To detect EGFR and BRAFV600E mutations, the PANAMutyper EGFR Kit (Panagene) and the PNA Clamp BRAF Mutation Detection Kit (Panagene) were used according to the manufacturer’s instructions, respectively. Additionally, we performed real-time PCR using the ROS1 Gene Fusions Detection Kit (AmoyDx, Xiamen, China) to detect ROS1 rearrangements.
Panamutyper egfr kit
Manufactured by Panagene
The PANAMutyper EGFR Kit is a molecular diagnostic test designed to detect mutations in the EGFR gene. It is intended for use in clinical laboratories to assist in the identification of EGFR gene mutations in tumor samples.
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6 protocols using panamutyper egfr kit
1
Targeted Mutation Detection Assays
2
EGFR Mutation Detection by PNA-based PCR
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A peptide nucleic acid (PNA) Clamp™ EGFR Mutation Detection Kit (PANAGENE Inc., Daejeon, Korea) was used to detect EGFR mutations in sample tissues via a real-time PCR, as previously described.15 (link) Likewise, EGFR mutations were detected in plasma samples via the PANAMutyper method, using a PANAMutyper™ EGFR kit (PANAGENE Inc.), as previously described.16 (link) Briefly, these techniques used specific PNA detection clamp probes to tightly bind, and thereby suppress the amplification of wild-type (WT) DNA sequences, and fluorescent dye- and quencher-conjugated PNA detection probes to specifically detect target mutant DNA sequences. Mutations were genotyped via a melting peak analysis.
3
EGFR Mutation Detection via PNA-Mediated Real-Time PCR
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To detect EGFR mutations, peptide nucleic acid (PNA)-mediated real-time PCR-based methods were performed using the PNAClamp EGFR Mutation Detection Kit (Panagene, Daejeon, Korea) or PANAMutyper EGFR Kit (Panagene) according to manufacturer’s instructions. In PNA-Clamp method, the efficiency and results of the test is determined by measuring threshold cycle (Ct) value. Ct value is a PCR cycle number at which the fluorescent signal of the reaction crosses the threshold and it is inversely related to the starting amount of target DNA. For data interpretation, PNA clamped Ct value and non-PNA Ct value of patient samples are measured. If non-PNA Ct value is between 22 and 30, the sample is regarded to have an appropriate quality. In addition, delta Ct (ΔCt) values (ΔCt1=standard Ct−sample PNA Ct, ΔCt2=sample PNA Ct−sample non-PNA Ct) are calculated. ΔCt1 < 0 indicates target mutation wild-type of tested samples, while (1) ΔCt1 ≥ 2, or (2) 0 < ΔCt1 < 2 and ΔCt2 ≤ 3 is regarded presence of targeted mutation. The manufacturer also described a possibility of suboptimal tests, if ΔCt1 is between 0 and 2 and non-PNA Ct value is between 24 and 30. In this case, the sample might have a low mutation rate that re-test by using twice as high concentration of the sample is recommended.
4
EGFR Mutation Screening Protocol
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All patients were required to provide a blood sample at screening to test for activating EGFRm in the plasma. To this end, 10 mL blood samples were withdrawn, centrifuged at 4°C to obtain the plasma (for 10 minutes at 2000 × g to remove cells, and then for 10 minutes at 3000 × g using the supernatant to deplete platelets), and then stored at −20°C until delivery to the Central Laboratory (Panagene Inc., Daejeon, South Korea). EGFRm tests for ctDNA were performed at the Central Laboratory using a PANA Mutyper R EGFR assay (Panagene Inc.). In‐house EGFRm testing for tumor DNA was performed by each hospital using a PNA clamp EGFRm or PANA Mutyper EGFR kit (Panagene Inc.).
5
Molecular Profiling of Lung Cancer
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To detect EGFR mutations, peptide nucleic acid-mediated real-time PCR was performed using the PANA Mutyper EGFR Kit (PANAGENE, Daejeon, Korea). To identify ALK rearrangements, the VENTANA ALK (D5F3) CDx Assay (Ventana Medical Systems, Tucson, AZ, USA) was performed. ROS1 rearrangements were detected with real-time PCR using the ROS1 Gene Fusions Detection Kit (AmoyDx, Xiamen, China). Their detection was also possible by next-generation sequencing, which will be described as follows.
6
EGFR Mutant Lung Adenocarcinoma Resection Protocol
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Patients with lung cancer who underwent complete tumor resection and were histologically confirmed to have stage I lung adenocarcinoma between 2005 and 2013 were screened for inclusion in this study. Of these patients, those with EGFR-mutant stage I adenocarcinoma were selected based on a review of electronic pathological reports and additional confirmation of EGFR mutation status using the PNAClamp EGFR Mutation Detection Kit (Panagene Inc., Daejeon, Korea) or the PANAMutyper EGFR Kit (Panagene Inc.), according to the manufacturer’s instructions. Electronic medical records were reviewed, and age, sex, smoking history, pathologic stage, lymphovascular invasion, visceral pleural invasion, and dates of tumor recurrence and death/last follow-up were collected. Patients with non-adenocarcinoma histology, insufficient tumor specimens for complete molecular analysis, and those who received neoadjuvant treatment were excluded. Pathological stages were reviewed and assigned according to the 8th American Joint Committee on Cancer (AJCC) criteria. Further, the International Association for the Study of Lung Cancer (IASLC) grades were determined according to previously described criteria [10 (link)]. This retrospective study was approved by the institutional review board (approval No. 4-2021-1181).
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