TheraScreen®: K-RAS Mutation Kit
Contents
1. ABI 7500 Breakthrough has been assessed using pooled cell line DNA from K RAS mutation negative cell lines at 3 different levels of DNA Three identical PCR plates were tested Each plate included 3 lots of reagents The Mixed Standard was used as the positive control In addition positive FFPE samples were tested in duplicate The majority of mutation positive samples are positive for one mutation only The other mutation assays can be used to assess breakthrough although some cross reactivity is expected between some assays and positive mutations However this produces a known pattern Ct values and ACt values have been obtained and compared to the cut off values The data from cell line DNA and FFPE samples gave ACt values that were all above the I cut off values apart from one FFPE sample that gave a ACt just below 6 84 the cut off of 7 5 As the 1 cut off determination data range covered 6 84 the 1 ACt value for the 12VAL assay was changed from 7 5 to 6 5 to avoid the possibility of a false positive result Table 18 below shows the current ACt values based on the Breakthrough validation results Page 28 of 38 For Diagnostic Use Only TheraScreen K RAS Mutation Kit Table 18 Breakthrough Validation Results 1 ACt values Precision Validation LightCycler 480 Instrument Precision was tested using 1 synthetic controls in 3 different levels of K RAS mutation negative2. 6 3 The Ct and ACt values are reported 6 4 The following Flags Warnings are reported for the run controls in columns and 2 Table 8 LightCycler Adapt Software Flags Warnings for the Run Controls Flags Warning Meaning CT_OUT_OF_RANGE FAM Ct for Control assay is out of range EXO FAIL For the mixed standard the exogenous control Ct lt 30 or is negative when the FAM result is negative DELTA_CT OUT OF RANGE Mutation ACt values are outside of the set range EXO_CONTROL_INVALID The exogenous control reaction has failed in an NTC TARGET_CHANNEL_INVALID The FAM reaction has a positive Ct value in an NTC Page 7 of 38 For Diagnostic Use Only TheraScreen K RAS Mutation Kit 6 5 6 6 6 4 1 The meanings of the Flags Warnings are given in more detail below 6 4 1 1 CT _OUT_OF_RANGE The control assay for the mixed standard must have a Ct value of 26 60 2 If the assay is out of this range this Flag Warning is displayed for all mixed standard wells as ACt values are not calculated and the mixed standard is invalid This indicates that the control assay is not working correctly 6 4 1 2 EXO_FAIL This Flag Warning is displayed if the exogenous control assay in any of the mixed standard wells is below 30 as this may indicate that PCR contamination is present in this mix If the exogenous control assay fails when a FAM reaction fails in any of the mixed standard wells the EXO_FAIL Flag Warning is
3. Page 22 of 38 For Diagnostic Use Only TheraScreen K RAS Mutation Kit Table 12 ABI7500 K RAS Plate Layout 96 well layout Aswy _ 47 5 6 7 8 9 0 Control standard A Mixed Sample Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 2 ALA standard Mixed Samplel Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 2ASP standard C Mixed Samplel Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 l 2ARG standard D Mixed Samplel Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 2CYS standard E Mixed Samplel Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 l 2SER standard F Mixed Samplel Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 2VAL standard G Mixed Sample Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 3ASP standard H Mixed Sample Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 7 Seal and spin the PCR plate briefly to collect the reagents at the bottom of the wells 8 Immediately place the plate into the ABI7500 instrument ABI7500 Instrument Setup Select the 7500 System Software icon
4. Purchase of this product is accompanied by a limited non transferable licence to use ARMS and Scorpions technologies for in vitro diagnostic use only ARMS is covered by US patent 5 595 890 and EP 332435 Scorpions by US patent 6 326 145 and EP1088102 Information in this document is subject to change DxS Limited assumes no responsibility for any errors that may appear in this document In no event shall DxS Limited be liable in any way whether in contract tort including negligence or otherwise for any claim arising in connection with or from the use of this product Nothing in this document excludes or limits any liability which it is illegal for DxS Limited to exclude or limit Copyright 2009 DxS Limited All rights reserved DxS Limited 48 Grafton Street Manchester M13 9XX UK www dxsdiagnostics com Page 38 of 38
5. 10 Standard curves were set up using a 5 fold serial dilution and one PCR plate containing three batches of kit reagents was run for each assay The 100 mutation standards were tested at 50 ng 10 ng 2 ng and 0 4 ng of DNA on each of the mutation assays by diluting into water The mutation assays were also tested using a 5 fold serial dilution into 10 ng ul of cell line DNA to assess the effect of breakthrough Page 31 of 38 For Diagnostic Use Only TheraScreen K RAS Mutation Kit Standard curves were generated for each mutation assay from the experimental data The PCR efficiencies were calculated using the equation 00 ores The higher level of breakthrough in the 10 ng ul cell line dilutions gave efficiencies of above 100 Overall efficiencies across reagent batches were calculated All values were similar and within 10 of the control assay efficiency Parallel standard curves between the control assay and mutation assays means that the ACt method of analysis is suitable for use Linearity was maintained acceptably over the range of 0 4 ng DNA to 50 ng DNA Cross reactivity Validation LightCycler 480 Instrument Cross reactivity validation has not been performed since the LightCycler Adapt Software will only report a single mutation with the smallest ACt ABI7500 Since all the mutations detected by the K RAS Kit occur with a 5 base pair region it is expected that some cross reactivity between prim
6. LightCycler 480 Instrument These studies determined the tolerance of the mutation assays to variations in cycling temperature Since the annealing temperature is the most critical temperature that affects assay performance the annealing temperature is the parameter that was varied Cycling tolerance has been assessed using 1 synthetic standards in 3 different levels of DNA For each mutation these samples have been run in triplicate alongside duplicate mixed standard and single NTC reactions to ensure that the assays were performing within the specifications Each plate was tested at 59 C 60 C and 61 C annealing temperature 60 C is the optimal temperature for the assay to be run at but variation across or between PCR blocks mean that the assay must be robust over the range given Each plate was run in triplicate at each temperature Ct values were obtained from the LightCycler Adapt Software and all combinations of control and mutation Ct values for each sample were used to calculate ACt values All assays met the acceptance criteria that the values at 59 C and 61 C must not differ from the mean value at 60 C the standard deviation at 60 C This indicates that the assays are robust enough to withstand a I C deviation in annealing temperature ABI7500 Three identical PCR plates each containing 3 kit reagent lots were run at each annealing temperature The Mixed Standard was tested as was 50 ng of pooled mutation negative c
7. once thawed to avoid localised concentrations of salts Prepare sufficient mixes for the DNA samples the mixed standard and no template controls plus an excess of 2 reactions per mix as shown in Table 5 Table 5 K RAS Master Mix volumes Master Mixes Assay Reaction Mix ul Zag ul per plate per plate x14 x14 Reaction Mix ul xl Taq ul x Control assay 19 8 0 2 277 2 2 8 Mutation assays 19 8 0 2 277 7 2 8 2 DO NOT vortex the Taq or any reaction mixes that contain Tag as this may cause inactivation of the enzyme 3 Ensure that the 7aq is at room temperature before use Spin the vial to ensure that all the 7aq is collected at the bottom then pipette by placing the pipette tip just under the surface of the 7ag to minimise the risk of the tip getting coated in excess 7ag 4 Mix the master mixes by gently pipetting up and down 5 Immediately add 20 ul of the master mixes to the reaction wells 6 Immediately add 5 ul of sample mixed standard or water for the no template controls to the reaction wells Each DNA sample must be tested with both the control and all mutation assays The plate setup is given in Table 6 Page 14 of 38 For Diagnostic Use Only TheraScreen K RAS Mutation Kit Table 6 K RAS Kit Plate Layout 96 well layout wy _ 47 5 6 7 8 9 0 A Mixed Control standard Sample Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Samp
