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1. fe 7500 Fast meg 5 Syste bie 5 351 Ensure names of V600E and V600K reactions for the same sample are strictly identical Make sure that each sample has a unique name Do not modify the selection of the detectors for the clinical samples Do not modify the name and the selection of the detectors for the Positive Control and Negative Control e Delete unused clinical sample wells from the run layout if any delete sample name and uncheck selected detectors Note Unused wells and detectors not deleted may create background noise which would alter results Do not change other run layout settings Save the modified SDS run file Select a name for the SDS run file that clearly identifies the run The run layout of the SDS run file can be printed and used as a guide for the dispensing of the reagent mix and eluates e The run is ready to start For complete instructions refer to the 7500 Fast Dx Real Time PCR instrument User s Manual QUALITY CONTROL A Positive Control is included in each THxID BRAF kit A Negative Control must be performed at the same time as the samples starting from sample preparation These controls must be performed in each run to ensure that the reagents have not been altered and to check the absence of contamination The instrument will be able to check the control value or the validity only if their positioning corresponds to the plate layout Moreover the integrity of2. BRAF assay yielded 29 invalid final results after applying the troubleshooting instructions i e 3 2 2 7 5 2 CLINICAL PERFORMANCE Clinical validation of the THxID BRAF assay to select patients for dabrafenib and trametinib treatment Clinical validation of the THxID BRAF assay was applied retrospectively to the available samples used for selecting patients for treatment with dabrafenib BRAF inhibitor in GlaxoSmithKline GSK sponsored clinical trial BRF113683 dabrafenib trial 1 or with trametinib MEK inhibitor in clinical trial MEK114267 study trametinib trial 1 Enrollment in the clinical trials was limited to patients whose melanoma tissue tested positive by the clinical trial assay CTA and if they met other eligibility criteria The therapeutic outcome of the trials was linked to the result of THxID BRAF in order to establish the clinical efficacy of the drugs when using this test for the selection of patients retrospectively The primary endpoint for both trials was investigator assessed Progression Free Survival PFS All available specimens from patients screened for the GSK trials were evaluated in a blinded manner Dabrafenib clinical efficacy The safety and efficacy of dabrafenib was evaluated in an international multi center randomized 3 1 open label phase III study in patients with advanced Stage III or metastatic Stage IV melanoma whose melanoma tissue harbors a BRAF V600E mutation Patients were
3. e 30 minutes at 18 25 C e 1 month at 19 C to 31 C Re use frozen solution a maximum of 2 times After each thawing mix and spin down briefly before use Stability 3 months at 18 25 C 5 and 10 um thick FFPE eae tied in tube or mounted on 3 months at 19 C to 31 C CONT solution Samples FFPE specimens can be transported at 18 25 C e 2 hours at 18 25 C e 48 hours at 2 8 C e 7 months at 19 C to 31 C Eluate e 7 months at lt 60 C Re use frozen solution a maximum of 4 times After each thawing mix and spin down briefly before use bioM rieux SA English 6 THxID BRAF INSTRUCTIONS FOR USE Sample requirements e Standard formalin fixation and _ paraffin embedding procedures should be followed To limit the extent of DNA fragmentation Fix tissue samples in 10 formalin as quickly as possible after surgical removal Use a fixation time of 14 24 hours longer fixation times lead to more severe DNA fragmentation resulting in poor performance in THxID BRAF assay Thoroughly dehydrate samples prior to embedding residual formalin can inhibit the Proteinase K digestion Sections will be processed according to the pathologist s indications 1 If the sample section contains more than 80 of tumor cells and does not contain a distinct area of necrotic tissue fatty tissue hemorrhagic tissue or non tumoral melanin rich area then the entire section can be placed in a tu
4. BRAF test demonstrated a similar statistically significant improvement median PFS 5 0 vs 2 7 hazard ratio 0 34 The results demonstrate that the observed clinical benefit with the CTA results is observed with THxID BRAF assay and support the use of the THxID BRAF test to aid in the identification of patients for dabrafenib therapy Table 2 efficacy in subjects testing positive V600E with CTA vs THxID BRAF assay in dabrafenib trial 1 THxID BRAF CTA Dabrafenib Dacarbazine Dabrafenib Dacarbazine Number of subjects 177 55 187 63 Hazard Ratio Estimate 0 34 0 33 95 Confidence Interval 0 20 0 57 0 20 0 54 p value lt 0 0001 lt 0 0001 Estimates for PFS months Median 5 0 2 1 5 1 2 1 95 Confidence Interval 4 9 6 8 1 5 3 2 4 9 6 9 1 5 3 2 PFS Progression free Survival Additional efficacy analysis was conducted to consider the impact of discordance between the THxID BRAF assay and the CTA i e patients who were tested positive by the THxID BRAF assay but were tested negative or invalid by the CTA In the worst case scenario assuming a hazard ratio of 1 for patients positive by the THxID BRAF test and negative by the CTA the hazard ratio was 0 34 95 Cl 0 23 0 50 and similar to the results in the trial Kaplan Meier Curves of Investigator Assessed Progression Free Survival tl TAFINLAR n 187 Median 5 1 months 0 9 D
5. CI Positive Percent Agreement 403 418 403 411 PPA for V600E and V600K 96 4 94 2 97 8 98 1 96 2 99 0 Negative Percent Agreement 417 464 417 444 NPA 89 9 86 8 92 3 93 9 91 3 95 8 820 882 820 855 92 3 91 1 94 5 95 9 94 4 97 0 The accuracy of the V600E and V600K was individually assessed THxID BRAF invalids were included in this analysis QNS and Sanger invalids excluded The results in the table below demonstrate that the THxID BRAF assay has high accuracy for the V600E and V600K allele V600E including THxID BRAF invalids Overall Agreement V600K including THxID BRAF invalids No of concordance No of tests No of concordance No of tests 95 Cl 95 Cl Positive Percent 341 354 59 64 Agreement PPA 96 3 93 8 97 8 92 2 79 7 94 7 Negative Percent 503 528 813 817 Agreement NPA 99 2 93 1 96 8 99 5 98 8 99 8 1 Negative agreement for V600E was based on the total non V600E alleles Negative agreement for V600K was based on the total non V600K alleles Two samples with a V600E and K THxID BRAF status were detected V600K by Sanger sequencing Since Sanger sequencing can only report one mutation these 2 samples were included in the calculation Agreement was not impacted by specimen type data not shown bioM rieux SA English 18 Invalid rate Out of 896 tested clinical samples the THxID
6. KARASARIDES M MARAIS R The RAF proteins take center stage Nature Reviews Molecular Cell Biology 2004 vol 5 p 875 885 2 DAVIES H BIGNELL G R COX C et al Mutations of the BRAF gene in human cancer Nature 2002 vol 417 p 949 954 3 WAN P T GARNETT M J ROE S M et al Mechanism of activation of the RAF ERK signaling pathway by oncogenic mutations of B RAF Cell 2004 vol 116 p 855 867 4 RUBINSTEIN J C SZNOL M PAVLICK A C et al Incidence of the V600K mutation among melanoma patients with BRAF mutations and potential therapeutic response to the specific BRAF inhibitor PLX4032 Journal of translational medicine 2010 vol 8 p 67 5 NEWTON C R GRAHAM A HEPTINSTALL L E et al Analysis of any point mutation in DNA The amplification refractory mutation system ARMS Nucleic Acids Research 1989 vol 17 p 2503 2516 6 QUERINGS S ALTMULEER J ANSEN S et al Benchmarking of Mutation Diagnostics in Clinical Lung Cancer Specimens PLoS ONE DOI 10 1371 journal pone 0019601 2011 vol 6 n 5 7 PICHLER M BALIC M STADELMEYER E et al Evaluation of High Resolution Melting Analysis as a Diagnostic Tool to Detect the BRAF V600E Mutation in Colorectal Tumors Journal of Molecular Diagnostics March 2009 vol 11 n 2 p 140 147 8 TSIATIS A C NORRIS KIRBY A RICH R G et al Comparison of Sanger Sequencing Pyrosequencing an
