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14 protocols using adx egfr mutations detection kit

1

EGFR Autoantibody Detection in Lung Cancer

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Serum from lung adenocarcinoma cancer patients treated in Shanghai Pulmonary Hospital were banked according to protocols approved by Institutional Review Board (K16–245-1). EGFR mutations were detected by ARMS method using ADx EGFR Mutations Detection Kit (Amoy Diagnostics, Xiamen, China) [40 (link)].
The recombinant EGFR protein (extracellular part aa 1–645 and intracellular part aa 668–1210) was from Sinobiologicals, China. The EGFR protein was bound to ELISA plates (1 μg/ml) for overnight at 4 °C. 100 μl serum of lung cancer patients were added and incubated for 1 h at room temperature. The plates were washed for 3 times by washing buffer (PBS with 0.05% Tween-20), and incubated with HRP labeled goat anti-human IgG for 1 h, followed by colorimetric detection. PBS 1% BSA was used as blank for determining the cutoff value.
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2

EGFR Mutation Detection in Tumors

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Direct sequencing or amplification refractory mutation system using ADx EGFR mutations detection kit (Amoy Diagnostics, Xiamen, People’s Republic of China) was applied to determine EGFR genotype. All procedures were performed following the user manual. Sensitive genotypes, including exon 19 deletion, exon 21 L858R and L861, exon 18 G719X and G719, and exon 20 S768I mutations, can be detected. Patients who underwent EGFR determination by paraffin-embedded tissue after initial diagnosis or even after the occurrence of BM were also eligible.
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3

EGFR Mutation Detection in Tissue Specimens

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The EGFR status of all specimens mentioned before was carried out using amplification refractory mutation system-PCR (ARMS-PCR) technology on a Bio-Rad CFX96 machine (Bio-Rad, American), which is fully evaluated on histological tissue. The ADx EGFR Mutations Detection Kit (Amoy Diagnostics, China) was subjected to this procedure. There are 29 known mutations in exons 18–21 of EGFR, including G719X in exon 18, deletion in exon 19, S768I and T90M in exon 20, and L861Q in exon 21.
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4

Molecular Biomarker Detection in Lung Cancer

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Tumor specimens used for epidermal growth factor receptor (EGFR) mutation, anaplastic lymphoma kinase (ALK), proto-oncogene receptor tyrosine kinas (ROS-1) rearrangements, and programmed death-ligand 1 (PD-L1) expression detection were obtained from surgery, bronchoscopy, computed tomography (CT)-guided biopsy, and pleural effusion. EGFR mutation detection was carried out using Amplification Refractory Mutation System technology as the ADx EGFR Mutations Detection Kit (Amoy Diagnostics, China) had been approved for clinical application by the State Food and Drug Administration in China. ALK and ROS-1 gene testing and PD-L1 expression was conducted on biopsy samples using immunostaining or the fluorescence in situ hybridization (FISH) method.
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5

EGFR Mutation Detection Protocol

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The isolated DNA samples were re-tested with ADx EGFR Mutations Detection kit (Amoy Diagnostics, Xiamen, China), which has received China Food and Drug Administration's (CFDA) approval for clinical usage in mainland China. The kit covered the 29 EGFR mutation hotspots from exon 18 to 21. The assay was carried out according to the manufacturer's protocol with the MX3000P (Stratagene; Agilent Technologies, Inc., Santa Clara, CA, USA) Real-Time PCR system.
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6

Screening for EGFR Mutations in MPE

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Screening for wild-type EGFR 18–21 exons in the 313 MPE samples was performed using the amplification refractory mutation system (ARMS). The ADx EGFR Mutations Detection Kit (Amoy Diagnostics, Xiamen, China) was employed to perform this analytical procedure. This kit covers 29 EGFR mutation hotspots from exons 18 to 21, consisting of G719X (3 types), 19 deletions (19 types), 20 inserts (3 types), T790M, S768I, L858R and L861Q. Real-time direct sequencing PCR experiments were carried out using the ABI 7500 Fast PCR system (Applied Biosystems Inc., CA, USA). The Ct values used to determine whether a sample was positive or negative were based on extensive validation.
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7

EGFR Mutation Detection in NSCLC Tissues

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All tumor tissue samples had routine pathological evaluations to confirm the diagnosis of NSCLC. Tumor tissue (drug-resistant specimens) DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) specimens. All tumor samples were routinely assessed by sectioning, hematoxylin–eosin staining, and visualization under a microscope to ensure tumor content by two pathologists. FFPE sections and smear slides were deparaffinized in xylene and rehydrated in descending grades of absolute ethanol. DNA was extracted using an AmoyDx FFPE DNA Kit (Amoy Diagnostics, Xiamen, China) according to the manufacturer’s instructions.
A Human EGFR Gene Mutation (exons 18–21) Fluorescence Polymerase Chain Reaction (PCR) Diagnostic Kit (Amoy Diagnostics), which was based on ARMS technology, was used to analyze the DNA from the tissue samples. An ADx EGFR Mutations Detection Kit (Amoy Diagnostics) has received China Food and Drug Administration (CFDA) approval for clinical usage since 2010. We defined a cut-off of 2% tumor cell content as a sample quality check according to the minimum requirement of ARMS technology (approximately 1% analytical sensitivity). Samples below this threshold were rejected.
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8

EGFR and KRAS Mutation Detection

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In all histologic and cytologic samples, EGFR gene exons 18-21 and KRAS gene exon 2 mutations were detected using the amplification refractory mutation system (ARMS). The ADx EGFR Mutations Detection kit and the ADx KRAS Mutations Detection kit (Amoy Diagnostics, Xiamen, China) were employed to perform this analytical procedure. Quantitative real-time PCR experiments were carried out using the ABI 7500 Fast PCR system (Applied Biosystems Inc., CA, USA). Ct values used to determine whether a sample was positive or negative were based on extensive validation.
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9

EGFR Mutation Analysis in NSCLC

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Mutation analysis was conducted separately at the two hospitals and confirmed centrally at Beijing Tiantan Hospital. EGFR mutations were analysed in paraffin-embedded tissue sections. Tumour tissue was scraped from the glass slides under direct visualisation or under a dissecting microscope. DNA was then extracted using a QIAamp DNA Mini Kit (Qiagen Inc., Valencia, CA, USA). EGFR mutations were detected with an ADx EGFR Mutations Detection Kit (Amoy Diagnostics, Xiamen, China), which employs an amplification refractory mutation system. This assay was performed according to the manufacturer’s protocol using an ABI 7500 real-time polymerase chain reaction system (Applied Biosystems, Foster City, CA, USA).
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10

EGFR Mutation Detection in PC-9-GR Cells

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Genomic DNA was extracted from the PC-9-GR cells using DNeasy Blood& Tissue Kit (Qiagen, Venlo, Netherland) per manufacturer's instruction. EGFR hotspot mutations were examined by ADx EGFR Mutations Detection Kit (Amoy Diagnostics, Xiamen, China). This kit uses the principle of Amplified Refractory Mutation System and covers the 29 EGFR mutation hotspots from exon 18 to 21. The assay was carried out according to the manufacturer's protocol with the ABI7900 (Applied Biosystems, USA) real-time PCR system. Direct DNA sequence analysis for Exon 18-21 of EGFR was also applied for detection of potential mutations in tyrosine kinase domain of EGFR gene.
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