8. Note The cross reactivity pattern may be different on DNA samples e g paraffin embedded DNA samples The K RAS Kit is designed to detect one mutation ina DNA sample If a second mutation assay gives a positive result this is likely to be cross reactivity However although rare double mutants have been observed If the pattern does not fit the cross reactivity pattern further investigation may be required l Assay Performance Characteristics Assay performance has been characterised for the K RAS Kit on the ABI7500 and the LightCycler 480 Instrument using the LightCycler Adapt Software to obtain Ct values A variety of tests have been performed on each instrument These results of these tests are summarised below 1 Cut off Validation LightCycler 480 Instrument The detection of 1 mutation in a background of K RAS mutation negative cell line DNA was determined over a range of three different concentrations of cell line DNA to assign a cut off ACt value for each mutation assay For each assay 1 standards were tested in triplicate on 3 separate runs for both the control assay and mutation assay This was repeated by 3 different operators Ct values were obtained from the LightCycler Adapt Software The 1 ACt values were calculated from all combinations of mutation and control Ct values over the replicates within a run Page 26 of 38 For Diagnostic Use Only TheraScreen K RAS Mutation Kit The cut off ACt valu
9. occurs Scorpions Detection of amplification is performed using Scorpions Scorpions are bi functional molecules containing a PCR primer covalently linked to a probe The fluorophore in this probe interacts with a quencher also incorporated into the probe which reduces fluorescence During a PCR reaction when the probe binds to the amplicon the fluorophore and quencher become separated This leads to an increase in fluorescence from the reaction tube Data Analysis ACt Method Scorpions real time assays use the number of PCR cycles necessary to detect a fluorescent signal above a background signal as a measure of the target molecules present at the beginning of the reaction The point at which the signal is detected above background fluorescence is called the cycle threshold Ct Sample ACt values are calculated as the difference between the mutation assay Ct and control assay Ct from the same sample Samples are classed as mutation positive if they give a ACt less than the 1 ACt value for that assay Page 4 of 38 For Diagnostic Use Only TheraScreen K RAS Mutation Kit Above this value the sample may either contain less than 1 mutation beyond the limit of the assays or the sample is mutation negative When using ARMS primers some inefficient priming may occur giving a very late background Ct from DNA not containing a mutation All ACt values calculated from background amplification will be greater than t
10. 38 the DNA is limiting and data must not be used Page 24 of 38 For Diagnostic Use Only TheraScreen K RAS Mutation Kit 2 Mixed Standard ACt values 2 1 The mixed standard ACt values should be as given in Table 14 but variations of 2 may occur due to different threshold settings on different instruments Table 14 ABI7500 Mixed Standard ACt values and Cut off Points Mixed Standard Mixed Standard Sample 1 ACt acceptable range ACt 0 70 2 70 to 1 30 6 5 1 04 3 04 to 0 96 8 0 02 2 02 to 1 98 8 0 98 2 98 to 1 02 7 0 02 1 98 to 2 02 9 0 19 2 19 to 1 81 6 5 1 12 3 12 to 0 88 9 3 Sample ACt values 3 1 If the sample ACt value is greater than the I value as indicated in Table 14 the DNA sample is classed as mutation negative or below the limits of the K RAS Kit 3 2 If the sample ACt is less than the 1 value the DNA sample is classed mutation positive 3 3 Mutation Ct values of 38 or above must be scored as negative or below the limits of the kit 10 Limitations of the Test It is necessary to have a sample control assay Ct of lt 29 on the ABI7500 and 28 9 on the LightCycler 480 Instrument to detect 1 mutation in a background of wild type DNA The assays will not be able to detect mutation in samples with greater control Ct values and sensitivity of mutation detection will decrease as the control Ct increases above these values The Control Ct val
11. 4 of the K RAS gene The primers and probe have been designed to avoid any known K RAS polymorphisms Mutation assays Each mutation assay labelled with FAM contains one Scorpion plus one ARMS primer for discrimination between the wild type DNA and the mutant DNA detected by a real time PCR assay Page 5 of 38 For Diagnostic Use Only TheraScreen K RAS Mutation Kit 4 Reagents This K RAS Kit contains sufficient reagents to perform quality assessment of samples and run the K RAS assays for up to 20 or 80 reactions dependent on kit size Kit Size DxS Product Code Roche Diagnostics Ordering Number 20 Reactions KR 21 05366216190 80 Reactions KR 22 05366224190 The number of samples that can be tested is dependent on sample batch size Table 2 K RAS Kit Contents 20 reactions 80 reactions Volume Volume Control Reaction Mix 1300 ul 5200 ul I2ALA Reaction Mix 650 ul 2600 ul 12ASP Reaction Mix 650 ul 2600 ul 12ARG Reaction Mix 650 ul 2600 ul 12CYS Reaction Mix 650 ul 2600 ul I2SER Reaction Mix 650 ul 2600 ul I2VAL Reaction Mix 650 ul 2600 ul 13ASP Reaction Mix 650 ul 2600 ul Mixed standard 300 ul 1000 ul polymerase 60 ul 240 ul Reagents Supplied Equipment and Reagents Not Supplied With K RAS Kit The user will require the following equipment and consumables e The LightCycler 480 Instrument or the ABI7500 Real time PCR Machine capabl
12. Ah Im Kyung Hun Lee Jee Hyun Kim Dong Wan Kim Dae Seog Heo Noe Kyeong Kim Doo Hyun Chung Yung Jue Bang 2006 Optimization of Patient Selection for Gefitinib in Non Small Cell Lung Cancer by combined analysis of Epidermal Growth Factor Receptor Mutation K ras Mutation and AKT Phosphorylation Clin Cancer Res 2 8 2538 2544 William Pao Theresa Y Wang Gregory J Riely Vincent A Miller Qiulu Pan Marc Ladanyi Maureen SF Zakowski Robert T Heelan Mark G Kris Harold E Varmus 2005 KRAS Mutations and Primary Resistance of Lung Adenocarcinomas to Gefitinib or Erlotinib PloS Medicine 2 1 57 61 Astrid Lievre Jean Baptiste Bachet Delphine Le Corre Valerie Boige Bruno Landi Jean Francois Cote Gorana Tomasic Christophe Penna Michel Ducreux Philippe Rougier Frederique Penault Llorca Pierre Laurent Puig 2006 KRAS Mutation Status is Predictive of Response to Cetuximab Therapy in Colorectal Cancer Cancer Res 66 8 3992 3995 Silvia Benvenuti Andrea Sartore Bianchi Federica Di Nicolantonio Carlo Zanon Mauro Moroni Silvio Veronese Salvatore Siena Alberto Bardelli 2007 Cancer Res 67 6 2643 2648 W De Roock J De Schutter G De Hertogh M Jannsens B Biesmans N Personeni K Geboes C Verslyp E Van Cutsem S Tejpar 2007 Journal of Clinical Oncology 25 18S 4132 G Finocchiaro F Cappuzzo P A Janne K Bencardino C Carnaghi W A Franklin M Roncalli L Crino A Santoro M Varell
13. also displayed This indicates that there is a problem with this assay which could lead to false negative results The mixed standard is already invalidated due to the FAM failure 6 4 1 3 DELTA_CT_OUT_OF_RANGE If the Mixed Standard ACt values are within 2 00 of the values given in Table 7 the software will report a valid status If the ACt is beyond the expected range the status will be invalid and this Flag Warning will be displayed This indicates a problem with an assay that could lead to false results 6 4 1 4 EXO _CONTROL_INVALID In the no template control wells the exogenous control assay must give a positive result in all 8 wells Ct lt 41 If a negative result is obtained this Flag Warning is displayed and an invalid status shown This indicates a problem with the assay which could lead to false results 6 4 1 5 TARGET CHANNEL_INVALID In the 8 no template control wells the FAM result must be negative Ct gt 38 Any amplification indicates contamination A positive result will lead to this Flag Warning being displayed and will invalidate the no template control In the case of an invalid status for any of the mixed standard wells or the no template control wells the Run Summary Result will be Run Invalid In the Sample Result Table sample names and control Ct values will be reported but the mutation status will be reported as invalid The mixed standard indicates that all assays are working correctly If thi
14. for individual instruments and the overall mean were 0 097 and the difference between means for individual instruments was no worse than 0 186 demonstrating that the inter instrument variation was no greater than the variation within the whole data set and the assays are robust enough to withstand inter instrument variability Page 29 of 38 For Diagnostic Use Only TheraScreen K RAS Mutation Kit For every test the biggest difference between a single lot and the overall mean was 0 195 and the worst inter lot differences were 0 38 demonstrating that the inter lot variation was no greater than the variation within the whole data set and the assays are robust enough to withstand inter lot variability ABI7500 Experiments were performed to establish the precision performance of each assay by repeating one PCR plate with a range of samples at high and low DNA concentration and high and low mutation level for 3 reagent lots within a day between days between operators and between instruments A one way ANOVA was used to assess the variation between replicates lots of reagent runs day of testing instrument and operator The null hypothesis was that the mean values across categories were equal Total means SD and CV were also calculated for the Ct values None of the assays showed significant deviation at the p 0 05 level from the null hypothesis of the means being equal across categories This indicates that the assays are robu
