Prognostic and predictive values of baseline and mid-treatment FDG-PET in oropharyngeal carcinoma treated with primary definitive (chemo)radiation and impact of HPV status: Review of current literature and emerging roles.

BACKGROUND AND PURPOSE: This study provides a review of the literature assessing whether semiquantitative PET parameters acquired at baseline and/or during definitive (chemo)radiotherapy ("prePET" and "iPET") can predict survival outcomes in patients with oropharyngeal squamous cell carcinoma (OPC), and the impact of human papilloma virus (HPV) status. MATERIAL AND METHODS: A literature search was carried out using PubMed and Embase between 2001 to 2021 in accordance with PRISMA. RESULTS: The analysis included 22 FDG-PET/CT studies [1-22], 19 pre-PET and 3 both pre-PET and iPET, The analysis involved 2646 patients, of which 1483 are HPV-positive (17 studies: 10 mixed and 7 HPV-positive only), 589 are HPV-negative, and 574 have unknown HPV status. Eighteen studies found significant correlations of survival outcomes with pre-PET parameters, most commonly primary or "Total" (combined primary and nodal) metabolic tumour volume and/or total lesional glycolysis. Two studies could not establish significant correlations and both employed SUVmax only. Two studies also could not establish significant correlations when taking into account of the HPV-positive population only. Because of the heterogeneity and lack of standardized methodology, no conclusions on optimal cut-off values can be drawn. Ten studies specifically evaluated HPV-positive patients: five showed positive correlation of pre-PET parameters and survival outcomes, but four of these studies did not include advanced T or N staging in multivariate analysis, and two studies only showed positive correlations after excluding high risk patients with smoking history or adverse CT features. Two studies found that prePET parameters predicted treatment outcomes only in HPV-negative but not HPV-positive patients. Two studies found that iPET parameters could predict outcomes in HPV-positive patients but not prePET parameters. CONCLUSION: The current literature supports high pre-treatment metabolic burden prior to definitive (chemo)radiotherapy can predict poor treatment outcomes for HPV-negative OPC patients. Evidence is conflicting and currently does not support correlation in HPV-positive patients.

Cellular states are coupled to genomic and viral heterogeneity in HPV-related oropharyngeal carcinoma.

Head and neck squamous cell carcinoma (HNSCC) includes a subset of cancers driven by human papillomavirus (HPV). Here we use single-cell RNA-seq to profile both HPV-positive and HPV-negative oropharyngeal tumors, uncovering a high level of cellular diversity within and between tumors. First, we detect diverse chromosomal aberrations within individual tumors, suggesting genomic instability and enabling the identification of malignant cells even at pathologically negative margins. Second, we uncover diversity with respect to HNSCC subtypes and other cellular states such as the cell cycle, senescence and epithelial-mesenchymal transitions. Third, we find heterogeneity in viral gene expression within HPV-positive tumors. HPV expression is lost or repressed in a subset of cells, which are associated with a decrease in HPV-associated cell cycle phenotypes, decreased response to treatment, increased invasion and poor prognosis. These findings suggest that HPV expression diversity must be considered during diagnosis and treatment of HPV-positive tumors, with important prognostic ramifications.

Correlation of immune makers with HPV 16 infections and the prognosis in oropharyngeal squamous cell carcinoma.

