Methods correlation between RT-PCR test and Sanger at Site 1 and after 454 sequencing of discrepant specimens.

<p>CI = confidence interval; MD = mutation detected; MND = mutation not detected.</p>*<p>One sample subsequently confirmed as V600E by 454; Two samples subsequently confirmed as V600K;</p>†<p>Subsequently confirmed as non-V600E/wild-type by 454.</p

Example of Summary Table for Evaluation of Percent Agreement.

<p>In the table:</p><p><i>a</i> = number of specimens tested positive by both Method 1 and Method 2.</p><p><i>b</i> = number of specimens tested positive by Method 1 and negative by Method 2.</p><p><i>c</i> = number of specimens tested negative by Method 1 and positive Method 2.</p><p><i>d</i> = number of specimens tested negative by both Method 1 and Method 2.</p><p>The following statistics will be calculated:</p><p>•Overall Percent agreement between Methods = .</p><p>•Positive Percent Agreement between Methods = .</p><p>•Negative Percent Agreement between Methods = .</p><p>95% confidence intervals for the above percent agreements will be calculated using methods described in CLSI EP12-A, User Protocol for Evaluation of Qualitative Test Performance; Approved Guideline, 2002.</p

Distribution of pathological characteristics.

<p>NA = Not applicable.</p>a<p>Low = <10%; high = ≥10%.</p>b<p>Low = <50%; high = ≥50%.</p

Invalid test rates.

*<p>Retesting was permitted according to the manufacturer’s or procedure’s instructions as follows: RT-PCR test: <50% tumour content, insufficient DNA concentration, or invalid initial test result; Sanger: no PCR amplification or difficult sequence interpretation; FA test: fluorescence signal too strong, background noise, extra peaks that did not match any peaks from controls, or small mutation peaks that were difficult to identify as mutation signals.</p>†<p>The same sample was invalid when tested at the two sites.</p

Methods correlation between RT-PCR test and FA test sequencing at Site 2 and after 454 sequencing of discrepant specimens.

<p>CI = confidence interval; MD = mutation detected; MND = mutation not detected.</p>*<p>Seven samples were reported as wild type by FA test and ‘mutation detected’ by RT-PCR test of which four were subsequently found to be V600E by 454, and three to be V600K. One sample was reported as V600G by FA test and ‘mutation detected’ by the RT-PCR test and was subsequently found to be V600K by 454.</p>†<p>Twelve samples subsequently reported as wild type, three as V600E2, and one as V600R by 454.</p

Study design and specimen selection.

<p>FFPET = formalin-fixed paraffin-embedded tissue. * Low tumor content (<50%); high levels of necrosis (≥50%); significant pigmentation (<10%); or non-V600E mutations.</p

Key milestones in the co-development of vemurafenib and the cobas® 4800 BRAF V600 Mutation Test.

<p>Phases of companion diagnostic (CoDx) development in green, drug (Rx) development in blue. IDE = Investigational Device Exemption; IND = Investigational New Drug Application; MAA = Marketing Authorisation Application; NDA = New Drug Application; PMA = Premarket Approval Application; RMS = Roche Molecular Systems, Inc.</p

Correlation of NGS <i>KRAS</i> mutant allele frequency with digital PCR <i>KRAS</i> mutant allele frequency detected in the appropriate multiplex assay for FFPE tissue DNA and cell line gDNA samples.

<p>Correlation of NGS <i>KRAS</i> mutant allele frequency with digital PCR <i>KRAS</i> mutant allele frequency detected in the appropriate multiplex assay for FFPE tissue DNA and cell line gDNA samples.</p

<i>KRAS</i> mutant FFPE tissue DNA analysis using multiplex and duplex assays to detect <i>KRAS</i> mutant clones.

<p>All samples, except for S011, were analysed with multiplexes A, B and C (upper panels) and the <i>KRAS</i> mutation detected was subsequently confirmed with the appropriate duplex assay (lower panels). Mutant DNA droplet populations are highlighted with a red dashed square. Droplet populations caused by cross-reactivity with a <i>KRAS</i> mutant DNA species not present in the multiplex are indicated by a yellow dashed square.</p

<i>KRAS</i> multiplex digital PCR assays A-C and corresponding duplex assays.

<p>Multiplex A (top left panel) is an assay combination of 900 nM primers and 500 nM G13C probe (red dashed square), 450 nM primers and 250 nM G12C probe (blue dashed square) and 225 nM primers and 125 nM G12V probe (yellow dashed square). Multiplex B (top middle panel) is an assay combination of 675 nM primers and 375 nM G12S probe (red dashed square), 450 nM primers and 250 nM G12D probe (blue dashed square) and 225 nM primers and 125 nM G13D probe (yellow dashed square). Multiplex C (top right panel) is an assay combination of 675 nM primers and 375 nM G12R probe (red dashed square), 450 nM primers and 250 nM G12A probe (blue dashed square) and 900 nM primers and 500 nM Q61H probe (yellow dashed square). Multiplex C has 900 nM primers and 500 nM Q61H wild-type probe in addition to a G12C wild-type assay. All other wild-type droplet populations shown, except in the Q61H duplex assay, are 450 nM primers and 250 nM G12C wild-type probe. All panels in the left and centre columns show a FAM amplitude up to 18000 and an HEX amplitude up to 6000. Panels in the right column have a FAM amplitude up to 18000 and a HEX amplitude up to 11000.</p