Defining the relationship of salivary gland malignancies to novel cell subpopulations in human salivary glands using single nucleus RNA-sequencing.

Salivary glands have essential roles in maintaining oral health, mastication, taste and speech, by secreting saliva. Salivary glands are composed of several types of cells, and each cell type is predicted to be involved in the carcinogenesis of different types of cancers including adenoid cystic carcinoma (ACC), acinic cell carcinoma (AciCC), salivary duct carcinoma (SDC), myoepithelial carcinoma (MECA) and other histology. In our study, we performed single nucleus RNA-seq on three human salivary gland samples to clarify the gene expression profile of each complex cellular component of the salivary glands and related these expression patterns to expression found in salivary gland cancers (SGC) to infer cell of origin. By single nucleus RNA-seq, salivary gland cells were stratified into four clusters: acinar cells, ductal cells 1, ductal cells 2 and myoepithelial cells/stromal cells. The localization of each cell group was verified by IHC of each cluster marker gene, and one group of ductal cells was found to represent intercalated ductal cells labeled with HES1. Furthermore, in comparison with SGC RNA-seq data, acinar cell markers were upregulated in AciCC, but downregulated in ACC and ductal cell markers were upregulated in SDC but downregulated in MECA, suggesting that markers of origin are highly expressed in some SGC. Cell type expressions in specific SGC histology are similar to those found in normal salivary gland populations, indicating a potential etiologic relationship.

High-risk HPV infection-associated hypermethylated genes in oropharyngeal squamous cell carcinomas.

BACKGROUND: HPV-positive oropharyngeal squamous cell carcinomas (OPSCCs) are sensitive to chemo-radiation therapy and have favorable survival outcomes compared with HPV-negative cancers. These tumors are usually not related to tobacco and alcohol exposure. Therefore, diagnosing HPV-positive OPSCCs for the appropriate disease management is crucial, and no suitable markers are available for detecting early malignancies in HPV-infected tissues. In this study, we attempt to find HPV-specific epigenetic biomarkers for OPSCCs. METHODS: A total of 127 surgical samples were analyzed for HPV positivity and promoter methylation of a panel of genes. HPV detection was performed by PCR detection of HPV E6 and E7 viral oncoproteins. In addition, promoter methylation of a total of 8 genes (DAPK, FHIT, RASSF1A, TIMP3, AGTR1, CSGALNACT2, GULP1 and VGF) was analyzed by quantitative-methylation specific PCR (QMSP), and their associations with HPV positivity or RB/p16 expressions were evaluated. RESULTS: AGTR1 and FHIT were frequently methylated in HPV-positive OPSCC samples with a good area under the curve (AUC over 0.70). In addition, these genes' promoter methylation was significantly associated with p16 positive and RB negative cases, which were the characteristics of OPSCC cases with favorable survival outcomes. Either AGTR1 or FHIT methylated cases were significantly associated with HPV-positive cancers with 92.0% sensitivity (P < 0.001). Also, they had significantly better overall survival (P = 0.047) than both unmethylated cases. CONCLUSIONS: A combination of AGTR1 and FHIT methylation demonstrated a suitable detection marker of OPSCCs derived from the HPV-infected field, familiar with p16-positive and RB-negative phenotypes.

A Hospital-Wide Intervention to Improve Compliance With TNM Cancer Staging Documentation.

BACKGROUND: Accurate oncologic staging meeting clinical practice guidelines is essential for guideline adherence, quality assessment, and survival outcomes. However, timely and uniform documentation in the electronic health record (EHR) at the time of diagnosis is a challenge for providers. This quality improvement project aimed to increase provider compliance of timely clinical TNM (cTNM) or pathologic TNM (pTNM) staging for newly diagnosed oncologic patients. METHODS: Providers in the following site-specific oncologic teams were included: head and neck, skin, breast, genitourinary, gastrointestinal, lung and thoracic, gynecologic, colorectal, and bone marrow transplant. Interventions to facilitate timely cTNM and pTNM staging included standardized EHR-based workflows, learning modules, stakeholder meetings, and individualized provider training sessions. For most teams, staging was considered compliant if it was completed in the EHR within the first 7 days of the calendar month after the date of the patient visit. Factors associated with staging compliance were analyzed using logistic regression models. RESULTS: From January 1, 2014, to December 31, 2018, 7,787 preintervention and 5,152 postintervention new patient visits occurred. During the preintervention period, staging was compliant in 5.6% of patients compared with 67.4% of patients after intervention (P<.001). In the final month of the postintervention period, the overall staging compliance rate was 78.1%. At most recent tracking, staging compliance was 95%, 97%, and 93% in December 2019, January 2020, and February 2020, respectively. Logistic regression found that increasing years of provider experience was associated with decreased staging compliance. CONCLUSIONS: High rates of staging compliance in complex multidisciplinary academic oncologic practice models can be achieved via comprehensive quality improvement and structured initiatives. This approach serves as a model for improving oncologic documentation systems to facilitate clinical decision-making and multidisciplinary coordination of care.