OBJECTIVES: This study aims to investigate the association of immune markers with high risk human papillomavirus 16 (HPV 16) infection status and to evaluate the prognostic value of programmed death ligand-1 (PD-L1) in patients with oropharyngeal squamous cell carcinoma (OPSCC). MATERIALS AND METHODS: This retrospective study collected 50 cases of HPV positive and HPV negative OPSCC from January 2011 to December 2015. The correlation of CD8 + tumor infiltrating lymphocytes (TILs), programmed death-1 (PD-1), and PD-L1 expression with HPV 16 infection status was analyzed via immunofluorescent staining and quantitative real-time PCR. RESULTS: There was no significant difference in the baseline data between the two groups. Patients with HPV + OPSCC had better prognosis compared to HPV - patients (5-year overall survival [OS], 66% vs. 40%, P = 0.003; 5-year disease specific survival [DSS], 73% vs. 44%, P = 0.001). The expressions of immunity related makers were significantly higher in the HPV + group than the HPV - group (CD8 + TIL: P = 0.039; PD-L1: P = 0.005; PD-1: P = 0.044). Positive CD8 + TIL and PD-L1 were independent factors for better prognosis of OPSCC (DSS, P < 0.001; OS, P < 0.001, respectively). Kaplan-Meier survival analysis indicated that patients with TILs of high HPV + /CD8 + expression were more likely to have better prognosis than those with TILs of low HPV + /CD8 + expression (DSS, P < 0.001; OS, P < 0.001), TILs of high expression of HPV - /CD8 + (DSS, P = 0.010; OS, P = 0.032), and TILs of low expression of HPV - /CD8 + (DSS, P < 0.001; OS, P < 0.001). Furthermore, HPV + /PD-L1 + OPSCC patients had significant better prognosis compared to patients with HPV + /PD-L1 - (DSS, P < 0.001; OS, P = 0.004), HPV - /PD-L1 + (DSS, P = 0.010; OS, P = 0.048) and HPV - /PD-L1 - (DSS, P < 0.001; OS, P < 0.001). CONCLUSIONS: HPV + OPSCC had a significantly better prognosis, and PD-L1 expression was elevated in HPV + OPSCC. PD-L1 positivity might be related to the better prognosis of HPV + OPSCC. CLINICAL RELEVANCE: This study provides a theoretical basis and baseline data for the application of immune checkpoint inhibitors in head and neck tumors.

Loco-Regional Control and Sustained Difference in Serum Immune Protein Expression in Patients Treated for p16-Positive and p16-Negative Head and Neck Squamous Cell Carcinoma.

The main prognostic factors for patients with head and neck cancer are the tumour site and stage, yet immunological and metabolic factors are certainly important, although knowledge is still limited. Expression of the biomarker p16INK4a (p16) in oropharyngeal cancer tumour tissue is one of the few biomarkers for the diagnosis and prognosis of head and neck cancer. The association between p16 expression in the tumour and the systemic immune response in the blood compartment has not been established. This study aimed to assess whether there is a difference in serum immune protein expression profiles between patients with p16+ and p16- head and squamous cell carcinoma (HNCC). The serum immune protein expression profiles, using the Olink® immunoassay, of 132 patients with p16+ and p16- tumours were compared before treatment and one year after treatment. A significant difference in the serum immune protein expression profile was observed both before and one year after treatment. In the p16- group, a low expression of four proteins: IL12RB1, CD28, CCL3, and GZMA before treatment conferred a higher rate of failure. Based on the sustained difference between serum immune proteins, we hypothesise that the immunological system is still adapted to the tumour p16 status one year after tumour eradication or that a fundamental difference exists in the immunological system between patients with p16+ and p16- tumours.

External validation of an 18F-FDG-PET radiomic model predicting survival after radiotherapy for oropharyngeal cancer.