15. on the desktop of the workstation attached to the instrument Open a new run file from the file menu using the 7500 Sequence Detection Software version 1 4 Under Assay select Standard Curve Absolute Quantification Under Run Mode select Standard 7500 Move to the detector setup window by selecting the Next button Add a FAM and a JOE detector with quenchers set to none to the detectors list If these detectors do not already exist select the New Detectors button and set up a FAM and a JOE detector with the quencher set to none Set the Passive Reference to None and select the Next button Select the entire plate and check the tick boxes for the FAM and JOE detectors to ensure that both dyes are monitored in each well Select the Finish button Under the Instrument tab set up the cycling as given in Table 13 Page 23 of 38 For Diagnostic Use Only TheraScreen K RAS Mutation Kit Tablel3 ABI7500 Cycling Conditions Temperature Data Collection Stage 95 C Stage 2 95 C 60 C 7 Select the Start button to save the experiment and start the cycling ABI7500 Sample Analysis Ensure that the passive reference is set to none in the well inspector screen 2 Check that each well gives a JOE signal from the exogenous control assay a b If the exogenous control assay gives a positive
16. result continue with the analysis If the exogenous control assay has failed but the FAM reaction has amplified strongly continue with the analysis as the FAM reaction has out competed the exogenous control reaction If both the FAM and exogenous control reactions have failed the data must be discarded as there may be inhibitors present These inhibitors could lead to false negative results 3 In the Amplification Plot tab select all the wells in use and select the FAM dye from the detector drop down menu Use the automatic baseline setting and manual Ct and then set the threshold manually in the middle of the exponential phase using the log scale for the Y axis as described in the ABI 7500 user guide 4 Analyse the data and calculate the ACt value as follows sample mutation assay Ct sample control assay Ct ACt Data can be exported to Microsoft Excel for ease of analysis ABI7500 Data Interpretation Control Ct values I I 1 2 1 3 1 4 The control Ct value must be 24 to avoid overloading the assay Control Ct lt 29 the K RAS Kit is capable of detecting as little as 1 mutation in these samples In samples with a control Ct gt 29 the kit will not detect as low as 1 mutation but will still be able to detect higher level mutations Control Ct 35 only a few amplifiable copies of DNA are present in the sample and mutations are only likely to be seen if most copies are mutated Control Ct
17. technology This method is highly selective Providing there are enough copies of DNA detection of approximately 1 mutant in a background of wild type genomic DNA is possible The K RAS Kit will detect seven K RAS mutations in codons 12 and 13 of the K RAS oncogene as shown in Table I Page 3 of 38 For Diagnostic Use Only TheraScreen K RAS Mutation Kit Table K RAS Mutations Detected by the DxS Kit COSMIC ID s are taken from the Catalogue of Somatic Mutations in Cancer http www sanger ac uk genetics CGP cosmic Mutation Base Change Cosmic ID Gly 2Ala GGT gt GCT 522 Gly 12Asp GGT gt GAT 521 Gly12Arg GGT gt CGT 518 Glyl2Cys GGT gt TGT 516 Glyl2Ser GGT gt AGT 517 Glyl2Val GGT gt GTT 520 Glyl3Asp GGC gt GAC 532 3 Technological Principles The K RAS Kit combines two technologies ARMS and Scorpions 12 13 to detect mutations in real time PCR assays ARMS Allele or mutation specific amplification is achieved by ARMS 7ag DNA polymerase is extremely effective at distinguishing between a match and a mismatch at the 3 end of a PCR primer Specific mutated sequences can be selectively amplified even in samples where the majority of the sequences do not carry the mutation as e When the primer is fully matched the amplification proceeds with full efficiency e When the 3 base is mismatched only low level background amplification
18. the 7ag to minimise the risk of the tip getting coated in excess Tag 5 Mix the master mix by gently pipetting up and down 6 Immediately add 20 ul of the control master mix to each of the reaction wells 7 Immediately add 5 ul of sample mixed standard or water for the no template controls to the reaction wells 8 The plate must be set up with the mixed standard added to well Al and no template control water added to A2 All other wells in use must contain the samples 9 Seal and spin the PCR plate briefly to collect the reagents at the bottom of the wells Page 10 of 38 For Diagnostic Use Only TheraScreen K RAS Mutation Kit 10 Follow the instrument setup for the appropriate platform Sample Assessment Protocol LightCycler 480 Instrument Setup Immediately place the plate into the LightCycler 480 Instrument 2 Select the LightCycler 480 Software icon on the desktop of the workstation attached to the instrument Log into the software and from the Overview screen select New Experiment from Template From the run templates listed choose K RAS LC480II Run Template This template will have the following parameters I Detection Format is DxS IVD Assays 2 Reaction Volume is 25 3 Arn initial hold step at 95 C for 4 minutes 4 A 2 step amplification for 45 cycles with a denaturation at 95 C for 30 seconds and annealing at 60 C for minute The fluorescence acquisition
19. which is a standalone computer i e not part of a network The use of any other computer is prohibited as the software may not perform as intended Open the LightCycler Adapt Software by clicking on the LightCycler Adapt Software icon on the desktop of the workstation and fill in the username field This name will be entered as the report author In the main window use the browser button to select a single LightCycler 480 Instrument run file To remove a selection the software must be closed and re opened Select a report type pdf or csv If csv is chosen then a pdf version is automatically created in addition Select Analyse to create the results report The report is saved automatically in the folder containing the run file and is given the same name as the run file The report will be displayed automatically The reportable result is displayed in the Mutation Status column of the Sample Result Table LightCycler Adapt Software Report Interpretation The LightCycler Adapt Software report contains general information about the analysis 1 1 The report author is the person who has run the software and created the report 1 2 The date and time of analysis is given The report gives an analysis overview detailing the run file name used plus the algorithm definition file and algorithm sequence The algorithm details are fixed and cannot be altered The resu
20. If the exogenous control assay Ct is lt 30 in any of the 8 wells an EXO_FAIL Flag Warning will be displayed and the status will be invalid It is possible that there is PCR product contamination in this assay f The LightCycler 480 Instrument run file can be checked to establish the possible cause of exogenous control assay fails If all the exogenous control reactions have failed in a column an inhibitor could be present If it has failed in just well there may be a plate setup problem 7 5 2 5 FAIL A FAIL Flag Warning is displayed when the software is unable to determine an amplification curve as positive or negative for example if the curve shape is abnormal 7 5 3 The LightCycler Adapt Software checks that the sample name within a column is identical in order to ensure that the mutation and control assay Ct values used to calculate ACt values are from the same sample The sample names are taken from the LightCycler 480 Instrument sample editor screen in the experiment run file If there is a mismatch down the column the software reports SAMPLE MISMATCH in the Sample Name column in the Sample Result Table In the plate layout the software reports the sample name followed by MISMATCH If this is a typing error the name can be corrected in the LightCycler 480 Instrument run file and the data reanalysed with the LightCycler Adapt Software Page 21 of 38 For Diagnostic Use Only TheraScreen K RAS Mutation Kit ABI7500 Ex
21. Instrument sample assessment section for details 3 Select the Sample Editor tab and under Step l Select Workflow select the Abs Quant tick box Under Select Filter Combinations ensure both filters are selected 465 510 nm and 533 580 nm Set up the samples names under Step 2 and Step 3 In column the sample name must be Mixed Standard In column 2 the sample name must be NTC In columns 3 12 sample names should be entered The names must be identical for all wells in column The Quantification Sample Type should be set as unknown The replicate column should be left blank as replicates are not taken into account by the LightCycler Adapt Software 4 Select the Experiment button and click the Start Run button to save the experiment and start the cycling Page I5 of 38 For Diagnostic Use Only TheraScreen K RAS Mutation Kit LightCycler 480 Instrument Sample Analysis On completion of the LightCycler 480 Instrument run using the navigator window export the experiment ixo file to a suitable location that can be accessed by the LightCycler Adapt Software IMPORTANT The LightCycler Adapt Software is validated for use on a single computer system which is defined as a single control unit workstation provided by Roche Diagnostics for use with a single LightCycler 480 Instrument II This validation of the LightCycler Adapt Software is currently only for the control unit