7. dissolve by heating to 70 C with gentle agitation Preparing Buffer AW1 Add 25 ml ethanol 96 100 to the bottle containing 19 ml Buffer AW1 concentrate Write down the current date on the label after ethanol addition Note Before starting the procedure mix reconstituted Buffer AW1 by shaking bioM rieux SA 16464 C en 2013 09 Preparing Buffer AW2 Add 30 ml ethanol 96 100 to the bottle containing 13 ml Buffer AW2 concentrate Write down the current date on the label after ethanol addition Note Before starting the procedure mix reconstituted Buffer AW2 by shaking Sample preparation e Immediately place the dissected sections in a Lysis Tube 1 5 mL microtube not provided or 2 mL Lysis Tube LT Note electrostatic effect can be observed for dissected sample Transfer the whole dissected area inside the tube and briefly centrifuge e Prepare an empty Lysis Tube for the Negative Control which will undergo the whole process e Add 1 mL xylene to all microtubes samples and Negative Control e Close the tube and mix at full speed using a vortex type mixer for at least 10 seconds e Centrifuge at full speed approximately 20000 g for 2 minutes 30 seconds at 18 25 C e Remove the supernatant by pipetting Make sure to leave 50 100 uL of supernatant in order not to remove any of the pellet e Add 1 mL ethanol 96 100 to the pellet and mix briefly at full soeed using a vortex type mixer e Cent
8. A Negative Control starting from the extraction process and a Positive Control for amplification will be tested per run for each duplex reaction reagents in strips or For dispensing of the reagent mix and eluate respect the plate layout from the created run Note Do not invert the V600E and V600K reagent mixes English 8 THxID BRAF In each tube of an 8 tube strip or of a 96 well plate e Following the plate layout transfer 18 uL of the V600E or V600K reagent mix to the bottom of a single tube e Add 2 uL of Positive Control or Negative Control or sample eluate to the dedicated tube Mix by aspiration and dispensing Note After adding the reagent mix and controls or sample eluate s to all tubes visually check that the volume in each tube is identical Run amplification on the 7500 Fast Dx Real Time PCR instrument For 8 tube strips e Close the strips using the MicroAmp Cap Installing Tool Handle e Properly identify the strips on the extremity of the cap strip according to the position in the plate layout Note Do not write directly on the caps or tubes as ink interferes with fluorescence readings e Briefly centrifuge the strips e Place the strips on the Precision Plate Holder according to the plate layout Notes A minimum of 2 tube strips and a maximum of 6 tube strips can be run on the Precision Plate Holder at the same time Do not invert the V600E and V600K strips e Insert 2 fully cappe
9. Skin V600E 20 20 lower limit of DNA range Lymph node V600K 20 20 Skin V600K 20 20 High melanin content ea A A Lymph node V600K 20 20 Lymph node V600E 20 20 700 ng reaction Skin V600E 20 20 higher limit of DNA range Lymph node V600K 20 20 Skin V600K 20 20 1000 ng reaction Lymph node V600E 20 20 above DNA range Lymph node V600K 20 20 Melanoma V600E 20 20 cell line Melanoma cell line FFPE blocks Melanoma V600K 20 20 cell line DNA input for high melanin samples were at 60 ng reaction for the V600E sample and 526 ng reaction for the V600K sample and melanin content were respectively 80 and 75 After 1 4 dilution of the eluate Before dilution each of the 20 replicates was invalid In accordance with the troubleshooting table the eluate was diluted 1 4 in Buffer ATE and re tested with results as described in the table Inclusivity The THxID BRAF assay is designed to detect the V600E T1799A and V600K GT1798 1799AA mutations In addition the THxID BRAF assay was shown to detect a rare form of the V600E mutation i e rare codon GAA and the V600E K601E mutation also referred to as V600E2 using 2 FFPE lymph node specimens and plasmids Cross reactivity Cross reactivity of the THxID BRAF assay was assessed by testing e plasmids representing Wild Type V600D V600R V600L V600M V600G V600A and BRAF pseudogene plasmids BRAF homologue present on chromosome X e procured clinical s
10. all samples a ee Bi directional Sequencing _ and K mutations not BRAF V600 renee a te V600E V600K Invalid QNS Total V600D V600R WT V600E 341 2 2 0 213 7 0 373 V600K 1 57 0 0 a 0 0 60 THxID BRAF result V600E and K 2 J 4 E and K mutation negative Invalid te ee re res oe oe oe fe oe f fe No result was obtained _ QNS Quantity Not Sufficient for testing Sanger sequencing has a limit of detection of approximately 20 mutant alleles in FFPE specimens Therefore Sanger Sequencing may not be adequate to confirm mutation status at lower percentages of mutant alleles 6 7 8 9 Double mutants cannot be confirmed by the Sanger sequencing method These variants are not intended to be detected by the THxID BRAF For the purposes of analyzing agreement between the THxID BRAF assay and Sanger any specimen that was deemed E or K was considered mutation positive and any sequencing result not E or K was deemed E and K mutation negative Analyses were conducted with and without the THxID BRAF assay invalids Agreement between the THxID BRAF assay and Sanger sequencing for all samples excluding Sanger sequencing invalids and all QNS samples Including THxID BRAF invalids total of 27 samples Without THxID BRAF invalids No of concordance No of tests No of concordance No of tests 95 CI 95
11. or in the 7500 Fast Dx Real Time PCR instrument refer to the User s Manual LIMITATIONS OF THE METHOD THxID BRAF kit is to be used by professionals trained and skilled in molecular biology techniques The product must be used in strict accordance with the instructions for use Any deviation from the procedure should be validated by the end user Melanin is a known inhibitor of PCR reactions Melanin may interfere with the THxID BRAF assay in highly pigmented samples If melanin inhibition is suspected repeat testing using a 1 4 dilution as suggested in the troubleshooting table The claimed tumor area for the assay is 20 mm to 250 mm for a 10 um section Smaller tissue i e lt 20 mm areas cannot ensure reliable results The assay has been validated for a DNA input range of 10 350 ng uL Users should ensure thermocycler temperature is correctly calibrated as incorrect temperatures can lead to invalid results The test is designed to detect the BRAF V600E and V600K mutations Samples with results reported as BRAF mutation negative may harbor BRAF mutations not detected by the assay e g V600R Results that suggest E K heterogeneity of the sample may potentially come from a homozygous sample see accuracy section The V600E PCR in the THxID BRAF assay cross reacts with the V600D The assay has not been validated to reliably detect V600D Refer to the section on performance studies below The presence of A
12. stuffs 26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice 36 Wear suitable protective clothing 46 If swallowed seek medical advice immediately and show this container or label HARMFUL reagent R36 37 38 Irritating to eyes respiratory system and skin R42 May cause sensitization by inhalation 22 Do not breathe dust 24 Avoid contact with skin 26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice 36 37 Wear suitable protective clothing and gloves For further information refer to the Material Safety Data Sheet available on request bioM rieux SA English 3 THxID BRAF THxID BRAF AMP Blue Sphere containing e synthetic primers e synthetic fluorescent labeled probes Each tube packed in a foil pack with silica gel desiccant Lilac Sphere containing e synthetic primers e synthetic fluorescent labeled probes Each tube packed in a foil pack with silica gel desiccant Mix containing e Tris Buffer e PCR enzyme e UNG amperase e Glycerol e Nucleotides e Sodium Azide NaNs e Passive reference dye PRMdil Blue Diluent containing e RNase DNase free water e preservative C Yellow V600E Primers and Probes 6 x 5 2 mg lyophilized V600K Primers and Probes 6 x 5 2 mg lyophilized Master Mix 6 x 0 24 mL liquid Primers Diluent 6 x 0 5 mL liquid Positive Con
13. L Buffer in the eluate tested by adding 5 uL AL Buffer in the eluate may lead to a false result for a mutant sample which may then be reported as BRAF mutation negative The THxID BRAF kit is validated for FFPE skin and lymph node melanoma tissue The THxID BRAF kit is only validated for use with the THxID BRAF PUR kit and the ABI 7500 Fast Dx Real Time PCR instrument The THxID BRAF kit is not to be used for diagnosis English 5 STORAGE CONDITIONS THxID BRAF PUR Stability THxID BRAF PUR kit until the labeled expiration date at 2 8 C or 18 25 C The Columns must be stored at 2 8 C Reconstituted Buffer AW1 6 months at 18 25 C or until the expiration date of the kit Reconstituted Buffer AW2 6 months at 18 25 C or until the expiration date of the kit THxID BRAF AMP Stability Kit stability THxID BRAF AMP kit until the labeled expiration date at 2 8 C e 30 minutes at 18 25 C e 1 month at 19 C to 31 C Re use frozen solution a maximum of 2 times After each thawing mix and spin down briefly before use e 30 minutes at 18 25 C e 1 month at 19 C to 31 C Re use frozen solution a maximum of 2 times After each thawing mix and spin down briefly before use 30 minutes at 18 25 C Master Mix Re use solution a maximum of 2 times Reagent Mix solution primer probe solution Master Mix Kit stability PRM V600E solution PRM V600K solution 30 minutes at 18 25 C
