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A Multiplex Technology Platform for the Rapid Analysis of Clinically Actionable Genetic Alterations and Validation for BRAF p.V600E Detection in 1549 Cytologic and Histologic Specimens.

With the advent of personalized medicine, molecular testing plays a growing role in clinical oncology and clinical pathology practices. In many cases, the analysis of specific genetic alterations complements the cytopathologic or histopathologic evaluation of solid tumor specimens and provides additional valuable information to ascertain diagnosis or predict outcome and select optimal therapeutic options. For example, treatment of non-small cell lung cancer with tyrosine kinase inhibitors targeting the epidermal growth factor receptor (EGFR) requires previous knowledge of mutations associated with drug sensitivity or resistance. (1) In metastatic colorectal cancer (mCRC), the use of anti-EGFR monoclonal antibodies is restricted to patients with tumors wild type for KRAS in codons 12 and 13. (2,3) Other mutations in genes downstream of the EGFR signaling pathway, such as BRAF codon 600, are further associated with poor outcome and increased mortality in mCRC. (4,5) The mutated and activated BRAF kinase (p.V600E) is also a druggable target and a specific inhibitor is now approved in Europe and the United States for use in firstline treatment of patients with metastatic melanoma. (6,7) In thyroid cancer, testing for BRAF, KRAS, HRAS, and NRAS mutations, as well as chromosomal rearrangements involving the RET or PAX8 proto-oncogenes, increasingly contributes to the optimal surgical management of papillary thyroid carcinoma. (8,9)

Several technologies attuned to the clinical laboratory workflow and instrumentation have been developed over the years to achieve sensitive and specific detection of various clinically actionable genetic alterations. Most of these assays rely on end-point polymerase chain reaction (PCR) followed by Sanger sequencing, pyrosequencing, or amplicon detection by sizing or probe-hybridization techniques. (10,11) Another common method is to perform simultaneous amplification and fluorescence detection by using real-time PCR instruments and a variety of chemistries such as intercalating dyes, hydrolysis probes, or self-probing amplicons. (12-14) Because the multiplexing capability and throughput of these technologies are somewhat limited, alternative methods capable of detecting several mutations from distinct genes has also been developed. For example, multiplex primer extension reactions followed by capillary electrophoresis sizing (SNaPshot, Life Technologies, Carls-bad, California) have been successfully implemented in the clinical setting. (15,16) Likewise, solution hybridization of multiple PCR products on addressable microspheres, followed by flow cytometry fluorescence analysis, is broadly used in clinical diagnostic applications for bacterial, viral, genetic, or oncology targets. (17-21)

Regardless of the technology platform, molecular methods currently in clinical use have been optimized to be compatible with anatomic pathology specimens. Because of the breadth of specimen types and preanalytic parameters commonly encountered in the clinical setting, the quantity or quality of nucleic acids recovered from these specimens can vary greatly. For the preoperative molecular assessment of papillary thyroid carcinoma, specimens are in general fine-needle aspirates (FNAs) from suspicious nodules with an indeterminate cytologic diagnosis. (8,9) Depending on the cellularity and conditions of collection, storage, and extraction, the yield or integrity of the resulting purified genomic DNA (gDNA) can be severely compromised. For colorectal cancer, mutational analysis is performed on postsurgical formalin-fixed, paraffin-embedded (FFPE) tissues after histologic diagnosis. (2,3,10-12) The quality of gDNA extracted from these specimens can be dramatically affected by intramolecular and intermolecular cross-links and chemical modifications resulting in DNA fragmentation and sequence alteration. (15,22)

Preliminary studies have shown that distinct KRAS mutations can be detected by multiplex PCR and liquid bead array flow cytometry. (17) The goals of the present study were to expand the technology to relevant genetic alterations in BRAF and other genes in the EGFR pathway, to perform a complete analytic validation of the assay system, and to evaluate its performance with a large number of representative cytopathologic and histopathologic specimens.