PURPOSE/OBJECTIVE: The purpose of the study is to externally validate published 18F-FDG-PET radiomic models for outcome prediction in patients with oropharyngeal cancer treated with chemoradiotherapy. MATERIAL/METHODS: Outcome data and pre-radiotherapy PET images of 100 oropharyngeal cancer patients (stage IV:78) treated with concomitant chemotherapy to 66-69 Gy/30 fr were available. Tumors were segmented using a previously validated semi-automatic method; 450 radiomic features (RF) were extracted according to IBSI (Image Biomarker Standardization Initiative) guidelines. Only one model for cancer-specific survival (CSS) prediction was suitable to be independently tested, according to our criteria. This model, in addition to HPV status, SUVmean and SUVmax, included two independent meta-factors (Fi), resulting from combining selected RF clusters. In a subgroup of 66 patients with complete HPV information, the global risk score R was computed considering the original coefficients and was tested by Cox regression as predictive of CSS. Independently, only the radiomic risk score RF derived from Fi was tested on the same subgroup to learn about the radiomics contribution to the model. The metabolic tumor volume (MTV) was also tested as a single predictor and its prediction performances were compared to the global and radiomic models. Finally, the validation of MTV and the radiomic score RF were also tested on the entire dataset. RESULTS: Regarding the analysis of the subgroup with HPV information, with a median follow-up of 41.6 months, seven patients died due to cancer. R was confirmed to be associated to CSS (p value = 0.05) with a C-index equal 0.75 (95% CI=0.62-0.85). The best cut-off value (equal to 0.15) showed high ability in patient stratification (p=0.01, HR=7.4, 95% CI=1.6-11.4). The 5-year CSS for R were 97% (95% CI: 93-100%) vs 74% (56-92%) for low- and high-risk groups, respectively. RF and MTV alone were also significantly associated to CSS for the subgroup with an almost identical C-index. According to best cut-off value (RF>0.12 and MTV>15.5cc), the 5-year CSS were 96% (95% CI: 89-100%) vs 65% (36-94%) and 97% (95% CI: 88-100%) vs 77% (58-93%) for RF and MTV, respectively. Results regarding RF and MTV were confirmed in the overall group. CONCLUSION: A previously published PET radiomic model for CSS prediction was independently validated. Performances of the model were similar to the ones of using only the MTV, without improvement of prediction accuracy.

Oropharyngeal tumor cells induce COX-2 expression in peripheral blood monocytes by secretion of IL-1α.

Oropharyngeal squamous cell cancer (OPC) accounts for 3% of all cancers and greater than 1.5% of all cancer deaths in the United States, with marked treatment-associated morbidity in survivors. More than 80% of OPC is caused by HPV16. Tumors induced by HPV have been linked to impaired immune functions, with most studies focused on the local tumor microenvironment. Fewer studies have characterized the effects of these tumors on systemic responses in OPC, especially innate responses that drive subsequent adaptive responses, potentially creating feed-back loops favorable to the tumor. Here we report that elevated plasma levels of PGE2 are expressed in half of patients with OPC secondary to overexpression of COX-2 by peripheral blood monocytes, and this expression is driven by IL-1α secreted by the tumors. Monocytes from patients are much more sensitive to the stimulation than monocytes from controls, suggesting the possibility of enhanced immune-modulating feed-back loops. Furthermore, control monocytes pre-exposed to PGE2 overexpress COX-2 in response to IL-1α, simulating responses made by monocytes from some OPC patients. Disrupting the PGE2/IL-1α feed-back loop can have potential impact on targeted medical therapies.

Salivary Exosomal MicroRNA-486-5p and MicroRNA-10b-5p in Oral and Oropharyngeal Squamous Cell Carcinoma.

Background and Objectives: The research aimed at evaluating the capacity of salivary exosomal miR-10b-5p and miR-486-5p for oral and oropharyngeal cancer detection. Materials and Methods: The saliva samples were harvested from histopathological diagnosed oral and oropharyngeal squamous cell carcinoma patients and healthy volunteer subjects. The exosomes were isolated by differential ultracentrifugation and quantified by Nano Track Analysis. The microRNAs were extracted and quantified from salivary exosomes by quantitative Real-Time Polymerase Chain Reaction. Results: This research comprised fifty participants. When compared to healthy controls, salivary exosomal miR-486-5p was elevated and miR-10b-5p was reduced in oral and oropharyngeal squamous cell carcinoma. Moreover, miR-486-5p had a high expression level in stage II of cancer in comparison to the other cancer stages. The cancer samples presented an increased exosome dimension compared to the control samples. Conclusions: Salivary exosomal miR-10b-5p and miR-486-5p have an altered expression in oral and oropharyngeal cancer.

The immune microenvironment of HPV-positive and HPV-negative oropharyngeal squamous cell carcinoma: a multiparametric quantitative and spatial analysis unveils a rationale to target treatment-naïve tumors with immune checkpoint inhibitors.