14. M Agreements all 5x5 Agreements without THxID BRAF and CTA invalids No of Agreement No of Agreement concordance rate 95 CI concordance rate 95 CI No of tests No of tests V600E 232 240 96 70 93 6 98 3 232 236 98 30 95 7 99 3 V600K 45 50 90 00 78 6 95 7 45 48 93 80 83 2 97 9 Mutation negative Overall 536 565 94 90 92 7 96 4 522 538 Cl Confidence Interval 245 258 95 00 91 6 97 0 245 254 96 50 93 4 98 1 97 00 95 2 98 2 The primary endpoint of the trial was Progression Free Survival PFS which demonstrated median PFS of 5 1 months in the dabrafenib arm and 2 7 months in the chemotherapy arm HR 0 33 95 Cl 0 20 0 54 p value lt 0 00071 bioM rieux SA English 19 A total of 250 patients were enrolled in the trial Patients were randomized to receive dabrafenib n 187 or dacarbazine n 63 The trial results shows that patients who received dabrafenib had a statistically significant increase in median PFS as compared to patients who received dacarbazine 5 1 months vs 2 7 with a hazard ratio of 0 33 p value lt 0 0001 95 Cl 0 20 0 54 Of the 250 patients 237 177 in the dabrafenib arm and 55 in the dacarbazine arm were available for retesting with the THxID BRAF assay to demonstrate support of dabrafenib efficacy claims The reanalysis of PFS demonstrated that those patients who were V600E positive by the THxID
15. REF 410697 i6464 en 2013109 GD THxID BRAF IVD The THxID BRAF kit is an n Vitro Diagnostic device intended for the qualitative detection of the BRAF V600E and V600K mutations in DNA samples extracted from formalin fixed paraffin embedded FFPE human melanoma tissue The THxID BRAF kit is a real time PCR test on the ABI 7500 Fast Dx system and is intended to be used as an aid in selecting melanoma patients whose tumors carry the BRAF V600E mutation for treatment with dabrafenib Tafinlar and as an aid in selecting melanoma patients whose tumors carry the BRAF V600E or V600K mutation for treatment with trametinib Mekinist bioM rieux SA English 1 THxID BRAF SUMMARY AND EXPLANATION The RAS RAF MEK ERK pathway is a critical proliferation pathway in many human cancers This pathway can be constitutively activated by alterations in specific proteins including BRAF which phosphorylates MEK on 2 regulatory serine residues Over 45 cancer associated mutations have been identified in BRAF 1 BRAF mutations have been identified at a high frequency in specific cancers including approximately 50 to 60 of melanoma 2 Approximately 90 of all identified BRAF mutations that occur in human cancer are a 11 799A transversion mutation in exon 15 which results in a V600E amino acid substitution 3 This mutation appears to mimic regulatory phosphorylation locks the BRAF kinase in its active status and increas
16. TIC n 63 Median 2 7 months 0 8 Hazard Ratio 0 33 a 95 CI 0 20 0 54 2 P value lt 0 0001 ra c 07 w 0 6 2 2 0 5 j 0 4 a 03 M Z o 02 a 0 1 0 0 N atisk E TAFINLAR U EF t 184 173 412 99 41 34 5 3 DTIC _63 30 e 11 amp 4 F i D 1 2 3 4 g 4 8 9 Months bioM rieux SA English 20 Trametinib clinical efficacy The safety and efficacy of trametinid were evaluated in an international multi center open label randomized 2 1 phase Ill study in patients with advanced Stage Ill or metastatic Stage IV melanoma whose tumor tissue harbor a BRAF V600E or V600K mutation Patients were randomized to receive trametinib n 214 or chemotherapy n 108 consisting of dacarbazine or paclitaxel Enrollment in the study was limited to patients whose melanoma tissue tested positive for the V600E or V600K mutation as detected by a clinical trial assay CTA The ability of the THxID BRAF assay to support the safety and efficacy of trametinid was demonstrated in a bridging study that consisted of two components an assessment of analytical concordance between results obtained with the CTA and the THxID BRAF kit and analysis of the primary endpoint based on the V600E and V600K mutation positive subset identified by the THxID BRAF assay A total of 1108 patients were screened for the trial Of these a total of 808 specimens had CTA results including invalids Of the 808 specimens 766 we
17. amplification Results were obtained in duplicates on 8 panel members using 2 THxID BRAF lots 4 days per lot 2 runs per day 2 instruments 2 days per instrument 2 operators each performing 1 run per day The precision panel was comprised of FFRE melanoma specimens of skin or lymph node origin 1 specimen was from subcutaneous tissue The panel included e 2 negative samples Wild Type with a low and a high DNA input e 2 V600E samples close to LoD with a low and a high DNA input e 2 V600K samples close to LoD with a low and a high DNA input e 1 moderate V600K sample at a medium DNA input e 1 V600E high melanin containing sample at a medium DNA input Mutation status results of the THxID BRAF assay were compared to the expected results as determined by bi directional Sanger sequencing Panel member MOAN ade N correct calls N replicates Correct concentration lt n ae Call Skin Wild Type with low DNA input Wild Skin Wild Type with low DNA input with low DNA input 32 32 100 al node Wild Type with high DNA 3333 i007 Skin V600E close to LoD with low DNA 32 32 100 input Lymph node V600E close to LoD with a Skin V600E with medium DNA input 440 32 32 100 and high melanin content Lymph node V600K close to LoD with 44 39 32 100 low DNA input Lymph node V600K with medium DNA 85 32 32 100 input and moderate mutation content Lymph node V600K close to LoD with A had a moderate DNA concentration du
18. ate 1 6 sdt Browse Run Mode m Operator Your name Comments Reagents tracking information or comments Plate Name four plate name Cancel e Check the value of the following items Mandatory values Standard curve Absolute Quantitation 96 Well Clear Browse to one of the THxID BRAF templates provided 1 6 7 14 15 22 or 23 46 and open the selected template Template Standard 7500 e Fill in the Comments field with the lot numbers of the THxID BRAF PUR kit and the THxID BRAF AMP kit used in order to comply with the reagent tracking Change the name of the plate in the Plate Name field This field is used to suggest the name of the created SDS run file Click on Finish The SDS run file pre configured for a THxID BRAF run is created and automatically opened See figure below 2 7500 Fast System 05 Software DO Fie Vew Toots 21CFR11 Instrument Analysis Window Help Dekh S6R AGH ro at y 4 5 A Negstne Control Negstine Control 57 Positwe Contr asite C z v 516 s16 taj 3 amp aj 5 3 A s bioM rieux SA Setup Instrument Y Resuks Y Auda Trail 7 E Sgnalures P 4 1 1 16464 C en 2013 09 e Update the names of the clinical samples to be amplified using the Well Inspector icon from the top menu See figure below 2 7500 Fast System SDS Software Plate1 Standard Curve
19. be or if the sample is on a Slide it can be entirely scraped with a scalpel 2 If the sample section contains less than 80 of tumor cells then the section must be manually macro dissected in order to reach a final content of at least 80 tumor cells Use a dedicated sterile scalpel to select the tissue part in order to enrich the sample in tumoral cells 3 If the sample section contains necrotic tissue fatty tissue hemorrhagic tissue or non tumoral melanin rich area then the section should be manually macro dissected Use a dedicated sterile scalpel to select the tissue part in order to avoid the undesirable portion The minimum surface of tissue required for a 10 um section is 20 mm not counting the necrotic fatty hemorrhagic non tumoral melanin rich area if it is deemed dissectible see above If 5 um sections are prepared the minimum is then 40 mm Therefore a sufficient number of sections should be included to meet this requirement while not exceeding 8 x 10 um sections or 16 x 5 um sections to stay within the recommended limit of the purification column The total surface of tissue should not exceed 250 mm if 10 um sections are prepared or 500 mm if 5 um sections are prepared Note Use a single scalpel per sample Preparation of purification reagents e Equilibrate all buffers at 18 25 C e Before starting the procedure check whether precipitate has formed in Buffer AL or Buffer ATL If necessary