Preclinical experience with a novel single-port platform for transoral surgery.

BACKGROUND: We investigated a novel minimally invasive surgical platform for use in the oropharynx, hypopharynx, and larynx for single-port transoral surgery used in concert with standard transoral laryngeal and pharyngeal instrumentation. METHODS: The preclinical investigational device by Fortimedix Surgical B.V. (Netherlands) features two channels for manually controlled flexible articulating surgical instruments. A third central channel accepts both rigid and flexible endoscopes. The system is coupled to a standard laryngoscope for transoral access. In three cadaver models, we evaluated the surgical capabilities using wristed grasping instruments, microlaryngeal scissors, monopolar cautery, and a laser fiber sheath. Procedures were performed within the oropharynx, supraglottis, glottis, subglottis, and hypopharynx. RESULTS: Within the oropharynx, we found adequate strength, range of motion, and dexterity to perform lateral oropharyngectomy and tongue base resection. Within the larynx, visualization was achieved with a variety of instruments including a flexible, 0° and 30° rigid endoscope. The glottis, supraglottis, pyriform sinuses, post-cricoid space, and esophageal inlet were readily accessible. Visualization and manipulation of grasping, laser, and monopolar cautery instruments were also possible within the subglottis. Instrument reach and accuracy facilitated completion of a delicate micro-flap on the true vocal fold. Other procedures included vocal fold resection, cricopharyngeal myotomy, and resection of subglottic mucosa. CONCLUSIONS: From this initial proof of concept experience with this novel platform, we found a wide range of procedures within the oropharynx, larynx, and hypopharynx to be feasible. Further work is needed to evaluate its applicability to the clinical setting. The ability of this platform to be used with conventional instrumentation may provide an opportunity for complex transoral surgery to be performed in a facile manner at greatly reduced cost.

Identifying predictors of HPV-related head and neck squamous cell carcinoma progression and survival through patient-derived models.

Therapeutic innovation for human papilloma virus-related (HPV+) head and neck squamous cell carcinomas (HNSCCs) is impaired by inadequate preclinical models and the absence of accurate biomarkers. Our study establishes the first well-characterized panel of patient-derived xenografts (PDXs) and organoids from HPV+ HNSCCs while determining fidelity of the models to the distinguishing genetic features of this cancer type. Despite low engraftment rates, whole exome sequencing showed that PDXs retain multiple distinguishing features of HPV+ HNSCC lost in existing cell lines, including PIK3CA mutations, TRAF3 deletion and the absence of EGFR amplifications. Engrafted HPV+ tumors frequently contained NOTCH1 mutations, thus providing new models for a negatively prognostic alteration in this disease. Genotype-phenotype associations in the models were then tested for prediction of tumor progression and survival in published clinical cohorts. Observation of high tumor mutational burdens (TMBs) in the faster-growing models facilitated identification of a novel association between TMB and local progression in both HPV+ and HPV- patients that was prognostic in HPV- cases. In addition, reduced E7 and p16INK4A levels found in a PDX from an outlier case with lethal outcome led to detection of similar profiles among recurrent HPV+ HNSCCs. Transcriptional data from the Cancer Genome Atlas was used to demonstrate that the lower E2F target gene expression predicted by reduced E7 levels has potential as a biomarker of disease recurrence risk. Our findings bridge a critical gap in preclinical models for HPV+ HNSCCs and simultaneously reveal novel potential applications of quantifying mutational burden and viral oncogene functions for biomarker development.

