Analytical Performance of a Real-time PCR-based Assay for V600 Mutations in the BRAF Gene, Used as the Companion Diagnostic Test for the Novel BRAF Inhibitor Vemurafenib in Metastatic Melanoma : Diagnostic Molecular Pathology

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00019606-201203000-00001ArticleDiagnostic Molecular PathologyDiagnostic Molecular Pathology© 2012 Lippincott Williams & Wilkins, Inc.21March 2012 p 1–8Analytical Performance of a Real-time PCR-based Assay for V600 Mutations in the BRAF Gene, Used as the Companion Diagnostic Test for the Novel BRAF Inhibitor Vemurafenib in Metastatic MelanomaOriginal ArticlesHalait, Harkanwal MS; DeMartin, Kelli MS; Shah, Sweta BS; Soviero, Stephen BS; Langland, Rachel BS; Cheng, Suzanne PhD; Hillman, Grantland MS; Wu, Lin PhD; Lawrence, H. Jeffrey MDRoche Molecular Diagnostics, Pleasanton, CAThe authors declare no conflict of interest. All authors are employees of Roche Molecular Systems, Inc. Pleasanton, CA.Reprints: H. Jeffrey Lawrence, MD, Roche Molecular Diagnostics, Pleasanton, CA 94588 (e-mail: [email protected]).AbstractMelanomas frequently harbor BRAFV600 mutations. Vemurafenib (RG7204/PLX4032), a small-molecule inhibitor of mutant BRAF, has shown striking clinical efficacy in BRAFV600 mutant melanoma, creating the need for a well-validated companion diagnostic to select patients for treatment. We describe analytic performance characteristics of the cobas 4800 BRAF V600 Mutation Test, the test used to select patients for the pivotal vemurafenib trials. This real-time polymerase chain reaction assay was designed to detect the V600E (1799T>A) mutation DNA from formalin-fixed paraffin-embedded tissue samples. Sensitivity was assessed using blends of cell lines or tumor DNA, and tumor specimens with low levels of mutant alleles, as determined by 454 sequencing (a quantitative next-generation pyrosequencing method). A >96% hit rate was obtained across all specimen types with 5% mutant alleles at a DNA input of 125 ng, an amount readily obtained from one 5-μm section. The cobas test showed a higher sensitivity and specificity than direct bidirectional sequencing in a panel of 219 melanoma specimens. Cross reactivity with V600K and V600D was observed. Repeated testing of 5 specimens by 2 operators, using different instruments and reagent lots, yielded correct calls in 158/160 tests (98.8%). A set of 26 highly pigmented samples were identified that gave invalid test results. A simple 1:2 dilution resulted in a valid test result of 76% in such cases. The cobas test is a reproducible assay that detects some non-V600E mutations and is more accurate than direct sequencing in detecting BRAFV600E.Activating mutations of the proto-oncogene BRAF occur in many human cancers, including malignant melanoma, colorectal cancer, ovarian cancer, and thyroid cancer.1 BRAF mutations have been identified in 40% to 60% of melanomas,1–3 where they appear to be a very early event, as they are common in benign nevi.4 The discovery of such somatic mutations in the BRAF gene in melanoma and other human tumors has led to the clinical development of specific small-molecule inhibitors of the mutant BRAF kinase as a targeted therapy. One such molecule, vemurafenib (RG7204/PLX4032), has shown striking clinical efficacy in metastatic melanoma.5,6 However, preclinical and limited clinical data indicate that the compound has minimal activity in tumors lacking BRAF mutations.7 The majority of BRAF mutations in melanoma and other human tumors, including colorectal and papillary thyroid cancers, occur in codon 600, with the predominant mutation being the V600E (1799 T>A) single-nucleotide mutation.8The advent of effective targeted therapy against the mutant BRAF kinase has created the medical need for an accurate, rapid, and robust assay to detect BRAF V600 mutations. Traditional bidirectional direct (Sanger) sequencing has been commonly used for mutation testing in clinical laboratories, but it suffers from a limited sensitivity for low-level mutant alleles and a relatively slow turn-around time.9,10 We set out to develop a companion diagnostic assay for vemurafenib, which was used to select patients for