AmoyDx TM KRAS Seven Mutations Detection Kit Detection of seven mutations in KRAS codons 12 and 13 Instructions For Use Instructions Version: B1.0 Date of Revision: May 2012 Store at -20±2 o C 1/7
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 tests (Table2), and additional KRAS mixed standard DNA for positive control reactions. Table 2: Kit Contents Reagents Supplied Volume Channel #1: Gly12Asp Reaction Mixture 1250 µl/ tube FAM, VIC #2: Gly12Ala Reaction Mixture 1250 µl/ tube FAM, VIC #3:Gly12Val Reaction Mixture 1250 µl/ tube FAM, VIC #4:Gly12Ser Reaction Mixture 1250 µl/ tube FAM, VIC #5:Gly12Arg Reaction Mixture 1250 µl/ tube FAM, VIC #6:Gly12Cys Reaction Mixture 1250 µl/ tube FAM, VIC #7:Gly13Asp Reaction Mixture 1250 µl/ tube FAM, VIC #8:External Control Reaction Mixture 1250 µl/ tube FAM #9: KRAS Taq DNA Polymerase 75 µl/ tube / #10: KRAS Mixed Standard (Positive Control) 500 µl/ tube / Equipment and Reagents Not Supplied With Kit 1. The compatible PCR instruments is ABI StepOne. 2. Sterile, nuclease-free tubes. 2/7
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. 56404, for paraffin embedded specimens; DNeasy Blood & Tissue kit, cat. No. 69504 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 ddh 2O(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. 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 3/7
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 1 95 5min 1 Stage 2 95 25s 64 20s 15 72 20s Stage 3 93 25s 60 35s Data collection of FAM and VIC 31 72 20s Sample Data Analysis 1. The FAM signals of the mutation detection system indicate the mutation status of the sample. The 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 ~ 22 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 VIC signals in the internal control are also used as controls. If the VIC signal assay has failed but the FAM test has worked well, continue with the analysis. If both the 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. 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. 4/7
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 Strong Positive Weak Positive Negative Mutant Ct Value Mutant Content Mutant Ct Value Ct Cut-off value Mutant Content Mutant Ct Value Ct <25 Ct <26 Ct <25 Ct <26 Ct <26 Ct <24 Ct <26 >5% >5% >5% >5% >5% >5% >5% 25 Ct <28 26 Ct <29 25 Ct <28 26 Ct <29 26 Ct <29 24 Ct <28 26 Ct <29 8 9 7 8 9 8 9 1%~5% 1%~5% 1%~5% 1%~5% 1%~5% 1%~5% 1%~5% 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 1. Symbol for "In Vitro Diagnostic Medical Device". 2. Symbol for "Authorized Representative in the European Community". 5/7
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
Appendix 1 - Suggested PCR Plate Layout 96 well layout Assay 1 2 3 4 5 6 7 8 9 10 11 12 12-2-A Sample1 Sample2 Sample3 Sample4 Sample5 Sample6 Sample7 Sample8 Sample9 Sample10 STD NTC 12-2-C Sample1 Sample2 Sample3 Sample4 Sample5 Sample6 Sample7 Sample8 Sample9 Sample10 STD NTC 12-2-T Sample1 Sample2 Sample3 Sample4 Sample5 Sample6 Sample7 Sample8 Sample9 Sample10 STD NTC 12-1-A Sample1 Sample2 Sample3 Sample4 Sample5 Sample6 Sample7 Sample8 Sample9 Sample10 STD NTC 12-1-C Sample1 Sample2 Sample3 Sample4 Sample5 Sample6 Sample7 Sample8 Sample9 Sample10 STD NTC 12-1-T Sample1 Sample2 Sample3 Sample4 Sample5 Sample6 Sample7 Sample8 Sample9 Sample10 STD NTC 13-2-A 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. FDA website: http://www.fda.gov/aboutfda/centersoffices/cder/ucm172905.htm. 2. McGrath JP, Capon DJ, Smith DH, Chen EY, Seeburg PH, Goeddel DV, Levinson AD, 1983. Structure and organization of the human Ki-ras proto-oncogene and a related processed pseudogene. Nature 304 (5926): 501 6. 3. Lièvre A, Bachet JB, Le Corre D, et al. 2006. KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer. Cancer Res. 66 (8): 3992 5. 4. James RM, Arends MJ, Plowman SJ, et al. 2003. KRAS Proto-Oncogene exhibits tumor suppressor activity as its absence promotes tumorigeneis in Murine Teratomas. Mol Cancer Res. 1: 820 825. 7/7