Oropharyngeal Squamous Cell Carcinoma Tissues Research Articles

Abstract 3683: Prognostic value and biological correlates of LINE1 hypomethylation in oropharyngeal cancer

Abstract Background. We have recently demonstrated that LINE-1 hypomethylation is a negative prognostic factor in oropharyngeal squamous cell carcinoma (OPSCC). HPV-positive patients with LINE-1 methylation <55% showed an almost 5-fold higher risk of death than hypermethylated once, with an overall survival as poor as HPV-negative patients. Somatic TP53 mutations are a common feature in HPV-negative OPSCC, whereas their presence has been reported in only a small subset of HPV-related OPSCC. At present, little is known about the correlation between LINE-1 methylation and p53 expression status and/or TP53 mutation in OPSCC. Methods. Since 2019, an ongoing prospective study has been enrolling patients with OPSCC in nine cancer centers in Northern Italy. HPV16 E6 DNA was quantified using real-time quantitative PCR (qPCR). LINE-1 methylation was evaluated by methylation-specific qPCR. p53 expression was detected by immunohistochemical staining. TP53 exon mutations were analyzed by Next Generation Sequencing. Results. Up to date, 134 OPSCC patients have been enrolled, including cancer of the tonsil (n=74; 55.2%), base of tongue (n=47; 35.1%), and other subsites (n=13; 9.7%). HPV16 DNA was found in 54.3% of cases: 65.7% in base of tongue, 57.1% in tonsil, and 16.7% in other subsites (p<0.001). LINE-1 methylation was higher in HPV16-positive patients (median: 55.8%, interquartile rage [IQR]: 42.5-68.5) than HPV16-negative ones (median: 40.7; IQR: 23.9-59.1; p=0.003). Nevertheless, the median LINE-1 methylation was lower (50.7%) among HPV-16 OPSCC patients who relapsed than those who did not (56.0%), thus confirming the findings previously reported. However, data from the present investigation are presently not mature to purse the study aims (median follow-up: 12.3 months). The correlation between LINE-1 hypomethylation and p53 expression has also been evaluated in OPSCC patients. p53 expression was categorized into three groups (0%, 1-49%, and ≥50%) based on the overall intensity of nuclear staining of the tumor cells. p53 was considered null, overexpressed, or wild type when its nuclear staining was 0 %, ≥50%, or ranged between 1% and 49%, respectively. Of note, the highest level of LINE-1 methylation was observed in OPSCC tissues with nuclear p53 staining between 1% and 49% (57.8%), whereas OPSCC patients with p53 expression ≥50% showed a decline of LINE-1 methylation levels (40.6; p=0.026). Interestingly, LINE-1 methylation was lower in OPSCC patients harboring TP53 mutations in both HPV16-negative (24.8%) and HPV16-positive (24.0%) OPSCC patients, even if statistical significance was reached only in the HPV-negative group (p=0.015). Conclusion. Preliminary results confirm the potential of LINE-1 methylation to identify HPV16-positive patients with poor prognosis. In addition, this study suggest that TP53 mutations might influence LINE-1 methylation, irrespective of HPV status. Citation Format: Mariateresa Casarotto, Roberto Guerrieri, Paolo Boscolo-Rizzo, Monica Schiappacassi, Sara D'Andrea, Valentina Lupato, Susanna Chiocca, Marta Tagliabue, Rita De Berardinis, Anna Menegaldo, Alice Piccinato, Paola Stritoni, Mohssen Ansarin, Doriano Politi, Giuseppe Fanetti, Giorgio Giurato, Jerry Polesel, Elisabetta Fratta. Prognostic value and biological correlates of LINE1 hypomethylation in oropharyngeal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3683.

PSMD1 as a prognostic marker and potential target in oropharyngeal cancer

BackgroundDespite the diverse genetic mutations in head and neck cancer, the chemotherapy outcome for this cancer has not improved for decades. It is urgent to select prognostic factors and therapeutic targets for oropharyngeal cancer to establish precision medicine. Recent studies have identified PSMD1 as a potential prognostic marker in several cancers. We aimed to assess the prognostic significance of PSMD1 expression in oropharyngeal squamous cell carcinoma (OPSCC) patients using immunohistochemistry.MethodsWe studied 64 individuals with OPSCC tissue from surgery at Seoul National University Bundang Hospital between April 2008 and August 2017. Immunostaining analysis was conducted on the tissue microarray (TMA) sections (4 μm) for p16 and PSMD1. H-score, which scale from 0 to 300, was calculated from each nucleus, cytoplasm, and cellular expression. Clinicopathological data were compared with Chi-squared test, Fisher’s exact test, t-test, and logistic regression. Survival data until 2021 were achieved from national statistical office of Korea. Kaplan–Meier method and cox-regression model were used for disease-specific survival (DSS) analysis.ResultsH-score of 90 in nucleus was appropriate cutoff value for ‘High PSMD1 expression’ in OPSCC. Tonsil was more frequent location in low PSMD1 group (42/52, 80.8%) than in high PSMD1 group (4/12, 33.3%; P = .002). Early-stage tumor was more frequent in in low PSMD1 group (45/52, 86.5%) than in high PSMD1 group (6/12, 50%; P = .005). HPV was more positive in low PSMD1 group (43/52, 82.7%) than in high PSMD1 group (5/12, 41.7%; P = .016). Patients with PSMD1 high expression showed poorer DSS than in patients with PSMD1 low expression (P = .006 in log rank test). In multivariate analysis, PSMD1 expression, pathologic T staging, and specimen age were found to be associated with DSS (P = .011, P = .025, P = .029, respectively).ConclusionsIn our study, we established PSMD1 as a negative prognostic factor in oropharyngeal squamous cell carcinoma, indicating its potential as a target for targeted therapy and paving the way for future in vitro studies on drug repositioning.