A novel splice variant of LOXL2 promotes progression of human papillomavirus-negative head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is one of the most frequently diagnosed cancers worldwide. LOXL2 demonstrates alternative splicing events in patients with human papillomavirus (HPV)-negative HNSCC. The current study explored the role of a dominant LOXL2 variant in HPV-negative HNSCC. METHODS: Expression of the LOXL2 variant was analyzed using The Cancer Genome Atlas cohorts and validated using quantitative reverse transcriptase-polymerase chain reaction in a separate primary tumor set. The authors defined the effect of LOXL2 splice variants in assays for cell proliferation using a cell viability assay and colony formation assay. Cell migration and invasion were examined using a cell scratch assay and transwell cell migration and invasion assay in LOXL2 splice variant gain and loss of expression cells. Western blot analysis and gene set enrichment analysis were used to explore the potential mechanism of the LOXL2 splice variant in HPV-negative HNSCC. RESULTS: Expression of a novel LOXL2 variant was found to be upregulated in The Cancer Genome Atlas HPV-negative HNSCC, and confirmed in the separate primary tumor validation set. Analyses of loss and gain of function demonstrated that this LOXL2 variant enhanced proliferation, migration, and invasion in HPV-negative HNSCC cells and activated the FAK/AKT pathway. A total of 837 upregulated and 820 downregulated genes and 526 upregulated and 124 downregulated pathways associated with LOXL2 variant expression were identified using gene set enrichment analysis, which helped in developing a better understanding of the networks activated by this LOXL2 variant in patients with HPV-negative HNSCC. CONCLUSIONS: The novel LOXL2 variant can promote the progression of HPV-negative HNSCC, in part through FAK/AKT pathway activation, which may provide a new potential therapeutic target among patients with HPV-negative HNSCC.

Matrix stiffening promotes a tumor vasculature phenotype.

Tumor microvasculature tends to be malformed, more permeable, and more tortuous than vessels in healthy tissue, effects that have been largely attributed to up-regulated VEGF expression. However, tumor tissue tends to stiffen during solid tumor progression, and tissue stiffness is known to alter cell behaviors including proliferation, migration, and cell-cell adhesion, which are all requisite for angiogenesis. Using in vitro, in vivo, and ex ovo models, we investigated the effects of matrix stiffness on vessel growth and integrity during angiogenesis. Our data indicate that angiogenic outgrowth, invasion, and neovessel branching increase with matrix cross-linking. These effects are caused by increased matrix stiffness independent of matrix density, because increased matrix density results in decreased angiogenesis. Notably, matrix stiffness up-regulates matrix metalloproteinase (MMP) activity, and inhibiting MMPs significantly reduces angiogenic outgrowth in stiffer cross-linked gels. To investigate the functional significance of altered endothelial cell behavior in response to matrix stiffness, we measured endothelial cell barrier function on substrates mimicking the stiffness of healthy and tumor tissue. Our data indicate that barrier function is impaired and the localization of vascular endothelial cadherin is altered as function of matrix stiffness. These results demonstrate that matrix stiffness, separately from matrix density, can alter vascular growth and integrity, mimicking the changes that exist in tumor vasculature. These data suggest that therapeutically targeting tumor stiffness or the endothelial cell response to tumor stiffening may help restore vessel structure, minimize metastasis, and aid in drug delivery.

Mutation of chromatin regulators and focal hotspot alterations characterize human papillomavirus-positive oropharyngeal squamous cell carcinoma.

BACKGROUND: Human papillomavirus (HPV)-associated oropharyngeal cancer is a disease clinically and biologically distinct from smoking-related head and neck squamous cell carcinoma (HNSCC). Despite its rapidly increasing incidence, the mutational landscape of HPV+ oropharyngeal squamous cell carcinoma (OPSCC) remains understudied. METHODS: This article presents the first mutational analysis of the 46 HPV+ OPSCC tumors within the newly expanded cohort of 530 HNSCC tumors from The Cancer Genome Atlas. A separate exome sequencing analysis was also performed for 46 HPV+ OPSCCs matched to their normal lymphocyte controls from the Johns Hopkins University cohort. RESULTS: There was a strikingly high 33% frequency of mutations within genes associated with chromatin regulation, including mutations in lysine methyltransferase 2C (KMT2C), lysine methyltransferase 2D (KMT2D), nuclear receptor binding SET domain protein 1 (NSD1), CREB binding protein (CREBBP), E1A-associated protein p300 (EP300), and CCCTC-binding factor (CTCF). In addition, the commonly altered genes phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA) and fibroblast growth factor receptor 3 (FGFR3) showed distinct domain-specific hotspot mutations in comparison with their HPV- counterparts. PIK3CA showed a uniquely high rate of mutations within the helicase domain, and FGFR3 contained a predominance of hotspot S249C alterations that were not found in HPV- HNSCC. CONCLUSIONS: This analysis represents one of the largest studies to date of HPV+ OPSCC and lends novel insight into the genetic landscape of this biologically distinct disease, including a high rate of mutations in histone- and chromatin-modifying genes, which may offer novel therapeutic targets.

