AmoyDx KRAS/NRAS Mutations Detection Kit Detection of 19 KRAS mutations (exons 2, 3 and 4) and 13 NRAS mutations (exons 2, 3 and 4) Instruction for Use Instruction Version: P1.0 Revision Date: September 2014 Store at -20±5 Address: 5th Floor, No.4 Plant, Kechuang Center Building, 289 Wengjiao Road, Xinyang Street, Haicang District, Xiamen,
Background RAS protein is a GTPase and one of the key molecules in the downstream signaling pathway of epidermal growth factor receptor (EGFR). RAS protein transduces signals from membrane-bound receptors via multiple downstream effector pathways and thereby affects fundamental cellular processes, including proliferation, apoptosis, and differentiation. In total, activating KRAS and NRAS mutations occur in 20~50% and 1~6% of colorectal cancer respectively, mainly in exons 2, 3 or 4. The mutation status of the RAS gene is relevant to the primary drug resistance of colorectal cancers treated with tyrosine kinase inhibitors. Patients with wild-type KRAS and NRAS gene could benefit from Erbitux (Cetuximab) or Vectibix (Panitumumab), whereas, the patients with mutant KRAS or NRAS gene show poor response to this treatment. The NCCN Clinical Practice Guideline in Oncology for colorectal cancers and European Drug Administration Organization suggest the employment of KRAS and NRAS gene mutation detection prior to the use of targeted medicines Erbitux and Vectibix in the treatment of metastatic colorectal cancer. As a result, KRAS and NRAS mutations detection supplies evidence for targeted clinical therapy of tumor patients, which decreases cost and time of treatment. The AmoyDx KRAS/NRAS Mutations Detection Kit is a real-time PCR test intended for qualitative detection of 19 KRAS mutations (exons 2, 3 and 4) and 13 NRAS mutations (exons 2, 3 and 4). Our company s patented technology allows detection of 1~5% mutant DNA in a background of 95~99% normal DNA at 10 ng sample DNA amount, while ensuring that false negatives are minimized. The purpose of the kit is to aid doctors to identify lung cancer or colorectal cancer patient carrying mutations in KRAS/NRAS gene. Intended Use The AmoyDx KRAS/NRAS Mutations Detection Kit is a highly sensitive test designed to accurately identify 19 KRAS mutations (exons 2, 3 and 4) and 13 NRAS mutations (exons 2, 3 and 4) in human tissue samples. Kit Contents This kit contains sufficient reagents to carry out 6 tests (Table 1). Table 1 Kit Contents Contents KN PCR Reaction Mix KN Enzyme Mix KN Positive Control Volume 8 strips 45μL 250 μl One 12-tube strip is employed for each sample (Table 2). Table 2 Mutation information for each strip Tube No. Mutation Name Volume (μl) Channel KN Reaction Mix 1 35 FAM, HEX/VIC KN Reaction Mix 2 35 FAM, HEX/VIC KN Reaction Mix 3 35 FAM, HEX/VIC KN Reaction Mix 4 35 FAM, HEX/VIC KN Reaction Mix 5 35 FAM, HEX/VIC KN Reaction Mix 6 35 FAM, HEX/VIC 1/8
KN Reaction Mix 7 35 FAM, HEX/VIC KN Reaction Mix 8 35 FAM, HEX/VIC KN Reaction Mix 9 35 FAM, HEX/VIC KN Reaction Mix 10 35 FAM, HEX/VIC KN Reaction Mix 11 35 FAM, HEX/VIC KN External Control Reaction Mix 35 FAM Note: Distinguish Tube from Tube according to the trapezoid end of strip edge, described as follows. Equipment and Reagents Not Supplied With Kit 1. The compatible PCR instruments are: Stratagene Mx3000P /Mx3005P, ABI7500, ABI7900HT and LightCycler480. (1) This kit is compatible with LightCycler480 II instrument. Fluorescence calibration is required for LightCycler480 I instrument. If the fluorescence crossover occurs in the LightCycler480 II instrument, the fluorescence calibration is also needed prior to the operation. To run the assay on a LightCycler machine, please use the Roche 480 adaptor, available from BIOplastics, Cat No. B79480. It s essential to place a 12-tube strip on the first row (Row A) of the adaptor. (2) For ABI instruments please set