMOX with respect to response rate (RR) as compared with GEMOX alone (P = 0.04). CONCLUSION. This study demonstrates that KRAS mutational status might be considered a predictive biomarker for the response to erlotinib in biliary tract cancers (BTCs). Additionally, the mutation status of PIK3CA may be a determinant for adding erlotinib to chemotherapy in KRAS wild type BTCs. Keywords: KRAS, PIK3CA, Erlotinib Citation Format: Seung T. Kim, Joon Park. Molecular subgroup analysis of clinical outcomes in a phase 3 study of gemcitabine and oxaliplatin with or without erlotinib in advanced biliary tract cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 578. doi:10.1158/1538-7445.AM2015-578

Phase II study of erlotinib for previously treated patients with EGFR wild-type non-small-cell lung cancer, following EGFR mutation status reevaluation with the Scorpion Amplified Refractory Mutation System.

While assessing the efficacy of erlotinib in patients with epidermal growth factor receptor (EGFR) wild-type (WT) non-small-cell lung cancer (NSCLC), the sensitivity of the method used for the EGFR mutation analysis may affect the evaluation of the efficacy. We conducted a phase II study of erlotinib for previously treated patients with EGFR WT NSCLC screened by the peptide nucleic acid-locked nucleic acid (PNA-LNA) polymerase chain reaction (PCR) clamp method, which is known to be highly sensitive. The primary endpoint was the objective response rate (ORR). Preplanned reevaluation of the EGFR genotype as an exploratory endpoint was performed using the Scorpion Amplification Refractory Mutation System (S-ARMS) assay. Erlotinib was administered daily until disease progression or development of unacceptable toxicity. A total of 53 evaluable patients were enrolled. The histological subtypes were adenocarcinoma in 40 patients, squamous cell carcinoma in 9 patients and not otherwise specified NSCLC in 4 patients. Partial response (PR) was achieved in 6 patients (4 with adenocarcinoma and 2 with squamous cell carcinoma). The ORR was 11.3% [95% confidence interval (CI): 4.3-23.0]. The median progression-free survival (PFS) was 1.8 months (95% CI: 1.2-2.3). Samples from 26 of the 53 patients (49.0%) were available for EGFR mutation reanalysis with the S-ARMS assay. Of these 26 samples, only 1 sample of adenocarcinoma was found to be EGFR mutation-positive (exon 19 deletion) and the patient achieved a PR. The EGFR WT genotype was reconfirmed by the S-ARMS assay in the remaining 25 patients and 2 of these patients exhibited a PR. This study did not meet the primary endpoint, although erlotinib was found to be moderately effective in pretreated patients with EGFR WT NSCLC, even when the EGFR mutational status was confirmed by the highly sensitive PNA-LNA clamp PCR method.

DISSECT Method Using PNA-LNA Clamp Improves Detection of EGFR T790m Mutation

Non-small cell lung cancer (NSCLC) patients treated with small molecule EGFR inhibitors, such as gefitinib, frequently develop drug resistance due to the presence of secondary mutations like the T790M mutation on EGFR exon 20. These mutations may originate from small subclonal populations in the primary tumor that become dominant later on during treatment. In order to detect these low-level DNA variations in the primary tumor or to monitor their progress in plasma, it is important to apply reliable and sensitive mutation detection methods. Here, we combine two recently developed methodologies, Differential Strand Separation at Critical Temperature (DISSECT), with peptide nucleic acid-locked nucleic acid (PNA-LNA) polymerase chain reaction (PCR) for the detection of T790M EGFR mutation. DISSECT pre-enriches low-abundance T790M EGFR mutations from target DNA prior to implementing PNA-LNA PCR, a method that can detect 1 mutant allele in a background of 100–1000 wild type alleles. The combination of DISSECT and PNA-LNA PCR enables the detection of 1 mutant allele in a background of 10,000 wild type alleles. The combined DISSECT-PNA-LNA PCR methodology is amenable to adaptation for the sensitive detection of additional emerging resistance mutations in cancer.