22. Kit must be used to assess the total DNA in a sample The control assay amplifies a region of exon 4 of the K RAS gene Samples should be set up with only the control assay using the mixed standard as a positive control and water as the no template control Note that to obtain the optimal use of the reagents in the K RAS kit samples should be batched All experimental runs must contain controls If samples are tested individually this will use up more reagents and reduce the number of samples that can be tested with the K RAS Kit Sample Assessment Protocol Plate Setup Thaw the control reaction mix and mixed standard from the K RAS Kit at room temperature Mix each solution by inverting each tube 10 times once thawed to avoid localised concentrations of salts Prepare sufficient mixes for the DNA samples one mixed standard reaction and one no template control reaction plus an excess of 2 reactions 2 To make the master mix use the amounts of reagents per reaction as given in Table 3 Table 3 Control Assay Master Mix Volumes Master Mix Reaction Mix ul xl Tag ul x Control assay 19 8 0 2 Assay 3 DO NOT vortex the Taq or any reaction mixes that contain Tag as this may cause inactivation of the enzyme 4 Ensure that the Tag is at room temperature before use Spin the vial to ensure all the 7aq is collected at the bottom of the vial then pipette by placing the pipette tip just under the surface of
23. NITED Roche Diagnostics GERMANY Roche Diagnostics GmbH KINGDOM Charles Avenue Sandhoferstr 116 Burgess Hill Dept VM G West Sussex Mannheim D 68298 United Kingdom RHI5 9RY FRANCE Roche Diagnostics S A SWITZERLAND Roche Diagnostics Schweiz 2 Avenue du Vercors AG BP 59 Meylan Cedex 38240 Industriestr 7 Rotkreuz CH 6343 SPAIN Roche Diagnostics S L ITALY Roche Diagnostics Italy Av de la Generalitat s n Sant Cugat del Valles Barcelona E 08190 V le G B Stucchi 110 Monza MI I 20052 If you have purchased this kit in a country which is not included in the list of Roche Diagnostics offices above please contact Roche Diagnostics GmbH in Mannheim at the address given below MAIN DISTRIBUTION CENTRE Roche Diagnostics GmbH Sandhofer Strasse 16 68305 Mannheim 14 Date of issuance of last revision Date of Last Version January 2009 Change Summary Extensively re written to incorporate details for use on the Roche LightCycler 480 Real Time PCR System Instrument Il Page 35 of 38 For Diagnostic Use Only TheraScreen K RAS Mutation Kit 15 References 2 R A Hilger M E Scheulen D Strumberg 2002 The Ras Raf MEK ERK Pathway in the Treatment of Cancer Onkologie 25 511 518 Pavan Bachireddy Pavan K Bendapudi Dean W Felsher 2005 Getting at MYC through RAS Clin Cancer Res 1 12 4278 4281 Sae Won Han Tae You Kim Yoon Kyung Jeon Pil Gyu Hwang Seock
24. TheraScreen molecular diagnostics for personalised medicine TheraScreen K RAS Mutation Kit For the Detection of 7 Mutations in the K RAS Gene For use on the Roche LightCycler 480 Real Time PCR System Instrument II Catalogue 05015278001 And the Applied BioSystems 7500 Real Time PCR System Part Number 4351105 Includes the user manual for the LightCycler Adapt Software vl from Roche Diagnostics Catalogue 05474914001 for the TheraScreen K RAS Mutation Kit CE IVD Instructions for Use Product Codes Kit Size DxS Product Code Roche Diagnostics Ordering Number 20 Reactions KR 21 05366216190 80 Reactions KR 22 05366224190 Instructions Version DU00If Date of Revision February 2009 Store at 18 C to 25 C Diagnostic Innovations Page of 38 For Diagnostic Use Only TheraScreen K RAS Mutation Kit Contents l Intended Use Indications for Use u cccccsscssessssssscscsssssssssssscssesessssscscsssssssssseseeees 3 2 Summary and Explanation of the Test esessseeseseesereseseeesseeureeosseresseeererseesseressereresees 3 3 Technological Principles icamsnieniaintichinantacmsmamawanieaiaiiialusiads 4 Mh OPEC cesresrscucsinadi comesersaoauinasan aun EAN EENEN EOE 6 5 WARNINGS AND PRECAUTIONS cccccccsccsscssssesssssssscsssssssssssssssscsssssessseeseseess 7 6 Storage Stability and Shipping Conditions iscascsiessssscssscscsatecsssressessviessassccreesnsicane
25. a Garcia 2007 Journal of Clinical Oncology 25 18S 4021 F Di Fiore F Blanchard F Charbonnier F Le Pessot A Lamy M P Galais L Bastit A Killian R Sesboue J J Tuech A M Queuniet B Paillot J C Sabourin F Michot P Michel T Frebourg 2007 British Journal of Cancer 96 166 1169 Shirin Khambata Ford Christopher R Garrett Neal J Meropol Mark Basik Christopher T Harbison Shujian Wu Tai W Wong Xin Huang Chris H Takimoto Andrew K Godwin Benjamin R Tan Smitha S Krishnamurthi Howard A Burris Ill Elizabeth A Poplin Manuel Hidalgo Jose Baselga Edwin A Clark David J Mauro 2007 Journal of Clinical Oncology 25 22 3230 3237 Newton CR Graham A Heptinstall LE Powell SJ Summers C et al 1989 Analysis of any point mutation in DNA The amplification refractory mutation system ARMS Nucleic Acids Res 17 7 2503 16 Whitcombe D Theaker J Guy S P Brown T Little S 1999 Detection of PCR products using self probing amplicons and fluorescence Nature Biotech 17 804 807 Thelwell N Millington S Solinas A Booth J and Brown T 2000 Mode of Action and Application of Scorpion Primers to Mutation Detection Nucleic Acids Research 28 19 3752 3761 Page 36 of 38 For Diagnostic Use Only TheraScreen K RAS Mutation Kit 14 20 21 De Roock W Piessevaux H De Schutter J Janssens M De Hertogh G Personeni N Biesmans B Van Laethem JL Peeters M H
26. ated with irinotecan q2w and escalating doses of cetuximab qlw The EVEREST experience preliminary data J Clin Oncol 26 2008 May 20 suppl abstr 4001 10th World Congress on Gastrointestinal Cancer Abstract 0 037 Presented June 27 2008 KRAS mutation status is a predictive biomarker for cetuximab benefit in the treatment of advanced colorectal cancer Results from NCIC CTG CO 1I7 A phase Ill trial of cetuximab versus best supportive care Christos Karapetis et al Rapael G Amado Michael Wolf Marc Peeters Eric Van Cutsem Salvatore Siena Daniel J Freeman Todd Juan Robert Sikorski Sid Suggs Robert Radinsky Scott D Patterson and David D Chang Wild Type KRAS Is Required for Panitumumab Efficacy in Patients with Metastatic Colorectal Cancer J Clin Oncol 2008 26 1626 1634 Page 37 of 38 For Diagnostic Use Only TheraScreen K RAS Mutation Kit Notes To The Purchaser Neither this product the TheraScreen K RAS Mutation nor any of its components can be resold or otherwise transferred or modified for resale without the written approval of DxS Limited TheraScreen and Scorpions are registered trademarks of DxS Limited ARMS is a registered trademark of AstraZeneca UK Limited LIGHTCYCLER and ROCHE are trademarks of Roche ABI7500 is a trademark of Applied BioSystems This product is a CE marked diagnostic device in accordance with the European Union in vitro Diagnostic Medical Devices Directive 98 79 EC
27. cell line DNA Each control was tested in triplicate for the relevant assay on a plate that also contained duplicate mixed standard and single NTC reactions to ensure that the reactions were working within the specifications The same plate was repeated 54 times over 6 days Incorporated into this matrix of runs were the use of 3 instruments 3 reagent lots and 3 operators Ct values were obtained from the LightCycler Adapt Software and all combinations of Ct values were used to calculate ACt values Any ACt values that exceed three times the inter quartile range were considered outliers and removed from the analysis Means standard errors and standard deviations were calculated and the data was grouped by the kit lot instrument or operator to assess the variation caused by these variables The overall repeatability of the assays was also assessed The data indicates that overall variability within the assays is low and 2 standard deviations around the mean covers a range of 1 4 cycles from the mean for the assay with the maximum standard deviation For every test the mean ACt for each operator lay within 0 15 cycles of the overall mean for the whole data set Differences between operators were no more than 0 2 Ct demonstrating that the inter operator variation was no greater than the variation within the whole data set and the assays are robust enough to withstand inter operator variability For every test the difference between the means
28. classed as negative or beyond the limits of the kit when they are greater than or equal to 38 It is necessary to have a control Ct of 28 9 or below to provide enough DNA to enable 1 mutation to be detected given the 1 cut off values and Ct cut off value of 38 If the Control Ct is greater than 28 9 and a mutation is detected the positive mutation is displayed and this Flag Warning is not displayed as this sample contains a definite mutation However if the control Ct is above 28 9 and the sample appears to be mutation negative this Flag Warning is displayed to warn that low level mutations may have been missed As the control Ct increases above 28 9 the sensitivity of mutation detection decreases 7 5 2 3 LIMITED Control Ct gt 35 This indicates that there is very little amplifiable DNA available so only mutations present at a high percentage can be detected In LIMITED samples if a positive mutation gives a Ct lt 38 the mutation will still be given a valid status and will be reported The presence of low level mutations in a sample with a negative result and LIMITED Flag Warning cannot be discounted 7 5 2 4 EXO_FAIL The software checks for amplification of the exogenous control assay to establish whether there could be an inhibitor present which could lead to a false negative result The following logic is used a The exogenous control reaction will be assessed only in the mutation reaction wells not in the control assay well
