AmoyDx TM EGFR 29 Mutations Detection Kit

Transcription

1 AmoyDx TM EGFR 29 Mutations Detection Kit Detection of 29 mutations in exons Instructions For Use Instructions Version: B2.2 Date of Revision: April 2012 Store at -20±2 o C 1 / 8

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 TM EGFR 29 Mutations Detection Kit is highly selective and sensitive, detecting 29 of the most common somatic mutations (both activating and resistance-related) 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 TM EGFR 29 Mutations Detection Kit is a highly sensitive real-time PCR-based test designed to accurately identify 29 EGFR mutations in Exons (Table 1). It is SFDA approved for clinical use in China and CE marked for IVD use in Europe. Table 1 Details of 29 Somatic mutations in EGFR gene Name Mutation Exon Base Change Cosmic ID Ex18-mutant-1 G719A G>C 6239 Ex18-mutant-2 G719S G>A 6252 Ex18-mutant-3 G719C G>T 6253 Ex19-mutant-1 E746_A750del (1) _2249del Ex19-mutant-2 E746_A750del (2) _2250del Ex19-mutant-3 L747_P753>S _2257del Ex19-mutant-4 E746_T751>I _2252>AAT(complex) Ex19-mutant-5 E746_T751del _2253del Ex19-mutant-6 E746_T751>A _2251del Ex19-mutant-7 E746_S752>A _2254del Ex19-mutant-8 E746_S752>V _2250>T(complex) Ex19-mutant-9 E746_S752>D _2255del Ex19-mutant-10 L747_A750>P _2248>GC(complex) Ex19-mutant-11 L747_T751>Q _2252>GCA(complex) Ex19-mutant-12 L747_E749del _2247del Ex19-mutant-13 L747_T751del _2253del Ex19-mutant-14 L747_S752del _2256del Ex19-mutant-15 L747_A750>P _2248TTAAGAGAAG>C(complex) Ex19-mutant-16 L747_P753>Q _2258>CA(complex) Ex19-mutant-17 L747_T751>S _2251del Ex19-mutant-18 L747_T751del _2254del Ex19-mutant-19 L747_T751>P _2251>C(complex) Ex20-mutant-1 T790M C>T 6240 Ex20-mutant-2 S768I G>T / 8

3 Ex20-mutant-3 H773_V774insH _2320insCAC Ex20-mutant-4 D770_N771insG _2311insGGT Ex20-mutant-5 V769_D770insASV _2308insgccagcgtg Ex21-mutant-1 L858R T>G 6224 Ex21-mutant-2 L861Q T>A 6213 Kit Contents This kit contains sufficient reagents to carry out 24, 48 or 96 tests (Table 2), and additional EGFR mixed standard DNA for positive control reactions. The 19-Del reaction mixture can detect the presence of any of 19 deletions in exon 19. The Insertions reaction mixture can detect the presence of any of 3 insertions in exon 20. The G719X reaction mixture can detect the presence of G719S, G719A and G719C. Table 2. Kit Contents Kit Size Reagents Supplied Volume Channel reactions reactions reactions Volume Volume Volume #1: 19-Del Reaction Mixture 1000 μl/ tube FAM, HEX/VIC 1 tube 2 tubes 4 tubes #2: L858R Reaction Mixture 1000 μl/ tube FAM, HEX/VIC 1 tube 2 tubes 4 tubes #3: T790M Reaction Mixture 1000 μl/ tube FAM, HEX/VIC 1 tube 2 tubes 4 tubes #4: Insertions Reaction Mixture 1000 μl/ tube FAM, HEX/VIC 1 tube 2 tubes 4 tubes #5: G719X Reaction Mixture 1000 μl/ tube FAM, HEX/VIC 1 tube 2 tubes 4 tubes #6: S768I Reaction Mixture 1000 μl/ tube FAM, HEX/VIC 1 tube 2 tubes 4 tubes #7: L861Q Reaction Mixture 1000 μl/ tube FAM, HEX/VIC 1 tube 2 tubes 4 tubes #8:External Control Reaction Mixture 1000 μl/ tube FAM 1 tube 2 tubes 4 tubes #9: EGFR Taq DNA Polymerase 85 μl/ tube / 1 tube 2 tubes 4 tubes #10: EGFR Mixed Standard (Positive Control) 500 μl/ tube / 1 tube 2 tubes 4 tubes Equipment and Reagents Not Supplied With Kit 1. Compatible PCR instruments are: Stratagene Mx3000P, Stratagene Mx3005P, ABI7300, ABI7500, ABI7900, ABI StepOne, LightCycler480Ⅰ and Ⅱ, BioRad-CFX Sterile, nuclease-free tubes. 3. Dedicated pipette and filtered pipette tips for handling DNA. 4. Sterile, nuclease-free H2O. Shipping and Storage The kit requires cold-chain-transportation. The shelf-life of the kit is six months when the kit is stored immediately upon receipt at -20±2 o C in a constant-temperature freezer and protected from light. 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 3 / 8