up as follows: Reporter Dye: FAM, VIC; Quencher Dye: TAMRA; Passive Reference: NONE. (3) To run the assay on ABI 7500 machine, please use the 96-well plate adaptor. (4) To run the assay on ABI7900HT machine, please use the ABI7900 adaptor, available from BIOplastics, Cat No. B7900RAN. (5) For ABI7900HT, please set up as follows: Instrument: Standard. Ramp speed: Standard. Reaction volume: 40 μl. (6) For Stratagene Mx3000P /Mx3005P, if there s low net fluorescence signal (dr) but high background signal (R), please reduce the signal gain setting of instrument properly. (7) We recommend that all PCR instruments in use should be conducted fluorescence calibration once a year. 2. Sterile, nuclease-free tubes. 3. Dedicated pipettes and filtered pipette tips for handling DNA. 4. Sterile, nuclease-free H 2 O. Shipping and Storage The kit requires cold-chain-transportation. All contents of the kit should be stored immediately upon receipt at -20±5. Avoid unnecessary freezing and thawing of the contents of the kit. Do not use the reagent after five freeze-thaw cycles. Once opened, this reagent is stable at -20±5 until the expiry. Stability The shelf-life of the kit is 8 months when stored under the recommended conditions and in the original packaging. Do not use the kit after the stated expiry date. 2/8
Specimen Material Human genomic DNA should be extracted from tissue or formalin-fixed paraffin-embedded (FFPE) samples and stored at -20±5 prior to use. The FFPE samples should be made and stored following proper procedures. The slides should preferably be less than 3 years. Before extraction of DNA, it is very important to make sure that there is at least 30% tumor cells in the tissue samples. If the extracted DNA is not used immediately, it should be stored at below -20 for no more than 6 months. Selection of high quality DNA extraction reagents is essential for the kit. We recommend use of DNA extraction kit (AmoyDx FFPE DNA Kit, Cat No. ADx-FF01, for paraffin embedded specimens; AmoyDx Tissue DNA Kit, Cat No. ADx-TI01, for tissue and pleural effusion specimens). The OD value of DNA samples should be measured using the spectrophotometer after extraction. Make sure A 260 /A 280 value between 1.8 and 2.0. Technological Principles The kit uses ADx-ARMS technology, which comprises novel, specific primers and fluorescent probes to detect mutations and reference gene in a real-time PCR assay. The mutant DNA is amplified accurately by the specific primers, and detected by the novel probes. Highly Specific primers and probes, and a highly validated procedure based on Taq DNA polymerase contribute to outstanding assay sensitivity and selectivity. Protocol 1. The reaction mix tubes contain the reaction buffer, dntps, specific oligos and probes. 2. The KN Reaction Mix 1~11 includes a mutation detection system and an internal control system. The mutation detection system is used to detect the mutation status of KRAS and NRAS gene (positive or negative). The internal control system is designed to detect the presence of inhibitors and monitor the accuracy of experimental operation, which may lead to false negative results. 3. The KN External Control Reaction Mix is used to assess the DNA quality, that is, to reveal the presence of PCR inhibitors or compromised DNA integrity that may lead to false negative results. 4. The KN Positive Control contains a recombinant gene with the 19 KRAS mutations, 13 NRAS mutations, and normal human genomic DNA. 5. The KN Enzyme Mix contains the Taq DNA polymerase for PCR amplification and uracil-n-glycosylase which works at room temperature to prevent PCR amplicon carryover contamination. Experimental Procedure 1. Thaw KN Positive Control at room temperature. When the reagents completely thawed, mix the reagents by inverting the tube 10 times and centrifuge briefly to collect the contents at the bottom of the tube. 2. Centrifuge KN Enzyme Mix prior to use. 3. Add 4.2 μl KN Enzyme Mix into the following solutions individually: 65.8 μl DNA sample (see following for DNA sample concentrations), 65.8 μl KN Positive Control (PC) and 65.8 μl no-template controls (sterile water, NTC). 