MATERIALS AND METHODS

Clinical Specimens

All human specimens in this study were residual, deidentified gDNA samples for research purpose. No results were reported to physicians or patients or used for treatment decision and no protected health information or other information identifying patients was released. At both study sites, 10- to 20-[micro]m-thick sections from FFPE mCRC specimens were macrodissected to selectively exclude nonmalignant, stromal, and contaminating inflammatory cells and obtain a minimum of 40% tumor content. Genomic DNA was then extracted from the tumor-enriched specimens by using laboratory-developed methods based on the Recover All Total Nucleic Acid Isolation Kit for FFPE Tissues (Life Technologies, Carlsbad, California) at both sites. Genomic DNA from thyroid nodules was prepared at site 2 by using a laboratory-developed method based on the mirVana PARIS Kit (Life Technologies) and single-pass FNAs collected and stored in RNARetain, a single-use vial containing a nontoxic solution for the preservation and stabilization of intracellular nucleic acids (Asuragen, Austin, Texas). All human gDNA samples were quantified by using a GeneQuant pro (GE Healthcare Biosciences, Pittsburg, Pennsylvania) at site 1 or a NanoDrop ND1000 (NanoDrop Technologies, Waltham, Massachusetts) at site 2.

Plasmids, Cell Lines, and Dilution Samples

Plasmid DNA carrying specific mutations were prepared at site 2 by using the QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany) and characterized by bidirectional sequencing with the Sanger method. Genomic DNA from fresh cultured cell lines was prepared at site 2 by using the QIAamp DNA Mini Kit (Qiagen), and the presence of specific genetic alterations was verified by Sanger sequencing. The following amino acid changes were confirmed: BRAF p.V600E (HT29), EGFR p.L858R/p.T790M (H1975), EGFR p.E746_A750del (H1650), EGFR p.T790M/ p.E746_A750del (H820), KRAS p.G12A (SW1116), KRAS p.G12D (PL45), KRAS p.G12V (SW480), KRAS p.G13D (HCT116), HRAS p.G12V (T24), NRAS p.Q61K (HT1080), and NRAS p.Q61R (SKMEL2). Samples for analytic characterization experiments were prepared at site 2 by serial dilution based on the nucleic acid concentration as determined by NanoDrop ND1000 (NanoDrop Technologies) for each sample. Samples positive for a given mutation (plasmid, cell line, thyroid FNA, or mCRC FFPE) were diluted in a background of purified gDNA from a cell line confirmed negative for that specific mutation, keeping the total concentration of gDNA constant. Allele copy numbers in gDNA samples were estimated by using a rounded value of 3.3 pg for the mass of a haploid human genome, corresponding to about 300 allele copies per ng of gDNA.

Molecular Assays

DNA specimens were tested with the Signature BRAF Mutations Kit (for research use only; Asuragen) or prototype assays, following the protocol described in Laosinchai-Wolf et al. (17) Briefly, DNA samples were first amplified by multiplex PCR using gene-specific biotin-modified primers. Biotinylated amplified products were then directly hybridized to mutation-specific capture probes covalently conjugated to carboxylated microspheres. Following addition of fluorescent reporter, the probe-bound PCR products were detected by flow cytometry on a Luminex 200 system (Luminex, Austin, Texas). Fluorescence signals were calculated by using a minimum of 50 beads for each target. Primers and probe sequences were according to the Catalog of Somatic Mutations in Cancer Web site (www.sanger.ac.uk/genetics/CGP/cosmic, accessed November 16, 2012). Polymerase chain reaction amplifications and hybridization reactions were performed on a GeneAmp PCR System 9700 or a Veriti 96-Well Thermal Cycler (Life Technologies). The reference method at site 1 was the PCR/SNaPshot assay described in Lamy et al. (15) Amplifications were performed by using 100 to 1000 ng of gDNA and followed by primer extension reactions using sense or antisense SNaPshot primers and the ABI PRISM SNaPshot Multiplex Kit (Life Technologies). The purified labeled products were resolved on an ABI PRISM 3130x1 Genetic Analyzer (Life Technologies) and analyzed by using GeneMapper Software version 4.0 (Life Technologies). The reference method at site 2 was a laboratory-developed real-time PCR assay using the primers and hydrolysis probes described in Smyth et al. (23) Amplifications were performed with 10 ng of gDNA on a 7500 Real-Time PCR System (Life Technologies).

