Retrospective review of 11 discordant cases.

<p>Abbreviations: A, amplification; Eq, Equivocal; hg, heterogeneous; NA, no amplification; na, not assessable; nt, not tested.</p>a<p>Amplification status would not have been determined properly when applying the ASCO/CAP protocol.</p>b<p>True discordant cases.</p

Representative images of 2 discordant cases (B2 and A1).

<p>Upper left: IHC of case B2 scored as 3+. Upper right: no amplification with CISH in case B2. Lower left: IHC of case A1 scored as 1+. Lower right: amplification with CISH in case A1. IHC images: 10x magnification. CISH images: 40x magnification.</p

HER-2 co-testing protocol.

<p>IHC was performed on every case of invasive breast cancer. 129/230 cases with IHC score 0, 361/409 IHC 1+ cases, all 189 IHC 2+ cases and 105/110 IHC 3+ cases were tested by either MLPA, CISH or both. MLPA was performed in 758 cases and reflex CISH was performed in 86/96 cases with an equivocal MLPA result, a technical problem or a discordant result from IHC. The remaining 10/96 cases did not undergo reflex CISH either due to an insufficient amount of tumor tissue or an unknown reason. Furthermore, in 16 cases, CISH was performed beyond the protocol. Finally, CISH was performed instead of MLPA in 26 cases (4 were immunohistochemically heterogeneous, in 4 cases invasive tumor could not be physically separated from DCIS, in 12 cases tumor cell percentage was low, in 3 cases no MLPA was requested accidentally and in 3 cases the reason why no MLPA was performed could not be elucidated).</p

Concordance between MLPA and CISH in the validation study on 99 invasive breast cancer cases.

<p>Concordance between MLPA and CISH in the validation study on 99 invasive breast cancer cases.</p

Comparison of HER-2 IHC scoring percentages between 4 Dutch pathology laboratories.

*<p>statistically significant (p < 0.05).</p

Concordance between MLPA/CISH and IHC in the validation study on 99 invasive breast cancer cases.

<p>Abbreviations: na, not assessable.</p>a<p>6/53 (11.3%) lacked amplification by CISH following an equivocal or na MLPA result.</p>b<p>5/24 (20.8%) lacked amplification by CISH following an equivocal or na MLPA result.</p

Correlation between tumour percentage and allele frequency.

<p>The observed allele frequency for all variants detected using NGS is plotted against the tumour cell percentage as determined by a pathologist. The green line depicts the theoretical line of expected allele frequency of a heterozygous (somatic) mutation versus tumour cell percentage. A forced linear regression line (black line) was plotted to determine whether increased tumour percentage affects the mean allele frequency detected with a correlation coefficient of 0.041.</p

Next Generation Sequencing workflow for routine diagnostics.

<p>A) Number of samples included in this study. B) Ion Torrent NGS workflow analysis by which routine diagnostics from tissue arrival to reporting results to the clinician is performed within 5 working days. First, tumour percentage is determined in control sections, and subsequently tumour tissue is macro dissected, DNA is isolated and NGS is performed. If insufficient DNA is isolated and no further tissue is available, conventional techniques like Sanger sequencing is performed. Somatic variants are identified by the Torrent Variant Caller supplemented with variant annotation and filtering. Variants are manually checked in IGV and discussed in a multidisciplinary meeting with necessary clinicians. Finally, results are reported to the responsible clinician.</p

Run and library statistics.

<p>A) Boxplot of run statistics of FFPE (green) and FF (orange) samples for 4 variables: 1. the percentage of ISP (Ion Sphere Particle) density (the addressable wells on the chip which have detectable loading); 2. usable reads of the total number of reads (percentage of ISPs that pass the polyclonal, low quality, and primer dimer filters); 3. polyclonals, ISPs that contain more than one template sequence per ISP and 4. low quality, ISPs with a low or unrecognizable signal. The upper and lower “hinges” of the boxplots correspond to the first and third quartiles (the 25^th and 75^th percentiles). The upper “whisker” extends from the hinge to the highest value that is within 1.5*IQR of the line, where IQR is the inter-quartile range (the distance between the first and third quartiles). The lower “whisker” extends from the hinge to the lowest value within 1.5*IQR of the hinge. Data beyond the end of the vertical lines are outliers and plotted as points. B) Library statistics of FFPE (green) and FF (orange) samples; the mean target base read depth (including non-covered target bases); the number of reads mapped to the full reference genome; and the percentage of mapped reads which are aligned to the target region. Significant differences calculated by means of an independent t-test between FFPE and FF samples are depicted with ** p = 0.002 or ***p = 0.0009).</p

Variant distribution of the complete dataset.

<p>A) Heatmap of number of variants per tumour group. On the y-axis the different primary tumour site is depicted and on the x-axis all genes with mutational data are depicted. The relative number of mutations is defined as the number of mutations normalized per number of samples in the tumour group. B) co-occurrence of different variants in colorectal tumours. The size of the circle around a gene is indicative of the number of times a variant is identified in the gene. The lines represent co-occurrences between genes where the line thickness indicates the number of co-occurrences. The colour of the circles indicates the function of the gene: green–tumour suppressor genes and oncogenes, purple–receptor tyrosine kinases, pink–PI3K pathway, yellow–KRAS/BRAF pathway.</p