LAUSR

The annual incidence of head and neck squamous cell carcinoma (HNSCC) is approximately 600,000 cases worldwide and 60,000 cases in the United States; HNSCC accounts for 3% of cancers nationally. Despite advances in surgical techniques and radiotherapy, which are the mainstays of treatment, 5year overall survival remains below 50%, and this highlights the need for better patient stratification and targeted therapy. Over the past decade, nextgeneration sequencing has enabled rapid and costeffective profiling of human cancers and novel precision medicine approaches that aim to address these needs. One such profiling approach is RNA sequencing (RNAseq), which typically refers to the profiling of cellular messenger RNA but may include other regulatory noncoding RNAs as well. This approach represents an intermediate manifestation of the genetic and epigenetic background of cells and is closely related to protein expression, which ultimately drives biological phenotypes. In addition to quantifying gene expression, RNAseq can detect structural variants such as alternative splicing and fusions. The first reports of the use of RNAseq appeared in 2008 and described the profiling of Arabidopsis, yeast, and mouse transcriptomes. 5 Two years later, RNAseq was first used in HNSCC. Over the next few years, multiple largescale studies, including The Cancer Genome Atlas (TCGA), performed RNA and whole exome sequencing to characterize head and neck cancer. At a mutational level, these studies revealed that HNSCC is driven primarily by the inactivation of tumor suppressors, such as TP53, CDKN2A, FBXW7, and NSD1, and the disruption of squamous differentiation, including dysfunction of NOTCH1, IRF6, and TP63, with rare oncogenic alterations in PIK3CA, CCND1, and HRAS. 9 At the transcriptional level, these studies identified hallmarks of human papillomavirus (HPV) infection as well as intertumoral differences attributable to the anatomic subsite and the expression subtype. Among HPVrelated tumors, viral integration into the host genome was confirmed, with 80% having integration breakpoints within a gene. Transcription of the host genome was demonstrated across these loci, and amplified genes attributed to HPV infection included multiple transcription factors, cell cycle regulators (including CDKN2A), and multiple genes related to DNA replication and transcription. Overexpression of p16, encoded by CDKN2A, has become a hallmark of HPVrelated HNSCC, with downregulation having been demonstrated in HPV(– ) tumors. This reliable differential expression has led to the widespread utilization of p16 staining in diagnosis, including cytology specimens, as well as the newest American Joint Committee on Cancer staging guidelines. Differential expression by subsite demonstrated a number of markers related to the underlying pathophysiology. Not surprisingly, expression in oropharyngeal tumors was similar to that in HPV(+) tumors, with upregulation of a number of cell cycle regulators and DNA replication genes. Oral cavity tumors, subject to repetitive carcinogen exposure, demonstrated increased expression of several S100 family genes as well as stratified epithelial and keratinocyte markers KRT6, KRT14, and KRT17 and CD44, a putative cancer stem cell marker, with reduced expression found for a number of genes associated with DNA repair that were previously demonstrated to exhibit chromosomal loss in these tumors. Larynx tumors, typically related to a significant smoking history, demonstrated elevated expression of oxidative stress response genes AKR1C1, AKR1C2, and AKR1C4, which have been associated with chemoresistance; HNSCC stem cell markers ALDH1A1 and SOX2; and fibroblast growth factor and hedgehog signaling pathway genes. TCGA analysis also confirmed four previously described expression subtypes. The classic subtype was defined by a TP53 mutation, CDKN2A loss of function, chromosome 3q amplification, the alteration of oxidative stress genes (KEAP1, NFE2L2, and CUL3), a heavy smoking history, and enrichment for larynx subsite tumors. The basal subtype demonstrated inactivation of NOTCH1 with intact oxidative stress and fewer alterations of 3q; this suggests that disrupted squamous differentiation may be a prominent pathway in these tumors. The basal subtype also included most tumors with the HRAS– CASP8 comutation, and this suggests that a major alteration in this subtype may be disrupted cell death. Atypical tumors were enriched for HPV(+) tumors with activating mutations in PIK3CA. Mesenchymal tumors showed alterations in innate immunity, including high expression of natural