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Most laboratory models of head and neck squamous cell cancer (HNSCC) rely on established immortalized cell lines, which carry inherent bias due to selection and clonality. We established a robust panel of HNSCC tumor cultures using a "conditional reprogramming" (CR) method, which utilizes a rho kinase inhibitor (Y-27632) and co-culture with irradiated fibroblast (J2 strain) feeder cells to support indefinite tumor cell survival. Sixteen CR cultures were successfully generated from 19 consecutively enrolled ethnically and racially diverse patients with HNSCC at a tertiary care center in the Bronx, NY. Of the 16 CR cultures, 9/16 were derived from oral cavity, 4/16 were derived from oropharynx, and 3/16 were from laryngeal carcinomas. Short tandem repeat (STR) profiling was used to validate culture against patient tumor tissue DNA. All CR cultures expressed ΔNp63 and cytokeratin 5/6, which are markers of squamous identity. Human papilloma virus (HPV) testing was assessed utilizing clinical p16 staining on primary tumors, RT-PCR of HPV16/18 specific viral oncogenes E6 and E7 in RNA extracted from tumor samples, and HPV DNA sequencing. Three of 4 oropharyngeal tumors were p16 and HPV-positive and maintained HPV in culture. CR cultures were able to establish three-dimensional spheroid and murine flank and orthotopic tongue models. CR methods can be readily applied to all HNSCC tumors regardless of patient characteristics, disease site, and molecular background, providing a translational research model that properly includes patient and tumor diversity. This article is protected by copyright. All rights reserved.

INTRODUCTION: CDKN2A, coding for p16INK4a, a CDK4/6 inhibitory protein, is among the most commonly lost tumor suppressors in oral cavity squamous cell carcinoma (OCSCC). Drugs like palbociclib are CDK4/6 inhibitors that phenomenally also induce senescence. Senescence can be pro-tumorigenic. Studies suggests prosurvival BCL-2 family support tumor cell survival in senescent state. Agents targeting BCL-2 family e.g. navitoclax, are “senolytics” The current study examines combining palbociclib (P) with the senolytic agent navitoclax (N) as a treatment strategy. We demonstrate efficacy of combining P+N in OCSCC, and examine senescence and apoptosis processes as related mechanistic response of combination. EXPERIMENTAL PROCEDURE: We examined cell viability after sequential treatment: 72 h P followed by 72 h N (P+N) in 2-dimensional (2D) model of immortalized cell line HN5 and 3D model of a human OCSCC patient-derived conditional reprogrammed cell line (CR18). Flow cytometry measured apoptosis with Annexin V assay in 2D. Confocal imaging in 3D spheroids corroborated pro-survival protein levels. An in vivo xenograft study was performed with HN5 in 8 weeks old male nude mice to examine tumor responses to sequential P+N treatment. 3 million cells were injected in right flank. Tumors were treated at ~300 mm3 (P-100 mg/kg o.p; N -17 mg/kg i.p). After treatment termination, Bcl-2 family proteins (BcLxl); apoptosis markers (caspase 3, Cleaved PARP) and β-galactosidase (β -gal) for senescence were evaluated with western blot. QPCR measured expression of CDK4/6 target (FOXM1) to examine senescence and pro survival genes. RESULTS: Sequential treatment of P+N (1µM each) demonstrated consistent synergy in both 2D and 3D viability assays. Bright field images of P+N revealed senescence with P followed with apoptosis on N application. β -gal levels increased to 1.5 fold with P and Caspase 3 showed 2-fold increase with P+N. Flow cytometry data revealed 2 folds increase in the apoptotic population with P+N. Confocal imaging in organoids demonstrated increased levels of BcLxL with P, and repression of BCL-xL following N administration. Sequential P+N significantly reduced tumor growth in vivo. P+N demonstrated a robust 5-fold decrease in tumor volume (TV) with a complete cure (TV CONCLUSION: Our results suggest that CDK4/6 inhibition makes OCSCC undergo senescence and make it more susceptible to apoptosis when combined with the BCL2-family inhibitor, navitoclax. We propose a sequential P+N as a rational and potentially effective treatment strategy in OCSCC. Citation Format: Nitisha Shrivastava, Nicole Kawachi, Michael B. Prystowsky, Evripidis Gavathiotis, Chandan Guha, Thomas J. Ow. The CDK4/6 inhibitor palbociclib sensitizes oral cavity squamous cell carcinoma to navitoclax-induced apoptosis-An in vitro and in vivo study. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5986.