Splice Expression Variation Analysis (SEVA) for inter-tumor heterogeneity of gene isoform usage in cancer.

Motivation: Current bioinformatics methods to detect changes in gene isoform usage in distinct phenotypes compare the relative expected isoform usage in phenotypes. These statistics model differences in isoform usage in normal tissues, which have stable regulation of gene splicing. Pathological conditions, such as cancer, can have broken regulation of splicing that increases the heterogeneity of the expression of splice variants. Inferring events with such differential heterogeneity in gene isoform usage requires new statistical approaches. Results: We introduce Splice Expression Variability Analysis (SEVA) to model increased heterogeneity of splice variant usage between conditions (e.g. tumor and normal samples). SEVA uses a rank-based multivariate statistic that compares the variability of junction expression profiles within one condition to the variability within another. Simulated data show that SEVA is unique in modeling heterogeneity of gene isoform usage, and benchmark SEVA's performance against EBSeq, DiffSplice and rMATS that model differential isoform usage instead of heterogeneity. We confirm the accuracy of SEVA in identifying known splice variants in head and neck cancer and perform cross-study validation of novel splice variants. A novel comparison of splice variant heterogeneity between subtypes of head and neck cancer demonstrated unanticipated similarity between the heterogeneity of gene isoform usage in HPV-positive and HPV-negative subtypes and anticipated increased heterogeneity among HPV-negative samples with mutations in genes that regulate the splice variant machinery. These results show that SEVA accurately models differential heterogeneity of gene isoform usage from RNA-seq data. Availability and implementation: SEVA is implemented in the R/Bioconductor package GSReg. Contact: bahman@jhu.edu or favorov@sensi.org or ejfertig@jhmi.edu. Supplementary information: Supplementary data are available at Bioinformatics online.

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.

Tissue stiffness regulates serine/arginine-rich protein-mediated splicing of the extra domain B-fibronectin isoform in tumors.

Alternative splicing of proteins gives rise to different isoforms that play a crucial role in regulating several cellular processes. Notably, splicing profiles are altered in several cancer types, and these profiles are believed to be involved in driving the oncogenic process. Although the importance of alternative splicing alterations occurring during cancer is increasingly appreciated, the underlying regulatory mechanisms remain poorly understood. In this study, we use both biochemical and physical tools coupled with engineered models, patient samples, and a murine model to investigate the role of the mechanical properties of the tumor microenvironment in regulating the production of the extra domain-B (EDB) splice variant of fibronectin (FN), a hallmark of tumor angiogenesis. Specifically, we show that the amount of EDB-FN produced by endothelial cells increases with matrix stiffness both in vitro and within mouse mammary tumors. Matrix stiffness regulates splicing through the activation of serine/arginine rich (SR) proteins, the splicing factors involved in the production of FN isoforms. Activation of the SR proteins by matrix stiffness and the subsequent production of EDB-FN are dependent on intracellular contractility and PI3K-AKT signaling. Notably, matrix stiffness-mediated splicing is not limited to EDB-FN, but also affects splicing in the production of PKC ßII and the VEGF 165b splice variant. Together, these results demonstrate that the mechanical properties of the microenvironment regulate alternative splicing and establish a previously unidentified mechanism by which cells can adapt to their microenvironment.

Ephrin-B2 overexpression predicts for poor prognosis and response to therapy in solid tumors.

Ephrin B2 is variably expressed on tumor cells and its blockade has been shown to inhibit angiogenesis in animal models of pancreatic, colorectal, lung and head, and neck squamous cell carcinomas. However, the implications of ephrinB2 expression in cancer patients have remained elusive. In this study, we analyzed the cancer genome atlas (TCGA) for ephrinB2 expression. We report significant correlations between EFNB2 expression, overall survival and disease-free survival in head and neck squamous cell carcinoma (HNSCC, n = 519), pancreatic adenocarcinoma (n = 186), and bladder urothelial carcinoma (n = 410). In HNSCC patients, high-EFNB2 mRNA expression was associated with tumor HPV negativity, oral cavity location, alcohol intake, higher TP53 mutation, and EGFR amplification. EphrinB2 overexpression also correlated with worse response to chemotherapy and radiotherapy. The therapeutic potential of blocking ephrinB2 was validated in HNSCC patient-derived tumor xenografts and showed significant improvement in survival and tumor growth delay. Our data shows that ephrinB2 overexpression can serve as a critical biomarker for patient prognosis and response to therapy. These results should guide design of future clinical trials exploring EphrinB2 inhibition in cancer patients. © 2016 Wiley Periodicals, Inc.