BACKGROUND: Immune checkpoint inhibitors (ICI) are approved for treatment of recurrent or metastatic oropharyngeal head and neck squamous cell carcinoma in the first- and second-line settings. However, only 15-20% of patients benefit from this treatment, a feature increasingly ascribed to the peculiar characteristics of the tumor immune microenvironment (TIME). METHODS: Immune-related gene expression profiling (GEP) and multiplex immunofluorescence (mIF) including spatial proximity analysis, were used to characterize the TIME of 39 treatment-naïve oropharyngeal squamous cell carcinomas (OPSCC) and the corresponding lymph node metastases. GEP and mIF results were correlated with disease-free survival (DFS). HPV-positive tumors disclosed a stronger activation of several immune signalling pathways, as well as a higher expression of genes related to total tumor-infiltrating lymphocytes, CD8 T cells, cytotoxic cells and exhausted CD8 cells, than HPV-negative patients. Accordingly, mIF revealed that HPV-positive lesions were heavily infiltrated as compared to HPV-negative counterparts, with a higher density of T cells and checkpoint molecules. CD8+ T cells appeared in closer proximity to tumor cells, CD163+ macrophages and FoxP3+ cells in HPV-positive primary tumors, and related metastases. In HPV-positive lesions, PD-L1 expression was increased as compared to HPV-negative samples, and PD-L1+ tumor cells and macrophages were closer to PD-1+ cytotoxic T lymphocytes. Considering the whole cohort, a positive correlation was observed between DFS and higher levels of activating immune signatures and T cell responses, higher density of PD-1+ T cells and their closer proximity to tumor cells or PD-L1+ macrophages. HPV-positive patients with higher infiltration of T cells and macrophages had a longer DFS, while CD163+ macrophages had a negative role in prognosis of HPV-negative patients. CONCLUSIONS: Our results suggest that checkpoint expression may reflect an ongoing antitumor immune response. Thus, these observations provide the rationale for the incorporation of ICI in the loco-regional therapy strategies for patients with heavily infiltrated treatment-naïve OPSCC, and for the combination of ICI with tumor-specific T cell response inducers or TAM modulators for the "cold" OPSCC counterparts.

Human Papillomavirus Detected in Oropharyngeal Cancers from Chilean Subjects.

High-risk human papillomaviruses (HR-HPV) are the causal agents of an important subset of oropharyngeal cancers that has increased considerably in incidence in recent years. In this study, we evaluated the presence of HPV in 49 oropharyngeal cancers from Chilean subjects. The presence of HPV DNA was analyzed by conventional PCR, the genotypes were identified through sequencing, and the expression of E6/E7 transcripts was evaluated by a reverse transcriptase polymerase chain reaction (RT-PCR). Additionally, to determine p16 expression-a surrogate marker for oncogenic HPV infection-a tissue array was constructed for immunohistochemistry (IHC). HPV was detected in 61.2% of oropharyngeal carcinomas, the most prevalent genotype being HPV16 (80%). E6 and E7 transcripts were detected in 91.6% and 79.1% of the HPV16-positive specimens, respectively, demonstrating functional HPV infections. Furthermore, p16 expression was positive in 58.3% of cases. These findings show a high prevalence of HR-HPV in oropharyngeal tumors from Chile, suggesting the necessity of additional studies to address this growing public health concern.

A Contemporary Systematic Review on Repartition of HPV-Positivity in Oropharyngeal Cancer Worldwide.