Purpose: HPV(−) OCSCC resists radiation treatment. The CDKN2A gene, encoding p16INK4A, is commonly disrupted in OCSCC. p16 inhibits CDK4/CDK6, leading to cell cycle arrest, but the biological sequelae of CDK4/6 inhibition in OCSCC remains understudied. This study examines whether inhibition of CDK4/6 enhances radiation response in OCSCC. Methods: MTT assays were performed in OCSCC cell lines HN5 and CAL27 following treatment with palbociclib. Clonogenic survival and synergy were analyzed after radiation (RT-2 or 4Gy), palbociclib (P) (0.5 µM or 1 µM), or concurrent combination treatment (P+RT). DNA damage/repair and senescence were examined. CDK4/6 were targeted via siRNA to corroborate P+RT effects. Three-dimensional immortalized spheroids and organoids derived from patient tumors (conditionally reprogrammed OCSCC CR-06 and CR-18) were established to further examine and validate responses to P+RT. Results: P+RT demonstrated reduced viability and synergy, increased β-gal expression (~95%), and ~two-fold higher γH2AX. Rad51 and Ku80 were reduced after P+RT, indicating impairment of both HR and NHEJ. siCDK4/6 increased senescence with radiation. Spheroids showed reduced proliferation and size with P+RT. CR-06 and CR-18 further demonstrated three-fold reduced proliferation and organoids size with P+RT. Conclusion: Targeting CDK4/6 can lead to improved efficacy when combined with radiation in OCSCC by inducing senescence and inhibiting DNA damage repair.

Mechanisms of treatment resistance in head and neck squamous cell carcinoma (HNSCC) are not well characterized. In this study, HNSCC tumors from a cohort of prospectively enrolled subjects on an ongoing tissue banking study were divided into those that persisted or recurred locoregionally (n=23) and those that responded without recurrence (n=35). Gene expression was evaluated using llumina HumanHT-12-v3 Expression BeadChip microarrays. Sparse Partial Least Squares – Discriminant Analysis (sPLS-DA) identified 135 genes discriminating treatment-resistant from treatment-sensitive tumors. BCL-xL was identified among 23% of canonical pathways derived from this set of genes using Ingenuity Pathway analysis. The BCL-xL protein was expressed in 8 HNSCC cell lines examined. Cells were treated with the BCL-xL inhibitor, ABT-263 (navitoclax): the average half maximal inhibitory concentration (IC50) was 8.9μM (range 6.6μM – 13.9μM). Combining ABT-263 did not significantly increase responses to 2 Gy radiation or cisplatin in the majority of cell lines. MCL-1, a potential mediator of resistance to ABT-263, was expressed in all cell lines and HNSCC patient tumors, in addition to BCL-xL. Treatment with the MCL-1 inhibitor, A-1210477, in HNSCC cell lines showed an average IC50 of 10.7μM (range, 8.8μM to 12.7μM). Adding A-1210477 to ABT-263 (navitoclax) treatment resulted in an average 7-fold reduction in the required lethal dose of ABT-263 (navitoclax) when measured across all 8 cell lines. Synergistic activity was confirmed in PCI15B, Detroit 562, MDA686LN, and HN30 based on Bliss Independence analysis. This study demonstrates that targeting both BCL-xL and MCL-1 is required to optimally inhibit BCL-family pro-survival molecules in HNSCC, and co-inhibition is synergistic in HNSCC cancer cells.

Human papillomavirus positive (HPV+) oropharyngeal squamous cell carcinomas (OPSCC), associated with improved patient outcome, exhibit increased expression of CDKN2A tumor suppressor p16(INK4a), while inactivation of the CDKN2A locus is frequently observed in HPV negative HNSCC. We recently identified a novel region of intragenic DNA hypermethylation within the CDKN2A locus that correlates with RNA expression of two CDKN2A transcripts and improved patient outcome in oropharyngeal and laryngeal SCC. Given the importance of CDKN2A expression in OPSCC, we chose an in vitro model using primary keratinocytes transduced with HPV-16 oncogenes E6 and E7 to study the relationship between CDKN2A methylation and expression. RNA levels for CDKN2A transcripts, p14(ARF) and p16(INK4a), and intragenic CDKN2A methylation were assessed in the primary cells and after transduction with E6 and E7 at two time points. CDKN2A expression increased early post-transduction, while there was a clear delay in intragenic methylation. Methylation eventually increased suggesting that intragenic methylation is not required for transcription at the CDKN2A locus, and that methylation may be a consequence of open and active chromatin.