4 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 mixture tubes contain the reaction buffer, dntps, specific oligos and probes. 2. The mutation reaction mixtures include a mutation detection system and an internal control system. The mutation detection system is used to detect the mutation status of EGFR gene (positive or negative). The internal control system is designed to detect the presence of inhibitors, which may lead to false negative results. The external control reaction mixture is used to assess the DNA quality, that is, to detect the presence of inhibitors, which may lead to false negative results 3. 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. Weak positives can be confirmed by calculating ΔCt values and referring to Table 4 below. 4. The EGFR mixed standard contains a recombinant EGFR gene with the 29 mutations, and normal human genomic DNA. 5. The 8 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 8 reaction mixtures and the EGFR mixed standard. Only thaw 1 tube of each, unless more reagent is required, to avoid freeze-thawing. 2. Centrifuge reaction mixtures, EGFR Taq DNA polymerase and EGFR mix standard prior to use. 3. Accordingtotheratioof35μL reaction mixture to 0.3 μl Taq DNA Polymerase per sample, transfer the appropriate amount of reaction mixture and Taq DNA Polymerase into a clean tube. The volumes given for each reaction mix have been optimized and validated. Changing volumes of any reagent may result in a loss of performance. Do not store user-prepared mixes, use immediately. 4. Mix the solution thoroughly by gently pipetting it up and down. (Avoid vortexing solutions with Taq). 5. Centrifuge briefly. 6. Transfer 35.3 μl of the reaction mixture into the appropriate PCR tubes. 7. Add 4.7 μl sample DNA (see following for sample DNA concentrations), 4.7 μl EGFR mixed standard or 4.7 μl ddh2o (no-template control) to the appropriate PCR tubes. A recommended plate layout is given in Appendix 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. Seal the PCR tubes. 10. Spin the PCR tubes in order to collect the reagents at the bottom of wells. 4 / 8

5 a) This spin step is critical to the success of the procedure. 11. Place the PCR tubes into the real-time PCR instrument. 12. Carry out real-time PCR using the cycling conditions described in Table 3. For: ADx-EG07 ADx-EG08 ADx-EG09 Table3 Cycling Parameters Temperature Time Cycles Stage min 1 Stage s 64 20s s Stage s 60 35s Data collection of FAM and HEX/VIC s Sample Data Analysis 1. The FAM signals of the mutation detection system indicate the mutation status of the sample. The HEX/VIC signals indicate the internal control status. The internal control amplifies and detects a region of genomic DNA that has no known mutations or SNPs. 2. Check the FAM signal from the external control assay: i. The Ct value should be between 15 ~ 21 for paraffin embedded specimens; and between 13~19 for non-paraffin embedded specimens. ii. If the requirements of i) are satisfied, further analysis should be carried out. However, if Ct value is below the indicated range, the DNA is overloaded. The procedure should be repeated with reduced DNA. iii. If the external control assay has failed, the DNA template contains PCR inhibitors, indicting that the DNA needs to be re-extracted. 3. The HEX/VIC signals in the internal control are also used as controls. If the HEX/VIC signal assay has failed but the FAM test has worked well, continue with the analysis. If both the HEX/VIC and FAM signal tests have failed, the data should be discarded and the experiment should be repeated. 4. Instrument set-up: Ensure the calibration fluorescence is unselected, and select single mutation detection for each tube accordingly. It is necessary to choose reaction holes for positive control, no-template reference and samples simultaneously. Then, users may adjust the Threshold of FAM amplification curve, and obtain the Ct value of mutant group. 5. The EGFR mixed standard FAM Ct value should be less than 20, but variation may occur due to different threshold settings on different instruments. Analysis of mutation assay results. See Table Check the FAM Ct value for each sample. Based on different mutant Ct values, the detection results are divided into strong positive, weak positive or negative 2. Strong Positive: If the sample FAM Ct value is less than the Ct value shown in the Strong Positive row in Table4, the sample is classified as strong positive. 3. Weak Positive: If the sample FAM Ct value is in the range shown in the Weak Positive row in Table 4, the sample is provisionally classified as weak positive. a. If the FAM Ct value is in the Weak Positive range, the Ct of the reaction tube is calculated to confirm the result. b. If the Ct value is less than the corresponding Cut-off value of Ct, the sample is confirmed as weak positive. c. If the Ct value is greater than the Cut-off Ct value, the sample is classified as negative or below the limits of the kit. 5 / 8