Significant variation in human papillomavirus (HPV) prevalence in oropharyngeal squamous cell carcinoma (OPSCC) across countries ranging from 11% in Brazil to 74% in New Zealand has been reported earlier. The aim of this study was to systematically review the most recently published studies on the occurrence of HPV in OPSCC globally. PubMed and Embase were systematically searched for articles assessing the occurrence of HPV+ OPSCC published between January 2016 and May 2021. Studies with a study period including 2015 and the following years were included. Both HPV DNA and/or p16 were accepted as indicators of HPV+ OPSCC. 31 studies were enrolled comprising 49,564 patients with OPSCC (range 12-42,024 patients per study) from 26 different countries covering all continents. The lowest occurrences of HPV+ OPSCC were observed in India (0%) and Spain (10%) and the highest occurrences were observed in Lebanon (85%) and Sweden (70%). We observed great variation in HPV prevalence in OPSCC worldwide varying from 0% to 85%. The highest occurrences of HPV+ OPSCC were found in general in Northern European countries, USA, Lebanon, China, and South Korea. We observed a trend of increase in HPV-positivity, indicating a mounting burden of HPV+ OPSCC.

Oral secretory leukocyte protease inhibitor (SLPI): Associations with oropharyngeal cancer and treatment outcome.

BACKGROUND: Rates of oropharyngeal cancer (OPC) associated with alcohol & tobacco use have decreased, while human papillomavirus (HPV) associated OPC has increased among men in the US. Secretory leukocyte protease inhibitor (SLPI), detectable in a variety of secretions, has been implicated in cancers of the head and neck, associated with tumor progression and anti-viral activity. Using the recently verified oral gargle specimen, this study aimed to assess the association of salivary SLPI expression with risk of OPC and response to treatment. METHODS: A case-control study design compared levels of salivary SLPI among OPC cases to age and tobacco smoking matched healthy controls. Oral HPV DNA and SLPI was quantified from oral gargle specimens. Logistic regression estimated odds ratios (OR) and 95% confidence intervals (CI) for associations of oral SLPI and risk of OPC and treatment outcomes. RESULTS: In crude and adjusted analyses of 96 OPC cases and 97 age- and smoking-matched controls, OPC was not significantly associated with oral gargle SLPI levels. Among cases, oral SLPI was associated with tonsillectomy (p = 0.018) and among controls oral SLPI was associated with HPV in the oral gargle (p = 0.008). Higher concentrations of SLPI was significantly associated with increased odds of incomplete treatment response (T2: OR: 12.39; 95% CI: 1.44-106.72; T3: OR: 9.86; 95% CI: 1.13-85.90) among all cases, but not among P16+ cases. CONCLUSIONS: Salivary SLPI was not associated with OPC risk but was associated with higher odds of an incomplete treatment response.

Is There a Role for Sex Hormone Receptors in Head-and-neck Cancer? Links with HPV Infection and Prognosis.

BACKGROUND/AIM: Head-and-neck squamous cell carcinoma (HNSCC) is the fifth most common cancer in the world and human papillomavirus (HPV) is an important risk factor for this neoplasm. Recent studies showed an association between sex hormone receptors and pathogenesis and/or prognosis in patients with HNSCC. The aim of this study was to clarify the expression patterns of sex hormone receptors in HPV-positive and HPV-negative HNSCC and their associations with tumour biopathology and biological behaviour. MATERIALS AND METHODS: Scientific literature indexed in PubMed about sex hormone receptors in HNSCC was retrieved and critically analyzed, to obtain an overview of expression patterns and their possible implications for tumour biopathology and prognosis. RESULTS: Sex hormone receptors were more frequently detected in oropharyngeal tumours compared with HNSCC from other locations. ERα was associated with HPV-positive tumours. The androgen and progesterone receptors were associated with poor patient prognosis. Estrogen receptor alpha (ERα) is implicated in the biopathology of HNSCC in different ways, by promoting DNA hypermutation and facilitating HPV integration thus contributing to an immunogenic phenotype, but also by cooperating with the epithelial growth factor receptor (EGFR) to promote resistance to therapy. CONCLUSION: The expression of sex hormone receptors may be of prognostic value in specific tumour subgroups, but the use of hormonal therapies for HNSCC is still not in close sight.

Salivary High-Risk Human Papillomavirus (HPV) DNA as a Biomarker for HPV-Driven Head and Neck Cancers.