Abstract PO-049: The neural landscape is associated with functional outcomes for patients with oropharyngeal squamous cell carcinoma

Abstract Despite the excellent prognosis of patients with oropharyngeal squamous cell carcinoma (OPSCC), long-term functional impairment (e.g., dysphagia) is a major burden for many patients. Recent studies have explored the role of pharyngeal constrictors and muscle fibrosis in dysphagia. However, to date, no study has examined the association between the OPSCC neural microenvironment and posttreatment functional impairment. We hypothesized that cancer-related neuronal changes in the OPSCC tumor microenvironment (TME) would affect patients’ prognoses. Therefore, in the present study, we investigated whether cancer-related changes in the tumor neuronal microenvironment influence functional outcomes in patients with OPSCC. Prospectively collected, patient-reported outcomes (i.e., from the MD Anderson Symptom Inventory [MDASI] and the MD Anderson Dysphagia Inventory [MDADI]) and functional outcomes from the Dynamic Imaging Grade of Swallowing Toxicity [DIGEST] for patients with OPSCC were collected (n=29). OPSCC tissue samples were obtained from surgically treated patients, and multiplex/high-plex immunofluorescence and machine learning techniques were used to correlate neuronal changes with prospectively collected patient data. Electromyography was performed to assess the neuronal populations involved in neuropathies after treatment. A murine animal model of dysphagia was also used to correlate the neuronal changes. The results showed that sensory nerves with adrenergic features (CGRP+TH+) were associated with perineural and lymphovascular invasion. In addition, we found that human papillomavirus associated OPSCC was associated with an enrichment of parasympathetic progenitors (VIP+DCX+). Poor MDASI scores were associated with CGRP+, adrenergic (TH+), or CGRP+TH+ innervation, and growing progenitor neurons (GAP43+DCX+) were associated with improved oral symptoms. Swallowing, measured by MDADI scale revealed that patients with tumors enriched with growing (GAP43+), parasympathetic (VIP+), and neuronal progenitor (DCX+) neurons reported poor swallowing perception; subgroup electromyography analysis confirmed our findings. Quantitative swallowing assessment with DIGEST by speech language pathologists revealed that TMEs innervated by CGRP+, TH+, and MBP+ neurons were associated with poor swallowing functions. Finally, using a murine model of dysphagia we confirmed that sensory (CGRP+), myelinated (MBP+), and cholinergic parasympathetic (CHAT+) or progenitor (CHAT+DCX+) enrichment was associated with denervation and downstream dysphagia. In conclusion, our results show that neuronal changes are correlated with clinical features in OPSCC patients. Patients’ functional outcomes after therapy completion are influenced by distinct nerve populations in the TME. These benchmark data should be further evaluated as a potential target for cancer survivors with dysphagia and as a biomarker for patients who might need escalated swallowing rehabilitation during and after treatment for OPSCC. Citation Format: Shajedul Islam, Frederico O. Gleber-Netto, Collin F. Mulcahy, Mica D. E. Glaun, Patrick J. Hunt, Michelle D. Williams, Carly E. Barbon, Weilu Zhao, Adewale Adebayo, Shamima Akhter, Tongxin Xie, Ismail Yaman, Jared K. Burks, Javier Gomez, Karin Woodman, Teresa E. Lever, Katherine A. Hutcheson, Moran Amit. The neural landscape is associated with functional outcomes for patients with oropharyngeal squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-049.

Vascular permeability in HPV+ oropharyngeal cancers aids in fluorescent image-guided transoral robotic surgery using indocyanine green.

Indocyanine green (ICG) fluorescent image (FI)-guided surgery has demonstrated success in improving intraoperative visualization and tumor resections. The objectives were to evaluate the use of IGC in FI-guided transoral robotic surgery (TORS) and the underlying molecular mechanism. HPV+ oropharyngeal squamous cell carcinoma (OPSCCa) patient (n = 10) undergoing TORS were enrolled in this prospective study. Participants received intravenous ICG. Excised tissues were evaluated for ICG accumulation, tumor demarcation, and pathological characteristics using In-vivo imaging system (IVIS), histology, and RNA sequencing. ICG accumulation was significantly increased in primary tumor and pathological lymph nodes compared with normal tissues (p < 0.001). IVIS was 91.3% accurate in identifying OPSCCa in excised tissues; the correlation between IVIS- and histologically determined tumor tissues was significant (R2 = 0.8301; p = 0.001). Genes associated with vascular and angiogenic signaling pathways were significantly upregulated in OPSCCa tissues. ICG effectively demarcates tumor margins in OPSCCa, due to the increased upregulation of genes associated with vascular permeability.

