In recent years, our knowledge and understanding of head and neck squamous cell carcinoma (HNSCC) has expanded dramatically. New high-throughput sequencing technologies have accelerated these discoveries since the first reports of whole-exome sequencing of HNSCC tumors in 2011. In addition, the discovery of human papillomavirus in relationship with oropharyngeal squamous cell carcinoma has shifted our molecular understanding of the disease. New investigation into the role of immune evasion in HNSCC has also led to potential novel therapies based on immune-specific systemic therapies.
Key points
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Most head and neck squamous cell carcinomas are associated with smoking and alcohol, but an emerging subset of tumors is associated with human papillomavirus. These patients have improved clinical outcomes and a distinct genetic profile.
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Genetic sequencing of head and neck cancer revealed mutations in key cancer pathways, including p53, epidermal growth factor receptor (EGFR)/Ras/phosphatidylinositol 3-kinase (PI3K), Notch and apopototic pathways.
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Therapies targeted toward these pathways are limited but under investigation.
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Head and neck cancers may progress by immune evasion, which is another potential targetable mechanism under investigation.
In recent years, our knowledge and understanding of head and neck squamous cell carcinoma (HNSCC) has expanded dramatically. New high-throughput sequencing technologies have accelerated these discoveries since the first reports of whole-exome sequencing of HNSCC tumors in 2011. In addition, the discovery of human papillomavirus (HPV) in relationship with oropharyngeal squamous cell carcinoma (SCC) has shifted our molecular understanding of the disease. New investigation into the role of immune evasion in HNSCC has also led to potential novel therapies based on immune-specific systemic therapies.
Distinct etiologic subsets of head and neck squamous cell carcinoma
HNSCC forms after accumulation of genetic events, which are accelerated by genomic instability related to carcinogen exposures, particularly tobacco and alcohol. These tumors may occur throughout the upper aerodigestive tract (oral cavity, oropharynx, larynx) and are found in older patients, usually with history of smoking or alcohol use. They are also associated with p53 mutations and poor clinical outcomes, with 5-year survival of 33.8% to 66.8%, depending on subsite.
Recently, HPV has been associated with a subset of HNSCC, chiefly in the oropharynx and primarily in younger, white, nonsmokers. HPV is a double-stranded DNA virus that infects the squamous epithelium. High-risk subtypes, particularly HPV-16 and HPV-18, are associated with development of malignancy in both HNSCC and cervical cancer. The mechanism of oncogenesis is attributed to viral proteins E6 (which binds and degrades p53) and E7 (which inhibits retinoblastoma protein, a tumor suppressor gene that inhibits cell cycle progression). Patients with HPV-related HNSCC have improved prognosis with longer overall survival, decreased rate of recurrence, and improved response to chemoradiation.
Distinct etiologic subsets of head and neck squamous cell carcinoma
HNSCC forms after accumulation of genetic events, which are accelerated by genomic instability related to carcinogen exposures, particularly tobacco and alcohol. These tumors may occur throughout the upper aerodigestive tract (oral cavity, oropharynx, larynx) and are found in older patients, usually with history of smoking or alcohol use. They are also associated with p53 mutations and poor clinical outcomes, with 5-year survival of 33.8% to 66.8%, depending on subsite.
Recently, HPV has been associated with a subset of HNSCC, chiefly in the oropharynx and primarily in younger, white, nonsmokers. HPV is a double-stranded DNA virus that infects the squamous epithelium. High-risk subtypes, particularly HPV-16 and HPV-18, are associated with development of malignancy in both HNSCC and cervical cancer. The mechanism of oncogenesis is attributed to viral proteins E6 (which binds and degrades p53) and E7 (which inhibits retinoblastoma protein, a tumor suppressor gene that inhibits cell cycle progression). Patients with HPV-related HNSCC have improved prognosis with longer overall survival, decreased rate of recurrence, and improved response to chemoradiation.
Genetic alterations
In 2011, the first whole-exome sequencing of HNSCC was published. Recently, the Cancer Genome Atlas (TCGA) Research Network performed integrated genomic analysis, including genome sequencing, copy number and loss of heterozygosity arrays, whole-genome methylation, and RNA sequencing on 279 head and neck cancers, constituting the largest cohort of sequenced tumors studied.
Gene mutations were segregated by HPV tumor status. HPV-positive tumors harbored fewer mutations compared with HPV-negative tumors. TP53 mutations were found almost exclusively in HPV-negative tumors, whereas activating mutations and amplifications of PIK3CA (phosphatidylinositol 3-kinase, catalytic subunit alpha) were commonly seen in HPV-positive tumors ( Fig. 1 ). This finding is consistent with prior data showing the same distinct genetic alterations.
Beyond sequencing, gene promoter methylation of several genes, including CDKN2A (cyclin-dependent kinase inhibitor 2A), CDH1 (cadherin 1 type 1, E-cadeherin), MGMT (O-6-methylguanine-DNA methyltransferase), and DAPK1 (death-associated protein kinase 1), has been established in oral SCC. CDKN2A, a tumor suppressor gene, is one of the first genes in HNSCC to be associated with promoter methylation as a mechanism of downregulation.
Major pathways
TP53 and CDKN2A
The TP53 (tumor protein p53) gene encodes for the p53 protein, “guardian of the genome.” TP53 is one of the most frequently mutated genes in HNSCC tumors and even premalignant lesions. The p53 protein acts as a tumor suppressor that accumulates in response to stress, including DNA damage. Accumulation of p53 induces cell cycle arrest to allow the cell to perform DNA repair. If damage is beyond repair, p53 induces apoptosis. The expression of p53 is regulated by MDM2 (MDM2 proto-oncogene, E3 ubiquitin protein ligase), which inactivates and degrades p53. The CDKN2A locus at 9p21 codes for 2 alternatively spliced proteins p14ARF and p16INK4A, which both regulate p53 function ( Fig. 2 ).