Introduction Head and neck squamous cell carcinoma (HNSCC) most commonly arise in the oral cavity, pharynx, and larynx and exhibit diverse molecular characteristics and clinical behavior across sites. Current models of HNSCC largely rely on immortalized cell lines, which suffer from selection bias and clonality. Primary cell cultures, on the other hand, are difficult to maintain and are limited by their finite ability to proliferate. Here, we describe the establishment of a robust HNSCC cell line bank using a “conditional reprogramming” (CR) method which relies on treatment with a rho kinase inhibitor (Y-27632) and co-culture with irradiated fibroblast (J2 strain) feeder cells to support indefinite tumor cell survival. Methods HNSCC tumors were acquired on an IRB-approved prospective tissue banking protocol. Tumor tissue and blood were collected from each patient, and tumor cells were digested and cultured using previously described CR methods. DNA was collected from tumor tissue and CR cultures and short tandem repeat (STR) profiling was used to validate culture against patient tumor tissue. To verify that the cultures were of squamous origin, western blot analysis was used to detect cytokeratin 5, cytokeratin 6 and p63. Tumor cell phenotype and growth characteristics were examined with light microscopy. HPV testing was carried out on DNA from CR cultures and matched tumor samples using PCR and specific genotyping by oligonucleotide hybridization. HPV testing was correlated with clinical p16 testing results. Whole exome sequencing was carried out on a subset of blood/tumor/CR culture samples. Results Eighteen CR lines were successfully cultured and validated with STR genotyping from 25 sequentially procured tumors. 10/18 were derived from oral cavity squamous cell cancers (SCCs), 5/18 were derived from oropharyngeal SCCs, and 3/18 were derived from laryngeal SCCs. 18/18 lines were found to express p63 and either cytokeratin 5 or 6, verifying these cultures contained tumor cells of squamous origin. 4/5 tumors from oropharyngeal SCCs were p16-positive on clinical testing and considered HPV-mediated. These 4 tumors all tested positive for HPV DNA in both tumor samples and CR cultures. Preliminary comparison of exome sequencing results between CR cultures and primary tumors suggests that overall mutational profiles are preserved through the tumor “conditional programming” process. Tumor heterogeneity between original tumor and CR culture is being actively compared in ongoing analyses. Conclusion We have consistently generated primary tumor CR cultures from patients with HNSCC arising in three major anatomical subsites of HNSCC disease, including HPV-mediated tumors. CR methods can be readily applied to all HNSCC tumors regardless of disease site and molecular background, providing a translational research model that can capture the molecular and phenotypic breadth of HNSCC disease. Citation Format: Daniel Li, Carlos Thomas, Nitisha Shrivastava, Nicholas Gadsden, Nicole Kawachi, Bradley A. Schiff, Richard V. Smith, Nicolas F. Schlecht, Michael B. Prystowsky, Gregory Rosenblatt, Stelby Augustine, Chandan Guha, Evripidis Gavathiotis, Robert D. Burk, Vikas Mehta, Thomas J. Ow. Conditional reprogramming of primary head and neck tumor cells to establish consistent and diverse cell line models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2986.

Global proteomic analysis of oral cavity squamous cell carcinoma was performed to identify changes that reflect patient outcomes.To identify differentially expressed proteins associated with patient outcomes and to explore the use of imaging mass spectrometry as a clinical tool to identify clinically relevant proteins.Two-dimensional separation of digested peptides generated from 43 specimens with high-resolution mass spectrometry identified proteins associated with disease-specific death, distant metastasis, and loco-regional recurrence. RNA expressions had been correlated to protein levels to test transcriptional regulation of clinically relevant proteins. Imaging mass spectrometry explored an alternative platform for assessing clinically relevant proteins that would complement surgical pathologic diagnosis.Seventy-two peptide features were found to be associated with 3 patient outcomes: disease-specific death (9), distant metastasis (16), and loco-regional recurrence (39); 8 of them were associated with multiple outcomes. Functional ontology revealed major changes in cell adhesion and calcium binding. Thirteen RNAs showed strong correlation with their encoded proteins, implying transcriptional control. Reduction of DSP, PKP1, and TRIM29 was associated with significantly shorter time to onset of distant metastasis. Reduction of PKP1 and TRIM29 correlated with poorer disease-specific survival. Additionally, S100A8 and S100A9 reductions were verified for their association with poor prognosis using imaging mass spectrometry, a platform more adaptable for use with surgical pathology.Using global proteomic analysis, we have identified proteins associated with clinical outcomes. The list of clinically relevant proteins observed will provide a means to develop clinical assays for prognosis and optimizing treatment selection.