A diagnostic algorithm using EGFR mutation-specific antibodies for rapid response EGFR-TKI treatment in patients with non-small cell lung cancer

BackgroundWe previously reported that EGFR mutation-specific antibodies performed well in immunohisto/cytochemical analysis. Assessment of EGFR mutation status for EGFR-TKIs (tyrosin kinase inhibitors) treatment of non-small cell lung cancer (NSCLC) patients can be performed by DNA-based assay. To establish a testing algorithm for EGFR mutation status in NSCLC patients, we utilized the peptide nucleic acid-locked nucleic acid (PNA-LNA) PCR clamp assay to determine the EGFR mutation, and immunostaining to detect the delE746-A750 and L858R mutation protein. Patients and methodsOne hundred thirty-three patients with NSCLC were examined by immunostaining with EGFR mutation-specific antibodies, and by the PNA-LNA PCR clamp assay, using histological or cytological samples. The expression levels of immunostaining were classified as positive (score 2+), negative (score 0) or equivocal (score 1+), indicating questionable, negative or weak expression. Of these patients, 42 NSCLC patients received EGFR-TKIs treatment and we analyzed whether expression of mutant EGFR proteins was correlated with progression-free survival in patients with NSCLC treated with EGFR-TKIs. ResultsIn the 133 samples, positive, negative and equivocal results for EGFR mutation-specific antibodies were observed in 37 patients (27.8%), 85 patients (63.9%) and 11 patients (8.3%), respectively. Thirty-seven patients showed positive EGFR expression by immunostaining, and 35 (94.6%) of these also tested positive in the DNA-based assay. Of 85 EGFR-negative patients by immunostaining, 77 (90.6%) also tested negative in the DNA-based assay. Of the 11 patients with equivocal immunostaining results, DNA-based assay detected EGFR mutation in 8 (72.7%) patients. Immunostaining for the EGFR mutation-specific antibodies showed a sensitivity of 81.4%, specificity of 97.5%, positive predictive value of 94.6% and negative predictive value of 90.6%, excluding the 11 patients with equivocal results. In NSCLC patients treated with EGFR-TKIs, the progression-free survival after the start of EGFR-TKIs treatment was significantly longer in patients with EGFR positive expression than in those with equivocal and negative expression (P=0.002). ConclusionsOur results suggest that patients testing positive for EGFR mutations by immunostaining are good candidates for rapid response EGFR-TKI therapy. Immunostaining for EGFR mutation-specific antibodies is a new test for EGFR-TKI inhibition and could be a useful indicator with regard to patient management.

Analysis of the EGFR mutations in non-small cell lung cancer patients after EGFR-TKI treatment.

e18065 Background: Gefitinib and erlotinib are the epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) that have the dramatically effects in selective patients with non-small cell lung cancer (NSCLC). EGFR mutations predict response and survival in patients treated with TKIs. However, most patients have a relapse within 1 year after the initiation of therapy. The most important mechanism of resistance to TKIs is a secondary EGFR mutation, threonine to methionine at codon 790 in exon 20 (T790M). Therefore, we analyzed EGFR mutations after TKIs treatment in addition to before treatment. Methods: 99 patients were analyzed mutation status between July 2004 to December 2009 in our institution. 71 patients were treated with TKIs (gefitinib 67 cases, erlotinib 9 cases), and mutations were detected in 31 patients. When tumor was relapsed, we got written informed consent, and re-examination of mutations was performed. Mutations were examined by mutant-enriched polymerase chain reaction method and/or peptide nucleic acid-locked nucleic acid (PNA-LNA) PCR clamp method. Samples were biopsied tissues, surgically resected tissues, bronchial washing liquids, and pleural effusion, and we used relapsed tumor samples at secondary analysis. In addition, we evaluated the response to treatment and progression-free survival. Results: 31 patients with NSCLC harboring mutations treated with TKIs. Response rate was 74.2% (23/31). Within 1 year, most patients were relapse (83.9%, median 149 days). In 10 patients, we could analyze mutations after TKIs treatment. T790M was detected in 4 patients, and EGFR status was maintained in 6 patients. In 2 of 6 patients without T790M, readministration of gefitinib was effective, and 1 of 6 patients responded to erlotinib. Despite emergence of T790M, readministration of gefitinib and switch to erlotinib was effective in 1 patient. Conclusions: These results suggested that we cannot decide next treatment after relapse to TKIs by reason of only T790M. We must consider the mechanism of resistance to TKIs and the new therapeutic strategy. No significant financial relationships to disclose.