enrollment in the pivotal clinical trials of this compound in metastatic melanoma.The design of robust, accurate molecular diagnostic tests for clinical use is not straightforward, and is complicated by a number of specimen attributes, such as specimen age, tumor content, degree of necrosis, and the presence of endogenous and exogenous inhibitors, all of which may confound assay results.11 In addition, the damaging effects of formalin fixation can limit the amplifiability of the DNA. In the case of malignant melanoma, melanin itself may inhibit DNA polymerases, thus resulting in invalid test results.12 These specimen attributes may account for the variable mutation results observed in a number of quality control studies of assays performed by different clinical laboratories testing the same panel of samples.13,14 Thus, it is important to fully assess these specimen attributes as part of the validation of any companion diagnostic assay to ensure that its performance characteristics are well defined. Diagnostic testing methods may also be influenced by work flow issues such as specimen selection, reagents and testing platforms used, and the result-reporting process.A number of less common dinucleotide mutations affecting codon 600 [V600K (1798 1799 GT>AA), V600R (1798 1799 GT>AG), V600D (1799 1800AG>AT), “V600E2” (1799 1800AG>AA)] have also been observed in melanoma, along with rare mutations affecting other codons of the BRAF gene. According to the COSMIC database, the most prevalent of these variant mutations in melanoma is V600K, which is reported to represent 6.5% of all codon 600 mutations. Preclinical studies show that cell lines harboring V600K, V600D, and V600R mutations are sensitive to vemurafenib,7 and limited clinical data suggest that patients with V600K-mutant melanomas may be sensitive to vemurafenib.5The cobas 4800 BRAF V600 Mutation Test (cobas test; Roche Molecular Systems, Pleasanton, CA) is a real-time polymerase chain reaction (PCR) assay designed to detect the presence of the V600E (1799 T>A) mutation. Figure 1 depicts the work flow for the assay, which can be performed in approximately 8 hours. The testing process consists of 2 main steps: (1) a manual specimen preparation to obtain genomic DNA from formalin-fixed, paraffin-embedded tissue (FFPET); and (2) PCR amplification and detection of target DNA using a complementary primer pair and 2 oligonucleotide probes labeled with different fluorescent dyes. One probe is designed to detect the wild-type (WT) BRAF V600 sequence (GTG) and one is designed to detect the V600E mutation sequence (GAG). The WT allele serves as an internal full-process control. Two external run controls are provided, including one BRAF WT and one V600E mutant control. The analysis and result reporting are fully automated. In this report, we present the results of several nonclinical studies performed to evaluate the performance characteristics of the cobas test.JOURNAL/dimp/04.03/00019606-201203000-00001/figure1-1/v/2021-02-17T200041Z/r/image-jpegCobas 4800 BRAF V600 Mutation Test workflow. H&E indicates hematoxylin and eosin; PCR, polymerase chain reaction.MATERIALS AND METHODSTumor and Cell Line Specimen SourcesFFPET specimens of malignant melanoma were purchased from external commercial vendors. Two human melanoma cell lines, SK-MEL-28 and SK-MEL-2, were obtained from the American Type Culture Collection (Manassas, VA).Specimen PreparationFFPET specimens were processed, and genomic DNA was isolated using the cobas DNA Sample Preparation Kit (Roche Molecular Systems, Branchburg, NJ, USA), a manual specimen preparation based on nucleic acid binding to glass fibers. One 5-μm section of the FFPET was deparaffinized as per the instructions for use with xylene and ethanol. The deparaffinized section was subjected to lysis with proteinase K and further heated to a high temperature. It was then purified by centrifugation through a column with a glass fiber filter insert and subsequently eluted with an aqueous solution. The amount of genomic DNA was determined from duplicate readings at 260 nm, obtained with a Nanodrop ND-1000 or ND-2000 UV–vis Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE) according