Invasion is a hallmark of advanced head and neck squamous cell carcinoma (HNSCC). We previously determined that low relative miR-375 expression was associated with poor patient prognosis. HNSCC cells with increased miR-375 expression have lower invasive properties and impaired invadopodium activity. Using stable isotope labeling with amino acids in cell culture and reverse-phase liquid chromatography mass spectrometry, we assessed the impact of miR-375 expression on protein levels in UM-SCC-1 cells. Increased miR-375 expression was associated with down-regulation of proteins involved in cellular assembly and organization, death and survival, and movement. Two invasion-associated proteins, vimentin and L-plastin, were strongly down-regulated by miR-375. Luciferase reporter assays demonstrated that high miR-375 expression reduced vimentin promoter activity, suggesting that vimentin is an indirect target of miR-375. Runt-related transcription factor 1 (RUNX1) is a potential miR-375 direct target, and its knockdown reduced vimentin and L-plastin expression. Data in The Cancer Genome Atlas HNSCC database showed a significant inverse correlation between miR-375 expression and RUNX1, vimentin, and L-plastin RNA expression. These clinical correlations validate our in vitro model findings and support a mechanism in which miR-375 suppresses RUNX1 levels, resulting in reduced vimentin and L-plastin expression. Furthermore, knockdown of RUNX1, L-plastin, and vimentin resulted in significant reductions in cell invasion in vitro , indicating the functional significance of miR-375 regulation of specific proteins involved in HNSCC invasion.

While its prognostic significance remains unclear, p16(INK4a) protein expression is increasingly being used as a surrogate marker for oncogenic human papillomavirus (HPV) infection in head and neck squamous cell carcinomas (HNSCC). To evaluate the prognostic utility of p16 expression in HNSCC, we prospectively collected 163 primary tumor specimens from histologically confirmed HNSCC patients who were followed for up to 9.4 years. Formalin fixed tumor specimens were tested for p16 protein expression by immunohistochemistry (IHC). HPV type-16 DNA and RNA was detected by MY09/11-PCR and E6/E7 RT-PCR on matched frozen tissue, respectively. P16 protein expression was detected more often in oropharyngeal tumors (53%) as compared with laryngeal (24%), hypopharyngeal (8%) or oral cavity tumors (4%; p

Recent evidence suggests that human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) patients have better survival than HPV-negative patients. However, it is unclear if similar patterns for survival exist across different tumor sites, and whether HPV-associated prognosis is modified by type of treatment. We prospectively tested 222 histologically confirmed HNSCC primary tumors for HPV DNA by PCR and HPV E6/E7 RNA by RT-PCR prior to treatment at a large urban health center. Cox proportional hazard ratio models were constructed to assess HPV-associated differences in overall and disease-specific survival adjusting for clinical and demographic covariates. HPV detection varied significantly by primary HNSCC tumor site, from 35 % for oropharynx, to 25 % for hypopharynx, 5 % for larynx, and 3 % for oral cavity (p