6 4. The calculation of Ct: Formula 1. Ct = mutant FAM Ct value external control FAM Ct value. a. Where: i. The mutant FAM Ct value indicates the Ct value of the mutant FAM signal from a sample. ii. The external control FAM Ct value indicates the Ct value of the FAM signal in external control tube. 5. Negative: If the sample FAM Ct value is greater than or equal to the critical negative value shown in the Negative row in Table4, the sample is classified as negative or below the detection limit of the kit. Table 4 Results Determination Name of Mutation 19-Del L858R T790M Insertions G719X S768I L861Q Strong Positive Weak Positive Negative Mutant Ct Value Ct<26 Ct<26 Ct<26 Ct<26 Ct<26 Ct<26 Ct<26 Mutant Content >5% >5% >5% >5% >5% >5% >5% Mutant Ct Value 26 Ct <29 26 Ct <29 26 Ct <28 26 Ct <29 26 Ct <29 26 Ct <29 26 Ct <29 Ct Cut-off value Mutant Content 1%~5% 1%~5% 1%~5% 1%~5% 1%~5% 1%~5% 1%~5% Mutant Ct Value Ct 29 Ct 29 Ct 28 Ct 29 Ct 29 Ct 29 Ct 29 Warnings and Precautions 1. Please read the instruction carefully and become familiar with all components of the kit prior to use. 2. Do not exchange and mix up the kit contents with different batches. 3. The kit and its contents cannot be resold or modified for resale without the written approval of AmoyDx. 4. Using other sources of reagents is not recommended. Strictly distinguish the reagents from mixed standard to avoid contamination. Otherwise, false positive may be produced. 5. 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. 6. To optimize the activity and performance, mixtures should always be protected from light to avoid photo bleaching. 7. 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. 8. The product is CE-marked according to the European Union In Vitro Diagnostic Medical Devices Directive 98/79/EC. 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. Notes 1. Symbol for "In Vitro Diagnostic Medical Device". 2. Symbol for "Authorized Representative in the European Community". 3. Symbol for "Batch Code". 6 / 8

7 4. Symbol for "Used By", it indicates that the reagent should not be used after the end of the date as shown on box. 5. Symbol for "Attention, see instructions for use". 6. Symbol for "Temperature Limitation", the kits should be stored at -20±2. Information of European Authorised Representative Wellkang Ltd t/a Wellkang Tech Consulting Suite B, 29 Harley Street, London W1G 9QR United Kingdom 7 / 8

8 Appendix 1 - Suggested PCR Plate Layout 96 well layout Assay Del Sample1 Sample2 Sample3 Sample4 Sample5 Sample6 Sample7 Sample8 Sample9 Sample10 STD NTC L858R Sample1 Sample2 Sample3 Sample4 Sample5 Sample6 Sample7 Sample8 Sample9 Sample10 STD NTC T790M Sample1 Sample2 Sample3 Sample4 Sample5 Sample6 Sample7 Sample8 Sample9 Sample10 STD NTC Insertions Sample1 Sample2 Sample3 Sample4 Sample5 Sample6 Sample7 Sample8 Sample9 Sample10 STD NTC G719X Sample1 Sample2 Sample3 Sample4 Sample5 Sample6 Sample7 Sample8 Sample9 Sample10 STD NTC S768I Sample1 Sample2 Sample3 Sample4 Sample5 Sample6 Sample7 Sample8 Sample9 Sample10 STD NTC L861Q Sample1 Sample2 Sample3 Sample4 Sample5 Sample6 Sample7 Sample8 Sample9 Sample10 STD NTC External Control Sample1 Sample2 Sample3 Sample4 Sample5 Sample6 Sample7 Sample8 Sample9 Sample10 STD NTC 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): / 8