1. Supplementary Figure 1. A Case no. #1 #2 #3 #5 #6 #7 #8 #9 #10 #11 #13
#14
#15
#16 2 1 -1 -2
HPV status
= HPV(+)
= HPV(-)
One timepoint
= Diagnostic biopsy
or Surgical resection
DB/SR
Two timepoints DB
= Diagnostic biopsy
= Surgical resection SR DB SR 15 3 13 9 8 2 14 16 6 7 11 10 1 5

2. Supplementary Figure 1. B#1 #2 #3 #5 #6 #7 #8 #9
#10
#11
#13
#14
#15
#16
Case no.
2(11%)
3(8%)
1(24%)
Supplementary 1. Hierarchical clustering and Principle component analysis of all cases and all sequenced genes (n=18979). RNA-Seq analysis showing row wise z-scores of normalized read counts for all genes across all of the tumor replicates. Patient tumor replicates are color coded and displayed on the heatmap and PCA plot; HPV(+)=black and HPV(-)=beige; diagnostic biopsy (DB) and surgical resection (SR) replicates are annotated below. (A) Hierarchical clustering (distance measure = Pearson’s correlation metric; clustering = average linkage method) of tumor replicates displayed as a heatmap showing close clustering of replicates by case. (B) PCA was used to visualize the sample to sample distances and shows similarities between the tumor replicates.

3. Supplementary Figure 2. 1 2 8 11 15 16 3 5 6 7 9 10 13 14
Supplementary Figure 2. Comparison of minimum and maximum Euclidean distance from hierarchical clustering of tumor replicates. The distance between each tumor replicate in the hierarchical tree was calculated and used to assess how closely intrapatient replicates were compared to interpatient replicates. The minimum, maximum and median Euclidean distance (sqrt(1-pearson)⌃2) for intrapatient replicates was plotted alongside the median Euclidean distance for interpatient replicates. The smaller the distance and the shorter the line the more related the intra and interpatient samples are, conversely a larger distance or longer line identify lower homology between intra and interpatient samples. Overall intra patient tumor replicates are closer to each other than the interpatient replicates.

4. Supplementary Figure 3.
One timepoint
= Diagnostic biopsy
or Surgical resection
DB/SR
Two timepoints DB
= Diagnostic biopsy
= Surgical resection SR DB SR 1 2 8 11 15 16 3 5 6 7 9 10 13 14
Case no.
Spearman correlation r
Intrapatient
Interpatient
Median
0.96
0.92
Min
0.91
0.82
Max
0.98
0.97
Supplementary Figure 3. Spearman correlation analysis of all genes and all tumor replicates. Single timepoint (sampling across space) and two timepoint (sampling across time between diagnosis and resection) tumor replicates were assessed using a correlation matrix of gene expression from all sequenced genes (n=18979); each tumor replicates gene expression was correlated to each sample. Intrapatient tumor replicates were more correlated with a median correlation of r=0.96 compared to an interpatient median correlation r=0.92.

5. Supplementary Figure 4. A B C

6. D E
Supplementary Figure 4. Gene expression profiles of immune genes across the sample cohort. Gene expression (RPKM – Reads per kilobase per million mapped reads) of immunologically relevant genes for the single timepoint (sampling across space) and two timepoint (sampling across time between diagnosis and resection) tumor replicates. Samples from the same patient display a high level of similarity for the genes CD3E, GZMA, IFNG, CTLA4 and CD274 (PDL1), A-E respectively.

7. Exhaustion/ Regulatory
Supplementary Figure 5.
PTPRC
CD3E
CD4
CD8A
MS4A1
SELL
FOXP3
IL10
IL2RA
CD86
GZMA
IFNG
PRF1
TNFRSF9
CD274
PDCD1LG2
HAVCR2
LAG3
CLTA4
TIGIT
ICOS
CD27
Immune Cell
markers
Effector
Function 2 1 -1 -2
Imm gene list
Exhaustion/ Regulatory
Function DB SR 1 5 6 7 3 10 11 2 8 14 15 16 13 9
Case no.
HPV status #1 #2 #3 #5 #6 #7 #8 #9
#10
#11
#13
#14
#15
#16
= HPV(+)
= HPV(-)
One timepoint
= Diagnostic biopsy
or Surgical resection
DB/SR
Two timepoints DB
= Diagnostic biopsy
= Surgical resection SR

8. Supplementary Figure 5. Hierarchical clustering of immune gene markers across the tumor replicates. RNA-Seq analysis showing row wise z-scores of normalized read counts for genes associated with immune lineage markers, cytotoxic function, exhaustion and regulatory function. Samples were hierarchical clustered (distance measure = Pearson’s correlation metric; clustering = average linkage method). Tumor replicates cluster largely by patient displaying similar immune gene expression profiles.

9. Supplementary Figure 6. A B
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10. Supplementary Figure 6. Spearman correlation analysis of CD8A gene expression and CD8 immunohistochemistry (IHC). (A) CD8A gene expression (RPKM) across the cohort measured by RNA-Seq was compared to CD8 counts from IHC based on 10 high-powered fields (HPF) across full-face tumor sections. Spearman analysis shows a high level of correlation between CD8 IHC and CD8A gene expression with an r=0.82. (B) Correlation analysis (Spearman) of CD8 IHC between diagnostic biopsy and surgical resection (10 x HPF) in an independent cohort also displayed a high correlation between timepoints (r=0.95).
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