Members of the 4.1 superfamily of proteins, including ezrin, moesin, merlin, and willin regulate many normal physiologic processes such as cellular shape, motility, and proliferation. In addition, they contribute both to tumor development and tumor progression. We reported previously that strong cytoplasmic ezrin expression was independently associated with poorer patient survival. One hundred and thirty-one histologically confirmed primary head and neck squamous cell carcinomas were examined prospectively for cancer progression and survival at a large health care center in the Bronx, NY, USA. Immunohistochemical analysis of ezrin, moesin, merlin, and willin expression in tissue microarray samples of primary head and neck squamous cell carcinoma revealed a significant association of increased cytoplasmic ezrin with poor cancer survival. Global RNA analyses suggest that cancers with high cytoplasmic ezrin have a more invasive phenotype. This study supports our previous findings associating cytoplasmic ezrin with more aggressive behavior and poorer outcome and indicates the need for a multi-institutional study to validate the use of cytoplasmic ezrin as a biomarker for treatment planning in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma represents a complex set of neoplasms arising in diverse anatomical locations. The site and stage of the cancer determine whether patients will be treated with single or multi-modality therapy. The HDAC inhibitor LBH589 is effective in treating some haematological neoplasms and shows promise for certain epithelial neoplasms. As with other human cancer cell lines, LBH589 causes up-regulation of p21, G2/M cell cycle arrest, and cell death of human HNSCC cell lines, as measured using flow cytometry and cDNA microarrays. Global RNA expression studies following treatment of the HNSCC cell line FaDu with LBH589 reveal down-regulation of genes required for chromosome congression and segregation (SMC2L1), sister chromatid cohesion (DDX11), and kinetochore structure (CENP-A, CENP-F, and CENP-M); these LBH589-induced changes in gene expression coupled with the down-regulation of MYC and BIRC5 (survivin) provide a plausible explanation for the early mitotic arrest and cell death observed. When LBH589-induced changes in gene expression were compared with gene expression profiles of 41 primary HNSCC samples, many of the genes that were down-regulated by LBH589 showed increased expression in primary HNSCC, suggesting that some patients with HNSCC may respond to treatment with LBH589.

Epidemiological and laboratory evidence indicate that, in addition to tobacco and alcohol, human papillomaviruses (HPV) play an important aetiological role in a subset of head and neck squamous cell carcinoma (HNSCC). To evaluate the molecular pathogenesis of HPV-infected HNSCC, we compared gene expression patterns between HPV-positive and -negative HNSCC tumours using cDNA microarrays. Tumour tissue was collected from 42 histologically confirmed HNSCC patients from an inner-city area of New York. Total DNA and RNA were extracted and purified from frozen tumour samples and gene expression levels were compared to a universal human reference RNA standard using a 27 323 cDNA microarray chip. HPV detection and genotyping were performed using an MY09/11-PCR system and RT-PCR. HPV was detected in 29% of HNSCC tumours. Most harboured only HPV16 and expressed the HPV16-E6 oncogene. HPV prevalence was highest in pharyngeal tumours (45%). Gene expression patterns that differentiated HPV-positive from negative tumours were compared by supervised classification analysis, and a multiple-gene signature was found to predict HPV16 prevalence in primary HNSCC with a false discovery rate

Our group has initiated experiments to epigenetically profile CpG island hypermethylation in genomic DNA from tissue specimens of head and neck squamous cell carcinoma (HNSCC) using a microarray of 12,288 CpG island clones. Our technique, known as a methylation-specific restriction enzyme (MSRE) analysis, is a variation of the differential methylation hybridization (DMH) technique, in that it is not an array comparison of two DNA samples using methylation-specific restriction enzymes. Instead, it is a comparison of a single DNA sample’s response to a methylation-sensitive restriction enzyme (HpaII) and its corresponding methylation-insensitive isoschizomer (MspI). Estimation of the reproducibility of this microarray assay by intraclass correlation (ICC) demonstrated that in four replicate experiments for three tumor specimens, the ICC observed for a given tumor specimen ranged from 0.68 to 0.85 without filtering of data. Repeated assays achieved 87% concordance or greater for all tumors after filtering of array data by fluorescence intensity. We utilized hierarchical clustering on a population of 37 HNSCC samples to cluster tumor samples with similar DNA methylation profiles. Supervised learning techniques are now being utilized to allow us to identify associations between specific epigenetic signatures and clinical parameters. Such techniques will allow us to identify select groups of CpG island loci that could be used as epigenetic markers for both diagnosis and prognosis in HNSCC.