20. containing these buffers are spilt clean with liquid detergent and water If the spilt liquid contains potentially infectious agents clean the affected area first with liquid detergent and water and then with 1 sodium hypochlorite The Proteinase K reagent is a harmful agent Refer to the risk phrases R and the precautions S above Kit reagents contain sodium azide which can react with lead or copper plumbing to form explosive metal azides If any liquid containing sodium azide is disposed of in the plumbing system drains should be flushed with water to avoid build up Do not use reagents or Columns after the expiration date indicated on the label Do not mix reagents or disposables from different lots Check that the reagents are intact before use Make sure reagents and samples are at 18 25 C before use All centrifugation steps should be carried out at 18 25 C Make sure that the lyophilized material is at the bottom of the tube before opening the tube Ensure sample to result traceability by appropriate labeling of the container at each transfer step e g lysate transfer etc and at the level of the strips in the PCR instrument Avoid contamination or sample to sample carry over Never open more than one tube at a time to minimize the risk of cross contamination Perform FFPE extraction and amplification in separate dedicated laboratory areas preferably a self contained area or laminar fl
21. d Melting Curve Analysis for the Detection of KRAS Mutations Journal of Molecular Diagnostics July 2010 vol 12 n 4 p 425 432 9 OGINO S KAWASAKI T BRAHMANDAM M et al Sensitive Sequencing Method for KRAS Mutation Detection by Pyrosequencing Journal of Molecular Diagnostics August 2005 vol 7 no 3 p 413 421 16464 C en 2013 09 INDEX OF SYMBOLS Symbol In Vitro Diagnostic Medical Device Temperature limit gg fue OT Batch code m m Consult Instructions for Use a Contains sufficient for lt n gt tests km Write down the current date after adding ethanol to the bottle N e WARRANTY bioM rieux disclaims all warranties express or implied including any implied warranties of MERCHANTABILITY AND FITNESS FOR A PARTICULAR USE bioM rieux Shall not be liable for any incidental or consequential damages IN NO EVENT SHALL BIOMERIEUX S LIABILITY TO CUSTOMER UNDER ANY CLAIM EXCEED A REFUND OF THE AMOUNT PAID TO BIOMERIEUX FOR THE PRODUCT OR SERVICE WHICH IS THE SUBJECT OF THE CLAIM BIOMERIEUX the blue logo THxID are used pending and or registered trademarks belonging to bioM rieux or one of its subsidiaries or one of its companies Mekinist and Tafinlar are trademarks belonging to GlaxoSmithKline LLC CLSI is a trademark belonging to Clinical and Laboratory Standards Institute Inc Any other name or trademark is the property of its respective owner The purchase of this produc
22. d ACt results for V600E samples V600K samples internal control IC Ct values for the Wild Type WT samples and positive and negative controls were investigated as a measure of the variability of the assay For the Wild type panel samples the internal control Ct for the V600E multiplexes ranged from 24 4 to 28 7 with CV range 0 3 3 and the V600K multiplexes IC ranged from 24 2 to 28 5 with CV range 0 2 8 The mean ACt ranged from 1 9 to 6 1 for the V600E mutation positive samples with associated CV values ranging from 0 to 16 7 The mean ACt ranged from 1 2 to 4 9 for the V600K mutation positive samples with associated CV values ranging from 0 to 25 6 The higher imprecision was associated with the high melanin content sample For the V600E positive control the mean ACt was 3 7 and the CV values ranged from 0 to 12 8 The V600K positive control mean ACt value was 3 5 with associated CV ranging from 0 to 15 1 bioM rieux SA English 16 Sample handling variability 3 specimens for 3 different samples were studied Serial sections from a single block were used Three sections of each sample type were forwarded to each of the 3 external sites alternating the sections For example section 1 4 7 were sent to Site 1 Sections 2 5 8 were sent to Site 2 and similarly for Site 3 One specimen contained lymph node tissue the second contained skin tissue One of these specimens was positive for V600E and the other was positive for V600K T
23. d empty MicroAmp Fast 8 Tube Strips in the 1 left and last columns of the Precision Plate Holder e Start amplification on the instrument For 96 well plates e Cover the 96 well plates with the adhesive film using the Adhesive Film Applicator Note Do not write directly on the plate as ink interferes with fluorescence readings if necessary use the sides of the plate e Centrifuge the 96 well plates at 200 g for 3 minutes at 18 25 C e Transfer the plates to the Applied Biosystems 7500 Fast Dx Real Time PCR instrument e Start amplification on the instrument bioM rieux SA 16464 C en 2013 09 Generate an SDS result file e At the end of the run start the analysis on the instrument using Auto Ct Note Do not modify any other items The THxID BRAF software checks the instrument configuration If configuration is incorrect the software will not be able to interpret e At the end of the analysis save the SDS result file generated Generate a BRAF mutation test report e Transfer a copy of the SDS result file to the dedicated THxID BRAF computer using for instance a USB stick and the menu File Save As of the SDS software Warning If modifications need to be made to the SDS result file after it has been copied do the following steps Update the SDS run file on the SDS software Destroy all existing copies of the previous versions of the SDS result file recommended Destroy all the BRAF mutatio
24. detected the sample will be characterized as BRAF mutation negative The final result is determined based on results obtained for both multiplexes English 9 Valid results of clinical specimens BRAF mutation negative V600E and V600K mutations not detected V600E mutation V600E mutation detected V600K mutation V600K mutation detected V600E and V600K mutations V600E and V600K mutations detected Invalid results Invalid control An invalid Positive or Negative Control invalidates the complete run If one or more controls is invalid the results of the clinical specimen obtained in the run are not reported If an invalid result is obtained for a control refer to the troubleshooting table below Contamination with internal control DNA was detected in the Negative Control well The complete run must be repeated using l frozen sample eluates frozen Negative Amplification detected T N N Control eluate and either frozen Positive Negative Control in Negative Control well Control if available or a new preparation for V600E was detected in the if the Negative Control is still invalid Negative Control well repeat the complete procedure starting from the extraction Amplification detected in Negative Control well for IC Contamination with V600K mutation DNA was detected in the Negative Control well Amplification detected in Negative Control well for V600K Amplification for IC out of bounds in Positive Control wel
25. e Detection Systems SDS Software The templates configure the 7500 Fast Dx Real Time PCR instrument for a BRAF run Note On the Precision Plate Holder displayed on the screen numbers indicate the position of the Plate s columns from 1 to 12 letters indicate the position of the Plate s lines from A to H starting from the top 16464 C en 2013 09 Appropriate corrective measure The eluate has to be diluted 1 4 in Buffer ATE before amplification by adding 10 uL of eluate to 30 result is still uL of Buffer ATE If the invalid repeat the test starting from the extraction using lower tissue area If the tested sample has been characterized as containing no or a low level of melanin lt 10 by the pathologist repeat the test starting from the eluate If the result is still invalid repeat the test for the invalid sample starting from th tissue area If the tested e extraction using higher sample has been characterized as containing a medium to high level of melanin gt 10 by the pathologist the eluate has to be diluted 1 4 in Buffer ATE before amplification by adding 10 uL of eluate to 30 uL of Buffer ATE If the resu It is still invalid repeat the test starting from the extraction using higher tissue area Repeat the test starting from the eluate Please refer to the table for invalid control troubleshooting The proper template is chosen acco
