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.

Evaluation of white matter microstructural alterations in premature infants with necrotizing enterocolitis.

Background: Preterm infants with necrotizing enterocolitis (NEC) are at high risk of adverse neurodevelopmental outcomes. The aim of this study was to explore the value of diffusion tensor imaging (DTI) combined with serum C-reactive protein (CRP) and procalcitonin (PCT) in evaluating alterations of white matter (WM) microstructure in preterm infants with NEC. Methods: A retrospective cross-sectional study was conducted in which all participants were consecutively enrolled at The Third Affiliated Hospital of Zhengzhou University from June 2017 and October 2021. Data from 30 preterm infants with NEC [mean gestational age at birth 31.41±1.15 weeks; mean age at magnetic resonance imaging (MRI) 37.53±3.08 weeks] and 40 healthy preterm infants with no NEC were recorded (mean gestational age at birth 32.27±2.09 weeks; mean age at MRI 37.15±3.23 weeks). WM was used to obtain the fractional anisotropy (FA) and mean diffusivity (MD) values of the regions of interest (ROIs). Additionally, serum levels of CRP and PCT were determined. Spearman correlation analysis was performed between the WM-derived parameters, CRP level, and the PCT serum index. Results: Preterm infants with NEC had reduced FA values and elevated MD values in WM regions [posterior limbs of the internal capsule (PLIC), lentiform nucleus (LN), frontal white matter (FWM)] compared to the control group (P<0.05). Additionally, the FA of the PLIC was negatively correlated with serum CRP (r=-0.846; P<0.05) and PCT (r=-0.843; P<0.05). Meanwhile, the MD of PLIC was positively correlated with serum CRP (r=0.743; P<0.05) and PCT (r=0.743; P<0.05, respectively). The area under the curve (AUC) of FA and MD combined with CRP and PCT in the diagnosis of WM microstructure alterations with NEC was 0.968, representing a considerable improvement in predicted efficacy over single indicators, including FA [AUC: 0.938; 95% confidence interval (CI): 0.840-0.950], MD (AUC: 0.807; 95% CI: 0.722-0.838), CRP (AUC: 0.867; 95% CI: 0.822-0.889), and PCT (AUC: 0.706; 95% CI: 0.701-0.758). Conclusions: WM can noninvasively and quantitatively assess the WM microstructure alterations in preterm infants with NEC. WM combined with serum CRP and PCT demonstrated superior performance in detecting and evaluating WM microstructure alterations in preterm infants with NEC.

A Network Landscape of HPVOPC Reveals Methylation Alterations as Significant Drivers of Gene Expression via an Immune-Mediated GPCR Signal.

HPV-associated oropharynx carcinoma (HPVOPC) tumors have a relatively low mutational burden. Elucidating the relative contributions of other tumor alterations, such as DNA methylation alterations, alternative splicing events (ASE), and copy number variation (CNV), could provide a deeper understanding of carcinogenesis drivers in this disease. We applied network propagation analysis to multiple classes of tumor alterations in a discovery cohort of 46 primary HPVOPC tumors and 25 cancer-unaffected controls and validated our findings with TCGA data. We identified significant overlap between differential gene expression networks and all alteration classes, and this association was highest for methylation and lowest for CNV. Significant overlap was seen for gene clusters of G protein-coupled receptor (GPCR) pathways. HPV16-human protein interaction analysis identified an enriched cluster defined by an immune-mediated GPCR signal, including CXCR3 cytokines CXCL9, CXCL10, and CXCL11. CXCR3 was found to be expressed in primary HPVOPC, and scRNA-seq analysis demonstrated CXCR3 ligands to be highly expressed in M2 macrophages. In vivo models demonstrated decreased tumor growth with antagonism of the CXCR3 receptor in immunodeficient but not immunocompetent mice, suggesting that the CXCR3 axis can drive tumor proliferation in an autocrine fashion, but the effect is tempered by an intact immune system. In conclusion, methylation, ASE, and SNV alterations are highly associated with network gene expression changes in HPVOPC, suggesting that ASE and methylation alterations have an important role in driving the oncogenic phenotype. Network analysis identifies GPCR networks, specifically the CXCR3 chemokine axis, as modulators of tumor-immune interactions that may have proliferative effects on primary tumors as well as a role for immunosurveillance; however, CXCR3 inhibition should be used with caution, as these agents may both inhibit and stimulate tumor growth considering the competing effects of this cytokine axis. Further investigation is needed to explore opportunities for targeted therapy in this setting.