to the manufacturer’s instructions.Cobas 4800 BRAF V600 Mutation Test (the Cobas Test)Specimens were diluted to 5 ng DNA/µL. A working Master Mix was prepared as per the instructions for use and added at 25 µL to each reaction well on the 96-well amplification-detection plate. Reaction controls and diluted specimen were then added to the respective reaction wells at 25 µL/well and mixed. Plates were then covered with a sealing film. The sealed plate was loaded into the cobas 4800 System v2 analyzer to begin automated real-time PCR detection. The detection results (“Mutation Detected” or “Mutation Not Detected”) were automatically generated into a report through the cobas BRAF Analysis Software Package. The total test time including DNA isolation is approximately 8 hours.Preparation of the Melanoma Cell Line and FFPET DNA BlendsMutant-type and WT melanoma cell lines (SK-MEL 2, SK-MEL 28) and FFPET specimens were processed using the cobas DNA Sample Preparation Kit. Extracts from both mutant-type and WT samples were pooled separately, and the genomic DNA concentration of each pool was quantified by NanoDrop measurement. Sample pools were diluted with elution buffer to a final DNA concentration of 125 ng/25 µL (5 ng/µL). Subsequently, the mutant sample extract at 5 ng/µL was blended by volume with the WT sample extract at 5 ng/µL (0% mutation) to achieve the target BRAF V600E mutation percentage. After blending, the mutation percentage was verified by 454 GS Titanium Sequencing (454 LifeSciences, Branford, CT). If the target mutation percentage was not achieved, the mutant and WT extract blending ratio was adjusted, and the mutation percentage was reconfirmed by 454 GS Titanium Sequencing.Sanger SequencingDNA extraction and amplicon generation were performed at Roche Molecular Systems. 2X bidirectional sequencing was performed using a validated protocol for BRAF mutation detection by SeqWright (Houston, TX).454 SequencingIt is a quantitative massively parallel “deep”-sequencing method, which involves clonal amplification of target sequences by emulsion PCR followed by a massively parallel pyrosequencing. The assay has a validated limit of detection for V600E mutations of 1%. 454 sequencing was performed using a validated protocol for BRAF mutation detection at SeqWright (Houston, TX).Interfering SubstancesStock concentrations of hemoglobin and triglycerides were prepared in a saline background by Complex Antibodies (Coconut Creek, FL). Testing concentrations were prepared before testing by dilution of the stocks in saline (provided by Complex Antibodies.)Melanin QuantificationIn order to measure melanin in melanoma samples, a standard curve was generated from synthetic melanin (Sigma-Aldrich, St Louis, MO). The melanin was weighed and then solubilized with 1 N NaOH/10% dimethyl sulfoxide and heated at 80°C for 2 hours. Absorbance at 400 nM was measured on a NanoDrop ND-1000 (Thermo Scientific, Waltham, MA). To determine the concentration of melanin in specimens, 25 µL of sample was mixed with 25 µl of a 1 N NaOH/10% dimethyl sulfoxide solution and heated at 80°C for 2 hours. The absorbance at 400 nm was used in the standard curve equation to calculate the concentration.RESULTSAnalytical SensitivityThe analytical sensitivity based on hit rates (Table 1) was assessed using DNA blends from melanoma cell lines, DNA blends from FFPET melanoma specimens, and individual FFPET tumor specimens with low levels of BRAF mutant alleles (as determined by 454 sequencing).4 A correct mutation call rate of >96% was obtained across all specimen types, with 5% mutation sequences at a DNA input of 125 ng per PCR reaction. The calculated PROBIT values (95% probability) for the cobas test ranged from 0.6 to 30.3 ng per PCR genomic DNA input for specimens with 5% mutant sequences (data not shown).JOURNAL/dimp/04.03/00019606-201203000-00001/table1-1/v/2021-02-17T200041Z/r/image-tiffReplicates of Each Panel Member Were Run Using Each of the 3 cobas 4800 BRAF V600 Mutation Test kit LotsMinimal Tumor ContentThirty-three BRAF V600E-mutant, 24 BRAF WT, and 17 BRAF non-V600E mutant specimens (as determined by Sanger and/or 454 sequencing) were tested