26. e V600E or V600K solution with Master Mix according to the following table Number of V600E or V600K Master Mix samples and solution uL controls to be run uL H 3 27 36 8 85 110 16 170 220 24 255 330 If another number of samples is to be run make sure to use 8 uL of V600E or V600K solution per reaction and to leave a certain margin to calculate the volumes needed It is important to maintain a volume of Master Mix that is 1 3 times higher than the volume of primer solution rounding up to the nearest whole number e Mix at full speed briefly using a vortex type mixer e Centrifuge briefly Warning The remaining volume of reagent mix cannot be stored and should be used within 30 minutes Preparation of Positive Control solution e Add 150 uL of Positive Control Diluent to the Positive Control sphere e Mix at full speed using a vortex type mixer until a clear solution has been obtained e Centrifuge briefly Notes The remaining volume of Positive Control can be stored see Storage conditions Do not pool the remaining volumes of Positive Control solutions from different tubes Repartition of 8 tube 96 well plates Per single sample the DNA amplification is carried out in 2 wells each containing a duplex PCR reaction The V600E duplex PCR contains the internal control PCR and the V600E mutation specific PCR The V600K duplex PCR contains the internal control PCR and the V600K mutation specific PCR
27. each individual result can be monitored by reference to the performance of the internal control PCR Results cannot be validated if the control values are not valid English 12 THxID BRAF WASTE DISPOSAL e Unused AL AW1 and PK reagents must be disposed of 16464 C en 2013 09 e The other unused reagents may be considered as non hazardous waste and disposed of accordingly following procedures for hazardous chemical waste e Do not add bleach or acid solutions directly to the sample preparation waste containing Buffer AL and Buffer AW1 e Dispose of used or unused Master Mix reagents as well as any other contaminated disposable materials following procedures for infectious or potentially infectious products It is the responsibility of each laboratory to handle waste and effluents produced according to their nature and degree of hazardousness and to treat and dispose of them or have them treated and disposed of in accordance with any applicable regulations NON CLINICAL PERFORMANCE Sample characterization All FFPE samples used in the non clinical studies underwent a pathology review in order to determine the tumor content tumor cells the melanin content the presence of necrotic lipidic or hemorrhagic tissue in the stained sections The genetic status of the samples on the V600 locus was determined by bi directional Sanger sequencing Genomic input range The THxID BRAF assay was validated for a DNA inp
28. ement Cl Confidence Interval A total of 322 patients were enrolled in the trial Patients were randomized to receive trametinib n 214 or chemotherapy n 108 The trial results shows that patients who received trametinib had a statistically significant increase in median PFS as compared to patients who received dacarbazine 4 8 months vs 1 5 with a hazard ratio of 0 47 p value lt 0 0001 95 Cl 0 34 0 65 Of the 322 patients 289 196 in the trametinib arm and 93 in the chemotherapy arm were available for retesting with the THxID BRAF assay to demonstrate support of trametinib efficacy claims The reanalysis of PFS demonstrated that those patients who were V600E or V600K positive by the THxID BRAF test demonstrated the same statistically significant improvement median PFS 4 8 vs 1 5 hazard ratio 0 48 The results demonstrate that the observed clinical benefit with the CTA results is observed with the THxID BRAF assay and support the use of the THxID BRAF test to aid in the identification of patients for trametinib therapy The primary endpoint of the trial was Progression Free Survival PFS which demonstrated median PFS of 4 8 months in the trametinib arm and 1 5 months in the chemotherapy arm HR 0 47 Cl 0 34 0 65 p value lt 0 0001 bioM rieux SA English 21 Table 4 efficacy in subjects testing positive V600E or V600K with CTA vs THxID BRAF assay in trametinib trial 1 THxID BRAF CTA Tra
29. es BRAF activity approximately 10 fold compared to wild type 2 T1799A alteration V600E mutation accounts for 70 to 90 of BRAF mutant melanoma patients In addition the T1799A alteration could be associated with a second nucleotide mutation G1798A and leads to a V600K mutation in an additional 6 to 29 of patients with a BRAF mutation 4 Dabrafenib is a selective inhibitor of BRAF kinase activity and trametinib is a selective inhibitor of MEK activity for tumors that carry T1799A and or GT1798 1799AA alterations in the BRAF gene The clinical utility of this kit is to evaluate the BRAF V600E K T1799A GT1798 1799AA mutation status in order to screen patients for treatment with the dabrafenib or trametinib bioM rieux SA 16464 C en 2013 09 PRINCIPLE The THxID BRAF kit allows detection of the V600E and V600K mutations of the BRAF gene from FFPE sections For this the THxID BRAF kit makes use of 2 major processes e Nucleic acid isolation from FFPE sections through extraction purification steps The paraffin is removed The sample is lysed then heated to reverse formalin crosslinking The DNA is bound to a membrane After washing concentrated DNA is eluted from the membrane Real time PCR amplification and detection of target DNA BRAF gene present in the total nucleic acids Amplification Refractory Mutation Specific System ARMS 5 PCR technology is used In the PCR reaction primers specific f
30. he Column e Transfer the tubes with nucleic acid extracts to the amplification laboratory area or store see Storage Conditions Amplification Switch on the 7500 Fast Dx Real Time PCR instrument Preparation of the SDS run file on the SDS software Refer to the section Perform a run on the 7500 Fast Dx Real Time PCR instrument e Create a new run selecting the appropriate template e Adapt the created run to the plate layout Preparation of PCR reagents Equilibrate reagents and samples at 18 25 C before use Preparation of primer and probe solution e Add 85 uL of PRM diluent to the V600E and V600K primer sphere When more than 8 reactions are required spheres with the same lot number must be pooled in one tube and diluent volume must be adapted Number of spheres Maximum number of samples and controls to be run Volume of primer diluent uL e Immediately mix at full speed using a vortex type mixer until a clear solution has been obtained e Centrifuge briefly bioM rieux SA 16464 C en 2013 09 Notes e With one sphere it is possible to do 3 runs of one sample including the 2 controls e As long as the remaining volume of V600E and V600K solutions is sufficient for 3 reactions it can be stored see Storage Conditions e Do not pool the remaining volume of primer and probe solutions from different tubes Preparation of reagent mix e Briefly centrifuge the Master Mix e Mix th
31. he third specimen was Wild Type with a high melanin content A breakdown of the specimens provided is in the table below Wild Type with high melanin lymph nodes V600E skin The table below presents the results obtained for each site for each of the 3 specimens tested for each of the 3 FFPE tissue samples provided 9 9 100 9 9 100 9 9 100 27 27 100 The table above shows 100 agreement with the expected results for all sites This high agreement rate indicates the assay s ability to yield accurate consistent results even when there is variability in sample preparation due to multiple users Accuracy correlation to Reference Method for Clinical Samples The accuracy of the THxID BRAF assay was assessed relative to an analytical reference standard based on PCR amplification followed by bi directional Sanger DNA sequencing method on a representative sampling of clinical trial samples consecutively taken from the pool of clinical samples until a statistically significant sample size was reached for each allele in particular V600K There were 898 samples available for testing Excluding all invalids and QNS samples total 43 there were 35 discordant cases 35 898 43 4 1 Two samples determined to be V600D were detected by the THxID BRAF assay as V600E The overall results are shown in the tables below bioM rieux SA English 17 Agreement between THxID BRAF assay and Bi directional Sequencing for