Nickel adsorption from waters onto Fe3O4/sugar beet pulp nanocomposite.

In this study the magnetic nanocomposite material was synthesized with Fe3O4 impregnated to sugar beet pulp using chemical precipitation technique. Ni(II) removal performance of magnetic nanocomposite was investigated under different environmental conditions such as contact time, adsorbent dose, pH, initial heavy metal concentration, etc. The experimental studies have shown that, 81.2% Ni(II) removal efficiency was achieved at optimal conditions (25 mg/L initial Ni(II) concentration at 40 minute contact time, 200 rpm shaking speed, 5 g/L nanocomposite dose and pH 6.6). Freundlich and Langmuir isotherm experiments were performed and correlation coefficients were determined as 94.5% and 99.4%, respectively. The maximum adsorption capacity of material was achieved as 9.36 mg/g. These findings indicate that the adsorption that takes place is a monolayer process. The results of the pseudo-second order kinetic model (R2 = 0.9947) indicate the chemisorptions process is used for Ni(II) removal using the electrostatic interaction. Thermodynamic studies illustrated that Ni(II) adsorption onto nanocomposite are exothermic and causes a decrease in the entropy. The adsorption of Ni(II) ions is non-spontaneous except for at low temperature and low initial concentrations. Nanocomposite characterization was illuminated with XRD, FT-IR, BET, TGA, TEM, SEM/EDX analysis.

In this study, it was aimed to synthesis new adsorbent using sugar beet pulp together with Fe₃O₄ under suitable conditions, obtain a magnetic nanocomposite, and examine the reusability and recovery properties of the produced material. The use of industrial wastes as an adsorbent material provides both a solution to the problem of the removal of wastes and a reuse method for the use of wastes as a low cost adsorbent for a useful purpose. Therefore, it has two advantages: There is a need to investigate the feasibility of investigating all possible industry-based cheap adsorbent sources as well as the removal of heavy metals for the production of a reliable and harmless adsorbent.

Extrachromosomal DNA in HPV-Mediated Oropharyngeal Cancer Drives Diverse Oncogene Transcription.

PURPOSE: Human papillomavirus (HPV) plays a major role in oncogenesis and circular extrachromosomal DNA (ecDNA) is found in many cancers. However, the relationship between HPV and circular ecDNA in human cancer is not understood. EXPERIMENTAL DESIGN: Forty-four primary tumor tissue samples were obtained from a cohort of patients with HPV-positive oropharynx squamous cell carcinoma (OPSCC). Twenty-eight additional HPV oropharyngeal cancer (HPVOPC) tumors from The Cancer Genome Atlas (TCGA) project were analyzed as a separate validation cohort. Genomic, transcriptomic, proteomic, computational, and functional analyses of HPVOPC were applied to these datasets. RESULTS: Our analysis revealed circular, oncogenic DNA in nearly all HPVOPC, with circular human and human-viral hybrid ecDNA present in over a third of HPVOPC and viral circular DNA in remaining tumors. Hybrid ecDNA highly express fusion transcripts from HPV promoters and HPV oncogenes linked to downstream human transcripts that drive oncogenic transformation and immune evasion, and splice multiple, diverse human acceptors to a canonical SA880 viral donor site. HPVOPC have high E6*I expression with specific viral oncogene expression pattern related to viral or hybrid ecDNA composition. CONCLUSIONS: Nonchromosomal circular oncogenic DNA is a dominant feature of HPVOPC, revealing an unanticipated link between HPV and ecDNA that leverages the power of extrachromosomal inheritance to drive HPV and somatic oncogene expression.

Hybrid Percutaneous Brachiofemoral Shunt and Open Abdominal Aortic Aneurysm Repair in a Kidney Transplant Recipient.

Abdominal aortic aneurysm (AAA) repair in kidney transplant recipients may cause ischemia in the transplanted kidney. As a result, various techniques have been described for protection of the renal allograft during AAA repair including temporary shunt, extracorporeal bypass, cold renal perfusion, endovascular aortic aneurysm repair (EVAR), and operation without renal allograft protection. We successfully treated a 56-year-old man, a case of kidney transplantation with AAA, using a temporary hybrid percutaneous brachiofemoral shunt using vascular prosthesis with a long 7-French (Fr) catheter sheath introducer (CSI) in the aortic arch via the right brachial artery and 8 Fr CSI in the right femoral artery that were connected together with a 7 Fr guiding catheter, before aortic cross-clamping and repair of AAA using a Dacron tube graft. The patient recovered well from the surgery without any complication and was discharged on the 6th postoperative day. To our knowledge, this is the first report of using a temporary hybrid percutaneous brachiofemoral shunt for renal allograft protection in AAA repair surgery in a patient with kidney transplantation, and we think that this temporary shunt is an easy, safe, and rapid method for renal allograft protection from ischemia.