Tumor-specific T cells support chemokine-driven spatial organization of intratumoral immune microaggregates needed for long survival

BackgroundThe composition of the tumor immune microenvironment (TIME) associated with good prognosis generally also predicts the success of immunotherapy, and both entail the presence of pre-existing tumor-specific T cells. Here,...

Overexpression of HURP mRNA in head and neck carcinoma and association with in vitro response to vinorelbine.

HURP gene encodes the hepatoma upregulated protein (HURP), a microtubule associated protein regulating mitotic spindle dynamics, which promotes chromosomal congression and alignment during mitosis, with a potential role in tumorigenesis. In the present study, HURP mRNA expression was investigated by reverse transcription-quantitative PCR in oropharyngeal squamous cell carcinoma (OPSCC). Primary OPSCC tumors from 107 patients and 48 adjacent normal tissues, as well as 12 respiratory tract cancer cell lines (9 head and neck squamous cell carcinoma, 2 lung cancer and 1 normal bronchial) were utilised in the present study. mRNA expression levels of HURP were higher in malignant OPSCC tissues compared with in normal mucosa (P<1×10-5) and significantly associated with sex and smoking status (P<0.0001). Vinorelbine in vitro toxicity at half-maximal inhibitory concentration (IC50) was measured in the 11 cancer cell lines using an MTT assay. Sensitivity to vinorelbine was significantly correlated with HURP expression (r=0.636; P=0.035). The data indicated that HURP overexpression is frequent in OPSCC tissues and associated with smoking. The correlation between HURP mRNA expression and vinorelbine in vitro response suggests that HURP is a potential modulator of vinorelbine response; therefore, it should be explored for its possible predictive value for the efficiency of vinorelbine treatment in this type of cancer.

Discovery and development of differentially methylated regions in human papillomavirus-related oropharyngeal squamous cell carcinoma.

Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC) exhibits a different composition of epigenetic alterations. In this study, we identified differentially methylated regions (DMRs) with potential utility in screening for HPV-positive OPSCC. Genome wide DNA methylation was measured using methyl-CpG binding domain protein-enriched genome sequencing (MBD-seq) in 50 HPV-positive OPSCC tissues and 25 normal tissues. Fifty-one DMRs were defined with maximal methylation specificity to cancer samples. The Cancer Genome Atlas (TCGA) methylation array data was used to evaluate the performance of the proposed candidates. Supervised hierarchical clustering of 51 DMRs found that HPV-positive OPSCC had significantly higher DNA methylation levels compared to normal samples, and non-HPV-related head and neck squamous cell carcinoma (HNSCC). The methylation levels of all top 20 DNA methylation biomarkers in HPV-positive OPSCC were significantly higher than those in normal samples. Further confirmation using quantitative methylation specific PCR (QMSP) in an independent set of 24 HPV-related OPSCCs and 22 controls showed that 16 of the 20 candidates had significant higher methylation levels in HPV-positive OPSCC samples compared with controls. One candidate, OR6S1, had a sensitivity of 100%, while 17 candidates (KCNA3, EMBP1, CCDC181, DPP4, ITGA4, BEND4, ELMO1, SFMBT2, C1QL3, MIR129-2, NID2, HOXB4, ZNF439, ZNF93, VSTM2B, ZNF137P and ZNF773) had specificities of 100%. The prediction accuracy of the 20 candidates rang from 56.2% to 99.8% by receiver operating characteristic analysis. We have defined 20 highly specific DMRs in HPV-related OPSCC, which can potentially be applied to molecular-based detection tests and improve disease management.

Abstract 3320: Discovery and development of DNA methylation biomarkers in human papillomavirus related oropharyngeal squamous cell carcinoma