While its prognostic significance remains unclear, p16INK4a protein expression is increasingly being used as a surrogate marker for oncogenic human papillomavirus (HPV) infection in head and neck squamous cell carcinomas (HNSCC). To evaluate the prognostic utility of p16 expression in HNSCC, we prospectively collected 163 primary tumor specimens from histologically confirmed HNSCC patients who were followed for up to 9.4 years. Formalin fixed tumor specimens were tested for p16 protein expression by immunohistochemistry. HPV type-16 DNA and RNA was detected by MY09/11-PCR and E6/E7 RT-PCR on matched frozen tissue, respectively. P16 protein expression was detected more often in oropharyngeal tumors (53%) as compared with laryngeal (24%), hypopharyngeal (8%), or oral cavity tumors (4%; P

Work by JP is funded by a Royal Society University Research Fellowship award. Experiments carried out by MBP were supported by the Department of Pathology, Albert Einstein College of Medicine / Montefiore Medical Center. The development of clinically useful histone deacetylase inhibitors has expanded greatly. In a preclinical study, we showed that panobinostat (LBH589) inhibits cell cycle progression of human head and neck squamous cell carcinoma (HNSCC) cell lines at G(2)/M and an associated decrease in expression of particular genes required for passage through G(2) and mitosis. In this study we sought to analyse the mechanistic underpinnings of panobinostat-induced growth arrest. HNSCC cell lines were synchronised and progression through mitosis monitored. We demonstrate that panobinostat causes a marked G(2) delay and mitotic defects. A loss of G(2)-specific Plk1 and Cyclin B1 expression and co-incident increase in p21(Waf1/Cip1) expression is also shown. Furthermore, we show a significant loss of E2F1 recruitment to the promoters of these genes in response to panobinostat treatment. These data provide mechanistic evidence of panobinostat-induced cell cycle arrest and highlight its potential as a chemotherapeutic agent for HNSCC. Publisher PDF

The development of clinically useful histone deacetylase inhibitors has expanded greatly. In a preclinical study, we showed that panobinostat (LBH589) inhibits cell cycle progression of human head and neck squamous cell carcinoma (HNSCC) cell lines at G₂/M and an associated decrease in expression of particular genes required for passage through G₂ and mitosis. In this study we sought to analyse the mechanistic underpinnings of panobinostat-induced growth arrest. HNSCC cell lines were synchronised and progression through mitosis monitored. We demonstrate that panobinostat causes a marked G₂ delay and mitotic defects. A loss of G₂-specific Plk1 and Cyclin B1 expression and co-incident increase in p21(Waf1/Cip1) expression is also shown. Furthermore, we show a significant loss of E2F1 recruitment to the promoters of these genes in response to panobinostat treatment. These data provide mechanistic evidence of panobinostat-induced cell cycle arrest and highlight its potential as a chemotherapeutic agent for HNSCC.

Detection of human papillomavirus in head and neck cancer has therapeutic implications. In-situ hybridization and immuno-histochemistry for p16 are used by surgical pathologists. We compared the sensitivity and specificity of three popular commercial tests for human papillomavirus detection in head and neck squamous cell carcinomas to a “gold standard” human papillomavirus PCR assay. One hundred-and-ten prospectively collected, formalin fixed tumor specimens were compiled onto tissue microarrays and tested for human papillomavirus DNA by in-situ hybridization with two probe sets: a biotinylated probe for high-risk human papillomavirus types 16/18 (Dako, CA), and a probe cocktail for 16/18 plus 10 additional high-risk types (Ventana, AZ). P16INK4 expression was also assessed using a Pharmingen immuno-histochemistry antibody (BD Biosciences, CA). Tissue microarrays were stained and scored at expert laboratories. Human papillomavirus DNA was detected by MY09/11-PCR using Gold AmpliTaq and dot-blot hybridization on matched fresh frozen specimens in a research laboratory. Human papillomavirus 16 E6 and E7-RNA expression was also measured using RT-PCR. Test performance was assessed by receiver operating characteristic analysis. High-risk human papillomavirus DNA types 16, 18 and 35 were detected by MY-PCR in 28% of tumors, with the majority (97%) testing positive for type 16. Compared to MY-PCR, the sensitivity and specificity for high-risk human papillomavirus DNA detection with Dako in-situ hybridization was 21% (95%CI:7–42) and 100% (95%CI:93–100), respectively. Corresponding test results by Ventana in-situ hybridization were 59% (95%CI:39–78) and 58% (95%CI:45–71), respectively. P16 immuno-histochemistry performed better overall than Dako (p=0.042) and Ventana (p=0.055), with a sensitivity of 52% (95%CI:32–71) and specificity of 93% (95%CI:84–98). Compared to a gold standard human papillomavirus PCR assays, HPV detection by in-situ hybridization was less accurate for head and neck squamous cell carcinoma on tissue microarrays than p16 immuno-histochemistry. Further testing is warranted before these assays should be recommended for clinical human papillomavirus detection.