32. higher speed until the Column is empty e Place the Column in a clean 2 mL Wash Tube WT e Discard the Wash Tube containing the flow through e Carefully open the Column and add 500 uL of Buffer AW1 without wetting the rim English 7 THxID BRAF e Close the lid e Centrifuge at approximately 6000 g for 1 minute at 18 25 C e Place the Column in a clean 2 mL Wash Tube e Discard the Wash Tube containing the flow through e Carefully open the Column and add 500 uL of Buffer AW 2 without wetting the rim e Close the lid e Centrifuge at approximately 6000 g for 1 minute at 18 25 C e Place the Column in a clean 2 mL Wash Tube e Discard the Wash Tube containing the flow through e Centrifuge at full speed approximately 20000 g for 3 minutes at 18 25 C to dry the membrane completely Note Avoid contact between the Column and the flow through Take care when removing the Column and Wash Tube from the rotor so that flow through does not come into contact with the Column e Place the Column in a clean 1 5 mL Elution Tube ET e Discard the Wash Tube containing the flow through e Carefully open the lid of the Column and apply 60 uL of Buffer ATE to the center of the membrane e Close the lid e Incubate at 18 25 C for at least 1 minute e Centrifuge at full speed approximately 20000 g for 1 minute at 18 25 C Note The volume of eluate will be up to 5 uL less than the volume of elution solution applied to t
33. l Amplification for V600E This indicates that an out of bounds in error occurred during Positive Control Positive Control well the process or that Amplification for Ve00K agents failure was positive Control The complete run must be repeated using frozen sample eluates frozen Negative Control eluate and a new preparation of out of bounds in epee Positive Control well Delta Ct out of bounds in Positive Control well bioM rieux SA English 10 THxID BRAF Invalid clinical specimen If an invalid result is observed for a clinical specimen in a valid run refer to the troubleshooting table below Sample type Clinical specimen Message Sample IC amplification below minimum threshold in well XX Sample IC amplification above maximum threshold in well XX Sample delta Ct too low in well XX No available result due to control failure Description Too much DNA in the reaction possible PCR overloading Too low DNA in the reaction and or PCR inhibition An unexpected ratio between IC and mutant PCR is detected No result is reported for this sample due to an invalid Positive and or Negative Control PERFORM A RUN ON THE 7500 Fast Dx Real Time PCR INSTRUMENT Refer to the 7500 Fast Dx Real Time PCR instrument User s Manual Templates for the 7500 Fast Dx Real Time PCR instrument bioM rieux provides 4 templates for the 7500 Fast Dx Real Time PCR instrument and its associated Sequenc
34. metinib Chemotherapy Trametinib Chemotherapy Estimates for PFS months Median 95 Confidence Interval PFS Progression free Survival Additional efficacy analysis was conducted to consider the impact of discordance between the THxID BRAF assay and the CTA i e patients who were tested positive by the THxID BRAF assay but were tested negative or invalid by the CTA In the worst case scenario assuming a hazard ratio of 1 for patients positive by the THxID BRAF test and negative by the CTA the hazard ratio was 0 48 95 Cl 0 35 0 63 and similar to the results in the trial Kaplan Meier Curves of Investigator Assessed Progression Free Survival 1 0 4 3 Censored 0 9 i S _ MEKINIST N 214 4 Median 4 8 months 0 8 i Be O E Eeee L Chemotherapy N 108 Median 1 5 months 0 7 i Hazard Ratio 0 47 95 CI 0 34 0 65 o gos P value lt 0 0001 g o LL E gt o D w a U4 gt 2 Sr Eia L a T T T en 2 a th n 0 3 0 2 0 1 0 0 Number at risk MEKINIST 214 205 163 100 88 Chemotherapy 108 88 a 21 ns ah now o oN wih He U Months Refer to the most recent Mekinist trametinib and Tafinlar dabrafenib drug labels available at Drugs FDA available on the FDA website for more information regarding dabrafenib and trametinib indications bioM rieux SA English 22 THxID BRAF LITERATURE REFERENCES 1 WELLBROCK C
35. n test reports corresponding to the previous version of the SDS result file recommended Resume the instructions for the modified SDS result file from the section Generate an SDS result file e Generate a BRAF mutation test report for the SDS result file using the THxID BRAF software For complete instructions refer to the 7500 Fast Dx Real Time PCR instrument User s Manual or and to the THxID BRAF software User s Manual RESULTS AND INTERPRETATION The THxID BRAF software interprets the results automatically and highlights the presence of valid or invalid results in the generated report The 2 possible outcomes for Positive and Negative Controls are valid or invalid The result validity of clinical specimens is determined first by the internal control Ct Crossing threshold values that should fall within pre specified limits A result is invalid if one of the delta Ct or internal control Ct values falls outside the expected limits If a specific amplification is detected for the mutant target the result of each reaction V600E or V600K is based on the delta Ct value Ct mutant Ct ic e If the delta Ct value is below a threshold value then a V600E or V600K BRAF mutation is present e If the delta Ct value is above a threshold value then no V600E or V600K BRAF mutation is present or it is below the limit of detection lf no amplification is detected for the mutant targets V600E and V600K not
36. or the BRAF gene allow the amplification of a non polymorphic gene area which is used as an internal control The primers specific for the mutations V600E and V600K allow the amplification of mutated fragments leading to the identification of BRAF mutations Target specific probes bind instantaneously to the newly synthesized complementary DNA In the THxID BRAF kit 2 different probes labeled with 2 different dyes allow the simultaneous detection of the BRAF internal control and a BRAF mutation Kinetic analysis of the fluorescent signals and delta Ct Crossing threshold calculation reveal the presence of potential BRAF mutations English 2 CONTENT OF THE KIT 48 tests THxID BRAF PUR THxID BRAF Columns with Wash Tubes 50 Cou Columns in tubes THxID BRAF Wash Tubes Jm ukes 3 x 50 2 mL THxID BRAF Elution Tubes ee eng EE 1 5 mL tubes THxID BRAF Lysis Tubes Loe ES To THxID BRAF Tissue Lysis Buffer Edetic acid 10 mL Sodium dodecyl sulphate TML j ee LYSIS SUNET Guanidine salt GuHCI 25 50 12 mL THxID BRAF Wash Buffer 1 concentrate Guanidine salt GUHCI 50 100 19 mL THxID BRAF Wash Buffer 2 concentrate Sodium azide NaN3 13 mL THxID BRAF Elution Buffer 14 mL THxID BRAF Proteinase K 1 25 mL Sodium azide NaN3 lt 1 HARMFUL reagent R22 Harmful if swallowed R36 38 Irritating to eyes and skin 13 Keep away from food drink and animal feeding
37. ow hood using dedicated material Use a fresh aerosol resistant pipette tip or equivalent for each pipetting action Wear disposable gloves when working with amplified material Change gloves after contact with nucleic acids Wash hands thoroughly after completion of the test procedure Resuspend the V600E and V600K spheres before the control sphere To avoid amplicon contamination and to reduce the risk of evaporation ensure amplification strips remain closed Use powder free gloves as powder may inhibit the PCR reaction and cause invalid results bioM rieux SA 16464 C en 2013 09 Handling of Columns Carefully apply the sample or solution to the Column Pipet the sample into the Column without wetting the rim Avoid touching the Column membrane with the pipet tip After all pulse vortexing steps briefly centrifuge the microcentrifuge tubes to remove drops from the inside of the lids Open only one Column at a time and avoid generating aerosols Collect used disposable materials in a sealable container Close and remove the container after each test run Soak tube racks in a suitable detergent after each test run for at least one hour All materials and instruments should be regularly cleaned and decontaminated Immediately clean up any spillage containing tissue lysate with liquid detergent or a solution of household bleach containing at least 1 sodium hypochlorite For cleaning spills on