Reciprocal activation of HEY1 and NOTCH4 under SOX2 control promotes EMT in head and neck squamous cell carcinoma.

Several comprehensive studies have demonstrated that the NOTCH pathway is altered in a bimodal manner in head and neck squamous cell carcinoma (HNSCC). In a previous study, it was found that the NOTCH4/HEY1 pathway was specifically upregulated in HNSCC and promoted epithelial­mesenchymal transition (EMT), and that HEY1 activation supported SOX2 expression. However, the interactions in this pathway have not yet been fully elucidated. The present study investigated the NOTCH4/HEY1/SOX2 axis in HNSCC using in vitro models and the Cancer Genome Atlas (TCGA) database. To explore the association, reporter and ChIP RT­qPCR assays using SOX2­overexpressing (SOX2­OE) cells were performed. The association between NOTCH4 and HEY1 was examined in the same manner using HEY1­overexpressing (HEY1­OE) cells. The results of the in vitro experiments indicated that HEY1 promoted EMT in the HNSCC cells. Furthermore, the overexpression of HEY1 also promoted sphere formation and increased murine xenograft tumorigenicity. Reporter assays and ChIP RT­qPCR experiments indicated that SOX2 regulated HEY1 expression via direct binding of the HEY1 promoter. HEY1 expression significantly correlated with SOX2 expression in primary lung SCC and other SCCs using the TCGA database. HEY1 also regulated NOTCH4 expression to create a positive reciprocal feedback loop. On the whole, the present study demonstrates that HEY1 expression in HNSCC is regulated via the promotion of SOX2 and promotes EMT. The NOTCH4/HEY1 pathway is specifically upregulated via a positive reciprocal feedback loop mediated by the HEY1­medaited regulation of NOTCH4 transcription, and SOX2 correlates with HEY1 expression in SCC from other primary sites.

HPV E2, E4, E5 drive alternative carcinogenic pathways in HPV positive cancers.

The dominant paradigm for HPV carcinogenesis includes integration into the host genome followed by expression of E6 and E7 (E6/E7). We explored an alternative carcinogenic pathway characterized by episomal E2, E4, and E5 (E2/E4/E5) expression. Half of HPV positive cervical and pharyngeal cancers comprised a subtype with increase in expression of E2/E4/E5, as well as association with lack of integration into the host genome. Models of the E2/E4/E5 carcinogenesis show p53 dependent enhanced proliferation in vitro, as well as increased susceptibility to induction of cancer in vivo. Whole genomic expression analysis of the E2/E4/E5 pharyngeal cancer subtype is defined by activation of the fibroblast growth factor receptor (FGFR) pathway and this subtype is susceptible to combination FGFR and mTOR inhibition, with implications for targeted therapy.

Aberrant expression of CPSF1 promotes head and neck squamous cell carcinoma via regulating alternative splicing.

Alternative mRNA splicing increases protein diversity, and alternative splicing events (ASEs) drive oncogenesis in multiple tumor types. However, the driving alterations that underlie the broad dysregulation of ASEs are incompletely defined. Using head and neck squamous cell carcinoma (HNSCC) as a model, we hypothesized that the genomic alteration of genes associated with the spliceosome may broadly induce ASEs across a broad range of target genes, driving an oncogenic phenotype. We identified 319 spliceosome genes and employed a discovery pipeline to identify 13 candidate spliceosome genes altered in HNSCC using The Cancer Genome Atlas (TCGA) HNSCC data. Phenotypic screens identified amplified and overexpressed CPSF1 as a target gene alteration that was validated in proliferation, colony formation, and apoptosis assays in cell line and xenograft systems as well as in primary HNSCC. We employed knockdown and overexpression assays followed by identification of ASEs regulated by CPSF1 overexpression to identify changes in ASEs, and the expression of these ASEs was validated using RNA from cell line models. Alterations in expression of spliceosome genes, including CPSF1, may contribute to HNSCC by mediating aberrant ASE expression.

Targeting Viral DNA and Promoter Hypermethylation in Salivary Rinses for Recurrent HPV-Positive Oropharyngeal Cancer.