29. e of cycling as defined in Section 9 K RAS Mutation Detection Protocol e The LightCycler Adapt Software v1 1 from Roche Diagnostics Catalogue 05474914001 e 0 2 ml DNAse free PCR plates LightCycler 480 Instrument Multiwell Plate 96 catalogue number 04729692 001 or ABI MicroAmp Optical 96 well reaction Plate part number 4306737 with MicroAmp Optical Adhesive Film part number 4311971 Sterile tubes for preparing master mixes Dedicated pipettes for PCR mix preparation Dedicated pipettes for dispensing of DNA template Sterile nuclease free H 0 Page 6 of 38 For Diagnostic Use Only TheraScreen K RAS Mutation Kit 5 WARNINGS AND PRECAUTIONS For In Vitro Diagnostic Use The K RAS Kit is not intended to screen for or to diagnose any type of cancer including colorectal cancer It is intended to be used as an adjunct to other prognostic factors currently used to select patients who would not benefit from anti EGFR cancer therapies Therapy for cancer patients should not be based on K RAS gene mutation status alone A clinician should consider the mutation status of the patient alongside other disease factors The contents of the K RAS Kit may be freeze thawed up to 8 times without an adverse effect on assay performance DO NOT freeze thaw the K RAS Kit reagents more than 8 times Note that tumour samples are non homogeneous and data from a sample of tumour may not be concordant with other sections from the same tu
30. ed to the NTC an inhibitor may be present It is possible to reduce the effect of an inhibitor by diluting the sample although this will also dilute the DNA Sample Dilution A Control Ct of lt 24 will overload the mutation assays Samples with a control Ct of lt 24 must be diluted It should be noted that for the LightCycler 480 Instrument Ct values obtained through the 2 derivative method may be slightly different to those obtained with LightCycler Adapt Software It is recommended that concentrated samples are diluted to fall within the gt 24 but lt 29 range Ct values based on the 7500 Sequence Detection Software or LightCycler 480 Instrument Software using the basis that diluting 2 will increase the Ct by Page 13 of 38 For Diagnostic Use Only TheraScreen K RAS Mutation Kit 9 K RAS Mutation Detection Protocol Read these instructions carefully and become familiar with all components of the K RAS Kit prior to use To use the K RAS Kit efficiently samples must be batched into a batch size of 10 to fill one 96 well plate Smaller batch sizes will mean that fewer samples can be tested with the K RAS Kit LightCycler 480 Instrument Experimental Setup For each DNA sample the control and mutation assays must be analysed on the same PCR run to avoid run to run variations Thaw the reaction mixes and mixed standard from the K RAS Kit at room temperature Mix each solution by inverting each tube 10 times
31. ell line DNA Since 60 C is the recommended annealing temperature the plates were run at 58 C 60 C and 62 C If the assays were robust enough to tolerate the experimental temperatures the Mixed Standard ACt values should be within 1 5 of the set values for the Mixed Standard and the pooled cell line ACt values should be above the I cut off points 2ASP showed the most susceptibility to temperature variation however all of the assays performed acceptably at the 3 temperatures showing that the assays are robust when run at temperatures 2 C higher or lower than optimal Page 33 of 38 For Diagnostic Use Only TheraScreen K RAS Mutation Kit Taq Tolerance Validation LightCycler 480 Instrument These studies determined the tolerance of the mutation assays to variations in the level of 7aq polymerase a potential source of user variation since this is added by the user The tolerance of the assays to different levels of 7aq was assessed using synthetic standards in 3 different levels of DNA Each sample was tested in triplicate for a high normal and low level of 7aq high level is 20 extra 7aq low level is 20 less 7ag than normally used Duplicate mixed standard and single NTC reactions were also run to ensure that the assays were working within the specifications Triplicate runs were performed for each plate and 3 different lots of 7aq were tested Ct values were obtained from the LightCycler Adapt Software and all combi
32. emplate Tubes should never be opened after a PCR reaction Page 7 of 38 For Diagnostic Use Only TheraScreen K RAS Mutation Kit s Each assay included in the K RAS Kit has its own characteristics Calculation of the result must be made with reference to the correct assay parameters see Report Data Interpretation section Mutation Ct values of 38 or above must be scored as negative or below the limits of the kit The assays contain an exogenous control reaction internal control in addition to the reaction of interest see Technological Principles section If both assays have failed the data must be discarded as there may be inhibitors present that could lead to false negative results Diluting the sample may reduce the effect of inhibitors but it should be noted that this would also dilute down the DNA General laboratory precautions should be used including but not limited to a Do not pipette by mouth b Do not smoke eat or drink in areas where specimens or kit reagents are being handled c Wash hands after performing the test Only use the 7ag polymerase Taq that is provided in the kit do not substitute with 7ag from other kits of the same or any other type or with Tag from another supplier Only thaw the reagents required for each run do not thaw the whole kit each time in order to minimise the amount of freeze thaw cycles Safety Information Caution All chemicals and biological mat
33. entical to the case where a single mutation is present 7 4 If the sample ACt values are greater than the 1 ACt values the sample is reported as mutation negative may contain a mutation but this is at a level that is below the limits of the kit 7 5 Flags Warnings 7 5 1 The LightCycler Adapt Software will report a number of Flags Warnings for the samples in the Sample Result Table as described in Table 10 Table 10 LightCycler Adapt Software Sample Flags Warnings Meaning REP_DILUTION The Control Ct is less than 24 CONF_LEVEL Control Ct is greater than 28 9 and no mutation is reported LIMITED The control Ct is greater than 35 EXO FAIL The exogenous control assay has failed when the FAM reaction has also failed a in a mutation reaction or the exogenous control Ct is less than 30 FAIL The software is unable to call a curve as positive or negative 7 5 2 The meanings of the Flags Warnings are given in more detail below Page 19 of 38 For Diagnostic Use Only TheraScreen K RAS Mutation Kit 7 5 2 1 REP_DILUTION Control Ct lt 24 The assays have been validated to a level of DNA that gives a control Ct of 24 or above If a sample gives a lower control assay Ct it should be diluted to ensure that it falls within the valid working range 7 5 2 2 CONF_LEVEL Control Ct gt 28 9 A CONF_LEVEL Flag Warning will be displayed in a negative sample with a control Ct gt 28 9 Mutation Ct values are
34. ents please contact your local Roche Diagnostics Office see section 2 Technical Assistance for Contact Details 7 Instrument Refer to the instrument user manual for complete instructions on the installation and use of the real time PCR instrument 8 Specimens Specimen material must be human genomic DNA extracted from formalin fixed paraffin embedded colorectal tumour samples Specimen Collection and Preparation Specimen Transport Standard pathology methodology to ensure specimen quality 2 Recommended Sample Extraction Process DNA extraction using the Qiagen QlAamp DNA FFPE tissue kit catalogue number 56404 The following amendments to the Qiagen protocol must be used e FFPE sections must be collected onto glass slides e Excess paraffin must be scraped away from around the tissue sections using a fresh sterile scalpel e Scrape tissue section material into micro centrifuge tubes using a fresh scalpel for each sample to be extracted e Proteinase K digestion must be continued until completion This may take up to 48 hours e The samples must be eluted into 200 ul of ATE buffer from the Qiagen extraction kit Any alternative methods of sample preparation must be validated by the end user 3 Extracted DNA storage Store at 20 C prior to analysis Page 9 of 38 For Diagnostic Use Only TheraScreen K RAS Mutation Kit Sample Assessment Protocol The extra control assay mix supplied with the K RAS
