Tagged: head and neck cancer

New Tool Uses NF-κB Activity to Classify HPV+ Head and Neck Cancer

Researchers developed a new tool aimed at better classifying HPV+ HNSCC patients with good or poor prognosis in an effort to personalize treatment and improve patient outcomes.

New Tool Uses NF-κB Activity to Classify HPV+ Head and Neck Cancer
New Tool Uses NF-κB Activity to Classify HPV+ Head and Neck Cancer

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Over the last 10 years in the United States, the human papillomavirus (HPV) has caused more head and neck squamous cell carcinomas (HNSCC) than uterine cervical cancers. Primarily caused either by exposure to HPV or to ethanol or tobacco, HNSCC is a disease that impairs fundamental tissues involved in respiration, speech and digestion. HPV-positive and -negative HNSCC have contrasting clinical, epidemiological and histological features. 

“A major discovery in the recent past is that HPV associated HNSCC have improved survival compared to tobacco associated tumors.”

Therefore, treating HNSCC in accordance with HPV status is crucial for avoiding unnecessarily harsh therapeutic side effects in HPV+ HNSCC patients. However, while oncologic outcomes among patients with HPV+ HNSCC are generally favorable, approximately 30% experience a more aggressive disease course and recurrence. Coupled with increasing incidence worldwide, this highlights a growing need for the development of effective clinical stratification tools to accurately identify HPV+ HNSCC patients who have a good or poor prognosis. 

In a new study, researchers—from Columbia UniversityUniversity of Illinois Cancer CenterUniversity of North Carolina at Chapel Hill, and Yale School of Medicine—developed a new tool aimed at better classifying HPV+ HNSCC patients with good or poor prognosis in an effort to personalize treatment and improve patient outcomes. Their trending research paper was published in Oncotarget on May 24, 2022, and entitled, “NF-κB over-activation portends improved outcomes in HPV-associated head and neck cancer.”

“To improve on genomic classification, we designed this study to provide a foundation for development of NF-κB related, RNA based classification strategies to better identify HPV+ HNSCC patients with good or poor prognosis that could potentially aid in future efforts towards treatment personalization.”

The Study

The researchers from this study previously found that TRAF3 and CYLD genes are negative regulators of a family of inducible transcription factors involved in inflammation, called nuclear factor kappa B or NF-κB. The team found that somatic mutations or deletions in either TRAF3 or CYLD (not commonly found in uterine cervical cancer or HPV-negative HNSCC) lead to increased NF-κB pathway activation in HPV+ HNSCC. NF-κB overactivity may lead to cancer cell growth and survival. Alterations in these NF-κB related genes may be potential therapeutic targets in HPV+ HNSCC, and their expression may be capable of predicting treatment outcomes.

“[…] we hypothesized that tumor groups based on NF-κB related gene expression may correlate with treatment outcome, considering that tumors lacking defects in TRAF3 and CYLD may have unrecognized mechanisms driving constitutive NF-κB activation.”

In the current study, the researchers developed an RNA-based NF-κB classification tool called the NF-κB Activity Classifier, or NAC. They used bioinformatics and machine learning techniques, expression-based classification, principal component (PC) analysis, gene set enrichment analysis, and weighted gene correlation network analysis (WGCNA) to verify that the NAC is indeed capable of identifying tumors with high or low NF-κB activity and tumors with good and poor survival. 

“This report validates and expands on our findings that significant expression changes related to NF-κB activity occur in the subset of HPV+ HNSCC tumors marked by TRAF3 or CYLD mutations. We are planning future studies investigating the importance of ‘long-tail’ mutations in the NF-κB pathway which might further illuminate the origins of NF-κB dysregulation in HPV+ HNSCC.”


“Here we present data that these subclasses may also be identified by direct assessment of NF-κB activity; as demonstrated by gene expression differences highlighted by the NF-κB Activity Classifier.”

In summary, the researchers identified genomic differences within subclasses of HPV+ HNSCC. They found that defects in TRAF3 and CYLD genes and NF-κB activity were correlated with survival. Therefore, the NF-κB Activity Classifier could be a useful guide for clinicians who make therapeutic decisions for patients with HPV+ HNSCC.

“Future applications of the NF-κB Activity Classifier may be to identify HPV+ HNSCC patients with better or worse survival with implications for treatment strategies.”

Click here to read the full research paper published by Oncotarget.

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Gene Mutations and Neoantigens in Head and Neck Tumors

The aim of this exploratory study was to characterize the genomic and neoantigen changes in 23 paired primary and recurrent head and neck cell squamous-cell carcinomas.

X-Ray film of neck - front and side
True colour X-Ray film of neck – front and side
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Head and neck cancer is a group of various tumors located in the oral cavity, oropharynx, larynx, and hypopharynx. Head and neck cell squamous-cell carcinomas (HNSCC) often result from tobacco use or human papillomavirus (HPV+) infection. In locally advanced HNSCC, the current therapies used are combined surgery, radiotherapy and chemotherapy. Despite the use of traditional treatments, up to 50% of patients relapse due to the increase in mutational burden as HNSCC advances. Few studies have investigated the therapeutic potential of neoantigens in HNSCC tumors.