Data Analysis

Percentage agreements were calculated by using qualitative assay results (positive or negative) before discrepancy analysis. Quantitative analyses of signals were performed in Excel 2010 (Microsoft Corp, Redmond, Washington). Analysis of the positive and negative signal distributions at site 1 was performed by using the retest results for the 3 samples initially discrepant. Limit of blank (LOB) values were calculated according to current guidelines (mean plus 1.645 times the standard deviation), assuming 1-sided normal-like distribution of negative signals. (24) Confidence intervals were calculated by using the Wilson score interval for percentage agreements or the method described by Armitage and Berry for diagnostic odds ratios. (25-27)

RESULTS

Assay Workflow

The qualitative assay system consisted of 3 steps: (1) multiplex PCR amplification on purified gDNA, (2) hybridization of PCR products onto probes conjugated to spectrally distinct beads, and (3) simultaneous bead sorting and fluorescence analysis by flow cytometry (Figure 1). For 48 reactions in a 96-well plate format, the total assay time was less than 4 hours with approximately 1 hour of labor time and 3 hours of instrument time. The subsequent data analysis step was straightforward with qualitative interpretation of the median fluorescence intensity (MFI) signals reported for each target-specific bead population relative to a single positive/negative cutoff value (500 MFI). Representative examples of assay output, including amplification and detection of an endogenous control sequence and 3 plate/ run controls, are presented in Table 1.

Qualitative Analysis of Distinct Gene Mutations

Evaluation of the BRAF assay with 20 ng of gDNA extracted from well-characterized cell lines or 6000 copies of plasmids spiked into 20 ng of gDNA from the BRAF mutation-negative cell line DU145 showed specific detection of the mutation c.1799T>A (p.V600E) with a high signal-to-noise ratio (Table 1). With the same multiplex technology and workflow, mutational analysis of BRAF codon 600 was effectively combined with the detection of 12 distinct mutations in KRAS codons 12 and 13 (Supplemental Tables 1 through 3; see supplemental digital content in the March 2014 table of contents at www.archivesofpathology. org). No cross-detection was observed between BRAF and 7 KRAS mutations in codons 12/13 or 5 additional rare mutations in KRAS codon 13. Similarly, 6 distinct mutations in HRAS codons 12/61 or NRAS codon 61 could be detected in a single-well assay (Supplemental Table 4; see supplemental digital content at www.archivesofpathology.org). In addition to single-nucleotide substitutions, other types of genetic abnormalities, such as deletions in EGFR exon 19, could be assessed in the same reaction (Supplemental Table 5; see supplemental digital content at www archivesofpathology.org). The presence of multiple genetic alterations in a given test sample, for example EGFR c. T790M plus p.L858R or p.T790M plus p.E746_A750del, did not interfere with the assay. Overall, multiplex assays for 22 relevant mutations in the BRAF, HRAS, KRAS, NRAS, or EGFR genes were successfully established (Table 2). To fully characterize the technology platform, extensive validation studies were next performed with the prototypic BRAF assay.

Normal Range and LOB

The range of MFI signals in well-characterized, true negative samples was determined by repetitively testing 5 sample types (Table 3). A no-DNA template control and a plasmid DNA containing no BRAF-related sequences were tested in triplicate in 15 runs and BRAF-negative cell line, thyroid nodule FNAs, and mCRC FFPE tissue samples were tested in triplicate in 9 runs. The distributions of negative signals generated by the BRAF c.1799T>A probe across 171 independent measures were similar in the absence or presence of gDNA (Table 3). Analysis at the sample level also showed similar performance for different sample types (Figure 2). Mean signals (73 to 88 MFI), standard deviations (35 to 47 MFI), and maximum signals (170 to 259 MFI) were all in the same range. The calculated LOB values, representing 95% of the signal distribution for each sample type, were at least 3-fold lower than the 500 MFI cutoff value (3X LOB = 421 to 492 MFI and 447 MFI overall; Figure 2). These data indicate that a false-positive qualitative result (c.1799T>A probe signal >500 MFI) is not expected in true negative samples (P < .001).