4. Mix each solution thoroughly by inverting 10 times and then centrifuge briefly. (1) According to different sources, samples can be divided into two groups: paraffin embedded and non-paraffin embedded specimens. (2) Non-paraffin embedded specimens include fresh tissue, frozen pathological sections, non-heparin anticoagulant plasma, serum and non-heparin anticoagulant blood. a) For non-paraffin embedded samples, the recommended DNA amount in each PCR tube is 1.88 ~ 4.7 ng 3/8
(concentration 0.4~1 ng/μl). (3) For paraffin embedded samples, we recommend use of 7.05~14.1 ng template DNA (concentration 1.5~3 ng/μl) in each PCR tube depending on storage times. a) Use 7.05 ng template DNA (concentration 1.5 ng/μl) for samples with less than 3 months storage time. b) Use 9.4 ng template DNA (concentration 2 ng/μl) for samples with storage time between 3 months and 1 year. c) Use 11.75~14.1 ng template DNA (concentration 2.5~3 ng/μl) with storage time between 1 year and 3 years. d) Avoid use DNA for samples with more than 3 years storage time. Note: We recommend use of TE (ph = 8.0) for extracted DNA dilution. Suggest using at least 5 μl DNA for dilution within ten-fold series, to ensure the accuracy of final concentration. Since Enzyme Mix is viscous, please pay attention to the centrifugation and pipetting process. Minimize the contact interface between the pipette tip and Enzyme Mix to avoid adding excess enzyme. Avoid vortexing solutions with Enzyme Mix. 5. Take out the KN PCR Reation Mix strips and centrifuge the strips if there are any reagent droplets in the caps of the PCR tubes. Then briefly uncover the caps prior to use. 6. Transfer 5 μl of the above mixed solution to each PCR tube of 12-tube strip. Add the solution to the side of the tube wall above the reagents in the tube, and then seal the strips. 7. Spin or centrifuge the PCR tubes in order to collect the reagents at the bottom of wells. Note: this spin or centrifuge step is essential for proper mixing of the reagents. 8. Place the PCR tubes into the real-time PCR instrument. A recommended plate layout is shown in Table 3. Note: place the PCR tubes into the real-time PCR instrument and start to run immediately. If not, please store the PCR tubes at 4 for no more than 12 hours. Table 3 Suggested PCR Plate Layout Well 1 2 3 4 5 6 7 8 9 10 11 12 A Sample 1 Sample 1 Sample 1 Sample 1 Sample 1 Sample 1 Sample 1 Sample 1 Sample 1 Sample 1 Sample 1 Sample 1 B Sample 2 Sample 2 Sample 2 Sample 2 Sample 2 Sample 2 Sample 2 Sample 2 Sample 2 Sample 2 Sample 2 Sample 2 C Sample 3 Sample 3 Sample 3 Sample 3 Sample 3 Sample 3 Sample 3 Sample 3 Sample 3 Sample 3 Sample 3 Sample 3 D Sample 4 Sample 4 Sample 4 Sample 4 Sample 4 Sample 4 Sample 4 Sample 4 Sample 4 Sample 4 Sample 4 Sample 4 E Sample 5 Sample 5 Sample 5 Sample 5 Sample 5 Sample 5 Sample 5 Sample 5 Sample 5 Sample 5 Sample 5 Sample 5 F Sample 6 Sample 6 Sample 6 Sample 6 Sample 6 Sample 6 Sample 6 Sample 6 Sample 6 Sample 6 Sample 6 Sample 6 G PC PC PC PC PC PC PC PC PC PC PC PC H NTC NTC NTC NTC NTC NTC NTC NTC NTC NTC NTC NTC 9. Carry out real-time PCR using the cycling conditions described in Table 4. Note: The reaction volume is 40 μl per well. Please pack the post-pcr tubes with two disposable gloves and discard properly. Do NOT open the post-pcr tubes to avoid contamination. 4/8