High-risk human papillomavirus (HR-HPV) infection is a major risk factor of head and neck cancers (HNCs). Despite the rising prevalence of HPV-driven HNC (HPV-HNC), biomarkers for detection, prognostication, and disease monitoring are lacking. To evaluate the capacity of salivary HR-HPV DNA as a biomarker of HPV-HNC, the salivary HR-HPV statuses of 491 and 10 patients with primary and recurrent HNC, respectively, were determined at diagnosis, using quantitative real-time PCR and MassARRAY. Tumor cyclin-dependent kinase inhibitor 2A (p16) expression was determined by IHC analysis. Patients with oropharyngeal cancer (OPC) (n = 215) were followed up for ≤5 years. Survival characteristics were evaluated in terms of event-free and cause-specific survival. Of the primary-HNC cohort, 43.2% were positive for salivary HR-HPV DNA, with most having OPC. Salivary HR-HPV DNA was detected in 81.4% of tumor p16-positive OPC patients at diagnosis. Prognosis in salivary HR-HPV-positive OPC patients was favorable compared with that in salivary HR-HPV-negative patients (event-free survival, hazard ratio = 0.42 [95% CI, 0.21-0.81, P = 0.010]; cause-specific survival, hazard ratio = 0.39 [95% CI, 0.18-0.86, P = 0.019]). In the recurrent-HNC cohort, salivary HR-HPV DNA was detected in 83.3% of those who previously had tumor p16-positive HNC. These findings indicate that this liquid biopsy-based, noninvasive biomarker can play an essential role in the detection and management of HPV-HNC.

Proteomic Alterations in Salivary Exosomes Derived from Human Papillomavirus-Driven Oropharyngeal Cancer.

BACKGROUND: Increasing evidence supports the notion that human papillomavirus (HPV) DNA integration onto the human genome can influence and alter the molecular cargo in the exosomes derived from head and neck cancer cells. However, the molecular cargo of salivary exosomes derived from HPV-driven oropharyngeal cancer (HPV-driven OPC) remains unelucidated. METHODS AND MATERIALS: Salivary exosomes morphology and molecular characterizations were examined using the nanoparticle tracking (NTA), western blot analysis, transmission electron microscopy (TEM) and mass spectrometry analysis. RESULTS: We report that HPV16 DNA was detected (80%) in isolated salivary exosomes of HPV-driven OPC patients. Importantly, we demonstrate elevated protein levels of six main glycolytic enzymes [i.e., aldolase (ALDOA), glyceraldehye-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenase A/B (LDHA and LDHB), phosphoglycerate kinase 1 (PGK1) and pyruvate kinase M1/2 (PKM)] in isolated salivary exosomes of HPV-driven OPC patients, suggesting a novel mechanism underlying the potential role of salivary exosomes in mediating the reciprocal interplay between glucose metabolism and HPV-driven OPC. CONCLUSION: Our data demonstrate the potential diagnostic value of HPV16 DNA and glycolytic enzymes in salivary exosomes in discriminating healthy controls from HPV-driven OPC patients, thereby opening new avenues in the future for clinical translation studies.

Correlation of p16 immunohistochemistry with clinical and epidemiological features in oropharyngeal squamous-cell carcinoma.

BACKGROUND: Oropharyngeal cancer is an important public health problem. The aim of our study was to correlatep16 immunohistochemistry in oropharynx squamous cell carcinomas(OPSCC) with clinical and epidemiological features. MATERIAL AND METHODS: We conducted across-sectional study on patients with OPSCC treated at a single institution from 2014 to 2019. Epidemiological and clinical-pathological data were collected from medical records and a questionnaire was applied to determine alcohol consumption, smoking, and sexual behavior. The HPV status was determined by p16 immunohistochemistry. RESULTS: A total of 252 patients participated in the study, of these 221 (87.7%) were male. There were 81 (32.14%) p16 positive cases and 171 (67.85%) p16 negative cases. The p16positive group was significantly associated with younger patients (50-59 years), higher education level, lower clinical stage and patients who never drank or smoked. Through univariate logistic regression, we observed that female sex (OR, 3.47; 95% CI, 1.60-7.51) and higher education level (OR, 9.39; 95% CI, 2, 81-31,38) were significantly more likely to be p16 positive. Early clinical stage (AJCC8ed) was more associated with p16 positivity both in univariate (OR, 0.14; 95% CI, 0.07-0.26, p<0.001) and multivariate analysis (OR, 0.18; 95% CI, 0.06-0.49, p = 0.001). CONCLUSION: This study showed that drinkers and current smokers were less likely to be p16+. Female sex, higher education level and younger age at diagnosis were associated with a higher probability of being p16+. Additionally, there was a higher proportion of patients with early clinical stage (I or II) in the p16 positive group when compared to the p16 negative group.

