AmoyDx EGFR Three Mutations Detection Kit

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1 AmoyDx EGFR Three Mutations Detection Kit Detection of 3 mutations in exons 19 and 21 Instructions For Use Instructions Version: B1.1 Date of Revision: August 2012 Store at / 6

2 Background Due to its association with malignancies, epidermal growth factor receptor (EGFR) has become the target of an expanding class of anti-cancer therapies, such as gefitinib (Iressa) and erlotinib (Tarceva), which are tyrosine kinase inhibitors (TKIs). These drugs work best on patients whose cancer is driven by abnormal EGFR signaling. Lung cancer patients who experienced rapid, durable, complete or partial responses to TKIs therapy have been found to harbor somatic mutations in the EGFR gene. Cancer patients with somatic EGFR mutations have shown an impressive 60% response rate, much higher than that for conventional chemotherapy. Therefore, detection of the EGFR mutation status in tumor tissue is key to offering tailored, personalized treatment to cancer patients. Resistance to therapy, either in the primary tumor or acquired after TKI treatment, is also associated with somatic mutations. The AmoyDx EGFR Three Mutations Detection Kit is highly selective and sensitive, detecting 3 of the most common somatic mutations in the EGFR gene. AmoyDx s patented technology allows detection of 1% mutant DNA in a background of 99% normal DNA, while ensuring that false negatives are minimized. The procedure is easily adapted for use in high-throughput sample processing. The purpose of the kit is to aid physicians and clinical researchers in identifying non-small cell lung cancer patients whose tumors harbor EGFR mutations. Intended Use AmoyDx EGFR Three Mutations Detection Kit is a highly sensitive real-time PCR-based test designed to accurately identify 3 EGFR mutations in Exons 19 and 21 (Table 1). This kit is for research use only. Table 1 Details of 3 Somatic mutations in EGFR gene Name Mutation Exon Base Change Cosmic ID Ex19-mutant-1 E746_A750del (1) _2249del Ex19-mutant-2 E746_A750del (2) _2250del Ex21-mutant-1 L858R T>G 6224 Kit Contents This kit contains sufficient reagents to carry out 24 tests (Table 2), and additional EGFR Mixed Standard DNA for positive control reactions. The mutations of E746_A750del (1) and E746_A750del (2) are indicated by FAM signal, The mutation of L858R is indicated by ROX signal and the HEX signal indicates the internal control (Table 3) Table 2. Kit Contents Reagents Supplied Volume Channel #1: EGFR 3 Reaction Mix 650 μl/ tube FAM, HEX,ROX #2: EGFR 3 Taq DNA Polymerase 10 μl/ tube / #3: EGFR Mixed Standard (Positive Control) 250 μl/ tube / Table 3. Singal Mutation E746_A750del (1) and E746_A750del (1) L858R internal control Signal FAM ROX HEX Equipment and Reagents Not Supplied With Kit 1. Compatible PCR instrument is Rotor-Gene Sterile, nuclease-free tubes. 3. Dedicated pipette and filter pipette tips for handling DNA template. 4. Sterile, nuclease-free H2O. Shipping and Storage The kit requires cold-chain-transportation. The shelf-life of the kit is eight months when the kit is stored immediately upon receipt at -20 in a constant-temperature freezer and protected from light. Please aliquot the 2 / 6