to determine the sensitivity of the cobas test for detecting the mutation status in specimens with an estimated tumor content ranging from 5% to 50% (without macrodissection). Specimens were also subjected to 454 sequencing to quantify the percentage of mutant alleles, which was then correlated with the estimated tumor content (Fig. 2). A lack of correlation between the estimated tumor content of a FFPET section and the percent BRAF mutation, as measured by 454 sequencing, is evident.JOURNAL/dimp/04.03/00019606-201203000-00001/figure2-1/v/2021-02-17T200041Z/r/image-jpegPercent tumor and % mutation for 33 BRAF V600E-mutant specimens tested with the cobas 4800 BRAF V600 Mutation Test for the minimal tumor content sensitivity study test. PCR indicates polymerase chain reaction.The cobas test detected 29 BRAF V600E mutant specimens with tumor content ≥10% and percent mutation ≥2.3%; four mutants with tumor content ≤10% and percent mutation ≤4.7% were not detected (Fig. 2). For all 24 BRAF WT specimens tested (with tumor content ranging from <5% to 45%), Mutation Not Detected results were obtained. Among the 17 BRAF non-V600E mutant specimens tested, cross reactivity was observed with 2 BRAF V600K mutant specimens and 1 BRAF V600D mutant specimen. Mutation Not Detected results were obtained on the remaining 13 BRAF V600K specimens and a single BRAF V600R mutant specimen, with tumor content ranging from 5% to 45%, and percent mutant alleles ranging from 1.38% to 23.1% (median 14%). Thus, the assay reliably detects V600E mutation in samples with tumor content ≥10%, with some cross reactivity with non-V600E mutations.Method CorrelationThe cobas test was compared with 2x bidirectional Sanger sequencing for the detection of the predominant V600E (1799T>A) mutation, using a set of 219 FFPET melanoma specimens tested with 2 different lots of cobas reagents, with 1 specimen replicate per lot for a total of 438 replicates. The observed positive percent agreement between cobas test and Sanger sequencing was 96% (189/198), the negative percent agreement was 82% (196/240), and the overall percent agreement was 88%. Specimens that yielded discrepant results between the cobas test and Sanger were subjected to 454 sequencing. The agreement analysis between the cobas test and Sanger sequencing, including the discrepant resolution by 454, is depicted in Table 2.JOURNAL/dimp/04.03/00019606-201203000-00001/table2-1/v/2021-02-17T200041Z/r/image-tiffAgreement Analysis Between the cobas and Sanger Test Results Before and After Discrepant Resolution With 454 SequencingOf the 9 discrepant replicates determined to be V600E positive by Sanger but mutation negative by the cobas test, 454 sequencing detected no V600E mutation. In 6 of the 9 discrepant samples, 454 sequencing detected a WT result; in 2 replicates, 454 sequencing detected a “V600E2” mutation, and in 1 case a V600K mutation.Of the 44 discrepant replicates found to be V600E mutation negative by Sanger but mutation positive by the cobas test, Sanger sequencing yielded a WT result in 28 replicates and a non-V600E mutation in the other 16 replicates. 454 sequencing detected 26 V600E mutations and 2 V600D mutations in the 28 replicates that were WT by Sanger. For the remaining 16 replicates, 454 sequencing confirmed the presence of 14 V600K and 2 V600E mutations.After discrepant analysis with 454 sequencing, the positive percent agreement increased from 96% to 100%, negative percent agreement from 82% to 93%, and overall percent agreement from 88 to 96%. This study showed that (1) the cobas test detected a number of V600E mutations that were not detected by Sanger, (2) Sanger determined V600E mutations in a number of specimens that were not confirmed by either cobas or 454, and (3) the cobas test had substantial cross reactivity with V600K.Cross Reactivity With non-V600E MutationsGiven the observed cross reactivity of the cobas test for V600K mutations observed in the methods correlation study just described, we undertook a more detailed assessment of the cross reactivity of the assay for a number of variant codon 600 mutations and mutations in adjacent codons. According to COSMIC database v54,8 in tumors