AmoyDx TM EGFR 29 Mutations Detection Kit

Transcription

1 For: ADx-EG07 ADx-EG08 ADx-EG09 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 For: ADx-EG07 ADx-EG08 ADx-EG09 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 For: ADx-EG07 ADx-EG08 ADx-EG09 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 For: ADx-EG07 ADx-EG08 ADx-EG09 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 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 For: ADx-EG07 ADx-EG08 ADx-EG09 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 For: ADx-EG07 ADx-EG08 ADx-EG09 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 For: ADx-EG07 ADx-EG08 ADx-EG09 Appendix 1 - Suggested PCR Plate Layout 96 well layout Assay Del STD NTC L858R STD NTC T790M STD NTC Insertions STD NTC G719X STD NTC S768I STD NTC L861Q STD NTC External Control 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

9 For:ADx-KR02 KR03 KR04 AmoyDx TM KRAS Seven Mutations Detection Kit Detection of seven mutations in KRAS codons 12 and 13 Instructions For Use Instructions Version: B2.2 Date of Revision: April 2012 Store at -20±2 o C 1/7

10 For:ADx-KR02 KR03 KR04 Background KRAS protein is a GTPase and one of the key molecules in the downstream signaling pathway of epidermal growth factor receptor (EGFR). KRAS protein transduces signals from membrane-bound receptors via multiple downstream effector pathways and thereby affects fundamental cellular processes, including proliferation, apoptosis, and differentiation. In some cancer tissue, the KRAS protein is found to be in a permanently activated state due to somatic mutations. Most of these activating KRAS mutations are in exons 12 and 13. Point mutations in the KRAS gene have been found in a number of human tumor types, such as colorectal, pancreatic, and lung. The mutation status of the KRAS gene is relevant to the primary drug resistance of colorectal and non-small cell lung cancer treated with tyrosine kinase inhibitors. Patients with wild-type KRAS gene could benefit from Erbitux (Cetuximab) or Vectibix (Panitumumab), whereas, the patients with mutant KRAS gene show poor response to this treatment. The European Drug Administration Organization and US FDA approve the employment of KRAS gene mutation detection prior to the use of targeted medicines Erbitux and Vectibix in the treatment of metastatic colorectal cancer. The AmoyDx TM KRAS seven Mutations Detection Kit can detect seven mutations in KRAS gene. It is sensitive enough to detect mutant gene present at 1% in a background of 99% normal DNA. AmoyDx s patented technology uses proprietary probes in a real-time PCR format. The procedure is easily adapted for use in high-throughput sample processing. The purpose of the kit is to aid doctors to identify tumor samples carrying mutations in KRAS gene. Intended Use AmoyDx TM KRAS seven Mutations Detection Kit is SFDA approved for clinical use in China and CE marked for IVD use in Europe. It is a highly sensitive test designed to accurately identify seven KRAS mutations in codons 12 and 13 (Table 1). Table 1: KRAS mutations in codons 12 and 13 detected by the AmoyDx Kit Name Mutation Base Change 12-2-A Gly12Asp GGT>GAT 12-2-C Gly12Ala GGT>GCT 12-2-T Gly12Val GGT>GTT 12-1-A Gly12Ser GGT>AGT 12-1-C Gly12Arg GGT>CGT 12-1-T Gly12Cys GGT>TGT 13-2-A Gly13Asp GGC>GAC Kit Contents This kit contains sufficient reagents to carry out 24, 48 or 96 tests (Table2), and additional KRAS mixed standard DNA for positive control reactions. Table 2: Kit Contents Kit Size Reagents Supplied Volume Channel reactions reactions reactions Volume Volume Volume #1: Gly12Asp Reaction Mixture 1250 µl/ tube FAM, HEX/VIC 1 tube 2 tubes 4 tubes #2: Gly12Ala Reaction Mixture 1250 µl/ tube FAM, HEX/VIC 1 tube 2 tubes 4 tubes #3:Gly12Val Reaction Mixture 1250 µl/ tube FAM, HEX/VIC 1 tube 2 tubes 4 tubes #4:Gly12Ser Reaction Mixture 1250 µl/ tube FAM, HEX/VIC 1 tube 2 tubes 4 tubes #5:Gly12Arg Reaction Mixture 1250 µl/ tube FAM, HEX/VIC 1 tube 2 tubes 4 tubes #6:Gly12Cys Reaction Mixture 1250 µl/ tube FAM, HEX/VIC 1 tube 2 tubes 4 tubes #7:Gly13Asp Reaction Mixture 1250 µl/ tube FAM, HEX/VIC 1 tube 2 tubes 4 tubes #8:External Control Reaction Mixture 1250 µl/ tube FAM 1 tube 2 tubes 4 tubes #9: KRAS Taq DNA Polymerase 75 µl/ tube / 1 tube 2 tubes 4 tubes #10: KRAS Mixed Standard (Positive Control) 500 µl/ tube / 1 tube 2 tubes 4 tubes 2/7