Table 4 Cycling Parameters Sample Data Analysis Stage Temperature Time Cycles 1 95 5min 1 2 3 95 64 72 93 60 72 5/8 25s 20s 20s 25s 35s Data collection of FAM and HEX/VIC 1. The FAM signals of tubes 1~11 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 adjacent to the KRAS/NRAS gene. 2. Ensure the calibration fluorescence is unselected, and select single mutation detection for each tube accordingly. It is necessary to choose the reaction wells for positive control, no-template control and sample simultaneously. Then users could adjust the threshold of FAM amplification curve, and obtain the Ct value of mutant group. 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, the sample is classed as 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. Assess each NTC Ct value to ensure that there is no positive amplification, if the NTC has positive amplification, the data must be discarded as there is contamination. If the HEX/VIC signal of Tube 1~11 and FAM signal of Tube 12 occasionally rises, further analysis could be carried out. 5. The KN Positive Control FAM Ct value should be less than 20, but variation may occur due to different threshold settings on different instruments. 6. If the HEX/VIC signals of a sample fail but the FAM signal works well, continue with the analysis. If both the HEX/VIC and FAM signals fail, the obtained data must be discarded and the experiment should be repeated. 7. Check the FAM signal of the external control assay: (1) Ct value should be between 15~21. (2) If the requirements of item (1) are satisfied, further analysis shall be carried out. However, if Ct value is less than 15, it indicates the DNA is overloaded, so the amount of DNA should be reduced. If all the results of 11 tubes (1~11) are negative, the sample is classified as negative. (3) If the external control assay fails, it shows that the DNA template contains PCR inhibitors, indicating that the DNA needs to be re-extracted. If the sample FAM Ct value in any of the 11 tubes (1~11) is less than 26, the sample is classified as positive. 8. Analysis of mutation assay results (see Table 5): 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. (1) Strong Positive: if the sample FAM Ct value is less than 26, the sample is classified as strong positive. (2) Weak Positive: if the sample FAM Ct value is in the range shown in the Weak Positive in Table 5, the 20s 15 31
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. If the Ct value is less than the corresponding Ct Cut-off value, the sample is confirmed as weak positive. If the Ct value is greater than the Ct Cut-off value, the sample is classified as negative or below the limits of the kit. b) The calculation of Ct: Ct = mutant FAM Ct value external control FAM Ct value. The mutant FAM Ct value indicates the Ct value of the mutant FAM signal from a sample. The external control FAM Ct value indicates the Ct value of the FAM signal in external control tube of that sample. (3) Negative: if the sample FAM Ct value is greater than or equal to the critical negative value shown in the Negative in Table 5, the sample is classified as negative or below the detection limit of the kit. Table 5 Results Determination Strong Positive Weak Positive Negative Tube No. 1 2 3 4 5 6 7 8 9 10 11 Mutant Ct Value Ct <26 Ct <26 Ct <26 Ct <26 Ct <26 Ct <26 Ct <26 Ct <26 Ct <26 Ct <26 Ct <26 Mutant Ct Value 26 Ct <28 26 Ct <29 26 Ct <29 26 Ct <29 26 Ct <29 26 Ct <29 26 Ct <28 26 Ct <29 26 Ct <29 26 Ct <28 26 Ct <29 Ct Cut-off value 9 9 9 9 10 9 9 10 10 9 9 Mutant Ct Value Ct 28 Ct 29 Ct 29 Ct 29 Ct 29 Ct 29 Ct 28 Ct 29 Ct 29 Ct 28 Ct 29 9. The sample may contain two or more mutations simultaneously. Performance Characteristics The performance characteristics of this kit were validated on Stratagene Mx3000P /Mx3005P, ABI7500, ABI7900HT and LightCycler480. 