originating in the skin, V600E mutations accounted for 92.5%, V600K mutations for 5.6%, V600R mutations for 1.0%, “V600E2” for 0.7%, and all other codon 600 mutations 0.2%. Rare mutations have also been observed in adjacent codons (eg, D594G, G596R, K601E, L597Q, and L597S).BRAF non-V600E Plasmid BlendsPlasmid dilution panels with percent mutation ranging from 5% to 75% in a background of WT plasmid were prepared for 9 BRAF non-V600E mutations (D594G, G596R, K601E, L597Q, L597S, V600D, V600K, V600R, and “V600E2”). Cross reactivity was seen for BRAF V600D plasmid at ≥10% mutation, BRAF V600K plasmid at ≥35% mutation, and BRAF “V600E2” plasmid at ≥65% mutation. No cross reactivity was observed with plasmids from the remainder of the BRAF mutations tested.BRAF non-V600E Mutant FFPET SpecimensFourteen BRAF non-V600E mutant melanoma FFPET specimens were tested with the cobas test. (Table 3). No cross reactivity was observed for the BRAF V600R mutant specimen. Eight of the remaining 13 specimens [V600D (1/1), V600E2 (1/3), and V600K (6/9)] showed cross reactivity with the cobas test. A DNA dilution panel was tested in triplicate to determine the lowest DNA input that yielded a 100% Mutation Detected rate for those specimens (Table 3). The lowest DNA input detected ranged from 0.5 to 15.6 ng/PCR. These studies were consistent with the findings observed with the plasmid blends, and indicated that the cobas test can detect V600K mutations at >31% mutant alleles and “V600E2” at >68%.JOURNAL/dimp/04.03/00019606-201203000-00001/table3-1/v/2021-02-17T200041Z/r/image-tiffCobas 4800 BRAF V600 Mutation Test: the Lowest DNA Input Before the Loss of Cross Reactivity [<100% Mutation Detected (MD) Rate] Observed With 4 BRAF Variant Codon MutationsPlasmids of Other RAF Family MembersNo cross reactivity was seen with BRAF pseudogene, ARAF, and RAF-1. The BRAF V600E Mutant plasmid at 5% yielded Mutation Detected results in the presence of 95% of these other RAF plasmids, indicating that there was no interference from these plasmids in detecting the BRAF V600E mutation.Interference StudiesPotential Interference With MelaninBecause previous studies had demonstrated that melanin could bind to and interfere with DNA polymerases,12 there was concern that highly pigmented melanoma samples could result in invalid test results. Consistent with these previous studies, we observed that the addition of synthetic melanin could inhibit PCR amplification, starting at levels of >4.0 ug/ml (data not shown).We also noted that the cobas DNA Sample Preparation procedure, which utilizes a glass fiber capture column to purify DNA, was associated with a marked reduction in the degree of visible pigmentation of the crude lysate (Fig. 3). We measured the melanin concentration in 5 highly pigmented specimens both (a) in the crude lysate before column purification and (b) in the postcolumn eluate. We found that the DNA isolation procedure removed >89% of melanin in all 5 cases.JOURNAL/dimp/04.03/00019606-201203000-00001/figure3-1/v/2021-02-17T200041Z/r/image-jpegRemoval of melanin during DNA isolation. Each panel depicts the crude lysate (left) and postcolumn eluate (right) for individual specimens. The percent reduction in melanin for each specimen is represented by the percentage under each panel.Although the DNA isolation process removed a large fraction of the endogenous melanin, we nonetheless identified 26 specimens that yielded an initially invalid cobas test result, including 13 specimens identified at one of the clinical testing sites involved in the BRIM-3 clinical trial of vemurafenib and 13 vendor-purchased specimens. All 26 samples were found to be highly pigmented by visual inspection. Serial dilution studies of these samples revealed that 76% of the samples yielded a valid test result after a 1:2 dilution of the sample before amplification. The test results of all specimens were independently confirmed by 454 sequencing.Potential Interference With Other Endogenous SubstancesTriglycerides (≤74 mM), hemoglobin (≤2 mg/mL), and low levels of synthetic melanin (≤4 µg/mL) did not interfere with the test when the potential interfering substance was added at the tissue