11 For:ADx-KR02 KR03 KR04 Equipment and Reagents Not Supplied With Kit 1. The compatible PCR instruments are: Stratagene Mx3000P,Stratagene Mx3005P, ABI7300, ABI7500, ABI7900, 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 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 a 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, patented primers and probes to detect mutations in a real-time PCR assay. The mutant DNA is amplified accurately by the specific primers, and detected by the novel probes. Dedicated PCR analysis software and a highly validated procedure based on KRAS Taq DNA polymerase contribute to outstanding assay sensitivity and selectivity. Protocol Notes: 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 KRAS 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. 4. The KRAS mixed standard contains a recombinant KRAS gene with the seven mutations, and normal human genomic DNA. The eight 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 KRAS mixed standard should be analyzed during each PCR run, along with no-template controls. 1. Thaw the 8 reaction mixtures and the KRAS mixed standard. Only thaw 1 tube of each, unless more reagent is required, to avoid freeze-thawing. 2. Centrifuge reaction mixtures, KRAS Taq DNA polymerase and KRAS mixed standard prior to use. 3. According to the ratio of 45 µl reaction mixture to 0.25 µl Taq DNA Polymerase per sample, transfer the appropriate amount of reaction mixture 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 45 µl of the reaction mixture into the appropriate PCR tubes. 7. Add 5 µl sample DNA (see following for sample DNA concentrations), 5 µl KRAS mixed standard or 5 µl ddh2o(no-template control)to the appropriate PCR tubes. A recommended plate layout is given in Appendix1. 8. According to different sources, samples can be divided into two groups: paraffin embedded and non-paraffin embedded specimens. 3/7

12 For:ADx-KR02 KR03 KR04 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. b) 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. 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. Table 3 Cycling Parameters Temperature Time Cycle s Stage min 1 Stage s 64 20s s Stage s 60 35s Data collection of FAM and HEX/VIC s 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. 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 would be carried out. However, if Ct value is below the corresponding range, it indicates the DNA is overloaded, so the amount of DNA should be reduced. iii. If the external control assay has failed, it shows that the DNA template contains PCR inhibitors, thus, the DNA need 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 obtained data must be discarded and the experiment should be repeated. 4. 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 could adjust the Threshold of FAM amplification curve, and obtain the Ct value of mutant group. 4/7

13 For:ADx-KR02 KR03 KR04 5. The KRAS mixed standard FAM Ct value should be less than 20, but variation may occur due to different threshold settings on different instruments. 6. Analysis of mutation assay results. See Table 4: a) 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. b) Strong Positive: If the sample FAM Ct value is less than the Ct value shown in the Strong Positive row in Table 4, the sample is classified as strong positive. c) 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. d) If the FAM Ct value is in the Weak Positive range, the Ct of the reaction tube is calculated to confirm the result. If the Ct value is less than the corresponding Cut-off value of Ct, the sample is confirmed as weak positive. 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. e) The calculation of Ct: Ct = mutant FAM Ct value external control FAM Ct value. The mutant Ct value indicates the Ct value of the sample mutant FAM signal; the external control Ct value indicates the Ct value of external control FAM signal of the sample.. f) Negative: If the sample FAM Ct value is greater than or equal to the critical negative value shown in the Negative row in Table 4, the sample is classified as negative or below the limits of the kit. Table 4 Results Determination Name of Mutation Gly12Asp Gly12Ala Gly12Val Gly12Ser Gly12Arg Gly12Cys Gly13Asp Mutant Ct Strong Value Ct <24 Ct <26 Ct <24 Ct <26 Ct <26 Ct <24 Ct <26 Positive Mutant Content >5% >5% >5% >5% >5% >5% >5% Mutant Ct Value 24 Ct <28 26 Ct <29 24 Ct <28 26 Ct <29 26 Ct <29 24 Ct <28 26 Ct <29 Weak Ct Cut-off Positive value Mutant Content 1%~5% 1%~5% 1%~5% 1%~5% 1%~5% 1%~5% 1%~5% Negative Mutant Ct Value Ct 28 Ct 29 Ct 28 Ct 29 Ct 29 Ct 28 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, use separate, dedicated pipette 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 could use this kit. Please wear suitable lab coat and disposable gloves. The used kit should be disposed 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 for the information in this document is subject to change. Notes 5/7