Most p53 mutations in HNSCC (50%–63%) are missense mutations. Missense mutations in p53 can result in a stable protein with loss of key binding function or even act in a dominant negative fashion inactivating any remaining wild-type p53. Tobacco exposure is associated with increased rates of TP53 mutations. Mutations in TP53 have been associated with decreased overall survival, increased locoregional recurrence rates, and decreased response to therapy.
In recent sequencing data, it was found that CDKN2A was mutated in 9% to 12% of tumors. Loss of heterozygosity is frequently seen at the CDKN2A locus in HNSCC, including premalignant lesions. Additionally, p16 protein overexpression is consistently seen in HPV-related oropharyngeal cancers. The mechanism is related to the inactivation of retinoblastoma protein by the E7 viral protein, resulting in unregulated overexpression of p16.
Epidermal Growth Factor Receptor, Ras, and PI3K (Phosphatidylinositol 3-Kinase Pathways)
Epidermal growth factor receptor (EGFR) is part of the ErbB family of receptor tyrosine kinases. After ligand binding (ligands include epidermal growth factor or transforming growth factor-α), activated EGFR forms a dimer and activates downstream pathways. These pathways include phosphoinositide 3-kinase (PI3K), Akt and Ras pathways that promote cell growth, proliferation, and inhibit apoptosis ( Fig. 3 ). Activation of PI3K results in conversion of phosphatidylinositol biphosphate (PIP2) to phosphatidylinositol triphosphate (PIP3). PIP3 can then bind and phosphorylate Akt, triggering inhibition of apoptosis, mammalian target of rapamycin (mTOR) pathway activation, and activation of MDM2. PTEN (phosphatase and tensin homolog) negatively regulates this signaling by dephosphorylating PIP3 to PIP2, thus, preventing downstream signaling.
In HNSCC, overexpression of the EGFR gene is seen in about 90% of tumors. Increased EGFR expression correlates with increased local recurrence and worse overall survival. EGFR overexpression plays a clear role in HNSCC; interestingly, few mutations have been observed in EGFR (see Fig. 1 ). HRAS (Harvey rat sarcoma viral oncogene homolog), a target downstream of EGFR, is mutated in 4% to 5% of tumors.
In HNSCC, PI3KCA (catalytic subunit of PI3 kinase) is mutated in 6% to 21% of tumors. Advanced stage HNSCC harbor increased mutations along the PI3K pathway. PI3K pathway genes are the main genes mutated in HPV-related tumors (see Fig. 1 ).
Notch Signaling
Notch is a cell surface receptor that binds to ligands on an adjacent cell surface, such as Jagged or Delta. Next, proteolytic cleavage releases an intracellular fragment that travels to the nucleus, affecting gene transcription. Downstream targets include HES1 [hes family basic helix-loop-helix (bHLH) transcription factor 1] and HEY1 (hes-related family bHLH transcription factor with YRPW motif 1), which promote cell cycle progression and survival.
Inactivating mutations of NOTCH1 were found in 10% to 19% of head and neck tumors. This finding suggests that NOTCH1 acts as a tumor suppressor in HNSCC. In oral SCC cell lines, reactivation of the wild-type NOTCH1 gene blocked cell proliferation. However, NOTCH1 also acts as an oncogene in hematologic malignancies. Within sequencing data of HNSCC, some mutations in NOTCH1 were not inactivating, suggesting that its role in HNSCC may be mixed. Accordingly, recent data have shown that a subset of HNSCC tumors actually show downstream activation of NOTCH.
Apoptotic Pathways
Apoptotic pathways are regulated by intrinsic signals (such as p53) or extrinsic signals through cell surface receptors. Multiple signals, such as p53 response to DNA damage, UV radiation, or influx of calcium ions, can trigger apoptotic signaling. When the balance tips toward apoptosis, cytochrome c is released from the mitochondria, and the caspase cascade executes programmed cell death. Cell surface receptors, such as Fas and death receptors, may also trigger apoptosis through activation of caspase 8 (CASP8) and other downstream caspases.
With head and neck cancer, mutations in CASP8 have been observed in 8% to 9% of tumors (TCGA data) with most occurring in oral cavity SCC. TRAF3 (TNF receptor-associated factor 3), BIRC2 (baculoviral inhibitor of apoptosis protein repeat containing 2), and FADD (Fas-associated via death domain) interact with the cell surface death receptors and were found to harbor mutations in head and neck cancer. TRAF3 mutations were noted primarily in HPV-positive tumors.
Implications for Targeted Therapy
Current chemotherapy treatments for head and neck cancer are not targeted but instead primarily platinum based treatments (primarily cisplatin and carboplatin) with concurrent radiation are the mainstay of treatment.
Therapies Targeting Epidermal Growth Factor Receptor
The main targeted therapy currently available for HNSCC is cetuximab, an anti-EGFR immunoglobulin G1 (IgG1) antibody. Cetuximab was approved for the treatment of HNSCC after a study showed significantly improved progression-free and overall survival when concurrent cetuximab with radiation was compared with radiation alone. Recent data have not shown improved outcomes when combining cetuximab with concurrent cisplatin in primary chemoradiation. Questions still remain regarding the mechanism of cetuximab activity. Despite frequent EGFR overexpression, response rates to cetuximab as a single agent are around 10% to 15% in recurrent HNSCC ; efficacy has not been found to correlate with EGFR expression ( Fig. 4 ).