35. erial should be considered as potentially hazardous Specimens are potentially infectious and should be treated accordingly The K RAS Kit should be used only by those persons who have been trained in the appropriate laboratory techniques When working with the components of this K RAS Kit always wear a suitable lab coat disposable gloves and safety glasses After use K RAS Kit components should be disposed of as clinical waste 6 Storage Stability and Shipping Conditions Storage All the contents of the K RAS Kit should be stored immediately upon receipt at 18 C to 25 C in the dark in a constant temperature freezer Avoid unnecessary freeze thawing of the contents of the K RAS Kit Page 8 of 38 For Diagnostic Use Only TheraScreen K RAS Mutation Kit Stability Do not use the K RAS Kit after the stated expiry date The contents of the K RAS Kit are stable until the expiry date when stored under the recommended storage conditions and in the original packaging The contents of the K RAS Kit may be freeze thawed up to 8 times without an adverse effect on assay performance DO NOT freeze thaw the K RAS Kit reagents more than 8 times Shipping Conditions The contents of the K RAS Kit are shipped on dry ice and should still be frozen on arrival If the K RAS Kit is not frozen on arrival the outer packaging has been opened during transit the shipment does not contain a packing note instructions for use or the reag
36. ers would occur This study determined the cross reactivity pattern for the K RAS Kit Testing was performed using synthetic mutation controls to establish how many cycles after a true positive a cross reactivity signal could be detected Each mutation reaction mix was run with six replicates of the seven individual 100 mutation controls In addition each control was assayed with the matched reaction mix and all other reaction mixes on the same run The cross reactivity pattern was determined by calculating the difference between the real amplification Ct value from the matched cassette and the cross reactivity signal from every other primer The expected cross reactivity pattern is shown in Table 20 below The cross reactivity pattern has been shown to be consistent and will allow the user to read a pattern of amplification where more than one assay gives a positive result to obtain the correct assay result The numbers in each cell indicate the approximate number of cycles after the true positive signal is detected that a cross reactivity signal may be seen Values above 9 cycles have not been included because these are classified as mutation negative results Page 32 of 38 For Diagnostic Use Only TheraScreen K RAS Mutation Kit Table 20 ABI7500 Cross Reactivity Validation Results Positive Sample I2ALA 12ASP I2ZARG I2CYS 12SER I2VAL 13ASP Cycling Tolerance Validation
37. es were set as the mean value over all runs and concentrations The results of this testing are summarised in the data interpretation section ABI7500 For each assay dilutions were tested in triplicate for 3 kit lots on 3 separate runs for both the control assay and mutation assay The mixed standard was used on each plate to ensure that the assays were performing within the specified criteria The 1 ACt values were calculated from averaged Ct values over the replicates within a run and lot The cut off ACt values were set as the mean value to the nearest 0 5 over all lots runs and concentrations for concentrations where all replicates gave a Ct value The results of this testing are summarised in Table 16 below Table 16 ABI7500 1 Cut Off Values Validation Results 1 ACt values The 1 cut off for 12Val was changed to 6 5 based on the Breakthrough study See discussion below Mixed Standard ACt Determination LightCycler 480 Instrument The mixed standard ACt values are mean values taken from 50 runs on which the mixed standard was tested Ct values were obtained from the LightCycler 480 Adapt Software The expected ACt values for the mixed standard are shown in the data interpretation section ABI7500 Mixed standard values have been set using the mean of 422 ACt values These were calculated over many different kit lots and runs The expected ACt values for the mixed sta
38. he 1 ACt values and the sample will be classed mutation negative The K RAS Kit is CE marked for jn vitro diagnostic use on either the Roche Diagnostics LightCycler 480 Real Time PCR System Instrument Il LightCycler 480 Instrument 96 well format Roche Part Number 05015278001 or Applied BioSystems 7500 Real Time PCR System part number 4351105 ABI7500 For the LightCycler 480 Instrument the K RAS Kit must be used in combination with the LightCycler Adapt Software vl for the TheraScreen K RAS Mutation Kit CE IVD LightCycler Adapt Software This software has been developed to automate the calling of a positive or negative amplification plot and computes a suitable threshold from which to obtain Ct values These are used to calculate sample ACt values which are compared with the cut off values The software reports a positive or negative mutation result and removes any subjectivity from the analysis and interpretation of the K RAS Kit data K RAS Kit Format Eight assays are supplied in the K RAS Kit e One control assay e Seven mutation assays All reaction mixes contain an exogenous control internal control assay labelled with HEX to be detected by the JOE detector on the ABI7500 This controls for the presence of inhibitors which may lead to false negative results Control assay The control assay labelled with FAM is used to assess the total DNA in a sample The control assay amplifies a region of exon
39. ion Results The reduction in accuracy of the I2ALA assay is due to variation between replicates at one level of DNA Linearity Validation LightCycler 480 Instrument Linearity has been assessed using synthetic standards at 100 1 and 0 mutation levels for each assay At each percentage of mutation a serial dilution has been performed to maintain the mutation level but reduce the total amount of DNA Standard curves have been produced by running each dilution in triplicate on 3 replicate plates for the control assay and relevant mutation assay Ct values have been produced using the LightCycler Adapt Software For each mutation a graph has been produced data not shown where the control assay Ct and mutation assay Ct have been plotted against one another and a regression performed for each mutation level The 95 confidence intervals have also been plotted for each mutation level There were no mutation Ct values obtained from the 0 testing The 100 and 1 graphs have shown that there was clear discrimination between the data sets and the slopes of the curves were substantially equivalent indicating similar amplification efficiency across a range of mutation concentrations ABI7500 This study assessed the efficiency of each of the mutation assays across a range of DNA concentrations in a background of wild type DNA and in water Each mutation assay should have an efficiency of 90 110 100
40. is a single acquisition at the 60 C step 3 Select the Sample Editor tab and under Step l Select Workflow select the Abs Quant tick box Under Select Filter Combinations ensure both filters are selected 465 510 nm and 533 580 nm Set up the samples names under Step 2 and Step 3 The Quantification Sample Type should be set as unknown The replicate column should be left blank 4 Select the Experiment button and click the Start Run button to save the experiment and start the cycling 5 On completion of the run select the Analysis tab and chose Abs Quant 2nd Derivative Max from the Create New Analysis window Accept the defaults from the Create New Analysis Screen Ensure that the Filter Comb button is 465 510 and select the Calculate button Ct values are displayed in the Samples table 6 Select the Filter Comb button and change the filters to 533 580 nm Select the Calculate button and obtain the exogenous control Ct values from the Samples table Page II of 38 For Diagnostic Use Only TheraScreen K RAS Mutation Kit Sample Assessment Protocol ABI7500 Instrument Setup 2 F Immediately place the plate into the ABI7500 instrument Select the 7500 System Software icon on the desktop of the workstation attached to the instrument Open a new run file from the file menu in the 7500 Sequence Detection Software versi
41. le 8 Sample 9 B Mixed 2 ALA standard C Mixed 2ASP standard D Mixed 2ARG standard E Mixed 2CYS standard F Mixed l 2SER standard G Mixed 2VAL standard H Mixed 3ASP standard Samplel Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 7 Seal and spin the PCR plate briefly to collect the reagents at the bottom of the wells 8 Immediately place the plate into the LightCycler 480 instrument LightCycler 480 Instrument Setup l Select the LightCycler 480 Instrument software icon on the desktop of the workstation attached to the instrument Log into the software and from the overview page select New Experiment from Template 2 Choose the K RAS LC480II Run Template from the Run Templates list see LightCycler 480
42. lts section details the full name and file path of the LightCycler 480 Instrument run plus the following details 3 1 The LightCycler 480 Instrument serial number 3 2 Instrument Name the identifier given to the LightCycler 480 Instrument in the LightCycler 480 Instrument software Page 16 of 38 For Diagnostic Use Only TheraScreen K RAS Mutation Kit 3 3 Run Date Date and time of the LightCycler 480 Instrument run 3 4 Run Operator the name of the person who was logged onto the LightCycler 480 Instrument software when the experiment was run 3 5 Experiment Name Run file name 3 6 Software Version version of the software used on the LightCycler 480 Instrument 3 7 Lot Number taken from the Lot No field in the experiment setup screen in the LightCycler 480 Instrument software 4 The plate layout is given with sample names taken from the LightCycler 480 Instrument software sample editor screen 5 A Run Summary Result is given where the run is called as Valid or Invalid This is dependent on the run controls in columns and 2 6 Run Controls 6 1 The LightCycler Adapt Software automatically calculates the ACt value for the mixed standard using the formula Mutation Ct Control Ct ACt 6 2 The LightCycler Adapt Software compares the values with the expected values given in Table 7 Table 7 LightCycler Adapt Software Expected ACt values Assay Mixed Standard A Ct