14 For:ADx-KR02 KR03 KR04 1. Symbol for "In Vitro Diagnostic Medical Device". 2. Symbol for "Authorized Representative in the European Community". 3. Symbol for "Batch Code". 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. 7. ADx KRAS Seven Mutations Detection Kit has also received market approval by SFDA for clinical usage in China Mainland. Information of European Authorised Representative Wellkang Ltd t/a Wellkang Tech Consulting Suite B, 29 Harley Street, London W1G 9QR United Kingdom 6/7

15 For:ADx-KR02 KR03 KR04 Appendix 1 - Suggested PCR Plate Layout 96 well layout Assay A STD NTC 12-2-C STD NTC 12-2-T STD NTC 12-1-A STD NTC 12-1-C STD NTC 12-1-T STD NTC 13-2-A STD NTC External Control STD NTC References: 1. FDA website: 2. McGrath JP, Capon DJ, Smith DH, Chen EY, Seeburg PH, Goeddel DV, Levinson AD, Structure and organization of the human Ki-ras proto-oncogene and a related processed pseudogene. Nature 304 (5926): Lièvre A, Bachet JB, Le Corre D, et al KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer. Cancer Res. 66 (8): James RM, Arends MJ, Plowman SJ, et al KRAS Proto-Oncogene exhibits tumor suppressor activity as its absence promotes tumorigeneis in Murine Teratomas. Mol Cancer Res. 1: /7

16 For:ADx-AE01 AmoyDx TM EML4-ALK Fusion Gene Detection Kit Instructions For Use Instructions Version: B1.4 Date of Revision: April 2012 Store at -20±2 o C Website: Tel: / 4