Analytic Sensitivity and Specificity

To confirm the analytic specificity of the BRAF assay and determine its limit of detection (LOD) relative to the 500 MFI cutoff, serial dilutions of gDNA extracted from c. 1799T>A-positive cultured cell line (HT29), thyroid nodule FNA, or mCRC FFPE tissue were tested (Figure 3, A). The LOD was between 0.1% and 1% dilution at 20-ng gDNA input for all sample types. Additional mapping experiments suggested a LOD between 0.4% and 0.8% dilution (Supplemental Table 6; see supplemental digital content at www.archivesofpathology.org) and repeated testing with independent dilutions of HT29 gDNA showed reproducible detection at 0.5% dilution (Figure 3, B). Coamplification of BRAF and KRAS in a single reaction format resulted in a minimal decrease in c.1799T>A probe signal and a LOD of 1% (Supplemental Figure 1; see supplemental digital content at www.archivesofpathology. org). To further investigate analytic specificity, a series of plasmids carrying individual mutations were tested in multiple runs (Supplemental Table 7; see supplemental digital content at www.archivesofpathology.org). The plasmids c.1799_1800delinsAT (p.V600D), c.1799_1800delinsAA (p.V600E2), and c.1798_1799delinsAA (p.V600K) reproducibly generated MFI signal above the 500 MFI cutoff. No cross-detection was observed with plasmids carrying other mutations affecting BRAF codon 600 or surrounding codons, such as single-nucleotide substitutions (p.A598V, p.V600A, or p.K601E), insertions (p.A598_T599insV), or deletions (p.V600_K601>E) (Supplemental Table 8; see supplemental digital content at www.archivesofpathology.org).

Method Precision

In the analytic experiments described above, all replicates across all runs generated the same qualitative positive/ negative results. To fully validate the method precision, 8 additional studies were performed with the BRAF assay (Table 4). Within-run precision (repeatability) was evaluated by testing 4 medium to low positive samples (20%, 10%, 4%, and 2% HT29 dilutions) in quadruplicate and 2 low positive samples (2% and 1% HT29 dilutions) in 12 replicates (n = 40). Between-run precision (reproducibility) was evaluated by testing the positive, negative, and no-DNA template controls provided with the kit in 26 runs with 3 different operators (n = 78). Total precision (within laboratory precision) was further evaluated by testing (1) high, medium, and low positive samples (100%, 20%, and 2% HT29 dilutions) and 1 negative sample (DU145) in triplicate in 5 runs with 3 operators (n = 60), (2) six thyroid nodule FNAs or mCRC FFPE samples (3 positive and 3 negative) in triplicate in 3 runs with the same operator (n = 54), and (3) five thyroid nodule FNAs or mCRC FFPE samples (3 positive and 2 negative) in quadruplicate in 3 runs with 3 operators (n = 60). There was 100% qualitative agreement between all replicates and all runs for a total of 292 individual measures (95% confidence intervals: 98.7%100%) (Table 4). A precision of 100% (97.9%-100%) was also observed for the hybridization step only by performing 3 independent hybridizations on the same day with 2 instruments and 7 samples amplified in quadruplicate (n = 84) and 4 independent hybridizations on 4 different days on the same instrument with 8 samples amplified in triplicate (n = 96) (Table 4).