43. mour Tumour samples may also contain non tumour tissue DNA from non tumour tissue would not be expected to contain the K RAS mutations detected by the K RAS Kit All assays in the K RAS Kit generate short PCR products However the K RAS Kit will not work on heavily fragmented DNA DNA assessment should be based on PCR and may differ from quantification based on Optical Density readings Additional control reaction mix is supplied to allow assessment of quality and quantity of the DNA in samples before running the K RAS Kit Reagents for the K RAS Kit have been diluted optimally Further dilution of the reagent is not recommended and will result in a loss of performance Use of less than 25 ul reaction volumes is not recommended and will increase the risk of false negatives All reagents in the K RAS Kit are formulated specifically for use with the stated tests No substitutions should be made to the K RAS Kit reagents if optimal performance is to be maintained To ensure optimal activity and performance Scorpions primers as with all fluorescently labelled molecules should be protected from light to avoid photo bleaching Use extreme caution to prevent contamination of PCR reactions with synthetic control material It is recommended that separate dedicated pipettes be used for setting up reaction mixes and adding DNA template The preparation and dispensing of reaction mixes should be carried out in a separate area to the addition of t
44. nations of Ct values from replicates on a plate were used to calculate ACt values All assays met the acceptance criteria that the means from the high 7ag and low Tag samples do not differ from the mean of the normal level of 7ag by more than standard deviation of the normal 7ag data ABI7500 Three identical PCR plates each containing 3 reagent lots were run for each mutation assay with the 7ag polymerase varied at 10 and 10 from the recommended amount the recommended amount was also tested The Mixed Standard was tested as were standards in 2 levels of DNA and 50 ng mutation negative cell line DNA The assay data were analysed for mean standard deviation and coefficient of variation The results showed little variation equivalent SD between assays CV lt 10 in assay results when the 7aq level was varied by 10 showing that the assays could tolerate as much as a 10 variation in 7ag polymerase levels 12 Technical Assistance For Technical Assistance and further information please contact your Roche distributor A list of contact details for the main Roche distributors can be found in Section 13 Page 34 of 38 For Diagnostic Use Only 13 Manufacturer and Distributor Details TheraScreen K RAS Mutation Kit sal DxS Limited 48 Grafton Street Manchester M13 9XX United Kingdom D Roche Molecular Systems Inc Branchburg NJ 08876 USA Distributedby A Member of the Roche Group U
45. ndard are shown in the data interpretation section Breakthrough Validation Breakthrough is defined as the non specific amplification in the mutation assays of a wild type DNA target present in a particular sample This causes a measurable level of background noise The breakthrough from wild type DNA was assessed for each mutation assay The breakthrough study ensured that the 1 cut off values previously established were below the breakthrough level and would prevent reporting of a false positive result Page 27 of 38 For Diagnostic Use Only TheraScreen K RAS Mutation Kit LightCycler 480 Instrument Ten K RAS mutation negative cell line DNA samples at different concentrations 5 FFPE K RAS mutation negative samples and 5 FFPE K RAS mutation positive samples were tested in duplicate across 5 plates The mutation positive samples were positive for a single mutation and the other mutations were used to test breakthrough Ct values were obtained from the LightCycler Adapt Software and all combinations of Ct values were used to calculate ACt values The results from cell line DNA and FFPE samples mutation negative assays gave ACt values that were all above the 1 cut off values indicating that the 1 cut off values are robust Table 7 shows the worst breakthrough levels from the 5 runs Table 17 Breakthrough Results Worst breakthrough ACt values 12 35 11 36 No breakthrough No breakthrough 11 74 12 29 11 29
46. on 1 4 Under Assay select Standard Curve Absolute Quantification Under Run Mode select Standard 7500 Move to the detector setup window by selecting the Next button Add a FAM and a JOE detector with quenchers set to none to the detectors list If these detectors do not already exist select the New Detectors button and set up a FAM and a JOE detector with the quencher set to none Set the Passive Reference to None and select the Next button Select the entire plate and check the tick boxes for the FAM and JOE detectors to ensure that both dyes are monitored in each well Select the Finish button Under the Instrument tab set up the cycling as given in Table 4 Table 4 ABI7500 Cycling Conditions Temperature Data Collection Stage 95 C Stage 2 95 C 60 C Select the Start button to save the experiment and start the cycling On completion of the run ensure that the passive reference is set to none in the well inspector screen In the Amplification Plot tab select all the wells in use and select the JOE dye from the detector drop down menu 10 Check the JOE signal from each sample and compare to the JOE signal in the NTC well Note any samples that have a later amplification curve or failed amplification for the exogenous control as compared to the NTC In the Amplification Plot tab select all the wells in
47. perimental Setup For each DNA sample the control and mutation assays must be analysed on the same PCR run to avoid run to run variations I Thaw the reaction mixes and mixed standard from the K RAS Kit at room temperature Mix each solution by inverting each tube 10 times once thawed to avoid localised concentrations of salts Prepare sufficient mixes for the DNA samples the mixed standard and no template controls plus an excess of 2 reactions per mix as shown in Table I 1 Table I 1 K RAS Master Mix Volumes Master Mixes Reaction Mix ul 7aq pl per plate per plate x14 x14 Assay Reaction Mix ul xl Taq ul x Control assay 19 8 0 2 277 2 2 8 Mutation assays 19 8 0 2 277 7 2 8 2 DO NOT vortex the Taq or any reaction mixes that contain Tag as this may cause inactivation of the enzyme 3 Ensure that the 7aq is at room temperature before use Spin the vial to ensure that all the 7aq is collected at the bottom then pipette by placing the pipette tip just under the surface of the Tag to minimise the risk of the tip getting coated in excess Tag 4 Mix the master mixes by gently pipetting up and down 5 Immediately add 20 ul of the master mixes to the reaction wells 6 Immediately add 5 ul of sample mixed standard or water for the no template controls to the reaction wells Each DNA sample must be tested with both the control and mutation assays The plate setup is given in Table 12
48. s is shown not to be the case it can lead to a false positive or false negative mutation call The no template control indicates that there is no contamination in the master mixes and that the exogenous control is working as expected In the unlikely event that the LightCycler Adapt Software develops an error message please refer to Technical Assistance Section 12 Page 18 of 38 For Diagnostic Use Only TheraScreen K RAS Mutation Kit 7 Sample Results 7 1 The Sample Result Table reports sample names taken from the LightCycler 480 Instrument software and control assay Ct values 7 2 The LightCycler 480 Adapt Software will calculate ACt values and determine whether a sample is mutation positive or negative based on 1 cut off values given in Table 9 Table 9 LightCycler Adapt Software 1 Cut off Values 1 delta Ct 6 25 7 72 6 83 6 95 8 95 6 5 9 09 7 3 If a mutation ACt value for a sample is below the corresponding 1 ACt cut off value the sample is called mutation positive The LightCycler Adapt Software will report which mutation is present but will only report a single mutation If there are 2 positive ACt values the smallest value will be reported It is assumed that the second value has been produced through cross reactivity of one mutation primer binding and priming from a different mutation In rare cases where a double mutation is present the clinical decision will be id