1. Sensitivity: (1) For Stratagene Mx3000P /Mx3005P, the kit allows detection of 1% mutant DNA in a background of 99% normal DNA at 10 ng sample DNA amount. (2) For other PCR instruments, the kit allows detection of 1% mutant DNA in a background of 99% normal DNA at 10 ng sample DNA amount. Except: the sensitivity of NRAS-M1, NRAS-M2 and NRAS-M12 mutations are 2% at 10 ng sample DNA amount, the sensitivity of KRAS-M22 and KRAS-M23 mutations are 5% at 10 ng sample DNA amount. 2. Productivity: the kit can be used to analysis 6 samples maximum. (see Table 6) Table 6 Sample Qty detected with the Kit PCR Run(s) Control Qty Total samples can be detected 1 run 2 (2 controls/run * 1) 6 2 runs 4 (2 controls/run * 2) 4 3. Accuracy: Accuracy of the kit was established by testing 32 mutant positive reference controls and 10 negative reference controls, the detection concordance rate are 100%. 4. Precision: Precision of the kit was established by performing a certain mutant positive reference control for 10 repeats; all the controls can be detected. 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 6/8
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 manufacturer. 5. Using other sources of reagents is not recommended. Strictly distinguish the reagents from Positive Control 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, use separate, dedicated pipettes and filtered pipette tips to add DNA template 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 could use this kit. Please wear suitable lab coat and disposable gloves. The used kit should be disposed properly. 9. The kits can t detect KRAS and NRAS mutations other than those listed in Appendix I. 10. Wipe down the work area, spray down the pipettes and equipment with 75% ethanol or 10% hypochlorous acid. 11. The flow of tubes, racks, pipets and other equipment used should be from pre-amplification to post-amplification, and never backwards. Notes 1. Symbol for "AUTHORISED REPRESENTATIVE IN THE EUROPEAN COMMUNITY" 2. Symbol for "IN VITRO DIAGNOSTIC MEDICAL DEVICE" 3. Symbol for "KEEP DRY" 4. Symbol for "THIS WAY UP" 5. Symbol for "FRAGILE HANDLE WITH CARE" Information of European Authorised Representative Wellkang Ltd t/a Wellkang Tech Consulting Suite B, 29 Harley Street, London W1G 9QR United Kingdom References 1. Douillard JY, Oliner KS, Siena S, et al. 2013. Panitumumab-FOLFOX4 Treatment and RAS Mutations in Colorectal Cancer. The New England Journal of Medicine, 369 (11):1023-1034. 2. Di M, Pietrantonio F, Perrone F, et al. 2013. Lack of KRAS, NRAS, BRAF and TP53 mutations improves outcome of elderly metastatic colorectal cancer patients treated with cetuximab, oxaliplatin and UFT. Targeted Oncology, 1-8. 3. Roock WD, Claes Bart, Bernasconi D, et al. 2010. Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: a retrospective consortium analysis. Lancet Oncol, 11: 753-762. 7/8
Appendix I KRAS and NRAS Mutations detected with the Kit Tube No. Reagent Supplied Exon Mutation Base change Cosmic ID Name 1 KN Reaction Mix 1 KRAS 2 G12S 34G>A 517 KRAS-M4 G12D 35G>A 521 KRAS-M1 G12C 34G>T 516 KRAS-M6 G12R 34G>C 518 KRAS-M5 2 KN Reaction Mix 2 KRAS 2 G12V 35G>T 520 KRAS-M3 G12A 35G>C 522 KRAS-M2 G13C 37G>T 527 KRAS-M14 3 KN Reaction Mix 3 KRAS 2 G13D 38G>A 532 KRAS-M7 4 KN Reaction Mix 4 KRAS 3 A59T 175G>A 546 KRAS-M25 Q61K 181C>A 549 KRAS-M19 Q61L 182A>T 553 KRAS-M15 5 KN Reaction Mix 5 KRAS 4 Q61R 182A>G 552 KRAS-M16 Q61H 183A>C 554 KRAS-M17 Q61H 183A>T 555 KRAS-M18 K117N 351A>C 19940 KRAS-M20 K117N 351A>T 28519 KRAS-M21 6 KN Reaction Mix 6 KRAS 4 Al46T 436G>A 19404 KRAS-M22 A146V 437C>T 19900 KRAS-M23 A146P 436G>C 19905 KRAS-M24 7 KN Reaction Mix 7 NRAS 2 G12D 35G>A 564 NRAS-M3 G12S 34G>A 563 NRAS-M10 8 KN Reaction Mix 8 NRAS 2 G13D 38G>A 573 NRAS-M4 G13R 37G>C 569 NRAS-M6 G12C 34G>T 562 NRAS-M7 9 KN Reaction Mix 9 NRAS 2 G12V 35G>T 566 NRAS-M9 G12A 35G>C 565 NRAS-M11 G13V 38G>T 574 NRAS-M14 Q61R 182A>G 584 NRAS-M1 10 KN Reaction Mix 10 NRAS 3 Q61K 181C>A 580 NRAS-M2 Q61L 182A>T 583 NRAS-M5 Q61H 183A>C 586 NRAS-M8 11 KN Reaction Mix 11 NRAS 4 A146T 436G>A 27174 NRAS-M12 8/8