38. pecimens representing V600R specimens Plasmids were tested in triplicate at a concentration of 2 x 10 copies reaction No cross reaction with V600E nor V600K PCR was reported on Wild Type V600E V600K V60OR plasmid and clinical samples V600L V600M V600G and V600A mutants and the pseudogene at 2 x 10 copies reaction and on V600G with up to 3 x 10 copies reaction V600E cross reaction was reported for V600D Interfering substances Hemoglobin and triglycerides 2 concentrations of hemoglobin 4 mg mL and 2 mg mL or triglycerides 74 mM and 37 mM were added to 11 FFPE samples during the lysis step i e directly in the lysis buffer between deparaffinization and extraction Each condition was tested in 3 replicates from 3 extractions with one lot of THxID BRAF assay The tested concentrations of hemoglobin and triglycerides reflect 2 x and 1 x the CLSI recommended high concentration respectively The same FFPE specimens were also tested without interfering substance as a reference Neither hemoglobin nor triglycerides interfered with the THxID BRAF assay Necrotic tissue 21 melanoma FFPE specimens with necrotic tissue concentrations ranging from 15 to 60 were tested with the THxID BRAF assay Necrotic tissue content was determined by pathologist review Each sample was tested in 3 replicates from 3 extractions with one lot of THxID BRAF assay The correct allele was called in all instances including one sample wi
39. randomized to receive dabrafenib n 187 or dacarbazine n 63 The ability of the THxID BRAF assay to support the safety and efficacy of dabrafenib was demonstrated in a bridging study that consisted of two components an assessment of analytical concordance between results obtained with the CTA and the THxID BRAF kit and analysis of the primary endpoint based on the V600E mutation positive subset identified by the THxID BRAF assay A total of 734 patients were screened for the trial Of these a total of 584 specimens had CTA results including invalids Of the 584 specimens 565 were available for retesting 96 7 Table 1 shows the analytical concordance between the CTA results and the THxID BRAF results with specimens available for retesting Dabrafenib is indicated for patients whose melanoma harbor V600E mutations The agreement for the V600E mutation was 96 7 95 Cl 93 6 98 3 when including the test invalids Agreement for non V600E mutations was 95 95 Cl 92 7 97 0 Overall agreement between the assays was approximately 95 95 Cl 92 7 96 4 Table 1 agreement between the THxID BRAF assay and CTA for all subjects all testing sites Clinical Trial Assay CTA V600E V600K V600E and K WT Invalid Total V600E 232 1 0 8 1 242 V600K 0 45 0 0 46 THxID BRAF V600 E and K 0 0 0 0 assay WT 0 245 2 253 Invalid 0 4 14 24 Total 240 50 0 258 17 565 j T
40. rding to the number of clinical samples to be tested in the run Number of clinical samples Position of the strips on the plate POSON Orie CONNAIS on the plate Negative A6 and A7 template sdt 2 strips PCOM to oe and Positive B6 and B7 7 14 THxID BRAF Negative AS and A6 template sdt 4 strips POM LORNA Poni aioe Positive B5 and B6 1 6 THxID BRAF 15 22 THxID BRAF template sdt 6 strips 23 46 THxID BRAF template sdt plate Non applicable Negative A4 and AS From 15 to 22 Columns 4 to 9 Positive B4 and B5 From 23 to 46 Negative A1 and A2 Positive B1 and B2 The templates also include positions and settings for the Positive Control and Negative Control Strips cannot be used for the 23 46 layout configuration If 23 or more samples are tested use a plate bioM rieux SA English 11 THxID BRAF Run layout Creation of a new SDS run file for the 7500 Fast Dx Real Time PCR instrument e Open the Sequence Detection Systems SDS Software e Log in as a routine user e Create a new document Select New in the file menu or Create New Document in the Quick Startup dialog A New Document Wizard dialog box is displayed New Document Wizard Define Document Select the assay container and template for the document and enter the operator name and comments Assay Standard Curve Absolute Quantitation v Container 36 Well Clear xl Template TH ID BRAF templ
41. re available for retesting 94 8 Table 3 shows the analytical concordance between the CTA results and the THxID BRAF results with specimens available for retesting Trametinib is indicated for patients whose melanoma harbor V600E and V600K mutations The PPA for the V600E mutation was 93 5 95 Cl 90 1 95 7 when including the test invalids PPA for the V600K mutation was 86 8 95 Cl 72 7 94 2 MNPA was 96 95 Cl 93 8 97 5 Overall agreement between the assays was 95 95 Cl 93 3 96 4 Table 3 Agreement between the THxID BRAF assay and CTA for all subjects all testing sites Clinical Trial Assay CTA V600E V600K V600E and K WT Invalid Total V600E 252 1 0 11 0 264 V600K 0 33 0 0 0 33 THxID BRAF V600 E and K 1 0 assay WT 0 0 Invalid 0 8 Total 1 8 Agreements without THxID BRAF and CTA Agreements all 5x5 invalids No of Agreement No of Agreement concordance rate 95 Cl concordance rate 95 Cl No of tests No of tests 286 306 93 50 90 1 95 7 286 294 97 30 94 7 98 6 252 267 94 40 90 9 96 6 252 257 98 10 95 5 99 2 33 38 86 80 72 7 94 2 33 36 91 70 78 2 97 1 434 452 96 00 93 8 97 5 434 445 97 50 95 6 98 6 728 766 95 00 93 3 96 4 720 739 97 40 96 0 98 3 PPA Positive Percent Agreement MNPA Mutation Negative Percent Agreement OPA Overall Percent Agre
42. rifuge at full speed approximately 20000 g for 2 minutes 30 seconds at 18 25 C e Remove the supernatant by pipetting Do not remove any of the pellet Carefully remove any residual ethanol using a fine pipet tip e Open the tube and dry at 37 2 C for 10 1 minutes or until all residual ethanol has evaporated e Add 180 uL Buffer ATL to the dry pellet e Add 20 uL proteinase K e Mix briefly at full soeed using a vortex type mixer e Incubate at 56 3 C for 1 hour 5 minutes e Incubate at 90 5 C for 1 hour 5 minutes If using only one heating block leave the sample at 18 25 C after the 56 C incubation until the heating block has reached 90 C e Briefly centrifuge the Lysis Tube to remove drops from inside the lid e Add 200 uL Buffer AL to the sample and mix at full speed using a vortex type mixer e Add 200 uL ethanol 96 100 and mix again at full speed using a vortex type mixer A white precipitate may form upon addition of Buffer AL and ethanol This precipitate does not interfere with the extraction procedure e Briefly centrifuge the Lysis Tube to remove drops from inside the lid e Carefully transfer the entire lysate with the white precipitate if any to the Column in a 2 mL Wash Tube without wetting the rim e Close the lid e Centrifuge at approximately 6000 g for 1 minute at 18 25 C If the lysate has not completely passed through the membrane after centrifugation centrifuge again at a
43. ring the study 174 ng uL on average rather than high as originally determined before the study for the Lymph node V600K sample close to the LoD with a high DNA input a first sample was tested and the observed results agreed with the expected result for 17 of 32 replicates 53 1 while a Wild Type result was observed for the remaining 15 replicates The presumptive cause for the low positive rate of this panel is a mutation content below the 5 LoD of the THxID BRAF assay and slight variations in mutation content around the positivity cut off that can be present in different sections of this FFPE specimen The second sample was tested with one run per day hence the 16 replicates bioM rieux SA English 15 As a conclusion 100 agreement with expected results was obtained for 7 of 8 original panel members as well as for the additional V600K panel member tested demonstrating the precision of the THxID BRAF assay across runs operators instruments days and lots Reproducibility between laboratory precision Precision using DNA eluates analysis of quantitative results obtained for all sites instruments This precision study was evaluated on all pooled data of each panel member and positive control N 72 per panel member and per control performed at 3 external sites via multiple runs over multiple non consecutive days by 2 operators at each site using 3 lots of the THxID BRAF assay total only 2 lots were used at any one