Prophylactic Swallow Therapy for Patients with Head and Neck Cancer Undergoing Chemoradiotherapy: A Randomized Trial.

Evidence supporting prophylactic swallow exercises for patients with head and neck cancer (HNC) has not been universally demonstrated. This RCT examined diet level, feeding tube use, swallow function, and quality of life (QOL) of patients undergoing chemoradiotherapy who performed prophylactic swallowing exercises. Sixty HNC patients were randomized into exercise versus control groups. Swallowing, oromotor, toxicity, and QOL data were recorded (baseline, 3, 6, 12, 24 months). Physiological swallow function was examined at baseline and 3 months. Swallow exercises were completed twice daily. Oral intake at 3 months was 10% better in the exercise group, which was not statistically significant (p = 0.49). Significant (p < 0.05) differences in secondary outcomes including oromotor function, pharyngeal impairment, oral pharyngeal swallow efficiency, and incisal opening were noted at early time points (3-6 months) in the exercise group. Possible positive early improvements in swallow function are associated with swallowing exercises, although these improvements are not significant longer term.

mTOR inhibition prevents rapid-onset of carcinogen-induced malignancies in a novel inducible HPV-16 E6/E7 mouse model.

The rising incidence of human papillomavirus (HPV)-associated malignancies, especially for oropharyngeal cancers, has highlighted the urgent need to understand how the interplay between high-risk HPV oncogenes and carcinogenic exposure results in squamous cell carcinoma (SCC) development. Here, we describe an inducible mouse model expressing high risk HPV-16 E6/E7 oncoproteins in adults, bypassing the impact of these viral genes during development. HPV-16 E6/E7 genes were targeted to the basal squamous epithelia in transgenic mice using a doxycycline inducible cytokeratin 5 promoter (cK5-rtTA) system. After doxycycline induction, both E6 and E7 were highly expressed, resulting in rapid epidermal hyperplasia with a remarkable expansion of the proliferative cell compartment to the suprabasal layers. Surprisingly, in spite of the massive growth of epithelial cells and their stem cell progenitors, HPV-E6/E7 expression was not sufficient to trigger mTOR activation, a key oncogenic driver in HPV-associated malignancies, and malignant progression to SCC. However, these mice develop SCC rapidly after a single exposure to a skin carcinogen, DMBA, which was increased by the prolonged exposure to a tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Thus, only few oncogenic hits may be sufficient to induce cancer in E6/E7 expressing cells. All HPV-E6/E7 expressing SCC lesions exhibited increased mTOR activation. Remarkably, rapamycin, an mTOR inhibitor, abolished tumor development when administered to HPV-E6/E7 mice prior to DMBA exposure. Our findings revealed that mTOR inhibition protects HPV-E6/E7 expressing tissues form SCC development upon carcinogen exposure, thus supporting the potential clinical use of mTOR inhibitors as a molecular targeted approach for prevention of HPV-associated malignancies.

Characterization of functionally active gene fusions in human papillomavirus related oropharyngeal squamous cell carcinoma.

The Cancer Genome Atlas (TCGA) sequencing analysis of head and neck squamous cell carcinoma (HNSCC) recently reported on gene fusions, however, few human papillomavirus (HPV) positive samples were included, and the functional relevance of identified fusions was not explored. We therefore performed an independent analysis of gene fusions in HPV-positive oropharyngeal SCC (OPSCC). RNA sequencing was performed on 47 HPV-positive OPSCC primary tumors and 25 normal mucosal samples from cancer unaffected controls on an Illumina TruSeq platform. MapSplice2 was used for alignment and identification of fusion candidates. Putative fusions with less than five spanning reads, detected in normal tissues, or that mapped to the same gene were filtered out. Selected fusions were validated by RT-PCR and Sanger sequencing. Within 47 HPV-positive OPSCC tumors, 282 gene fusions were identified. Most fusions (85.1%) occurred in a single tumor, and the remaining fusions recurred in 2-16 tumors. Gene fusions were associated with significant up regulation of 16 genes (including EGFR and ERBB4) and down regulation of four genes (PTPRT, ZNF750, DLG2, SLCO5A1). Expression of these genes followed similar patterns of up regulation and down regulation in tumors without these fusions compared to normal tissue. Five of six gene fusions selected for validation were confirmed through RT-PCR and sequencing. This integrative analysis provides a method of prioritizing functionally relevant gene fusions that may be expanded to other tumor types. These results demonstrate that gene fusions may be one mechanism by which functionally relevant genes are altered in HPV-positive OPSCC.