Compatibility With Pathologic Specimens

To assess the multiplex technology with different tissue types, specimen processing methods, and testing sites, the BRAF assay was next evaluated with a set of 1549 representative cytologic and histologic specimens (Figure 4, A). A total of 416 residual mCRC FFPE gDNA samples were tested retrospectively at 2 sites, one in Europe and one in the United States. The sample set was enriched for known BRAF c.1799T>A-positive specimens to obtain a statistically significant number of positive samples (n = 88 or 21.2% of the mCRC samples). The 1133 residual gDNA samples from thyroid nodule FNAs collected in 26 states across the United States were tested consecutively at 1 site without knowledge of the cytologic or histologic diagnoses. As the set contained a mixture of lesions with benign, suspicious, malignant, or indeterminate cytology, the prevalence for BRAF c.1799T>A was 13.1% (148 of 1133). Quantitative analysis of the 1549 c.1799T>A probe signals generated with the BRAF assay during the study showed a similar distribution of negative signals across samples and sites (Figure 4, B). The median positive signals were consistent with the differences in specimen types and gDNA inputs (11.25 ng at site 1; 20 ng at site 2). Overall, 95% of the positive signals were at least 3-fold above the cutoff (>1533 MFI) and 95% of the negative signals were at least 3-fold below the cutoff (<159 MFI). These data confirmed the high signal-to-noise ratio of the technology regardless of the potential preanalytic differences between sites or sample types.

Agreement With Reference Methods

The qualitative positive/negative BRAF assay results were also compared against independent molecular data obtained with reference methods based on multiplex PCR/SNaPshot technology at site 1 or real-time quantitative PCR technology at site 2 (Table 5). For mCRC FFPE specimens, the overall agreement between methods was 99.0% (412 of 416). Testing at site 1 initially resulted in 2 false-negative results with c.1799T>A signals just below the cutoff (431 and 344 MFI) and 1 false-positive result with a low mutant signal (744 MFI). The 3 discrepancies were resolved after retesting (data not shown). At site 2, one apparent false-positive result was observed (1193 MFI) and could not be ruled out after retesting. For thyroid nodule FNA specimens, the overall agreement between methods was 98.9% (1121 of 1133) (Table 5). Twelve apparent false positives were observed and were all confirmed positive after retesting (data not shown). Overall, the positive and negative agreements for the combined 1549 specimens were 99.2% (234 of 236) and 98.9% (1299 of 1313), respectively, with a corresponding diagnostic odds ratio of 10 856 (Table 5).

COMMENT

Molecular methods designed to complement the cytopathologic or histopathologic evaluation of solid tumor lesions should be compatible with anatomic pathology specimens. In the present study, the BRAF assay was validated relative to established reference molecular methods (15,23) by using 1549 residual gDNA samples extracted from representative thyroid nodule FNAs or mCRC FFPE tissues. The positive, negative, and overall agreements were all greater than 97.7% with lower bound confidence intervals greater than 92.1% in both the cytologic and histologic sample sets. The 12 apparent false-positive results observed with thyroid nodule FNA samples likely reflected the difference in analytic sensitivity between the 2 assays at site 2 (0.5% for the BRAF assay, approximately 5% for the reference method). Retesting of these samples and more than 90% of the true-positive samples for which enough residual gDNA was available (n = 134) ruled out a potential technical error or cross-contamination issue. Overall, calculation of the diagnostic odds ratio for the combined 1549 specimens showed an excellent assay performance with a lower bound confidence interval greater than 2451. This metric is a single global indicator of assay performance and measures the effectiveness of a test to predict a disease independently of its prevalence. (27) In our study, it can further be interpreted as the odds of a true-positive BRAF assay result (234/2) being 10 856 times higher than the odds of a false-positive result (14/1299).

For routine use in clinical pathology laboratories, novel molecular methods should also be compatible with the clinical laboratory workflow and demonstrate robust and reliable performance. Comprehensive analytic characterization studies showed reproducible detection of BRAF c.1799T>A with different sample types, thermocyclers, flow cytometers, and operators. Both within-run and between run experiments for a total of 38 runs (1 repeatability run and 1 reproducibility run were confounded) resulted in 100% qualitative agreement. Other studies, such as DNA input range (5 to 20 ng recommended), interference by potential residual protein or RNA contaminants, and reagents' or PCR products' stability, further confirmed the BRAF assay robustness (data not shown). The LOD was 0.5% dilution, corresponding to 0.1-ng positive gDNA in a background of 20-ng negative gDNA or approximately 15 copies of mutant allele detected in a background of 6000 wild-type alleles (the cell line HT29 is heterozygote for BRAF c.1799T>A). Although a lower LOD may be desirable to protect from potential false-negative results, comparative validation studies for such an assay would be extremely challenging owing to the lack of accepted reference methods reaching this level of sensitivity. For example, independent studies have shown that 10% to 20% of mCRC FFPE samples positive by molecular methods with an LOD of approximately 1% were negative by the less sensitive Sanger sequencing method. (12,17,28)