44. site and all panel members were run in duplicate for each run including the controls The panel members are representative of the main possible sample types that can be analyzed with the THxID BRAF assay skin and lymph node Wild Type V600E or V600K low to high DNA concentration low to high mutation content presence or absence of melanin The results from the qualitative analysis coupled with information from the quantitative analysis demonstrate that the THxID BRAF assay shows a high degree of reproducibility at each study site across 3 instruments and with different operators with near 100 correct identification of each panel member The following table describes the overall agreement estimate across the sites by panel member No of valid tests all three sites Total number of tests 95 Cl Wild Type gt a l Med n a Diluted 72 72 94 9 100 high melanin V600E nin nies Med Med Diluted 72 72 94 9 100 high melanin Panel epecinen ine DNA Percent Dilution Member p yP input mutant step vo veo vo veo Cl Confidence Interval n a not applicable a high melanin content may interfere with the THxID BRAF assay see paragraph on melanin interference Dilution per the instructions restored the PCR signal An estimate of the within run precision between run operators between days between lots between sites instruments and the total precision was conducted The standard deviation and CV for Ct an
45. systems 4330015 e Precision Plate Holder for 0 1 mL tube strips for 7500 Fast System Ref Applied Biosystems 4388506 For 96 well plates e Centrifuge for 96 well plates e MicroAmp 96 and 384 well Optical Adhesive Film Ref Applied Biosystems 4311971 e MicroAmp Fast Optical 96 Well Reaction Plate with Barcode 0 1 mL Ref Applied Biosystems 4346906 e MicroAmp Adhesive Film Applicator Ref Applied Biosystems 4333183 Interpretation of results with the THxID BRAF software e Workstation with THxID BRAF software e THxID BRAF templates e Printer optional English 4 THxID BRAF WARNINGS AND PRECAUTIONS For In Vitro Diagnostic use only For professional use only This kit contains products of animal origin Certified knowledge of the origin and or sanitary state of the animals does not totally guarantee the absence of transmissible pathogenic agents It is therefore recommended that these products be treated as potentially infectious and handled observing the usual safety precautions do not ingest or inhale When working with chemicals for example Xylene consult the appropriate Material Safety Data Sheet available from the product supplier The Buffer AL and Buffer AW1 contain a harmful and irritant agent guanidine hydrochloride Refer to the risk phrases R and the precautions S above Guanidine hydrochloride can form highly reactive compounds when combined with bleach If liquids
46. t 350 ng uL The THxID BRAF assay did not show any background amplification in all tested conditions Analytical sensitivity Limit of Detection for V600E or V600K mutations The Limit of Detection LoD for the THxID BRAF assay is defined as the lowest mutation level in a specimen for which the assay yields a positive result in 95 of the tests DNA was extracted from FFPE melanoma skin and lymph node specimens with either the V600E or V600K mutation and blended with wild type FFPE DNA from the same clinical specimen type DNA input concentrations spanned the claimed input range high medium and low and a total of 24 replicates 12 replicates per lot for each condition were evaluated LoD was determined by Probit analysis the calculation was based on the assumption that the starting material extracted from the mutant specimens contained 100 mutant DNA The data support a claimed LoD of 5 mutant DNA in a background of wild type DNA for V600E and V600K positive FFPE skin and lymph node specimens across the DNA input range bioM rieux SA English 13 This 5 LoD was subsequently confirmed with 20 replicates on a sample panel including FFPE skin and lymph node specimens with high melanin and FFPE cell lines on a third lot as described in the following table Condition Sample Mutant allele at 5 Mutation positive tested 10 ng reaction Lymph node V600E 20 20 below DNA range Skin V600K 20 20 Lymph node V600E 20 20 20 ng reaction
47. t grants the purchaser rights under certain Roche patents to use it solely for providing human in vitro diagnostic services No general patent or other license of any kind other than this specific right of use from purchase is granted hereby ual bioM rieux SA Chemin de l Orme 3 M RIEUX 69280 Marcy l Etoile France RCS LYON 673 620 399 Tel 33 0 4 78 87 20 00 Distributed by bioM rieux Inc 100 Rodolphe Street Durham North Carolina 27712 USA www biomerieux com Fax 33 0 4 78 87 20 90 www biomerieux com
48. th high melanin content following the instruction to dilute showing that the presence of up to 60 of necrotic tissue does not interfere with the THxID BRAF assay bioM rieux SA English 14 Melanin Melanoma FFPE samples A population of 56 FFPE samples containing melanin levels ranging from 50 100 as determined by a pathology review were selected for the study A total of 16 specimens resulted in invalid results Nine of these samples remained unresolved after further dilution according to the troubleshooting table The distribution of melanin content for the 9 invalid specimens 4 lymph node and 5 skin is shown in the table below There were no false negative results e 1 invalid result was obtained for samples with a melanin content lt 80 1 25 4 0 e 8 invalid results were obtained on samples with a melanin content gt 80 8 31 25 8 Thus in the presence of a very high melanin content the risk of obtaining an invalid result is elevated Melanin content Number of final invalid Number of final valid Number of samples sampes samples Total 56 Dark skin FFPE samples 5 non melanoma dark skin samples were tested with the THxID BRAF assay The results were conform with the bi directional Sanger status of the samples i e Wild Type in all instances Repeatability within laboratory precision An internal study was conducted to evaluate the precision of the entire THxID BRAF workflow i e extraction and
49. trol Diluent 6 x 0 5 mL liquid Positive Control 6 x 6 4 mg lyophilized contains products of animal origin MATERIAL REQUIRED BUT NOT PROVIDED For information please contact the local bioM rieux representative General e Calibrated micropipettes with variable settings for 1 to 1000 uL delivery volumes e Timer e Vortex type mixer e Sterile disposable aerosol resistant tips e Waste container with cap or sealable e Powder free gloves Sample preparation e DNase and RNase free microtubes e Scalpel e Xylene 99 e Ethanol 96 100 molecular biology grade e 37 C 56 C and 90 C dry bath for incubation of 1 5 mL or 2 mL microtubes e Microcentrifuge 20000 g for 2 mL microtubes Amplification with Applied Biosystems 7500 Fast Dx Real Time PCR instrument e 7500 Fast Dx Instrument including Sequence Detection Software v1 4 Security Auditing and E Signature module Ref Applied Biosystems 4406984 or 4406985 bioM rieux SA 16464 C en 2013 09 Diluent containing e RNase DNase free water e preservative CONT Yellow Sphere containing synthetic DNA Each tube packed in a foil pack with silica gel desiccant For 8 tube strips e Mini Strip Centrifuge Ref bioM rieux 285056 or equivalent e MicroAmp Fast 8 Tube Strip 0 1 mL Ref Applied Biosystems 4358293 e MicroAmp Optical 8 Cap Strip Ref Applied Biosystems 4323032 e MicroAmp Cap Installing Tool Handle Ref Applied Bio
50. ut range of 10 350 ng uL i e 20 700 ng PCR reaction This range was used for most performance evaluation studies Among the 891 clinical samples included in the accuracy study see Accuracy study section with a Sanger sequencing result and a THxID BRAF result 94 6 DNA values were within 10 350 ng uL DNA values ranged from 2 to 1764 ng uL Frequency 300 f ESENE E o aM NN ee a a am 120 xD 30 4 amp 0 4D 5o 50 SD0 S0 700 759 8 amp 9 2775 WVS 2 SE xs E7 w5 675 7S 7 E 0 5 100 150 ww 25 75 1 25 1 75 225 4 7 J 425 4 7 gt i ars 5 6 DNA pornn ng L Figure 1 Distribution of the DNA concentrations observed for the 891 clinical specimens measured The x axis represents the concentration in ng uL 2 uL input are used per reaction and the y axis the number of samples per concentration One sample which has a concentration of 1764 ng uL is not represented in this plot to allow the reduction of the scale Analytical sensitivity Limit of Blank Potential background amplification Ct value was assessed on 6 clinical procured specimens at high DNA input target of 350 ng uL skin V600K at 150 ng uL covering all testing conditions i e Wild Type V600E and V600K for skin and lymph node and tested over 3 runs total of 60 replicates 20 for skin V600K samples Background amplification was also evaluated on DNA extracted from 3 cell lines Wild Type V600E homozygous V600K heterozygous at high DNA inpu
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