Silencing of high-mobility group box 2 (HMGB2) modulates cisplatin and 5-fluorouracil sensitivity in head and neck squamous cell carcinoma.

Dysregulation of protein expression is associated with most diseases including cancer. MS-based proteomic analysis is widely employed as a tool to study protein dysregulation in cancers. Proteins that are differentially expressed in head and neck squamous cell carcinoma (HNSCC) cell lines compared to the normal oral cell line could serve as biomarkers for patient stratification. To understand the proteomic complexity in HNSCC, we carried out iTRAQ-based MS analysis on a panel of HNSCC cell lines in addition to a normal oral keratinocyte cell line. LC-MS/MS analysis of total proteome of the HNSCC cell lines led to the identification of 3263 proteins, of which 185 proteins were overexpressed and 190 proteins were downregulated more than twofold in at least two of the three HNSCC cell lines studied. Among the overexpressed proteins, 23 proteins were related to DNA replication and repair. These included high-mobility group box 2 (HMGB2) protein, which was overexpressed in all three HNSCC lines studied. Overexpression of HMGB2 has been reported in various cancers, yet its role in HNSCC remains unclear. Immunohistochemical labeling of HMGB2 in a panel of HNSCC tumors using tissue microarrays revealed overexpression in 77% (54 of 70) of tumors. The HMGB proteins are known to bind to DNA structure resulting from cisplatin-DNA adducts and affect the chemosensitivity of cells. We observed that siRNA-mediated silencing of HMGB2 increased the sensitivity of the HNSCC cell lines to cisplatin and 5-FU. We hypothesize that targeting HMGB2 could enhance the efficacy of existing chemotherapeutic regimens for treatment of HNSCC. All MS data have been deposited in the ProteomeXchange with identifier PXD000737 (http://proteomecentral.proteomexchange.org/dataset/PXD000737).

NF-κB and stat3 transcription factor signatures differentiate HPV-positive and HPV-negative head and neck squamous cell carcinoma.

Using high-throughput analyses and the TRANSFAC database, we characterized TF signatures of head and neck squamous cell carcinoma (HNSCC) subgroups by inferential analysis of target gene expression, correcting for the effects of DNA methylation and copy number. Using this discovery pipeline, we determined that human papillomavirus-related (HPV+) and HPV- HNSCC differed significantly based on the activity levels of key TFs including AP1, STATs, NF-κB and p53. Immunohistochemical analysis confirmed that HPV- HNSCC is characterized by co-activated STAT3 and NF-κB pathways and functional studies demonstrate that this phenotype can be effectively targeted with combined anti-NF-κB and anti-STAT therapies. These discoveries correlate strongly with previous findings connecting STATs, NF-κB and AP1 in HNSCC. We identified five top-scoring pair biomarkers from STATs, NF-κB and AP1 pathways that distinguish HPV+ from HPV- HNSCC based on TF activity and validated these biomarkers on TCGA and on independent validation cohorts. We conclude that a novel approach to TF pathway analysis can provide insight into therapeutic targeting of patient subgroup for heterogeneous disease such as HNSCC.

Surgical salvage improves overall survival for patients with HPV-positive and HPV-negative recurrent locoregional and distant metastatic oropharyngeal cancer.