3 reagents if necessary, and store the left reagents at -20 immediately. The opened reagent is still valid until the expiry. Specimen Material Human genomic DNA must be extracted from tissue or blood, or fixed paraffin-embedded tissue prior to use and stored at -20±2. Good DNA quality is essential and we recommend use of Qiagen DNA extraction kit (QIAamp DNA FFPE Tissue Kit, cat No , for paraffin embedded specimens; DNeasy Blood & Tissue kit, cat. No or 69506, for tissue and blood specimens). The OD value of DNA samples should be measured using the spectrophotometer after extraction. The Thermo Fisher NanoDrop 1000 /2000 spectrophotometer is recommended. Make sure A260/A230value is greater than 2.0 anda260/a280 value between 1.8 and 2.0. Technological Principles The kit uses novel, proprietary primers and probes in a real-time PCR assay to detect EGFR mutations in human genomic DNA. The mutant EGFR gene DNA is amplified by the specific primers, and detected by the novel probes. Real time PCR analysis software and a highly validated procedure based on EGFR Taq DNA polymerase contribute to outstanding assay sensitivity and selectivity. Notes on Protocol 1. The reaction mix tubes contain the reaction buffer, dntps, specific oligos and probes. 2. The threshold at which the signal is detected above background fluorescence is called the Cycle threshold (Ct). The Ct values used to determine if a sample is positive or negative are based on extensive validation. If the Ct value falls within the appointed range (see below), the sample is classed as mutation positive. If the Ct value is outside the appointed range, the sample is classed as negative or below the detection limit of the kit. 3. The EGFR Mixed Standard contains a recombinant EGFR gene with the 3 mutations, and normal human genomic DNA. 4. The reactions for each sample must be analyzed within the same PCR run to avoid run-to-run variations in threshold settings. It is recommended that the EGFR Mixed Standard should be analyzed during each PCR run, along with no-template controls. Protocol 1. Thaw the reaction mixes and the EGFR Mixed Standard. 2. Centrifuge reaction mixes, EGFR 3 Taq DNA polymerase and EGFR Mixed Standard prior to use. 3. Accordingtotheratioof20µLreaction mix to 0.2 µl Taq DNA Polymerase per sample, transfer the appropriate amount of reaction mix and Taq DNA Polymerase into a clean tube. 4. Mix the solution thoroughly by gently pipetting it up and down. (Avoid vortexing solutions with Taq). 5. Centrifuge briefly. 6. Transfer 20 μl of the reaction mix into the appropriate PCR tubes. 7. Add 5 μl sample DNA (see following for sample DNA concentrations), 5 μl EGFR Mixed Standard or 5 μl ddh2o (no-template control) to the appropriate PCR tubes. 8. According to different sources, samples can be divided into two groups: paraffin embedded and non-paraffin embedded specimens. a) Non-paraffin embedded specimens include fresh tissue, frozen pathological sections, non-heparin anticoagulant blood plasma, blood serum and non-heparin anticoagulant blood. i. For non-paraffin embedded samples, the recommended DNA amount in each test tube is 2 ~ 5 ng. a) For paraffin embedded samples, we recommend use of 15 ~ 20 ng template DNA in each PCR tube based on different storage times. i. Use 15 ng of template DNA for samples with less than 3 years storage time. ii. Use 20 ng of template DNA for samples with more than 3 years storage time. Notes: We recommend use of TE (ph = 8.0) for extracted DNA dilution. Since Taq DNA polymerase is viscous, please pay attention to the centrifugation and pipetting process. Minimize the contact interface between the pipette tip and Taq DNA polymerase to avoid adding excess enzyme. 9. Place the PCR tubes into the real-time PCR instrument. 3 / 6