Small noncoding microRNAs (miRNAs) have been shown to be abnormally expressed in every tumor type examined. The importance of miRNAs as potential cancer prognostic indicators is underscored by their involvement in the regulation of basic cellular processes such as cell proliferation, differentiation, and apoptosis. In this study, miRNA expression profiles of head and neck squamous cell carcinoma (HNSCC) tumor and adjacent normal tissue were examined by microarray analysis and validated by quantitative TaqMan real-time polymerase chain reaction. Using TaqMan real-time polymerase chain reaction we measured the quantitative associations between a subset of miRNAs identified on microarrays in primary tumors at diagnosis and cancer survival in a cohort of 104 HNSCC patients undergoing treatment with curative intent. The majority of miRNAs exhibiting altered expression in primary human HNSCC tumors (including miR-1, miR-133a, miR-205, and let-7d) show lower expression levels relative to normal adjacent tissue. In contrast, hsa-miR-21 is frequently overexpressed in human HNSCC tumors. Using univariate and multivariable statistical models we show that low levels of hsa-miR205 are significantly associated with loco-regional recurrence independent of disease severity at diagnosis and treatment. In addition, combined low levels of hsa-miR-205 and hsa-let-7d expression in HNSCC tumors are significantly associated with poor head and neck cancer survival Our results show that miRNA expression levels can be used as prognostic markers of head and neck cancer.

It is known that head and neck squamous cell carcinomas (HNSCC) originating from different anatomic locations can exhibit varying behavior that is not predictable by histopathology of the primary tumor. Using a microarray containing 27,323 cDNA clones, we generated sets of gene expression profiles for 36 HNSCC primary tumors (12 oral cavity, 12 oropharynx, and 12 larynx/hypopharynx). From these datasets, we ranked genes according to their ability to differentiate between patients whose disease progressed within a 24 month period (aggressive phenotype) and those that did not (non-aggressive phenotype) based on levels of gene expression. A merging of datasets from the three sites revealed that only a fraction of identified genes were shared between any two sites. This contrasted greatly with the significant overlap (approximately 50%) in down-regulated genes identified in tumor/normal comparisons using cases both from oropharynx and larynx/hypopharynx. From these data, we conclude that HNSCC tumors originating from different anatomic sites share consistent changes in gene expression when comparing primary tumors to normal adjacent mucosa; these common changes most likely reflect alterations required for tumor development. In contrast, once a tumor has developed, tumor-host interactions at the different anatomic sites are likely responsible for the site-specific signatures associated with aggressive versus non-aggressive disease. Predictions of outcome based on gene expression profiling are therefore heavily influenced by the anatomic site of the primary tumor.

The goal of this study was to establish a method for detecting biologically significant differences in protein expression of head and neck squamous cell carcinoma (HNSCC) obtained from the same samples utilized in gene expression analyses. Proteins from two head and neck tumor cell lines, SCC-25 and FaDu, were isolated from the denatured protein solution remaining from the TRIzol extraction procedure used for isolation of total RNA for microarray analysis. Peptides resulting from chemical and enzymatic digestion of the proteins were first separated by strong cation-exchange chromatography, followed by liquid chromatography-mass spectrometry (LC-MS) analysis on a QqTOF mass spectrometer. Stable isotope-labeled synthetic peptides were added to each ion-exchange fraction as internal standards, for reversed-phase HPLC retention time alignment. Protein extraction and digestion were repeated three times for each cell line and each extract was analyzed three times by LC-MS. To discriminate between technical vs biological variation, the ion-exchange fraction, retention time, normalized mass and signal intensity of these nine data sets were constructed into numerical arrays for statistical analysis. Of the approximately 50,000 signals, 90 peptide ions were found to discriminate the two cell lines with high stringency. Of those, six peptides were derived from vimentin and four peptides were derived from annexin II; both expressed more in SCC-25. Follow-up analysis of some of these signals by LC-MS/MS and RNA expression profiling revealed both concordance and discordance of RNA and protein expression. This study demonstrates that this procedure is highly reliable for identifying peptides that distinguish biological variability among samples, indicating that this method can be applied to study clinical samples, to identify potential prognostic biomarkers for HNSCC.