BACKGROUND: Human papillomavirus (HPV) tumor status and surgical salvage are associated with improved prognosis for patients with recurrent oropharyngeal squamous cell carcinoma (OPSCC). Current data regarding types of surgery and the impact of surgery for patients with distant metastatic disease are limited. METHODS: A retrospective analysis of patients with recurrent OPSCC from 2 institutions between 2000 and 2012 was performed. p16 immunohistochemistry and/or in situ hybridization, as clinically available, were used to determine HPV tumor status. Clinical characteristics, distribution of recurrence site, and treatment modalities were compared by HPV tumor status. Overall survival (OS) was examined using Kaplan-Meier and Cox proportional hazards methods. RESULTS: The current study included 108 patients with 65 locoregional and 43 distant metastatic first recurrences. The majority of patients were HPV-positive (80 patients). HPV-positive tumor status was associated with longer time to disease recurrence (P<.01). Anatomic site distribution of disease recurrences did not differ by HPV tumor status. HPV-positive tumor status (adjusted HR [aHR], 0.23; 95% confidence interval [95% CI], 0.09-0.58 [P = .002]), longer time to disease recurrence (≥ 1 year; aHR, 0.36; 95% CI, 0.18-0.74 [P = .006]), and surgical salvage (aHR, 0.26; 95% CI, 0.12-0.61 [P = .002]) were found to be independently associated with OS after disease recurrence. Surgical salvage was independently associated with improved OS compared with nonsurgical treatment among patients with both locoregional (aHR, 0.15; 95% CI, 0.04-0.56 [P = .005]) and distant (aHR, 0.19; 95% CI, 0.05-0.75 [P = .018]) metastatic disease recurrences. CONCLUSIONS: Surgical salvage was found to be associated with improved OS for patients with recurrent locoregional and distant metastatic OPSCC, independent of HPV tumor status. Further prospective data are needed to confirm the role of surgical salvage for distant metastases.

Implanted adipose progenitor cells as physicochemical regulators of breast cancer.

Multipotent adipose-derived stem cells (ASCs) are increasingly used for regenerative purposes such as soft tissue reconstruction following mastectomy; however, the ability of tumors to commandeer ASC functions to advance tumor progression is not well understood. Through the integration of physical sciences and oncology approaches we investigated the capability of tumor-derived chemical and mechanical cues to enhance ASC-mediated contributions to tumor stroma formation. Our results indicate that soluble factors from breast cancer cells inhibit adipogenic differentiation while increasing proliferation, proangiogenic factor secretion, and myofibroblastic differentiation of ASCs. This altered ASC phenotype led to varied extracellular matrix (ECM) deposition and contraction thereby enhancing tissue stiffness, a characteristic feature of breast tumors. Increased stiffness, in turn, facilitated changes in ASC behavior similar to those observed with tumor-derived chemical cues. Orthotopic mouse studies further confirmed the pathological relevance of ASCs in tumor progression and stiffness in vivo. In summary, altered ASC behavior can promote tumorigenesis and, thus, their implementation for regenerative therapy should be carefully considered in patients previously treated for cancer.

Pharyngocutaneous fistula after total laryngectomy: a single-institution experience, 2001-2012.

PURPOSE: The purpose of this study was to determine the incidence of and risk factors for pharyngocutaneous fistula in patients undergoing total laryngectomy at a single institution. MATERIALS AND METHODS: The records of 59 patients undergoing primary or salvage total laryngectomy at our institution from 2001 to 2012 were retrospectively reviewed. Data collected included patient, tumor and treatment characteristics, and surgical technique. Risk factors were analyzed for association with pharyngocutaneous fistula formation. RESULTS: Twenty patients (34%) developed fistulas. Preoperative tracheostomy (OR 4.1; 95% CI 1.3-13 [p=0.02]) and low postoperative hemoglobin (OR 9.1; 95% CI 1.1-78 [p=0.04]) were associated with fistula development. Regarding surgical technique, primary sutured closure of the total laryngectomy defect had the lowest fistula rate (11%). In comparison, primary stapled closure and pectoralis onlay flap over primary closure had nonsignificantly increased fistula rates (43%, OR 6.0; 95% CI 1.0-37.3 [p=0.06] and 25%, OR 2.7; 95% CI 0.4-23.9 [p=0.38], respectively). Pectoralis flap incorporated into the suture line had a significantly increased fistula rate (50%, OR 7.1; 95% CI 1.4-46 [p=0.02]). After stratification for salvage status, patient comorbidities were associated with fistula in non-salvage cases whereas disease-related characteristics were associated with fistula in salvage cases. Fistula development was associated with increased length of hospital stay (p<0.001) and increased time before oral diet initiation (p<0.001). CONCLUSIONS: Pharyngocutaneous fistula is a common complication of total laryngectomy. Preoperative tracheostomy, postoperative hemoglobin, and surgical technique are important in determining the risk of fistula.