The EGFR mutation analysis was performed from July 2015 to June 2017 using the therascreen EGFR RGQ PCR Kit (Qiagen). The therascreen EGFR RGQ PCR Kit is a molecular diagnostic kit for detecting specific mutations, insertions, and deletions in the EGFR gene using realtime PCR on the Rotor-Gene Q 5plex HRM instrument (Qiagen). The kit includes all necessary reagents optimized for rapid and sensitive detection of a low percentage of mutant DNA in a background of wild-type genomic DNA. The kit provides exceptionally high sensitivity and specificity for the detection of specific mutations. This technique allows the detection of tumor cells as little as 1% of a mixture of tumor cells with normal tissue. The procedure and interpretation of results are performed per the user's manual provided by Qiagen without any modification. Idylla (Biocartis) was used to confirm the results to rule out false positivity. Idylla EGFR mutation test is a fully automated molecular diagnostic kit.
Therascreen egfr rgq pcr kit
Manufactured by Qiagen
Sourced in Germany, United Kingdom, Italy, United States
The Therascreen EGFR RGQ PCR kit is a real-time PCR-based diagnostic assay designed to detect specific mutations in the epidermal growth factor receptor (EGFR) gene. The kit utilizes a quantitative PCR (qPCR) method to identify the presence of these mutations in DNA samples extracted from tumor tissue.
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Lab products found in correlation
Egfr rgq pcr kit, by Qiagen (2 mentions)
Rotor gene q 5plex hrm instrument, by Qiagen (2 mentions)
Qiaamp dna micro kit, by Qiagen (2 mentions)
Cobas egfr mutation test v2, by Roche (1 mentions)
Oncoguide ncc oncopanel system, by Sysmex (1 mentions)
Ventana alk d5f3 cdx assay, by Roche (1 mentions)
Vysis alk break apart fish probe kit, by Abbott (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Rotor gene q series software, by Qiagen (1 mentions)
Cobas kras mutation test, by Roche (1 mentions)
Bigdye terminator kit, by Thermo Fisher Scientific (1 mentions)
Cobas 4800 braf v600 mutation test, by Roche (1 mentions)
Lightcycler 480 instrument 2, by Roche (1 mentions)
Therascreen braf rgq pcr kit, by Qiagen (1 mentions)
Therascreen nras pyro kit, by Qiagen (1 mentions)
Therascreen ras extension pyro kit, by Qiagen (1 mentions)
Pyromark q24, by Qiagen (1 mentions)
Dneasy kit, by Qiagen (1 mentions)
Qiaamp circulating nucleic acid kit, by Qiagen (1 mentions)
28 protocols using therascreen egfr rgq pcr kit
1
Sensitive EGFR Mutation Detection
2
EGFR Mutation Detection Protocol
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The Therascreen EGFR RGQ PCR kit (Qiagen) detects a total of 29 (Figure 1 ) specific somatic mutations in exons 18–21 of the EGFR gene utilizing both ARMS® and Scorpion® technologies. Data analysis was performed by using Rotor gene proprietary software, and the mutation status was categorized qualitatively as positive and negative based on the Therascreen EGFR RGQ PCR kit guidelines.
3
EGFR Mutation Detection Protocol
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According to the manufacturer's protocol, Scorpions ARMS assays were performed on Rotor‐Gene Q Real‐time PCR platform using therascreen EGFR RGQ PCR kit (Qiagen, Hilden, Germany).7 The sequence‐specific PCR primers that allow the detection of the common hotspot mutations of EGFR gene as follows: exon‐19 deletion, exon‐21 L858R (2573T > G), exon‐20 T790M (2369C > T), exon‐20 L861Q (2582T > A), exon‐18G719A (2156G > C), exon‐20 S768I (2303G > T), and exon‐20 insertions (2319_2320insCAC and 2310_2311insGGT).
4
Tissue and Liquid Biopsy NGS Analysis
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The study was approved by the Ethical Committee of the Pascale Institute: protocol n. 16/14 OSS and protocol n. 8/14 OSS. Consecutive cases fulfilling the inclusion criteria (availability of both plasma and tissue samples for NGS analysis; informed consent) were included in the analysis. Plasma samples were obtained at diagnosis, prior to any systemic treatment. Patients with metastatic NSCLC received EGFR testing on tissue as part of their initial workout. EGFR tissue testing was performed with the Therascreen EGFR RGQ PCR Kit (Qiagen, Milan, Italy) according to manufacturer's instructions. Patients with metastatic CRC received RAS testing on tissue as part of their routine diagnostic analysis. RAS mutational status of tissue samples was previously determined with the Therascreen KRAS and NRAS Pyro kit (Qiagen), according to manufacturer's instructions.