Abstract 1800: Analysis of EGFR mutations before and after EGFR-TKIs treatment

Abstract Background: Gefitinib is the epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) that has the dramatically effects in selective patients with non-small cell lung cancer (NSCLC). EGFR mutations predict response and survival in patients treated with gefitinib. However, almost of these patients have a relapse within a year after the start of therapy. Although the mechanisms of acquired resistance to Gefitinib were MET amplification, L861Q and hepatocyte growth factor(HGF), main reason was a secondary EGFR mutation, threonine to methionine at codon 790 in exon 20 (T790M). Therefore, we analyzed EGFR mutations after gefitinib treatment in addition to before treatment in patients with NSCLC. Materials and methods: From December 2004 to September 2009, 37 patients with advanced NSCLC harboring EGFR mutations were treated with gefitinib in our hospital. When tumor was relapsed, we got written informed consent from patients, and re-examination of EGFR mutations was performed in 9 patients by present. EGFR mutations were examined by mutant-enriched polymerase chain reaction (PCR) method and/or peptide nucleic acid-locked nucleic acid (PNA-LNA) PCR clamp method. Samples were the paraffin-embedded specimens, transbronchial biopsied tissues, surgically resected tissues, bronchial washing liquids, and pleural effusion, and we used relapsed tumor samples at secondary analysis. In addition, we evaluated the response to treatment according to the Response Evaluation Criteria in Solid Tumors (RECIST) and progression free survival defined as the time from the date of beginning treatment to the date of disease progression or death. Results: 37 patients treated by gefitinib, response rate was 60%(22/37) and disease control rate was 78%(29/37). Within a year, most patients were relapse. In 9 patients, we could analyze EGFR mutations again. Secondary analysis of EGFR mutations was performed in safety. T790M, a resistant mutation of EGFR, was detected in 4 patients. Samples from lymph node biopsy were used in 2 patients, pleural effusion was in a patient, and transbronchial biopsied tissue was in a patient. 1 patient was adenosquamous cell carcinoma, and all of the rest were adenocarcinoma. Despite emergence of T790M, in a patient, readministration of gefitinib was effective (stable disease), furthermore, treatment of erlotinib after gefitinib was also effective (partial response). In 1 patient, secondary analysis is performing at present. Conclusion: These results suggested the importance of EGFR analysis after relapse in patients treated with gefitinib. We must consider why erlotinib was effective in patients with T790M, and screen appropriate subjects of readministration of EGFR-TKIs. The new therapeutic strategy according to the mechanism of resistance to EGFR-TKI is expected. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1800.

Usefulness of EGFR mutation screening in pleural fluid to predict the clinical outcome of gefitinib treated patients with lung cancer

The importance of epidermal growth factor receptor (EGFR) gene mutation has been recognized in nonsmall cell lung cancer (NSCLC), requiring the standardization of mutation screening system including the kind of samples. Here, we examined the EGFR mutation status in 61 pleural fluid samples from NSCLC cases using direct sequencing, nonenriched PCR, mutant-enriched PCR and peptide nucleic acid-locked nucleic acid (PNA-LNA) PCR clamp assay. The mutant-enriched PCR assay detected 16 mutant cases. Among them, the nonenriched PCR assay failed to detect 3 mutant cases. Regarding the discrepancy between mutant-enriched PCR and PNA-LNA PCR clamp assays, 3 cases of exon19-deletions were detected only by mutant-enriched PCR assay and no difference at the L858R mutation. There was no difference in results between direct sequencing and nonenriched PCR assay. We also correlated the EGFR mutation with clinical outcome of gefitinib-treated 29 cases. EGFR mutations were present in 10 cases, revealing 7 partial response and 3 no change (NC). In EGFR wild-type cases, 10 revealed NC and 9 progressive disease. The responders were significantly more frequent among the EGFR mutant cases than among the wild-type (p < 0.0001). Overall survival (p = 0.0092) and progression-free survival (p = 0.018) were significantly longer among the EGFR mutant cases than among the wild-type. In summary, we evaluated the utility of EGFR mutation screening in pleural fluid using 4 assays that showed some discrepancies arising from the designs of the assays. As clinical importance, the EGFR mutation status in pleural fluid can be a biomarker for the favorable outcome of gefitinib-treated NSCLC cases.

Genetic Heterogeneity of the Epidermal Growth Factor Receptor in Non–Small Cell Lung Cancer Cell Lines Revealed by a Rapid and Sensitive Detection System, the Peptide Nucleic Acid-Locked Nucleic Acid PCR Clamp

Lung cancer is one of the leading causes of the cancer death worldwide. Gefitinib is an inhibitor of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR) and has been introduced in the treatment of advanced lung cancers. The responsiveness to gefitinib has been linked to the presence of EGFR mutations. Clinical samples contain many normal cells in addition to cancer cells. A method capable of detecting EGFR mutations in a large background of wild-type EGFR genes could provide a superior clinical test. We developed a rapid and sensitive detection system for EGFR mutations named the peptide nucleic acid-locked nucleic acid (PNA-LNA) PCR clamp that can detect EGFR mutations in the presence of 100-to 1,000-fold background of wild-type EGFR. We used this method to screen 30 non-small cell lung cancer cell lines established from Japanese patients. In addition to 11 cell lines that have mutations, we found 12 cell lines in which specific mutations are observed only in the subpopulation(s) of the cells. Genetic heterogeneity of EGFR suggests that the EGFR gene is unstable in established cancers and the heterogeneity may explain variable clinical responses of lung cancers to gefitinib.