49. sinens 8 Fee NUNS EM UNV Ms sss 504 ox cee EOT ATA ENTA OAT AEAEE A S 9 REST 117 0 ene eee een ee pee ree E eee eer re Sane Mart ere 9 9 K RAS Mutation Detection Protocol cc cssscsscsscssssssscssssssesssesscsssssenssssscessens 14 10 Limitations of the eS screcccscsesciceeveiavsarisssecsssossastevestaeeasaaiesassedniarvassnaamansmastecrontens 25 l Assay Performance Characteristics se eesesseeesreessesereesreesstesssesreesreesneesseesseesseesss 26 12 Technical Assistance zso2sesis texscescevcacseesievsssniedseeessesds states edt tviastenedestven eee 34 13 Manufacturer and Distributor Details ccccccssscessssecssscsescsscsssessestseeeessenees 35 14 Date of issuance of last REVISION si sisscsssccsceccassacdescseceseastssssesiaasacabicdsstscacatasscenscasaaacass 35 l5 RETCRGINCOS ies cccscccescaveccedsecsesvssvadsdeexscxtvce tesvscassovasert REENE EAEE 36 Notes To The Purchaser cccccssccsssssssssessessssssssssesssssssssssssssessesssssecsssessssesssesssessnsesseses 38 Page 2 of 38 For Diagnostic Use Only TheraScreen K RAS Mutation Kit A IMPORTANT Read these instructions carefully and become familiar with all components of the K RAS Kit prior to use Intended Use Indications for Use Intended Use The DxS TheraScreen K RAS Mutation Kit K RAS Kit is an n vitro diagnostic test intended for the detection of seven somatic mutations in the K RAS oncogene and will provide a qualitative assessment of mu
50. st enough to withstand instrument to instrument operator to operator lot to lot day to day and run to run variations Accuracy Validation LightCycler 480 Instrument Accuracy has been assessed using 92 FFPE samples and 28 mutation positive cell line dilutions These have been tested with the K RAS kit and have also been sequenced using Sanger sequencing K RAS mutation calls have been compared between the 2 methods and there were 18 discrepant results 5 FFPE s and 13 cell lines where sequencing indicated a negative results but the sample was positive using the DxS assay To confirm the presence of a mutation in these 18 samples each has had the region around the mutations cloned The presence of a mutation positive clone has been used to resolve the discrepancy ABI 7500 Accuracy was assessed using synthetic controls diluted into 2 levels of K RAS mutation negative cell line DNA to give 3 different percentages of mutation covering both positive and negative mutation levels Three identical plates each using 3 reagent lots were run for each mutation 25 5 and 0 5 mutation levels were tested The 25 and 5 samples should give a mutation positive result ACt below the cut off values the 0 5 sample should give a mutation negative result ACt above the 1 cut off values The results are summarized in Table 19 below Page 30 of 38 For Diagnostic Use Only TheraScreen K RAS Mutation Kit Table 19 ABI7500 Accuracy Validat
51. tation status The K RAS Kit is to be used by trained personnel in a professional laboratory environment with DNA samples extracted from formalin fixed paraffin embedded colorectal tissue Indications for use The results of the K RAS Kit are intended to aid the clinician in identifying colorectal cancer patients who may not benefit from anti epidermal growth factor receptor EGFR therapy such as panitumumab or cetuximab The K RAS Kit is not intended to diagnose colorectal cancer It is intended as an adjunct to other relevant prognostic factors used to select suitable patients for treatment with anti EGFR therapies based on the patient s mutation status The patient s mutation status will be considered by a Clinician alongside other disease factors to make a therapy decision No treatment decision for cancer patients should be based on K RAS mutation status alone 2 Summary and Explanation of the Test The K RAS kit is a CE marked diagnostic device in accordance with the European Union jn vitro Diagnostic Medical Device Directive 98 79 EC Mutations in the K RAS oncogene are frequently found in human cancers 4 The presence of these mutations correlates with a lack of response to certain EGFR inhibitor cancer therapies in metastatic colorectal cancer patients 5 0 14 2 Detection of seven mutations in the K RAS gene is possible in a background of wild type genomic DNA in a real time PCR assay based on DxS Scorpions
52. ue of sample DNA is based on the concentration as determined by PCR Values based on Optical Density readings do not correspond to control assay Ct values in fragmented DNA samples Additional control reaction mix is supplied to allow assessment of the sample Ct s before running the kit If a sample gives a mutation positive result where the mutation Ct gt 38 the assay must be scored as negative or below the limits of the kit This is carried out automatically by the LightCycler Adapt Software Page 25 of 38 For Diagnostic Use Only TheraScreen K RAS Mutation Kit Some cross reactivity may occur between mutation reactions For example if a high level 12ALA mutation is seen some of the other mutation reactions will also give a positive result This is due to the ARMS primers detecting other mutations within a few bases of one another On synthetic control material the cross reactivity forms a readable pattern on the ABI7500 that allows the true positive to be determined from several signals see Table I5 Table 5 Cross reactivity Pattern on the ABI7500 from Synthetic Control Material Yes indicates the true signal Numbers indicate the approximate number of cycles after the true signal that a cross reactivity signal may be seen Beyond 9 cycles values have not been entered as these will be in the negative zone Positive Sample I2ALA 2ASP I2ARG I2CYS 12SER I2VAL 13ASP
53. umblet Y Van Cutsem E Tejpar S KRAS wild type state predicts survival and is associated to early radiological response in metastatic colorectal cancer treated with cetuximab Ann Oncol 2007 Nov 12 Li vre A Bachet JB Le Corre D Boige V Landi B Emile JF C t JF Tomasic G Penna C Ducreux M Rougier P Penault Llorca F Laurent Puig P KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer Cancer Res 2006 66 3992 5 Li vre A Bachet JB Boige V Cayre A Le Corre D Buc E Ychou M Bouch O Landi B Louvet C Andr T Bibeau F Diebold MD Rougier P Ducreux M Tomasic G Emile JF Penault Llorca F Laurent Puig P KRAS mutations as an independent prognostic factor in patients with advanced colorectal cancer treated with cetuximab J Clin Oncol 2008 26 374 9 C Bokemeyer et al K RAS status and efficacy of first line treatment of patients with metastatic colorectal cancer mCRC with FOLFOX with or without cetuximab The OPUS experience J Clin Oncol 26 2008 May 20 suppl abstr 4000 E Van Cutsem et al K RAS status and efficacy in the first line treatment of patients with metastatic colorectal cancer mCRC treated with FOLFIRI with or without cetuximab The CRYSTAL experience Clin Oncol 26 2008 May 20 suppl abstr 2 S Tejpar et al Relationship of efficacy with K RAS status wild type versus mutant in patients with irinotecan refractory metastatic colorectal cancer mCRC tre
54. use and select the FAM dye from the detector drop down menu Use the automatic baseline setting and manual Ct and then set the threshold manually in the middle of the exponential phase using the log scale for the Y axis as described in the ABI7500 user guide Select the Analyse button to obtain Ct values Page 12 of 38 For Diagnostic Use Only TheraScreen K RAS Mutation Kit Sample Assessment Interpretation Assess the NTC Ct values to ensure that there is no contamination giving a positive amplification in the FAM channel Ct less than 40 or a failed exogenous control reaction in the HEX channel no Ct indicating a setup problem The mixed standard should give a control assay Ct FAM channel of 26 29 on the ABI7500 and lt 29 on the LightCycler 480 Instrument Data should not be used if either of these 2 run controls has failed Control assay Ct 29 35 Interpret with caution as very low level mutations may not be detected Control assay Ct 35 38 Only a few amplifiable copies of DNA are present in such samples and mutations are only likely to be seen if most copies are mutated Control assay Ct 38 Reject sample as there is minimal DNA present and the K RAS Kit will not be able to detect mutations Note that if a sample gives a late control assay Ct the sample exogenous control Ct should be compared with the exogenous control of the NTC If the exogenous control of the sample is delayed or negative as compar
55. with the exception of the mixed standard and NTC columns see point 6 or if there is an exogenous control Ct lt 30 b If there is a positive mutation call but the exogenous control assay has failed in the mutation positive well the EXO_FAIL Flag Warning is not displayed as there could be competitive inhibition from the FAM reaction The mutation status is valid c If there is a positive mutation call for a sample in one mutation well and an exogenous control assay failure in a different mutation well for the same sample the mutation in the first well will be called and the result is valid However an EXO_FAIL Flag Warning will be displayed This indicates that there is a problem with the assay in the exogenous control assay failure well and a mutation may have been missed in this well Page 20 of 38 For Diagnostic Use Only TheraScreen K RAS Mutation Kit It is possible that the mutation called is cross reactivity between the assays rather than the true mutation which has been missed However the mutation status in the assay reporting the positive mutation call remains valid as there is a clear mutation present and the clinical decision remains the same regardless of which mutation is present d If the sample is called negative but an exogenous control reaction has failed in any of the mutation assays the EXO_FAIL Flag Warning will be displayed and the status is invalid It is possible that a mutation may have been missed e
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