Abstract Purpose: Human papillomavirus (HPV) related oropharyngeal squamous cell carcinoma (OPSCC) has an improved prognosis compared to cancers associated with tobacco and alcohol exposure, and also exhibits a different composition of epigenetic alterations. In this methylome-wide association study, we identified DNA methylation-based biomarkers with potential utility in screening for HPV positive OPSCC. Experimental Design: In the discovery stage, genome wide DNA methylation was measured using methyl-CpG binding domain protein-enriched genome sequencing (MBD-seq) in 50 HPV positive OPSCC tissues and 25 normal tissues. We defined 51 differentially methylated regions (DMRs) with maximal methylation specificity to cancer samples. We used TCGA methylation array data to evaluate the performance of the proposed candidates. Top 20 candidates were validated using quantitative methylation specific PCR (QMSP) in independent 24 HPV related OPSCCs and 22 controls. Results: Supervised hierarchical clustering of 51 DMRs based on TCGA methylation data found that HPV positive OPSCC had significantly higher DNA methylation levels compared to normal samples and head and neck squamous cell carcinoma (HNSCC) without HPV or with different tumor sites. The methylation levels of all top 20 candidates in HPV positive OPSCC were significantly higher than those in normal samples in TCGA data. 16 of these 20 candidates were found by QMSP to have significant higher methylation levels compared with controls. 2 candidates (CCDC181 and OR6S1) had a sensitivity of 100%, while 10 candidates (KCNA3, ITGA4, BEND4, ELMO1, SFMBT2, HOXB4, ZNF439, ZNF93, VSTM2B and ZNF773) had a specificity of 100%. Our 20 candidates were found by the receiver operating characteristic analysis to have a prediction accuracy ranging from 56.3% to 99.8%. Conclusions: This study discovered 20 epigenetic markers for detection of HPV related OPSCC, which can potentially be applied to develop a population based screening test and improve disease management. Citation Format: Shuling Ren, Daria A. Gaykalova, Theresa W. Guo, Alexander V. Favorov, Elana J. Fertig, Zubair Khan, Peter M. DeJong, Mizuo Ando, Chao Liu, Akihiro Sakai, Takahito Fukusumi, Sunny Haft, Sayed Sadat, J Silvio Gutkind, Wayne M. Koch, Joseph A. Califano. Discovery and development of DNA methylation biomarkers in human papillomavirus related oropharyngeal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3320.

Altered Toll-like receptor expression and function in HPV-associated oropharyngeal carcinoma.

Toll-like receptors (TLRs) have been widely investigated due to their importance in the inflammatory response and possible links to tumor promotion/regression and prognosis. In cancers with an infective etiology, such as human papillomavirus (HPV)-associated Oropharyngeal Squamous Cell Carcinoma (OPSCC), TLR responses may be activated and play a role in tumorigenesis. Our aim was to assess the expression of all TLRs in OPSCC cell lines (both HPV+ and HPV–) by qPCR, Western Blot and flow cytometry and assess their response to TLR ligands lipopolysaccharide (LPS), LPS ultra-pure (LPS-UP) and peptidoglycan (PGN) by analyzing IL-8 and IL-6 production. We also immunostained 61 OPSCC tissue samples with anti-TLR4. Results showed lower TLR1 and TLR6 mRNA expression and higher TLR9 protein expression in HPV+ when compared to HPV–OPSCC cells. TLR4 expression did not vary by HPV status in OPSCC cells, but TLR4 expression was significantly lower in HPV+OPSCC tissues. After stimulation with PGN, only one cell line (HPV+) did not secrete IL-6 or IL-8. Furthermore, HPV+OPSCC lines showed no IL-6 or IL-8 production on treatment with LPS/LPS-UP. The data suggest changes in TLR4 signaling in HPV+OPSCC, since we have shown lower tissue expression of TLR4 and no pro-inflammatory response after stimulation with LPS and LPS-UP. Also, it suggests that OPSCC may respond to HPV infection by increased expression of TLR9. This study demonstrates differences in expression and function of TLRs in OPSCC, which are dependent on HPV status, and may indicate subversion of the innate immune response by HPV infection.

FGFR3 correlation with mutant p53 and its prognostic value in oropharyngeal squamous cell carcinoma (OPSCC).

6057 Background: Fibroblast growth factor receptor 3 (FGFR3) is expressed in squamous cell carcinoma of the head and neck (SCCHN) including oropharyngeal squamous cell carcinoma (OPSCC) and is a potential therapeutic target. Information on its prognostic value and its correlation with other relevant cancer related proteins is limited. Methods: We performed immunohistochemistry (IHC) analyses of p16, mutant p53 (mp53), and FGFR3 on 221 retrospectively collected OPSCC tissue samples. mp53, and FGFR3 were semi-quantified as weighted index [WI = % positive x intensity (0. 1+, 2+, and 3+)]. Correlations of FGFR3 WI with p16 status, and mp53 WI were analyzed. Association of FGFR3 with disease-free survival (DFS) or overall survival (OS) was assessed. Results: A total of 144/221 (65%) were p16 +, 93/172 (54%) had mp53, and 140/221 (63%) expressed FGFR3. FGFR3 was highly correlated with mp53 (p &lt; 0.001), which was true in both p16 + and – OPSCC (p &lt; 0.0001 and p = 0.0006, respectively).mp53 level was significantly lower in p16 positive versus p16 negative group (p &lt; 0.0001). Univariate analysis revealed an association of p16 negative and high mp53 with worse OS (p &lt; 0.001 and p &lt; 0.001, respectively) and DFS (p &lt; 0.001 and p = 0.004, respectively). FGFR3 was associated with worse OS and DFS (p = 0.014 and p = 0.047, respectively). On multivariable analysis FGFR3 was associated with worse DFS (p = 0.005), but not OS. Kaplan-Meier plot using medians of both FGFR3 and mp53 as the cut-off values showed that higher FGFR3 and mp53 correlated to worst DFS (p = 0.025) and OS (p = 0.009). Conclusions: Our results suggest that FGFR3 is associated with mp53 and p16 – OPSCC and correlates with worse clinical outcome. The biologic relation of FGFR3 and mp53 in OPSCC deserves further investigation. (This research was supported by a grant NCI R21 CA182661-01A1to NFS and GZC).