lysis step during the specimen preparation procedure (data not shown).Potential Interference With Exogenous Microbial SubstancesSix skin-related microorganisms (Staphylococcus epidermis, Staphylococcus aureus, Corynbacterium xerosis, Corynbacterium jeikeium, Corynbacterium minutissimum, and Corynbacterium ulcerans) were found to not cross react in the test when added to a WT specimen at 1×106 cfu during the tissue lysis step. The microorganisms tested did not interfere with the detection of the BRAF V600E mutation when 1×106 cfu were added during the tissue lysis step of a specimen containing a low level (7.8%) of the BRAF V600E mutation (data not shown).RepeatabilityRepeatability of the cobas test was assessed using five melanoma FFPET specimens (Table 4). Four of the specimens were BRAF V600E mutants, 2 with tumor content near 25% and 2 with mutation percentages near the analytical sensitivity of the test (∼5%). One specimen was a WT BRAF specimen. The specimens were tested in duplicate by 2 operators, using 2 different reagent lots and 2 cobas 4800 System v2.0 analyzers over 4 days (n=32/specimen). The cobas test had a correct call rate of 98.8% (158/160) across all days, specimens, replicates, operators, and reagent lots combined.JOURNAL/dimp/04.03/00019606-201203000-00001/table4-1/v/2021-02-17T200041Z/r/image-tiffCobas Test RepeatabilityDISCUSSIONThe number of malignancies that require molecular testing to select patients for specific targeted therapies is growing, and currently includes breast cancer (HER2 testing for trastuzumab), chronic myelogenous leukemia (bcr-abl testing for imatinib), and colorectal cancer (KRAS mutation testing for cetuximab and panitumumab). BRAF mutation testing has joined this list to select patients for therapy with specific BRAF inhibitors such as vemurafenib. The key features of a clinically useful mutation assay, in addition to diagnostic accuracy, include reproducibility, a rapid turn-around time, and the requirement for a small amount of DNA to perform the test. Another critical aspect of the validation of a mutation assay is a comprehensive understanding of the influence of potential endogenous and exogenous interfering substances and cross reactivity with related mutations on assay performance.The cobas test can be performed with a DNA input of 125 ng/PCR, an amount typically obtained from a single 5-µm FFPET section, and can be completed in approximately 8 hours. The assay was highly reproducible when tested by different operators on different instruments with different reagent lots, and using samples with <20% mutant alleles. The ability to detect low-level mutation is particularly valuable for tumor specimens that are heavily infiltrated with normal cells, such as lymphocytes, which cannot always be removed by macrodissection. The assay also showed no interference from common skin microbes that could be present in a biopsy specimen of a cutaneous melanoma or from endogenous substances such as hemoglobin and triglycerides.In the methods correlation study, the PCR assay showed superior analytic accuracy to 2X bidirectional Sanger sequencing, as determined by testing with a third more sensitive and quantitative deep sequencing method. It is noteworthy that Sanger not only missed 13% of V600E mutations but also yielded V600E mutation calls in cases where neither the cobas test nor 454 detected a mutation. In contrast, in specimens for which the cobas test detected a mutation, a codon 600 mutation was detected by Sanger and/or 454 in every case. Thus, the cobas test appeared to be more sensitive and more specific than Sanger sequencing.Although this assay was designed to detect the V600E (1799T>A) mutation, it showed varying degrees of cross reactivity with V600D, V600K, and “V600E2” in both plasmid blends and FFPET-derived DNA. The Catalog of Somatic Mutations in Cancer database indicates that approximately 90% of the codon 600 mutations in melanoma are of the V600E type.8 Data are currently very limited on the clinical importance of non-V600E mutations. In the phase III trial of vemurafenib, a subset of cobas test-positive specimens from enrolled patients was subjected to retrospective