5
Evaluating EGFR Mutation Detection
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Common EGFR mutations, including exon 19 deletion and L858R mutation, were assessed using PCR-based methods (Therascreen EGFR RGQ PCR kit [Scorpion-ARMS technology]; QIAGEN, Hilden, Germany, and Cobas EGFR Mutation Test v2; Roche Diagnostics, Basel, Switzerland) and next-generation sequencing testing (Oncomine™ Dx Target Test; Life Technologies Corporation, Carlsbad, CA and OncoGuide NCC Oncopanel System; Sysmex, Kobe, Japan).
6
Companion Diagnostics for EGFR, ALK, and ROS1
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Companion diagnostics approved in Japan in June 2022 were used. They included the cobas EGFR mutation test version 2.0 (Roche Diagnostics) and Therascreen EGFR RGQ PCR kit (Qiagen) for the EGFR mutation; VENTANA ALK (D5F3) CDx Assay (immunohistochemistry, IHC; Roche Diagnostics), Histofine ALK iAEP Kit (IHC, Nichirei Bioscience), and Vysis ALK Break Apart FISH Probe Kit (fluorescence in situ hybridization, FISH; Abbott) for the ALK fusion genes; and the OncoGuide AmoyDx ROS1 gene fusions detection kit for the ROS1 fusion gene. For the EGFR gene, the PNA‐LNA PCR clamp method6 and the PCR‐Invader method7 have been widely used in Japan, and some EGFR inhibitors were approved for prescription based on their results. Therefore, we included them in the companion diagnostics in the current study.
7
Serum EGFR Mutation Monitoring in Cancer
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Serum samples were analyzed using Scorpion-ARMS methodology in the central laboratory. Serum was extracted from 3.5 mL of blood collected at baseline screening, on day 190 of treatment, and at disease progression (as judged by the investigator). DNA was extracted from 0.5 mL of serum using the QIAamp DNA MinElute Virus Spin Kit (Qiagen, UK) as per the manufacturer's instructions. EGFR gene mutations in DNA from serum were analyzed by Scorpion-ARMS using the EGFR RGQ PCR kit or Therascreen EGFR RGQ PCR kit (Qiagen, UK) as per the manufacturer's instructions. Scorpion-ARMS detected exon 19 deletions, L858R mutation, T790M mutation, L861Q mutation, G719X mutations, S768I mutation, and exon 20 insertions. The limits of detection for each mutation were as follows: exon 19 deletions (1.64%), L858R (1.26%), T790M (7.02%), L861Q (0.50%), G719X (5.43%), S768I (1.37%), and exon 20 insertions (2.03%). This analysis evaluated agreement between EGFR mutations in serum and tumor tissue at screening and time course changes of EGFR mutation type in serum samples. The association between serum mutations and PFS outcomes was also evaluated.
8
Plasma-based EGFR T790M Mutation Assessment
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To assess the EGFR T790M mutation tumor status, mandatorily collected plasma from patients was processed, and isolated circulating cell-free DNA (cfDNA) was submitted for laboratory testing (Fluorescence Resonance Energy Transfer-based Preferential Formation Assay [F-PHFA] method) by central review. In some patients, collected tumor biopsies were submitted for EGFR genotyping (Therascreen® EGFR RGQ PCR kit [Qiagen, Venlo, Netherlands]).
9
EGFR Mutation Analysis in FFPE Tissue
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We extracted tumor DNA from formalin-fixed, paraffin-embedded tissue with the Qiagen QIAamp DNA Mini Kit (QIAGEN®, Crawley, UK). EGFR mutations were analyzed via direct sequencing and/or the Scorpion Amplification Refractory Mutation System-based EGFR mutation detection kit (Therascreen EGFR RGQ PCR kit; QIAGEN), which detects 29 mutations across the EGFR gene. The analyses were performed at 3 central laboratories at the Linkou, Kaohsiung, and Chiayi Chang Gang Memorial Hospital.
10
EGFR Mutation Detection in Cytological Samples
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For EGFR mutation detection, the Rotor-Gene Q 5plex HRM instrument was used with the therascreen EGFR RGQ PCR kit (therascreen EGFR assay; Qiagen, Inc.). This assay is approved for use in the United States, Europe, Japan, and China, and the kit is based on the amplification-refractory mutation system (ARMS) and Scorpion PCR technology, which enable the sensitive and selective site-specific detection of 29 types of somatic mutations in EGFR (24 (link)). The reaction conditions were as follows: 95°C for 15 min for 1 cycle; 95°C for 30 sec and 60°C for 60 sec for 40 cycles. The analysis was performed using the Rotor-Gene Q series software, version 2.0.2 (Qiagen, Inc.). For comparison between the three types of cytological samples (cell pellets, cell-free supernatants and FFPE cell blocks) using cell lines, the cycle quantification (Cq) value of the mutant allele in each sample was compared using the therascreen EGFR ‘Deletions’ assay, which detects EGFR E746_A750del. For the validation study comparing BLF specimens to FFPE tissue specimens, the manufacturer-supplied cut-off delta Cq (ΔCq) values were used to determine the result whether positive or negative for the mutation in each EGFR mutation reaction.
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