17 For:ADx-AE01 Background The EML4-ALK test provides information about the potential sensitivity of NSCLC to ALK inhibitor therapy. The fusion of the echinoderm microtubule-associated protein-like 4 (EML4) gene and the anaplastic lymphoma kinase (ALK) gene is present in a fraction of non-small cell lung cancer (NSCLC). The recombinant EML4-ALK activates the receptor tyrosine kinase s downstream signaling pathway, which includes PI3K/AKT, leading to carcinogenesis. It has been reported that the presence of the EML4-ALK fusion is correlated with the efficacy of ALK-targeted therapy. This kit contains reagents to detect EML4-ALK fusions, as shown in Table 1. The kit is based on analysis of tumor messenger RNA. Table 1. Fusions detected by EML4-ALK kit Fusion product Alternate name EML4 spliced exon Breakpoint variety(bp)* ALK spliced exon COSMIC ID EA-M1 EML4-ALK variant /-/ins69/-/-/ /489/1063/462/410/414 EA-M2 EML4-ALK variant 3a/b 6 -/-/ins33/ /734/476/493 EA-M3 EML4-ALK variant /-/ins18/ /490/731/464 EA-M4 EML4-ALK variant 4 15 del EA-M5 EML4-ALK variant 4 14 ins11del49/del12/del /1065/1128 EA-M6 EML4-ALK variant EA-M7/9 EML4-ALK variant 5a/b 2 -/ins /480 EA-M8 / 17 ins *The breakpoint variety leads to different cdna isoform: ins indicates insertion, while del indicates deletion. Intended Use AmoyDx EML4-ALK Fusion Gene Diagnostic Kit is intended for research use only. Kit Contents This kit contains sufficient reagents to carry out 24 tests (Table 2). Both the EML4-ALK fusion gene in Tube 1~3 and the reference gene (β-actin) expression in Tube 4 are indicated by FAM signals. Table 2 Kit Contents Tube No. Reagents Supplied Volume Quantity 1 EA-M1,2,3 Reaction Mix 650 µl 1 2 EA-M4,5 Reaction Mix 650 µl 1 3 EA-M6,7,8,9ReactionMix 650 µl 1 4 EA External Control Reaction Mix 650µL 1 5 EA Reverse Transcriptase 30 µl 1 6 EA Reverse Transcription Reaction Mix 550 µl 1 7 EA Positive Standard 250 µl 1 8 EA Taq DNA Polymerase 30 µl 1 Equipment and Reagents Not Supplied With Kit 1. Compatible PCR instruments are the Stratagene Mx3000P, Stratagene Mx3005P, ABI7300/7500, LightCycler480Ⅰ and Ⅱ. 2. Sterile, nuclease-free tubes. 3. Dedicated pipette and filter pipette tips for handling RNA&DNA template. 4. Sterile, nuclease-free H2O. 5. RNA extraction reagents. Shipping and Storage The kit requires cold-chain-transportation and be stored immediately upon receipt at -20±2 o Cina constant-temperature freezer and protected from light. The shelf-life of the kit is indicated on the package. Specimen Material Human messenger RNA must be extracted from materials prior to use. If it is not used immediately, it should be stored at below -70 o C for no more than six months. Good RNA quality is essential and we recommend use of Qiagen RNA extraction kits. The OD value of RNA samples should be measured using the spectrophotometer Website: Tel: / 4

18 For:ADx-AE01 after extraction. The Thermo Fisher NanoDrop 1000 /2000 spectrophotometer is recommended. The A260/A280 value should be between 1.9 and 2.1. Technological Principles The cdna is obtained from sample RNA through reverse transcription, then the target gene sequence is specifically amplified by novel, proprietary primers. The amount of target cdna is measured after each cycle in the data capture phase using a novel fluorescent probe. Note: Reporter Dye: FAM; Quencher Dye: TAMRA; Passive Reference: NONE. Protocol 1. Reverse Transcription (1) For each RNA sample, transfer 18 μl EA Reverse Transcription Reaction Mix and 1 μl EA Reverse Transcriptase to a sterile centrifuge tube; mix well by pipeting gently up and down. (2) Add 6 μl sample RNA into the appropriate centrifuge tube. The total amount of RNA should be within 0.1 ~ 5 μg. (3) Incubate the tubes at 42 o C for one hour. (4) Heat the tubes at 95 o C for five minutes, then transfer them to ice. The resulting cdna solutions are used for PCR amplification. 2. PCR amplification (1) According to the ratio of 20 μl Reaction Mix to 0.2 μl Taq DNA Polymerase(EA) per sample, transfer the appropriate amount of Reaction Mix and Taq DNA Polymerase(EA) into a sterile tube. (2) Mix well by pipeting up and down and centrifuge for 5 seconds. (3) Transfer 20 μl of the mixture solution into PCR reaction wells. (4) Add 5 μl of sample cdna, 5 μl of the various EA Positive Standard (STD) and 5 μl ddh2o (no-template control, NTC) solutions to the appropriate PCR reaction wells. (5) Centrifuge briefly. (6) An example of a plate layout is shown in Table 3. Tube No Table 3 Plate Layout (example for 96 well) NTC NTC NTC NTC STD STD STD STD (7) Seal the PCR tubes. (8) Spin the PCR tubes gently to collect the reagents at the bottom of wells. (9) Place the PCR tubes into the real-time PCR instrument. (10) Carry out real-time PCR using the cycling conditions described in Table 4. Website: Tel: / 4

