Level 3 RSEM normalized Illumina HiSeq RNA expression data for the TCGA head and neck cancer (HNSC) and cervical carcinoma (CESC) cohorts was downloaded from the Broad Genome Data Analysis Centers Firehose server (https://gdac.broadinstitute.org/). Although rare HPV+ samples have been identified in the TCGA bladder urothelial carcinoma (BLCA) and colon adenocarcinoma (COAD) cohorts, they were not present in sufficient numbers for any useful comparisons. For all genes except p16 and p14, the gene level Firehose dataset was used. For p16 and p14, the gene isoform level Firehose dataset was used (uc003zpk.2 and uc003zpl.2 respectively) to adequately discriminate between these two different products of the CDKN2A gene. Normalized expression data was extracted into Microsoft Excel and the HPV status was manually curated based on published datasets [40 (link), 41 (link), 61 (link), 62 (link)]. For each gene analyzed, primary patient samples with known HPV status were grouped as HPV+, HPV-, or normal control tissue. Patient samples with unknown HPV status were omitted from our calculations, as were samples obtained from secondary metastatic lesions. This resulted in 73 HPV+, 442 HPV-, and 43 normal control samples with data available for the HNSC gene expression analysis and 278 HPV+, 19 HPV-, and 3 normal control samples available for the cervical carcinoma gene expression analysis. Boxplot comparison of gene expression was performed using BoxPlotR (http://shiny.chemgrid.org/boxplotr/) and assembled into final form using CorelDRAW. For the box plots, center lines show the medians, box limits indicate the 25th and 75th percentiles as determined by R software and whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Statistical significance was calculated using Graphpad Prism v6.01. p-values were assigned using a one-tailed non-parametric Mann-Whitney U test. Post-hoc power calculations were performed with G*Power software version 3.1.9.2 [63 (link)], using post-hoc t-test family calculations, with effect size selected as 0.8 and α = 0.05. All comparisons achieved a power value >0.8, or demonstrated significant differences, unless otherwise noted in the text.
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