Utility of the Roche Cobas 4800 for detection of high-risk human papillomavirus in formalin-fixed paraffin-embedded oropharyngeal squamous cell carcinoma

Clinical laboratories are expected to reliably identify human papilloma virus (HPV) associated oropharyngeal squamous cell carcinoma (OPSCC) for prognostic and potential therapeutic applications. In addition to surrogate p16 immunohistochemistry (IHC) testing, DNA-based HPV-specific testing strategies are widely utilized. Recognizing the efficiency of the Roche Cobas 4800 platform for testing gynecological cytology specimens for high-risk HPV, we elected to evaluate the potential utility of this platform for testing formalin-fixed paraffin-embedded (FFPE) OPSCC tissue. Using the Roche Linear Array assay for comparison, we tested twenty-eight samples (16 primary OPSCC, 2 lymph node metastases from primary OPSCC, 1 oral tongue carcinoma, 3 benign squamous papillomas, and 3 non-oropharyngeal carcinoma tissues). Excluding two invalid results, the Roche Cobas 4800 testing resulted in excellent inter-assay concordance (25/26, 96.2%) and 100% concordance for HPV-16/HPV-18 positive samples. This data suggests that the Roche Cobas 4800 platform may be a cost-effective method for testing OPSCC FFPE tissues in a clinical molecular pathology laboratory setting.

FGFR Family Members Protein Expression as Prognostic Markers in Oral Cavity and Oropharyngeal Squamous Cell Carcinoma.

IntroductionFibroblast growth factor receptor family member proteins (FGFR1–4) have been identified as promising novel therapeutic targets and prognostic markers in a wide spectrum of solid tumors. The present study investigates the expression and prognostic value of four FGFR family member proteins in a large multicenter oral cavity squamous cell carcinoma (OCSCC) and oropharyngeal squamous cell carcinoma (OPSCC) cohort.MethodsProtein expression of FGFR1–4 was determined by immunohistochemistry on tissue microarrays containing 951 formalin-fixed paraffin embedded OCSCC and OPSCC tissues from the University Medical Center Utrecht and University Medical Center Groningen. Protein expression was correlated to overall survival using Cox regression models, and bootstrapping was performed as internal validation.ResultsFGFR proteins were highly expressed in 39–64 % of OCSCC and 63–79 % of OPSCC. Seventy-three percent (299/412) of OCSCC and 85 % (305/357) of OPSCC highly co-expressed two or more FGFR family member proteins. FGFR1 protein was more frequently highly expressed in human papillomavirus (HPV)-negative OPSCC than HPV-positive OPSCC (82 vs. 65 %; p = 0.008). Furthermore, protein expression of FGFR family members was not related to overall survival in OCSCC or OPSCC (p > 0.05).ConclusionFGFR family members are frequently highly expressed in OCSCC and OPSCC. These FGFR family member proteins are therefore potential targets for novel therapies that are urgently required to improve survival of OCSCC and OPSCC patients.Electronic supplementary materialThe online version of this article (doi:10.1007/s40291-016-0204-5) contains supplementary material, which is available to authorized users.

A novel prediction model for human papillomavirus-associated oropharyngeal squamous cell carcinoma using p16 and subcellular β-catenin expression.

p16 overexpression is a highly sensitive yet moderately specific biomarker for predicting human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC). Nuclear β-catenin translocation has been linked to HPV-positive OPSCC. However, whether the strategy of combining β-catenin with p16 can better predict HPV-associated OPSCC remains unknown. We evaluated the expression of p16 and β-catenin (nuclear and membrane) by immunohistochemistry staining in 101 OPSCC tissues and HPV status by HPV DNA in situ hybridization. Logistic regression models were used to evaluate single or multiple biomarkers for HPV prediction. The prediction power, sensitivity, and specificity were determined by receiver operating characteristic (ROC) analyses. Our data showed that upon univariate analysis, p16 and nuclear β-catenin were positively correlated with HPV status, while membrane β-catenin was inversely correlated with HPV status (P < 0.01). p16 showed the highest HPV predictive power, with area under the curve (AUC) of 0.9074 compared to 0.6762 for nuclear β-catenin and 0.7635 for membrane β-catenin, respectively, indicating differential accuracies for HPV prediction. Multivariable analysis showed that p16 was significantly correlated with HPV, while nuclear and membrane β-catenin showed marginal significance. The three-biomarker model was similarly sensitive (98.9% vs. 100%) but more specific (88.9% vs. 81%) than p16 alone, which also showed a good predictive value for overall (P = 0.0002) survival and disease-free (P = 0.0158) survival. Our study suggests a novel model of combining p16 and subcellular β-catenin for prediction of HPV-associatred OPSCC, and this finding deserves further validation.