“Prior work has characterized changes in the mutation burden between primary and recurrent tumors; however, little work has characterized the changes in neoantigen evolution.”

Neoantigens are new proteins/antigens that form on cancer cells after mutations occur in the tumor DNA. Certain neoantigens can promote anti-tumor immune responses and are potentially capable of controlling tumor progression. In an effort to characterize genomic and neoantigen changes in patients with HNSCC, researchers—from Washington University in St. LouisColumbia UniversitySt. Louis Children’s Hospital, and Siteman Cancer Center—investigated 23 paired primary and recurrent HNSCC tumors. Their paper, entitled, “Genomic and neoantigen evolution from primary tumor to first metastases in head and neck squamous cell carcinoma,” was chosen as the cover paper for Oncotarget’s Volume 12, Issue #6.

Patients and Samples

The researchers identified 23 biopsies from patients originally diagnosed with locally advanced HNSCC. Of the 23 patients in this study, 17 were male and 14 were tobacco smokers. The distribution of primary tumor location was nine in the oral cavity, seven in the oropharynx, six in the larynx, and one in the hypopharynx. The researchers note that all seven oropharyngeal primary tumor patients were HPV+. Each of the 23 patients received some combination of traditional treatment. Of these 23 patients, DNA and total RNA were independently extracted—totaling 69 samples. Twenty-three samples were from germlines, 23 were from primary tumors and 23 were from recurrent/metastatic tumors.

“To understand the recurrent mutation effect between primary and recurrent/metastatic tumors, we extract recurrently mutated genes (>1 sample mutated gene) from primary and recurrent/metastatic samples, separately.”

Recurrently Mutated Genes

The 23 germline blood samples were used in whole-exome sequencing (WES) data. The researchers also generated WES data using 46 paired primary and recurrent/metastatic samples from paraffin blocks and performed RNA sequencing successfully for 31 samples. After conducting RNA sequencing, they used Kallisto to predict gene expression in 16 primary tumors and 15 recurrent/metastatic tumors. A general trend showed that more mutations were within recurrent/metastatic tumors than in primary tumors. They performed KEGG pathways analysis to determine whether mutations occurred in pathways related to metastasis.

“Notably, ECM-receptor interaction pathway was extremely significant in recurrent/metastatic samples meaning that genes related to this pathway are more highly mutated than other pathway mutations in recurrent/metastatic samples.”

The TP53 gene was found to be the highest mutated driver gene in both sample groups, and the researchers identified BRCA1 and NOTCH1 as highly mutated driver genes in primary tumor samples. In recurrent/metastatic tumors, PIK3CA, ARID1A, RASA1, TSC2, and ERBB4 were mutated at higher rates than in primary tumor samples.

Infiltration of Immune Cells

To determine the infiltration of immune cells in primary tumors versus recurrent/metastatic tumors, the team performed immunohistochemistry. No significant differences in CD3+ cells, activated T cells, cytotoxic T cells, or  CD3+ FOXP3+ cells were found. A significant increase of PD-L1 (an immune checkpoint protein) was found in recurrent/metastatic tumors. Upon further examination of immune checkpoint molecules, the researchers found a decrease in the expression of PDCD1 and CTLA4, with PDCD1 significantly decreased.

“We next sought to determine if genes containing neoantigens were shared between patients. Most neoantigens were unique to an individual tumor.”

Neoantigen Trends

In order to predict neoantigens among 46 tumor samples, this team utilized OptiType and MuPeXI to define the candidate neoantigens. Most patients had unique neoantigens based on the individual tumor type, however, the researchers’ analysis identified multiple patients with neoantigens in shared genes. They found neoantigens in six genes shared between four or five patients. (Three genes were among primary tumors: RYR3, DNAH7 and TTN; and three genes were among recurrent tumors: TNN, PIK3CA and USH2A.) They found that, compared to patients without them, patients who shared neoantigens in these genes tended to have increased neoantigens, CD3+ CD8+ T cell infiltration and duration of survival with HNSCC.

“These patients have increased total neoantigens, and a trend toward increased duration of survival with disease, infiltration of CD8 cells, and CTL activity. This suggests HNSCC neoantigens can stimulate an anti-tumor immune response.”


In conclusion, six genes with predicted neoantigens were found in four or more HNSCC patients. The researchers explained that while there is considerably more work needed in order to expand on their results from this small sample, the observation of neoantigens in these shared genes is significant.

“This raises the possibility that the presentation of certain neoantigens are important for control of tumor growth. This small exploratory study will provide the justification for a larger study of neoantigens in HNSCC.”

Click here to read the full research paper, published by Oncotarget.

Behind the StudyDrs. Brian Van Tine and Charles Schutt discuss this research.

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