Prognostic significance of hypoxia-inducible factor-1α expression in advanced pharyngeal cancer without human papillomavirus infection.

OBJECTIVE: This study aimed to clarify the association between both hypoxia-inducible factor-1α and glucose transporter type-1 expression and survival outcome in advanced pharyngeal cancer without human papillomavirus infection. METHOD: Twenty-five oropharyngeal and 55 hypopharyngeal cancer patients without human papillomavirus infection were enrolled. All patients had stage III-IV lesions and underwent concurrent chemoradiotherapy or surgery. Hypoxia-inducible factor-1α and glucose transporter type-1 expression were investigated in primary lesions by immunohistochemistry. RESULTS: There were 41 and 39 cases with low and high hypoxia-inducible factor-1α expression, and 28 and 52 cases with low and high glucose transporter type-1 expression, respectively. There was no significant correlation between hypoxia-inducible factor-1α and glucose transporter type-1 expression. In univariate analysis, nodal metastasis, clinical stage and high hypoxia-inducible factor-1α expression, but not glucose transporter type-1 expression, predicted significantly worse prognosis. In multivariate analysis, hypoxia-inducible factor-1α overexpression was significantly correlated with poor overall survival, disease-specific survival and recurrence-free survival. CONCLUSION: High hypoxia-inducible factor-1α expression was an independent risk factor for poor prognosis for advanced human papillomavirus-unrelated pharyngeal cancer.

DYRK1A is required for maintenance of cancer stemness, contributing to tumorigenic potential in oral/oropharyngeal squamous cell carcinoma.

DYRK1A, one of the dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs), plays an important role in various biological processes by regulating downstream targets via kinase-dependent and independent mechanisms. Here, we report a novel role of DYRK1A in maintaining tumor growth and stemness of oral/oropharyngeal squamous cell carcinoma (OSCC) cells. Deletion of DYRK1A from OSCC cells abrogated their in vivo tumorigenicity and self-renewal capacity, the key features of cancer stem-like cells (CSCs; also referred to as tumor-initiating cells). The DYRK1A deletion also induced the suppression of CSC populations and properties, such as migration ability and chemoresistance. Conversely, ectopic expression of DYRK1A in OSCC cells augmented their CSC phenotype. Among five DYRK members (DYRK1A, 1B, 2, 3, and 4), DYRK1A is the most dominantly expressed kinase, and its expression is upregulated in OSCC compared to normal oral epithelial cells. More importantly, DYRK1A was highly enriched in various CSC-enriched OSCC populations compared to their corresponding non-CSC populations, indicating its pivotal role in cancer progression and stemness. Further, our study revealed that fibroblast growth factor 2 (FGF2) is a key regulator in the DYRK1A-mediated CSC regulation. Functional studies demonstrated that the loss of DYRK1A inhibits CSC phenotype via reduction of FGF2. Overexpression of DYRK1A promotes CSC phenotype via upregulation of FGF2. Our study delineates a novel mechanism of cancer stemness regulation by DYRK1A-FGF2 axis in OSCC. Thus, inhibition of DYRK1A would lead to a potential novel therapeutic option for targeting CSCs in OSCC.

Lack of CD44 overexpression and application of concurrent chemoradiotherapy with cisplatin independently indicate excellent prognosis in patients with HPV-positive oropharyngeal cancer.