HPV Status Determines the Efficacy of Adjuvant Chemotherapy With S-1, an Oral Fluorouracil Prodrug, in Oropharyngeal Cancer.

A subgroup of oropharyngeal squamous cell carcinoma (OPC) is infected with high-risk human papillomavirus (HPV). The object of this study is to evaluate the efficacy of adjuvant chemotherapy with S-1, an oral 5-fluorouracil prodrug, on survival of patients with OPC according to HPV status. Among OPC patients of stage III or IV who received definitive treatment from 1998 to 2008, 38 who were confirmed tumor-free after primary treatment were analyzed. Before 2003, none of the patients received S-1 adjuvant chemotherapy (S-1(-)-group); however, all patients who were eligible were administered S-1 (S-1(+)-group) after 2003. The expression of thymidylate synthase (TYMS) involved in 5-FU metabolism was also examined in protein and mRNA levels. Although there was a trend to disease-free and overall survival benefit in HPV-negative patients with S-1, it did not achieve statistical significance (P=.082 and P=.065, respectively). For the HPV-positive patients, the survivals were similar with or without S-1 administration. TYMS-expression in HPV-positive OPC tissues was significantly higher than in HPV-negative ones in both protein and mRNA levels (P=.0489 and P=.0446, respectively). The current study provides a rationale to plan a randomized trial to compare the efficacy of S-1 according to the HPV status in OPCs.

Abstract 1852: Enhancing specificity in predication of human papillomavirus associated oropharyngeal squamous cell carcinoma by combining biomarkers p16 and β-catenin

Abstract Background: p16 is a FDA approved surrogate biomarker for predicting human papillomavirus (HPV) associated oropharyngeal squamous cell carcinoma (OPSCC) with high sensitivity and moderate specificity. A p16 positive HPV negative phenotype indeed exists. Increased nuclear β-catenin expression has also been shown to correlate with HPV status in OPSCC. We, therefore, tested a novel model by combining β-catenin with p16 to increase the specificity for HPV prediction in OPSCC. Methods: Expression levels of β-catenin (nuclear and membrane immune-reactivity) and p16 were evaluated by immunohistochemistry (IHC) staining in 101 OPSCC tissues. HPV status was determined by HPV DNA in situ hybridization. Logistic regression models were used to estimate single or multiple biomarkers in HPV prediction. The prediction power, sensitivity, and specificity were determined by the Receiver Operating Characteristic (ROC) analysis. Results: All three biomarkers (p16, nuclear and membrane β-catenin) were significantly associated with HPV status (P &amp;lt; 0.01 for all). p16 alone showed the highest predictive power with area under the curve (AUC) of 0.9074 compared to 0.6762 for nuclear β-catenin and 0.7635 for membrane β-catenin. Multivariable analysis indicated that the odds ratios and 95% confidence interval (CI) for HPV positivity were 381.21 (17.14-8476.34), 4.93 (0.78-31.02), and 0.98 (0.97-1.00) for p16 (P &amp;lt; 0.001), nuclear β-catenin (P = 0.089) and membrane β-catenin (P = 0.085), respectively. The three-biomarker model had a sensitivity (99%) close to p16 alone (100%), but a higher specificity (89%) than p16 alone (81%) and showed prognosis value for overall survival (P = 0.0002) and disease-free survival (P = 0.0158). The model adjusting of clinical covariates only (AUC = 0.794) or adjusting of both clinical covariates and the three biomarkers (AUC = 0.852) did not produce a better HPV prediction as compared to the three-biomarker model (AUC = 0.962). Conclusion: Our findings suggests that the novel strategy of combining β-catenin with p16 increases the specificity for HPV prediction in OPSCC compared to p16 alone and deserves further validation in different clinical settings. (This study was supported by grants from Small Business Innovation Research (SBIR) Program (HHSN261201200097C) and National Institutes of Health (R33 CA161873) and Georgia Cancer Collation Distinguished Scholar Award to Dr. Zhuo Georgia Chen.) Citation Format: Guoqing Qian, Hong Xu, Zhongliang Hu, Sungjin Kim, Dongsheng Wang, Hongzheng Zhang, Zhengjia Chen, Susan Muller, Nabil Saba, Dong M. Shin, Andrew Wang, Zhuo Georgia Chen. Enhancing specificity in predication of human papillomavirus associated oropharyngeal squamous cell carcinoma by combining biomarkers p16 and β-catenin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1852. doi:10.1158/1538-7445.AM2014-1852

MET Genetic Abnormalities Unreliable for Patient Selection for Therapeutic Intervention in Oropharyngeal Squamous Cell Carcinoma