Specificity for the BRAF mutation c.1799T>A was confirmed with cell lines and residual clinical specimens, and potentially interfering mutations were evaluated with synthetic plasmids. The observed cross-detection with plasmids c.1799_1800delinsAT (p.V600D), c.1799_1800delinsAA (p.V600E2), and c.1798_1799delinsAA (p.V600K) was not surprising since these double-nucleotide substitutions result in a T to A change at position 1799 such as for p.V600E (c.1799T>A). Previous studies (13,29) have shown that p.V600D, E2, and K can also be sporadically detected by real-time PCR assays designed to detect BRAF p.V600E in melanoma specimens. These double mutations are relatively frequent in melanoma but could not be evaluated with representative clinical specimens in our study owing to their scarcity in colorectal and thyroid lesions. Specificity for distinct genes or distinct amplified regions within the same gene was however confirmed with the prototype BRAF/ KRAS, HRAS/NRAS, and EGFR assays. Our results indicate that a single technology platform can be used for the qualitative analysis of mutations in BRAF codon 600, HRAS codons 12/61, KRAS codons 12/13, NRAS codon 61, or EGFR codons 790/858, as well as deletions in EGFR exon 19. Each assay was performed in less than 4.5 hours in a 96-well plate format with a very favorable signal-to-noise ratio, enabling a simple and safe data interpretation relative to a single qualitative cutoff. Evaluation of serial dilutions with plasmid and cell line samples further suggested a similar LOD of at least 1% or 60 copies of mutant allele for HRAS, KRAS, NRAS, and EGFR (data not shown).

Multiplex assay systems provide obvious operational advantages relative to repeated series of individual PCR. In principle, all 22 mutations evaluated here could be further combined in a single assay. However, such a large panel would be extremely difficult to validate for every mutation and pathologic specimen type. Subpanels focusing on specific genes may be more practical, address most clinical needs for different tissue/specimen types, and prevent unnecessary testing. In addition, one advantage of the Luminex flow cytometer is the possibility to simultaneously analyze the fluorescence output from different assays using the multibatch mode. For example, the KRAS and BRAF assays can be performed in the same run to assess up to 45 mCRC FFPE specimens in a single 96-well plate, and up to 30 thyroid FNA specimens can be tested for BRAF, KRAS, and NRAS/HRAS in a single plate without significantly affecting the workflow or total assay time (data not shown).

Testing for oncogenic gene rearrangements in cytologic thyroid specimens also plays a key role in the management of patients with thyroid cancer to guide appropriate surgical therapy. (8,9) Independently from the present work, the multiplex assay system has been further optimized to detect fusion transcripts resulting from chromosomal inversions or translocations such as RET-PTC and PAX8-PPARG in thyroid nodule FNA specimens (unpublished results). In addition, preliminary studies suggest that the assays are compatible with histologic FFPE thyroid specimens as well as melanoma, lymph node, and lung tissues (unpublished results). We conclude that the multiplex assay system described here is a general technology platform suitable for the rapid analysis of relevant and clinically actionable genetic alterations in a variety of solid tumor specimens. Future validation studies for specific panels could provide additional speed and flexibility to clinical pathology laboratories and further advance the personalized molecular management of cancer patients.

The authors wish to thank Lauren Friar, BS, Andrew Hadd, PhD, Jeffrey Houghton, MS, Julie Krosting, MS, Maura Lloyd, MS, Rupali Shinde, MS, and Fei Ye, PhD, all from Asuragen Inc, for their technical expertise and contribution during the development of the multiplex assay system.

Please Note: Illustration(s) are not available due to copyright restrictions.

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David L. Smith, PhD; Aude Lamy, PhD; Sylvie Beaudenon-Huibregtse, PhD; Richard Sesboue, MD; Walairat Laosinchai-Wolf, PhD; Jean-Christophe Sabourin, PhD, MD; Emmanuel Labourier, PhD

Accepted for publication May 17, 2013.