BACKGROUND: HPV-16 positivity in patients with squamous cell carcinoma of oropharynx (OPSCC) is associated with better prognosis. However, in more than 40% of HPV infected patients progression of cancer disease is observed, which indicates the presence of cancer cells resistant to therapy. Some studies suggest that there may be a subpopulation of cancer stem cells (CSCs), which simultaneously exhibit unlimited ability to self-renew and differentiate towards neoplastic cells. The relation between HPV16 infection and biomarkers of CSCs is unclear. OBJECTIVE: The aim of the study was to compare the expression of CD44, CD98, ALDH1/2 and P16 in oropharyngeal cancer patients with or without HPV16 infection, as well as to analyze the prognostic potential of selected CSCs biomarkers in these two subgroups. METHODS: The study was performed in a group of 63 patients. HPV16 infection status was analyzed by quantitative polymerase chain reaction, while CD44, CD98, ALDH1/2 and P16 expression by immunohistochemistry. In survival analysis, two endpoints were applied: overall survival (OS) and disease-free survival (DFS). RESULTS: Among 63 cancers, HPV16 infection was found in 25 tumors (39.7%), overexpression of CD44, CD98, ALDH1/2 and P16 in 43 (68.2%), 30 (47.6%), 33 (52.4%) and 27 (42.9%) cancers, respectively. In the HPV16-positive subgroup, DFS rate of 100% was observed in patients with tumors characterized by lack of CD44 overexpression and those treated with concurrent chemoradiotherapy with cisplatin (CisPt-CRT). In the HPV16-negative subgroup 100% of DFS was noticed for patients (n = 6) with P16 immunopositive tumors. In this subgroup none of the CSCs biomarkers evaluated in the study had any impact on OS or DFS. In patients with HPV16-positive oropharyngeal cancer, lack of CD44 overexpression and application of CisPt-CRT were found to be positive prognostic factors.

ROC analysis of p16 expression in cell blocks of metastatic head and neck squamous cell carcinoma.

BACKGROUND: Oropharyngeal squamous cell carcinoma is associated with human papillomavirus (HPV) and often presents with early metastasis to cervical neck lymph nodes that are amenable to fine-needle aspiration (FNA). The most common method of HPV status determination is p16 immunohistochemistry (IHC). The literature suggests that a lower threshold is needed for p16 positivity on cell block. We examined and quantified p16 IHC staining on cell block and used receiver operating characteristics (ROC) curve analysis to determine an optimal cutoff value with high sensitivity and specificity. METHODS: Thirty-six FNAs of metastatic squamous cell carcinoma from cervical lymph nodes with p16 IHC were evaluated. The p16 stain was quantified in 5% increments and high-risk HPV mRNA in situ hybridization was performed as a gold standard test. Statistical analysis was performed. RESULTS: Interobserver variability was evaluated and was shown to be low with an intraclass correlation coefficient of 0.857. ROC analysis was performed and showed that a cell block p16 IHC cutoff of 15% yielded the highest sensitivity (80%) and specificity (81.8%). CONCLUSION: Our data show that a threshold of 15% p16 staining in cell block maximizes sensitivity and specificity.

MRI Dynamic Contrast Imaging of Oral Cavity and Oropharyngeal Tumors.

ABSTRACT: In the past decade, dynamic contrast-enhanced magnetic resonance imaging has had an increasing role in assessing the microvascular characteristics of various tumors, including head and neck cancer. Dynamic contrast-enhanced magnetic resonance imaging allows noninvasive assessment of permeability and blood flow, both important parametric features of tumor hypoxia, which is in turn a marker for treatment resistance for head and neck cancer.In this article we will provide a comprehensive review technique in evaluating tumor proliferation and application of its parameters in differentiating between various tumor types of the oral cavity and how its parameters can correlate between epidermal growth factor receptor and human papillomavirus which can have an implication in patient's overall survival rates.We will also review how the parameters of this method can predict local tumor control after treatment and compare its efficacy with other imaging modalities. Lastly, we will review how its parameters can be used prospectively to identify early complications from treatment.