4 10. Carry out real-time PCR using the cycling conditions described in Table 4. Note 1: Prior to the operation, please set up the PCR program by the following steps: 1select Gain Optimisation, then Auto Gain Optimisation Setup window will open; 2Click on Optimise Acquiring and Perform Calibration Before 1st Acquisition, Tube 1 is used as control. 3Adjust the temperature to 58, and click Start to continue. Please see the following Graph 1, Graph 2 and Graph 3 for more details. Note 2: Make sure the total volume of solution in each well is 25μl (20μlreagentplus5μl DNA). Graph 1 Table 4 Cycling Parameters Temperature Time Cycles Stage min 1 Stage s 60 20s s Stage s 58 35s Data collection of FAM,HEX and ROX s Graph2 Graph 3 Sample Data Analysis 1. The FAM and ROX signals of the mutation detection system indicate the mutation status of the sample. The HEX signals indicate the internal control status. 2. Make sure that each well gives a HEX signal : i. The Ct value of HEX signal should be between 13 ~ 22. ii. If the requirements of i) are satisfied, further analysis should be carried out. However, if the Ct value is below the indicated range, the DNA is overloaded. The procedure should be repeated with reduced DNA. iii. If the Ct value is above the indicated range, the DNA template contains PCR inhibitors or input DNA less than requirement. In this case, the DNA should be re-extracted and the whole experiment should be carried out again. iv. If the HEX signal assay has failed but the FAM or ROXsignaltesthasworked well, continue with the analysis. If the HEX, FAM and ROX signals tests have failed, the data should be discarded and the experiment should be repeated. 3. Analyze one sample at a time. It is necessary to choose reaction wells for positive control, no-template reference and samples simultaneously. Then, users can adjust the Threshold of FAM and amplification curve, and obtain the Ct value of mutant group. 4. The FAM and ROX Ct values of mixed standard should be less than 20; variation may occur in the Ct value due to different threshold settings on different instruments. 5. One sample may contain two mutant types, and the corresponding sample will have FAM and ROX Ct 4 / 6

5 values. i. If the sample FAM Ct value 28, the sample is classed as negative or below the detection limit of the kit. If the sample FAM Ct value <28, the sample is classed as mutation positive. ii. If the sample ROX Ct value 28, the sample is classed as negative or below the detection limit of the kit. If the sample ROX Ct value <28, the sample is classed as mutation positive. 6. The figures below shows a positive (Graph 4) and a negative result (Graph 5). Warnings and Precautions 1. Please read the instruction carefully and become familiar with all components of the kit prior to use. 2. The product specified above does not contain any virus, reagent by-product of the same, or metabolic by-product of Hepatitis A, B, C, D or HIV. 3. Do not exchange and mix up the kit contents with different batches. 4. The kit and its contents cannot be resold or modified for resale without the written approval of AmoyDx. 5. Using other sources of reagents is not recommended. Strictly distinguish the reagents from mixed standard to avoid contamination. Otherwise, false positive may be produced. 6. Do the experiments with attention to prevent exogenous DNA contamination to reagents. It is recommended that users have separate, dedicated pipettes and filter pipette tips to add DNA template and during the preparation of reagents. 7. To optimize the activity and performance, mixtures should always be protected from light to avoid photo bleaching. 8. All the chemicals are potential hazard, only trained professionals should use this kit. Please wear suitable lab coat and disposable gloves. The used kit should be disposed of properly. 9. AmoyDx grants customer a non-exclusive and non-transferable license to use AmoyDx technologies. 10. AmoyDx assumes no responsibility for any errors that may appear in this document. The information in this document is subject to change. Graph 4. Curve of DNA with mutant EGFR gene Mixed Standard Sample Non-template Control 5 / 6

6 Graph 5. Curve of DNA with wild-type EGFR gene Mixed Standard Non-template Control Sample References 1. Herbst RS, Review of epidermal growth factor receptor biology. Int. J. Radiat. Oncol. Biol. Phys. 59 (2 Suppl): Zhang H, Berezov A, Wang Q, Zhang G, Drebin J, Murali R, Greene MI, ErbB receptors: from oncogenes to targeted cancer therapies. J. Clin. Invest. 117 (8): Oda K, Matsuoka Y, Funahashi A, Kitano H, A comprehensive pathway map of epidermal growth factor receptor signaling. Mol. Syst. Biol. 1: Lynch TJ, Bell DW, Sordella R, et al, Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N.Engl.J.Med.350 (21): Seth D, Shaw K, Jazayeri J and Leedman PJ, Complex post-transcriptional regulation of EGF-receptor expression by EGF and TGF -α in human prostate cancer cells. Br J Cancer 80(5-6): Pao W, Miller VA, Politi KA, Riely GJ, Somwar R, Zakowski MF, Kris MG and Varmus H, Acquired resistance of Lung Adenocarcinomas to Gefitinib or Erlotinib is associates with a second mutation in the EGFR kinase domain. Plos Medicine 2(3): / 6