Published as an Early Online Release June 28, 2013.

From Asuragen Inc, Austin, Texas, (Drs Smith, Beaudenon-Huibregtse, Laosinchai-Wolf, and Labourier); the Department of Pathology, Rouen University Hospital, Rouen, France (Drs Lamy and Sabourin); and INSERM U1079, Faculty of Medicine, Rouen University, Rouen, France (Dr Sesboue).

Drs Smith, Beaudenon-Huibregtse, Laosinchai-Wolf, and Labourier are employees of Asuragen Inc, Austin, Texas. The other authors have no relevant financial interest in the products or companies described in this article.

Supplemental digital content is available for this article in the March 2014 table of contents at www.archivesofpathology.org.

Reprints: Emmanuel Labourier, PhD, Asuragen Inc, 2150 Woodward St, Suite 100, Austin, TX 78744 (e-mail: elabourier@asuragen. com).

Caption: Figure 1. Assay workflow and time-motion analysis. The graphic shows the steps and time requirements to perform 48 reactions (I kit equivalent) in a 96-well plate. The grey and black boxes represent the operator and instrument times (in hours), respectively. Abbreviation: PCR, polymerase chain reaction.

Caption: Figure 2. Limit of blank (LOB) studies. The graph shows the mean of the median fluorescence intensity (MFI) signals for the c.1799T>A probe, the maximum (MAX) MFI, and 3 times the calculated LOB value (3X LOB) for each of the 5 samples and overall relative to the qualitative 500 MFI cutoff value (dashed line). The error bars represent the standard deviations of each distribution. Abbreviations: mCRC FFPE, metastatic colorectal cancer formalin-fixed, paraffin-embedded tissue; Thyr FNA, thyroid nodule fine-needle aspirate.

Caption: Figure 3. Limit of detection studies. A, Representative example of median fluorescence intensity (MFI) signals for the c.1799T>A probe with genomic DNA (gDNA) extracted from BRAF mutation-positive cell line (HT29), thyroid nodule fine-needle aspirate (Thyr FNA), or metastatic colorectal cancer formalin-fixed, paraffin-embedded tissue (mCRC FFPE). The samples were tested undiluted (100%) or diluted at 10%, 5%, or 0.1% positive gDNA in a background of BRAF mutation-negative gDNA from the cell line DU145 (20-ng total input). The dashed line represents the qualitative 500 MFI cutoffvalue. B, Representative example of replicate assay results (Rep 1, Rep 2, Rep 3) for the c.1799T>A probe with 1%, 0.5%, or 0.1% dilutions of HT29 gDNA and undiluted DU145 gDNA (20-ng total input). The dashed line represents the qualitative 500 MFI cutoff value.
Table 1. Representative Examples of Median
Fluorescence Intensity Signal With the BRAF Assay

                         c.1799T>A     EC

Negative control               62     7168#
Positive control          10 952#     7822#
No DNA control                 78        76
DU145 cell line gDNA          115    10 490#
PL45 cell line gDNA            91    10 110#
HT29 cell line gDNA       13 457#    10 488#
HCT116 cell line gDNA         121    10 533#
SW1116 cell line gDNA         182    10 860#
BRAF c.1799T>A plasmid    15 137#    10 858#
BRAF wild-type plasmid        166    10 513#

Abbreviations: EC, endogenous control;gDNA, genomic DNA.

Values in bold indicate positive signals above
the qualitative cutoff.

Note: Value with positive signals above the
qualitative cutoff are indicated with #.

Table 2. Genes and Mutations (Amino Acid Changes) Evaluated

BRAF       EGFR      HRAS     KRAS     KRAS     NRAS

p.V600E   p.L858R   p.G12V   p.G12A   p.G13A   p.Q61K
          p.T790M   p.Q61K   p.G12C   p.G13C   p.Q61L
           DEL19    p.Q61R   p.G12D   p.G13D   p.Q61R
                             p.G12R   p.G13R

Abbreviation: DEL19, p.E746_A750del in exon 19.