testing with Sanger sequencing, which identified 10 specimens with V600K mutations in patients on the vemurafenib arm. Four of these 10 patients experienced objective responses from the drug, suggesting that vemurafenib may benefit patients with these mutations.5 No clinical outcome data are yet available for other non-V600E mutations such as V600D and V600R. Collecting clinical information on these other mutations will be challenging considering their rarity.A noteworthy observation from these studies is that the estimated tumor content showed little, if any, correlation with the percentage of mutant alleles detected in the specimens. These findings suggest that the tumor content assessment is highly observer dependent and intrinsically imprecise. The degree of infiltration by normal cells (lymphocytes, fibroblasts, endothelial cells, etc.) may be underestimated, as normal cells are typically much smaller than the malignant cells. Furthermore, underestimation of necrotic and apoptotic tumor cells may result in an exaggerated assessment of tumor content. Finally, it is hypothetically possible that tumors are heterogeneous with regard to their BRAF mutation status, although the evidence for this is limited.These studies indicate that endogenous melanin may be an important potential source for assay interference in some specimens, and this interference can be overcome in a majority of cases by a simple dilution step before amplification. In our clinical series and vendor-purchased samples, the phenomenon of melanin interference resulting in invalid test results was uncommon: approximately 2% of all cases. It is notable that the sample preparation kit used with this assay removes approximately 90% of the melanin in the tissue specimens (Fig. 3), perhaps reducing the likelihood of test interference. The small size of the BRAF amplicon generated for the cobas test may also explain the infrequency of melanin interference, as prior studies showed that the capacity of melanin to interfere with DNA polymerase increased with the size of the amplicon.12 In addition, a simple 1:2 dilution resulted in a valid cobas test result in those highly pigmented specimens that yielded an invalid result initially. The extent of inhibition of endogenous melanin in other commercial assays for BRAF mutations has not been reported.In summary, the analytical studies presented here demonstrate that this companion diagnostic assay for the BRAF inhibitor vemurafenib is a robust and reproducible test for the detection of the BRAF V600E (1799T>A) mutation. With a limit of detection of 5% mutant alleles, the test is more sensitive than conventional Sanger sequencing in the detection of V600E mutations and detects some non-V600E mutations. 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H&E indicates hematoxylin and eosin; PCR, polymerase chain reaction.Replicates of Each Panel Member Were Run Using Each of the 3 cobas 4800 BRAF V600 Mutation Test kit LotsPercent tumor and % mutation for 33 BRAF V600E-mutant specimens tested with the cobas 4800 BRAF V600 Mutation Test for the minimal tumor content sensitivity study test. PCR indicates polymerase chain reaction.Agreement Analysis Between the cobas and Sanger Test Results Before and After Discrepant Resolution With 454 SequencingCobas 4800 BRAF V600 Mutation Test: the Lowest DNA Input Before the Loss of Cross Reactivity [<100% Mutation Detected (MD) Rate] Observed With 4 BRAF Variant Codon MutationsRemoval of melanin during DNA isolation. Each panel depicts the crude lysate (left) and postcolumn eluate (right) for individual specimens. The percent reduction in melanin for each specimen is represented by the percentage under each panel.Cobas Test RepeatabilityAnalytical Performance of a Real-time PCR-based Assay for V600 Mutations in the BRAF Gene, Used as the Companion Diagnostic Test for the Novel BRAF Inhibitor Vemurafenib in Metastatic MelanomaHalait Harkanwal MS; DeMartin, Kelli MS; Shah, Sweta BS; Soviero, Stephen BS; Langland, Rachel BS; Cheng, Suzanne PhD; Hillman, Grantland MS; Wu, Lin PhD; Lawrence, H. Jeffrey MDOriginal ArticlesOriginal Articles121p 1-8

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