BackgroundIdentification of MET genetic alteration, mutation, or amplification in oropharyngeal squamous cell carcinoma (OPSCC) could lead to development of MET selective kinase inhibitors. The aim of this study was to assess the frequency and prognostic value of MET gene mutation, amplification, and protein expression in primary OPSCC.MethodsA retrospective chart review was conducted of patients treated for single primary OPSCC between January 2007 and December 2009. Pre-treatment OPSCC tissue samples were analyzed for MET mutations, gene amplification, and overexpression using Sanger sequencing, FISH analysis, and immunohistochemistry respectively. Univariate and multivariate analyses were used to analyze correlations between molecular abnormalities and patient survival.Results143 patients were included in this study. Six cases (4%) were identified that had a genetic variation, but previously described mutations such as p.Tyr1235Asp (Y1235D) or p.Tyr1230Cys (Y1230C) were not detected. There were 15 high polysomy cases, and only 3 cases met the criteria for true MET amplification, with ≥10% amplified cells per case. Immunohistochemistry evaluation showed 43% of cases were c-MET negative and in 57% c-MET was observed at the tumor cell level. Multivariate analysis showed no significant association between MET mutation, amplification, or expression and survival.ConclusionsOur study shows a low frequency of MET mutations and amplification in this cohort of OPSCC. There was no significant correlation between MET mutations, amplification, or expression and patient survival. These results suggest that patient selection based on these MET genetic abnormalities may not be a reliable strategy for therapeutic intervention in OPSCC.

Abstract B224: HPV-stratified analysis of prognostic role of miR-21 in oropharyngeal squamous cell carcinoma.

Abstract Background: Both smoking and human papillomavirus (HPV) infection play an significant role in the development and progression of oropharyngeal squamous cell carcinoma (OSCC). MicroRNAs are short noncoding RNAs that regulate multiple mRNAs and are frequently dysregulated in cancer. The miR-21 microRNA is overexpressed in a wide variety of cancers and has been causally related to cellular proliferation and apoptosis. In this study, we investigated the association between the expression of miR-21 and the clinicopathologic features by stratified analysis based on HPV infection status. Patients and Methods: The expression of miR-21 in the OSCC tissues of 137 patients was evaluated by in situ hybridization method. In addition, PDCD4 and PTEN were also examined by immunohistochemistry using tissue microarray. The clinicopathological implications of these molecules were analyzed statistically. Results: The up-regulation of miR-21 expression was 22.7% for HPV negative group and 42.1% for HPV positive group (P = 0.081), and significantly correlated with advanced lymph node metastasis and extracapsular extension (P = 0.005 and P = 0.014, respectively). There was a significant association between miR-21 and cytoplasmic PDCD4 overexpression (P = 0.027). Kaplan-Meier analysis showed that up-regulation of miR-21 was significantly related with shorter overall survival (OS; 77.0 v 193.0 months; P =0.05) in HPV-negative OSCC, but not in HPV-positive OSCC (P =0.95). At HPV-stratified multivariate analysis, up-regulation of miR-21 was independent prognostic factor for both relapse-free survival (RFS; HR, 2.681; 95% CI, 1.090 to 6.591; P = 0.032) and OS (HR, 6.286; 95% CI, 2.047 to 19.302; P = 0.001). Conclusion: Up-regulation of miR-21 expression is closely associated with poor prognosis in HPV-negative OSCC, but not in HPV-positive OSCC, suggesting that miR-21 may have a different role according to the HPV infection status. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B224. Citation Format: Yoon Ho Ko, Ho Jung An, Der Sheng Sun, Hye Sung Won, Jin Hyoung Kang, Chan-Kwon Jung. HPV-stratified analysis of prognostic role of miR-21 in oropharyngeal squamous cell carcinoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B224.

Abstract 4798: Subcellular translocation of β-catenin and its relationship with EGFR activation in HPV16 positive OPSCC.

