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Extensive analysis of the WGS data across several lymph node metastatic samples and the PDCCs has been performed by fellow PhD candidate Dr Bruce Ashford in a complimentary study. The implications of the WGS findings pertaining only to the novel PDCCs and their matched tumours are presented here.

Genome-wide molecular characteristics of the PDCCs compared to their clinical tumour of origin are globally summarised in the Circos plots shown in Figure 3.2 A-E. Purity adjusted allelic frequency of all observed SNV (the second outermost level in the circos plots) for UW- CSCC1, UW-CSCC1-R, and its clinical tumour are globally similar (Figure 3.2 A-C). The cell lines have expressed these frequencies more confidently as expected due to the purity of the cultures, whereas the clinical tumour is also calling some stromal contaminant SNVs. Of note is the conserved UV-associated (C>T) mutational signature of these SNV, as indicated by the red dots.

The pattern of copy number variants (both gains and losses; indicated by green or red bars, respectively) is very well conserved between UW-CSCC1/-R and their originating tumour (Figure 3.2 A-C). At most there is a subtle decrease in the PDCCs for the extent of copy number variation. This was most notably observed in chromosome 4 of UW-CSCC1-R compared to the other samples. Patterns of minor allele copy number were similarly well conserved between the UW-CSCC1/-R and the originating tumour. However, reductions in minor allele copy number were noticeable in chromosomes 9 and 11 for UW-CSCC1-R.

As expected with cancer, the originating tumour for UW-CSCC1/-R has a high coding and non-coding mutational burden, highlighted by the number of transitions and translocations present (Figure 3.2 A-C). Major translocation and transition events appear conserved in the cell lines, which themselves present with additional structural variants, likely relating to the

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purity of the cell line assisting variant detection. Many variants are further detected in UW- CSCC1-R, and are indicative of additional mutation incurred via irradiation or expansion of a previously undetectable subpopulation harbouring this genotype. Either case is consistent with the alterations in both major and minor allele copy number. As such, UW-CSCC1-R is less faithful to the originating tumour than UW-CSCC1.

The low cellularity of the tumour 658492 (origin of UW-CSCC2) is obvious by the lack of variant calling and copy number variations (Figure 3.2 D), matching instead with the germline background signal. As such, WGS data of this sample is detecting and reporting on normal skin tissue with sparing detections of cancer-associated genomic aberrations. Therefore comparisons cannot be drawn between UW-CSCC2 and its originating tumour. However, compared to the other cell lines, UW-CSCC2 does indeed harbour many copy number gains and structural variants relative to its germline control (Figure 3.2 E), which at the very least supports its validity as a pure culture of cancerous cells.

Greater than 75 % of mutations in these samples are C>T, typical of a UV-radiation effect (Figure 3.2). In vitro cell culture did not appear to drastically affect the distribution of these mutational signatures compared to their clinical counterpart. However, signature 58 was not called in UW-CSCC1, despite the fact it was called in both the clinical tumour and UW- CSCC1-R. Additionally, both UW-CSCC1 and UW-CSCC1-R had less of signature 7a than the clinical tumour, compensated by a greater detection of signature 7b and 7c; although these all represent signature 7 regardless.

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Figure 3.2 Genomic landscape of PDCCs and matched tumours. Circos plots showing overall pattern of genetic aberrations between A) Tumour 193958, B) UW-CSCC1, C) UW-CSCC1-R, D) Tumour 658492, E) UW-CSCC2. Matched tumours and cell lines are indicated by * for tumour 193958 and # for tumour 658492. The layers indicate the following: i) The outermost circle shows the chromosomes, with the darker shaded areas representing large gaps in the reference genome due to regions of centromeres, heterochromatin, and missing short arms. ii) The second circle shows the purity adjusted allelic frequency of all observed SNV (including introns and intergenic regions). The scale is from 0 to 100 % and each variant coloured according to its cosmic signature; red is C>T. iii) The third circle informs on all observed copy number changes; with losses indicated in red and copy number gain shown in green. This scale ranges from 0 (complete loss) to 6 (high level gains), with those >6 indicated with a green dot. iv) The fourth circle represents the minor allele copy number. Minor allele losses are indicated in orange, whilst blue shows regions of minor allele gain. This scale ranges from 0 (complete loss of heterozygosity) to 3 (high level gains in both chromosomes). v) the innermost circle displays the observed structural variants within or between the chromosomes. Translocations are indicated in blue, deletions in red, insertions in yellow, tandem duplications in green and inversions in black. F) Mutational signature frequency of PDCCs and one clinical tumour. Matched pairs indicated by *.

(D) 658492#

(E) UW-CSCC2

(F) Mutational

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Of 3,181 genes containing exonic variants identified in the tumour, 93.7 % of these were similarly identified in UW-CSCC1. An additional 1,298 affected genes were identified in UW-CSCC1, totalling 4,479 gene variants (Figure 3.3). Similarly, UW-CSCC1-R shared 92.7 % of the clinical tumour’s genes with variants, with an additional 1,586 mutated genes detected, totalling 4,767 genes with variants in UW-CSCC1-R.

Whilst the cell lines mostly shared the same variants between one another, genes with variants unique to either cell line were also identified (Figure 3.3). It is unknown if these additional variants are from new mutations manifested through cell culture or whether they already were present in the tumour, but their detection masked through stromal interference. Genes containing exonic variants private to UW-SCC1-R were most often associated with the pathways: immune system (REACT:R-HSA-168256), metabolism (REACT:R-HSA- 1430728), and signal transduction (REAC:R-HSA-12582).

Figure 3.3 Venn-diagram showing overlapping genes with coding variants. The numerical basis for the circle proportions are shown and correspond to the number of genes with an exonic variant. Created using BioVenn (www.biovenn.nl) (Hulsen et al., 2008).

TUMOUR (3181) UW-CSCC1 (4479) UW-CSCC1-R (4767) 175

Shared between cell lines only (1324) SHARED (2936) 494 188 44 13

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These above molecular data suggest that whilst additional mutations have been called in the cell lines, they also faithfully capture the genomic profile of the originating tumour. Major CNV and gene mutations appear to be well conserved, indicative of genotypic fidelity.