19 For:ADx-AE01 Table 4 Cycling Parameters Temperature Time Cycles Stage min 1 Stage s 64 20s s Stage s 60 35s Data collection of FAM s Data Analysis 1. The FAM signal indicates the fusion status of sample in Tube 1~3 and the reference gene (β-actin) expression status in Tube Make sure that the reference well of each sample gives a FAM signal. a) If the FAM signal gives a positive result, then continue with the analysis. b) If the FAM signal assay failed, it shows that the cdna template is insufficient or contains PCR inhibitors. The whole experiment should be carried out again with more cdna loaded in the case template insufficiency, or with re-purified cdna to remove PCR inhibitors. 3. Ensure the calibration fluorescence is unselected. Select the sample and control positions as a group. Then adjust the Threshold for FAM amplification curves to obtain the Ct values of the samples and controls. 4. The mixed standard FAM Ct value should be less than 24, but variation may occur due to different threshold settings on different instruments. 5. Analysis of fusion assay results. If the sample Ct value 30, the fusion assay result is classed as negative. If the sample Ct value < 30, the fusion assay result is classed as positive. 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. Only trained professionals should use this kit. Please wear suitable lab coat and disposable gloves. The used kit should be disposed of properly. 8. AmoyDx grants the customer a non-exclusive and non-transferable license to use AmoyDx technologies. 9. AmoyDx assumes no responsibility for any errors that may appear in this document. The information in this document is subject to change. References 1. Sasaki T, et al Eur J Cancer. 46: Kwak EL, et al, N Engl J Med. 363: Horn L. and Pao W, J Clin Oncol. 27: Website: Tel: / 4

20 For:ADx-BR01 ADx-BR02 ADx-BR03 AmoyDx TM BRAF V600E Mutation Detection Kit Detection of V600E mutation in the BRAF oncogene Instructions For Use Instructions Version: B3.1 Date of Revision: April 2012 Store at -20±2 o C 1/5

21 For:ADx-BR01 ADx-BR02 ADx-BR03 Background BRAF is a serine/threonine kinase that functions within the Ras-Raf-MEK-MAPK pathway. This pathway normally regulates cell proliferation and survival under the control of growth factors and hormones. Mutations in the BRAF gene have been associated with the development of cancer. The most common alteration in the BRAF gene is a mutation called V600E, which alters the valine at position 600 in the protein to a glutamic acid. The V600E mutation causes the BRAF protein to be permanently activated, even in the absence of growth factors. Aberrant BRAF signaling due to the V600E mutation can result in excessive cell proliferation and an adverse resistance to apoptosis. BRAF mutations occur in ~50% of melanoma tumors, ~40% of papillary thyroid tumors, ~30% of ovarian tumors, ~10% of colorectal tumors and ~10% of prostate tumors. The search for drugs that block oncogenic BRAF signaling is an active area of pharmaceutical research and development. The AmoyDx TM BRAF Mutation Detection Kit is highly selective and sensitive, detecting V600E mutation in the BRAF oncogene. 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. Intended Use AmoyDx TM BRAF V600E Mutation Detection Kit is SFDA approved for clinical use in China and CE marked for IVD use in Europe. Kit Contents This kit contains sufficient reagents to carry out 24, 48 or 96 tests (Table 1), and additional mixed standard DNA (genomic DNA plus V600E mutated plasmid DNA) for positive control reactions. Table 1. Kit Contents Kit Size Reagents Supplied 24 reactions 48 reactions 96 reactions Volume Volume Volume #1: BRAF Reaction Mixture (1150 µl/ tube) 1 tube 2 tubes 4 tubes #2: BRAF Taq DNA Polymerase 20 µl 40 µl 80 µl #3: BRAF Mixed Standard (Positive Control) 250 µl 250 µl 250 µl Equipment and Reagents Not Supplied With Kit 1. Compatible PCR instruments are: Stratagene Mx3000P, Stratagene Mx3005P, ABI7300, ABI7500, ABI7900, ABI StepOne, LightCycler480 I and II, BioRad-CFX 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 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 kits (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/A230 value is greater than 2.0 and A260/A280 value between 1.8 and /5