Abstract Background: The incidence of human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) has increased in recent years. HPV-associated OPSCC follows a distinct carcinogenic pathway from HPV-negative OPSCC. The molecular basis of the disease progression due to HPV remains to be elucidated. Clinical observations have revealed frequent and early lymph node metastasis and unusual distant metastasis in HPV-associated as compared with HPV-negative OPSCC. To understand the underlying molecular mechanism, we have studied whether β-catenin, one of the major proteins in the Wnt pathway, is involved in HPV-associated OPSCC metastasis. Methods: The study samples (n = 210) were collected from formalin-fixed and paraffin-embedded OPSCC tissues obtained from the Surgical Pathology files in the Department of Pathology at Emory University following the regulations of the Health Insurance Portability and Accountability Act (HIPAA). Expression levels of p16 as a surrogate marker for HPV-associated cancer, β-catenin, and epidermal growth factor receptor (EGFR) were determined by immunohistochemistry (IHC) analyses. Membrane expression of β-catenin and total EGFR level were scored as a weight index [WI = intensity (0, 1+, 2+, and 3+) x % of positive staining]. Nuclear β-catenin and p16 were recorded as positive and negative. Expression and subcellular localization of β-catenin as well as EGFR expression and activation were also studied in both HPV-positive and -negative head and neck SCC (HNSCC) cell lines (n =7) by Western blot analysis. SiRNA against HPV E6 was used to elucidate the effect of HPV on β-catenin translocation. Results: Our results showed that membrane WI of β-catenin was inversely correlated with p16 positivity (p &amp;lt; 0.0001), while nuclear staining of β-catenin was associated with p16 positive OPSCC (p = 0.007) and positive lymph nodes (p = 0.0304). A low level of membrane β-catenin (cut-off by the median value) was significantly associated with disease free survival and overall survival (p &amp;lt; 0.05 in both cases). Furthermore, WI EGFR was positively correlated with nuclear β-catenin (p = 0.0001). Our in vitro study showed that nuclear β-catenin level was higher in HPV16-positive HNSCC cell lines than in HPV16-negative cell lines. Furthermore, HPV16-positive cells showed higher EGFR activation than HPV16-negative cells. Reduction of HPV16 E6 led to the de-activation of EGFR and decreased level of β-catenin in the nucleus. Conclusion: Our findings suggest that HPV16 E6 mediates the translocation of β-catenin to the nucleus, which may be regulated by activated EGFR. The underlying mechanism deserves further investigation. (This study was supported by US National Institutes of Health (NIH/NCI) grant HHSN2610201000125C, R33 CA134392-01, and Georgia Cancer Coalition Distinguished Cancer Scholar Award to ZGC). Citation Format: Zhongliang Hu, Ning Jiang, Susan Muller, Nabil Saba, Dongsheng Wang, Hongzheng Zhang, Dong M. Shin, Zhuo Georgia Chen. Subcellular translocation of β-catenin and its relationship with EGFR activation in HPV16 positive OPSCC. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4798. doi:10.1158/1538-7445.AM2013-4798

Linking expression of FOXM1 , CEP55 and HELLS to tumorigenesis in oropharyngeal squamous cell carcinoma

To investigate the relationship between the expression of FOXM1, CEP55, and HELLS in oropharyngeal squamous cell carcinoma to human papillomavirus (HPV), smoking, and tumor stage. Retrospective cohort study. Transcriptome data were analyzed from matched tumor-normal samples taken from patients with oropharyngeal squamous cell carcinoma. Data were previously generated using deep-sequencing techniques (mRNA-Seq). Transcript levels of all three genes were validated using the NanoString nCounter system in a larger group of patients. Analyses were conducted to assess possible associations between expression levels and HPV infection status, smoking status, or tumor staging. FOXM1, CEP55, and HELLS were all overexpressed in oropharyngeal squamous cell carcinoma tissue when compared to normal tissue. Significant trends were found between expression levels of FOXM1, CEP55, and HELLS and tumor staging. Tumors staged 3 or greater showed significantly higher levels of expression compared with those staged 1. No significant association or trend was found between expression of any genes of interest and etiologic subgroupings (i.e., HPV, smoking). FOXM1, CEP55, and HELLS were all overexpressed in oropharyngeal squamous cell carcinoma. Gene expression is related to tumor stage. The significant association between the expression of these genes and advanced tumor stage suggest that they may play a role in tumorigenesis.

Genomewide Gene Expression Profiles of HPV-Positive and HPV-Negative Oropharyngeal Cancer

To study the difference in gene expression between human papillomavirus (HPV)-positive and HPV-negative oral cavity and oropharyngeal squamous cell carcinoma (OSCC). We used Affymetrix U133 plus 2.0 arrays to examine gene expression profiles of OSCC and normal oral tissue. The HPV DNA was detected using polymerase chain reaction followed by the Roche LINEAR ARRAY HPV Genotyping Test, and the differentially expressed genes were analyzed to examine their potential biological roles using the Ingenuity Pathway Analysis Software, version 5.0. Three medical centers affiliated with the University of Washington. A total of 119 patients with primary OSCC and 35 patients without cancer, all of whom were treated at the setting institutions, provided tissues samples for the study. Human papillomavirus DNA was found in 41 of 119 tumors (34.5%) and 2 of 35 normal tissue samples (5.7%); 39 of the 43 HPV specimens were HPV-16. A higher prevalence of HPV DNA was found in oropharyngeal cancer (23 of 31) than in oral cavity cancer (18 of 88). We found no significant difference in gene expression between HPV-positive and HPV-negative oral cavity cancer but found 446 probe sets (347 known genes) differentially expressed in HPV-positive oropharyngeal cancer than in HPV-negative oropharyngeal cancer. The most prominent functions of these genes are DNA replication, DNA repair, and cell cycling. Some genes differentially expressed between HPV-positive and HPV-negative oropharyngeal cancer (eg, TYMS, STMN1, CCND1, and RBBP4) are involved in chemotherapy or radiation sensitivity. These results suggest that differences in the biology of HPV-positive and HPV-negative oropharyngeal cancer may have implications for the management of patients with these different tumors.