A1 is an anti-apoptotic member of the Bcl-2 family that is up-regulated in inflammatory myeloid cells. In the present study, we investigated the role of A1 in the maintenance of acute inflammation in mice. Mice possess three genes encoding highly related isoforms of A1. A1-a isoform mRNA was minimally expressed in resident peritoneal macrophages, but was present at a 300-fold higher level in inflammatory macrophages elicited by i.p. infection with Toxoplasma gondii. In comparison, A1-b and A1-d levels were 3- and 10-fold higher, respectively. Peritoneal leukocytosis was decreased in infected A1-a-deficient mice compared with wild-type, and this reduction was associated with a small but reproducible enhancement of survival. These effects could not be explained by an alteration in peritoneal parasite load, nor by increased apoptosis of infected inflammatory cells, which were protected from cell death by an A1-a-independent mechanism. Increased apoptosis in inflammatory neutrophils was observed sporadically in A1-a-deficient mice. Regulation of apoptosis by A1-a may be an important proinflammatory event in acute host responses.

Head and neck squamous cell carcinoma (HNSCC) is a highly invasive cancer, with a five-year survival rate of around 50%. Our goal is to improve patient outcome through understanding the mechanisms determining HNSCC invasiveness. Our research group identified miR-375 as the most consistently down-regulated miRNA in tumor samples when compared to paired normal samples. Patients in the lowest quartile of miR-375 expression had significantly decreased disease-specific survival, increase incidence of loco-regional recurrence and distant metastasis. The highly significant association of lower miR-375 expression in patients with poor prognosis and distant metastasis led us to investigate the mechanism by which miR-375 expression causes alterations to the invasive properties of HNSCC. Stable transductant cell lines with increased expression of miR-375 have been generated from two oral cavity cell lines, UMSCC1 and UMSCC47, utilizing lentiviral constructs driving expression of the miR-375 precursor. We have not seen a significant change in proliferation rate for either of the miR-375 expressing lines compared to the empty vector control expressing lines. We have observed diminished invasion properties in vitro correlates with increased miR-375 expression. We have also identified that miR-375 expression in HNSCC result in impaired chemotactic migration properties and suppressed matrix degradation properties. To identify proteins regulated by miR-375, we are utilizing stable isotope labeling of cells in cell culture (SILAC) to identify candidate miRNA target proteins to identify differentially expressed proteins in control and miR-375 over-expressing lines. Candidate target proteins will be validated as targets of miR-375 and tested for involvement in the diminished invasive properties of HNSCC as a result of miR-375 expression. In conclusion, miR-375 represents a potential prognostic marker of poor outcome and metastasis in HNSCC and may function by suppressing the expression of genes and proteins which are involved in the invasive properties of HNSCC. Citation Format: Lizandra Jimenez, Thomas Harris, Nicole Kawachi, Thomas Belbin, Nicolas Schlecht, Jihyeon Lim, Ruth Angeletti, Michael B. Prystowsky, Geoffrey Childs, Jeffrey Segall. MicroRNA-375 regulates tumor invasion and metastasis phenotypes and is associated with poor outcome in head and neck squamous cell carcinoma. [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 4939. doi:10.1158/1538-7445.AM2013-4939

Small, noncoding microRNAs (miRNAs) have been shown to be abnormally expressed in every tumor type examined. We used comparisons of global miRNA expression profiles of head and neck squamous cell carcinoma (HNSCC) samples and adjacent normal tissue to rank those miRNAs that were most significantly altered in our patient population. Rank Consistency Score analysis revealed miR-375 to have the most significantly lowered miRNA levels in tumors relative to matched adjacent nonmalignant tissue from the same patient among 736 miRNAs that were evaluated. This result has been previously observed by other groups; however, we extend this finding with the unique observation that low miR-375 expression levels correlate significantly with cancer survival and distant metastasis. In a study of 123 primary HNSCC patients using multivariable Cox proportional hazard ratios (HR) and 95% confidence intervals (CI), both death from disease (HR: 12.8, 95% CI: 3 to 49) and incidence of distant metastasis (HR: 8.7, 95% CI: 2 to 31) correlated with lower expression levels of miR-375 regardless of the site or stage of the tumor. In addition, we found that oral cavity tumor cell lines (eg, UMSCC1 and UMSCC47) overexpressing miR-375 were significantly less invasive in vitro than their matched empty vector controls. We conclude that miR-375 represents a potential prognostic marker of poor outcome and metastasis in HNSCC and that it may function by suppressing the tumor's invasive properties.