22 For:ADx-BR01 ADx-BR02 ADx-BR03 Technological Principles The kit uses novel, proprietary primers and probes in a real-time PCR assay to detect the BRAF V600E mutation in human genomic DNA. The mutant BRAF V600E gene DNA is amplified by the specific primers, and detected by the novel probes. Protocol Notes: 1. Each reaction tube includes mutation detection system and internal control system. The mutation detection system is used to detect the mutation status of the BRAF gene (positive or negative). The internal control system is designed for detecting the presence of inhibitors, which may lead to false negative results. 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 BRAF mixed standard contains a recombinant BRAF gene with the V600E mutation, and normal human genomic DNA. 4. It is recommended that the BRAF mixed standard should be analyzed during each PCR run, along with no-template controls. 5. The amount of sample DNA used for each sample PCR reaction depends on the kind of sample. a. For non-paraffin embedded samples, the recommended DNA amount in each test tube is 2 ~ 5 ng. i. Non-paraffin embedded specimens include fresh tissue, frozen pathological sections, non-heparin anticoagulant blood plasma, blood serum and non-heparin anticoagulant blood. b. For paraffin embedded samples with less than 3 years storage, we recommend use of 10 ng of template DNA. c. For paraffin embedded samples with more than 3 years storage, we recommend use of 15 ng of template DNA. d. The volume of DNA added to the reaction tube is 5 µl. Experimental Procedure 1. Thaw the BRAF Reaction Mixture. 2. Centrifuge BRAF Taq DNA polymerase and BRAF Reaction Mixture prior to use. 3. According to the ratio of 35 µl Reaction Mixture to 0.4 µl Taq DNA Polymerase per sample, transfer the appropriate amount of Reaction Mixture and Taq DNA Polymerase into a clean tube. 4. Mix the solution thoroughly by gently pipetting it up and down. a) Avoid vortexing solutions with Taq. 5. Centrifuge briefly. 6. Transfer 35 µl of the solution into the appropriate PCR tubes. 7. Add 5 µl sample DNA (2 to 15 ng per reaction, see above), 5 µl BRAF mixed standard or 5 µl ddh 2O (no-template control) to the appropriate PCR tubes. The layout for 22 samples, a positive control and a no template control is shown in Table 2. Table 2 Plate Layout (example for 24 tests/kit) Code A B C STD NTC 8. Seal the PCR tubes. 9. Spin the PCR tubes gently to collect the reagents at the bottom of wells. 3/5

23 NOTE: This spin step is essential for proper mixing of the reagents. 10. Place the PCR tubes into the real-time PCR instrument. 11. Carry out real-time PCR using the cycling conditions described in Table 3. For:ADx-BR01 ADx-BR02 ADx-BR03 Table 3 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 Data Analysis 1. The FAM signal indicates the mutation status of sample and the HEX/VIC signal indicates the internal control status. 2. Make sure that each well gives a HEX/VIC signal. If the HEX/VIC signal gives a positive result, then continue with the analysis. If the Ct value <13, it indicates that the DNA was overloaded, and the amount of DNA should be reduced. If the HEX/VIC signal assay failed (Ct > 30), it shows that the DNA template contains PCR inhibitors. In this case, the DNA should be re-extracted and the whole experiment should be carried out again. 3. Ensure the calibration fluorescence is unselected, and select single mutation detection for each tube accordingly. It is necessary to choose reaction wells for positive control, no-template reference and samples simultaneously. Then, users can adjust the Threshold of FAM amplification curve, and obtain the Ct value of mutant group. 4. The mixed standard FAM Ct value should be less than 20; variation may occur in the Ct value due to different threshold settings on different instruments. 5. 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. 6. The figure below shows a positive (Figure 1) and a negative result (Figure 2). Mixed Standard Non-template Control Figure 1. Curve of DNA with mutant BRAF gene. 4/5

24 For:ADx-BR01 ADx-BR02 ADx-BR03 Mixed Standard Non-template Control Figure 2. Curve of DNA with wild-type BRAF gene. 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. Only trained professionals should use this kit. Please wear suitable lab coat and disposable gloves. The used kit should be disposed of properly. 8. AmoyDx grants the customer a non-exclusive and non-transferable license to use AmoyDx technologies. 9. AmoyDx assumes no responsibility for any errors that may appear in this document. The information in this document is subject to change. References 1. Dhomen N, Marais M, New insight into BRAF mutations in cancer. Current Opinion in Genetics and Development 17: Thomas NE, BRAF somatic mutations in malignant melanoma and melanocytic naevi. Melanoma Research 16(2): /5