Table 3. Median Fluorescence Intensity Signals for the
c.1799T>A Probe in 5 BRAF-Negative Samples

                   Replicate   Run   Measure   MIN   MAX   Median

No gDNA (n = 2)        3       15      90       0    259     78
With gDNA (n = 3)      3        9      81       0    237     82
Overall                3       24      171      0    259     82

                   Mean   SD

No gDNA (n = 2)     79    39
With gDNA (n = 3)   82    45
Overall             81    42

Abbreviations: gDNA, genomic DNA;MAX, maximum;MIN, minimum.

Table 4. Summary of Precision Studies

Precision               PCR and Hybridization

               Within-Run   Between-Run         Total
                                          (Within-Laboratory)

Run            1      1        26          5     3     3
Sample         4      2         3          4     6     5
Replicate      4     12         1          3     3     4
Measure        16    24        78         60    54    60
Operator       1      1         3          3     1     3
Instrument     1      1         2          2     1     1
Agreement, %   100   100       100        100   100   100

Precision        Hybridization Only

               Within-Day   Between-Day

Run                3              4
Sample             7              8
Replicate          4              3
Measure            84            96
Operator           1              1
Instrument         2              1
Agreement, %      100            100

Abbreviation: PCR, polymerase chain reaction.

Table 5. Summary of Comparative Studies

                                         BRAF Assay

                                            mCRC

                                Pos         Neg          Total

Reference methods
  Pos                           86           2            88
  Neg                            2          326           328
  Total                         88          328           416
Positive agreement, % (range)         97.7 (92.1-99.4)
Negative agreement, % (range)         99.4 (97.8-99.8)
Overall agreement, % (range)          99.0 (97.6-99.6)
Combined agreement, % (range)           99.0 (98.3-99.4)
Combined odds ratio                   10 856 (2451-48 078)

                                         BRAF Assay

                                          Thyroid

                                Pos         Neg          Total

Reference methods
  Pos                           148          0            148
  Neg                           12          973           985
  Total                         160         973          1133
Positive agreement, % (range)          100 (97.5-100)
Negative agreement, % (range)         98.8 (97.9-99.3)
Overall agreement, % (range)          98.9 (98.2-99.4)
Combined agreement, % (range)           99.0 (98.3-99.4)
Combined odds ratio                   10 856 (2451-48 078)

Abbreviations: mCRC, metastatic colorectal cancer; Neg, negative;
Pos, positive.

Values in parentheses represent the 95% confidence intervals.

Figure 4. Evaluation of representative clinical specimens. A,
Overview of study design. A total of 1549 residual genomic DNA (gDNA)
samples from metastatic colorectal cancer formalin-fixed, paraffin-
embedded tissue blocks (mCRC FFPE) or thyroid nodule fine-needle
aspirates (thyroid FNAs) were evaluated at 2 sites by using distinct
reference methods. PCR-SNaPshot, polymerase chain reaction followed
by primer extension using the SNaPshot (Life Technologies, Carlsbad,
California) technology. B, Quantitative analysis of signal output.
The boxes represent the 25th, 50th (median), and 75th percentiles
ofthe positive (Pos) and negative (Neg) c.1799T>A probe signal
distributions for each site and sample type. The tails of the
distributions are indicated by whiskers corresponding to 1.5 times
the interquartile range (75th percentile value minus the 25th
percentile value) or the maximum-minimum values ofthe distributions
ifwithin the interquartile range. The median fluorescence intensity
(MFI) values for each signal distribution and the qualitative 500 MFI
cutoff value (dashed line) are also shown. Abbreviation: Thyr,
thyroid nodule fine-needle aspirate.

Pos Neg   Pos Neg   Pos Neg
Sitel     Site 2    Site 2
mCRC       mCRC      Thyr
ling       20 ng     20 ng
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Title Annotation:Original Articles
Author:Smith, David L.; Lamy, Aude; Beaudenon-Huibregtse, Sylvie; Sesboue, Richard; Laosinchai-Wolf, Walair
Publication:Archives of Pathology & Laboratory Medicine
Date:Mar 1, 2014
Words:6027
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