OBJECTIVE: The incidence and survivorship of human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) are increasing. Presence of HPV DNA and epigenetic alterations in salivary rinses are independently associated with clinical prognosis. We evaluated the utility of a combined panel in detecting disease recurrence during surveillance. We also assessed the assay's applicability in screening for HPV+ OPSCC. STUDY DESIGN: Retrospective cohort study. SETTING: Two tertiary academic hospitals. SUBJECTS AND METHODS: Forty-nine patients with posttreatment OPSCC were enrolled. Separately, 21 treatment-naive patients and 40 controls were included in the screening analysis. Salivary rinses were obtained from these cohorts and biomarker levels were quantified. Receiver operative characteristic (ROC) curves and multivariate logistic models were used to assess performance of biomarker combinations. RESULTS: Eight patients (16.3%) in the posttreatment cohort developed locoregional recurrence. Recurrence was associated with alcohol use (odds ratio [OR], 6.12; 95% confidence interval [CI], 0.26-3.79) and advanced nodal disease (OR, 2.21; 95% CI, 1.52-3.01). A panel of HPV DNA and methylated EDNRB improved detection of recurrent disease (area under the curve [AUC], 0.88) compared to single markers (AUC, 0.69-0.78). Positive biomarkers preceded clinical detection by 2.4 ± 1.6 months and was associated with nearly 40-fold risk of recurrence (OR, 36.4; 95% CI, 1.15-45.22). Within the screening analysis, single biomarkers demonstrated moderate sensitivity and specificity (AUC, 0.59-0.83) in the detection of primary disease. A panel combining HPV DNA markers with methylated EDNRB and methylated PAX5 improved AUC to 0.93. CONCLUSION: Detection of high-risk HPV DNA or aberrant hypermethylation in oral rinses is associated with presence and recurrence of OPSCC. Targeting both markers in saliva may have utility in long-term surveillance.

Cannabinoids Promote Progression of HPV-Positive Head and Neck Squamous Cell Carcinoma via p38 MAPK Activation.

PURPOSE: Human papillomavirus (HPV)-related head and neck squamous cell carcinoma (HNSCC) is associated with daily marijuana use and is also increasing in parallel with increased marijuana use in the United States. Our study is designed to define the interaction between cannabinoids and HPV-positive HNSCC. EXPERIMENTAL DESIGN: The expression of cannabinoid receptors CNR1 and CNR2 was analyzed using The Cancer Genome Atlas (TCGA) HNSCC data. We used agonists, antagonists, siRNAs, or shRNA-based models to explore the roles of CNR1 and CNR2 in HPV-positive HNSCC cell lines and animal models. Cannabinoid downstream pathways involved were determined by Western blotting and analyzed in a primary HPV HNSCC cohort with single-sample gene set enrichment analysis (ssGSEA) and the OncoGenome Positioning System (Onco-GPS). RESULTS: In TCGA cohort, the expression of CNR1 and CNR2 was elevated in HPV-positive HNSCC compared with HPV-negative HNSCC, and knockdown of CNR1/CNR2 expression inhibited proliferation in HPV-positive HNSCC cell lines. Specific CNR1 and CNR2 activation as well as nonselective cannabinoid receptor activation in cell lines and animal models promoted cell growth, migration, and inhibited apoptosis through p38 MAPK pathway activation. CNR1/CNR2 antagonists suppressed cell proliferation and migration and induced apoptosis. Using whole-genome expression analysis in a primary HPV HNSCC cohort, we identified specific p38 MAPK pathway activation signature in tumors from HPV HNSCC patients with objective measurement of concurrent cannabinoid exposure. CONCLUSIONS: Cannabinoids can promote progression of HPV-positive HNSCC through p38 MAPK pathway activation.

Venoplasty and stenting in post-thrombotic syndrome and non-thrombotic iliac vein lesion.

Introduction: Venous outflow obstruction is a common condition among patients with chronic venous insufficiency. Endovascular treatment is favourable over open surgery. This study aimed to assess stent patency and clinical outcome in venous outflow obstruction of lower limbs, and also to compare it between post-thrombotic syndrome and non-thrombotic iliac vein lesions.Material and methods: The study was a historical cohort study. Patients with chronic deep venous insufficiency referred to our tertiary referral centre who underwent venoplasty were recruited. Patients were divided into two groups: non-thrombotic-iliac-vein-lesions and post-thrombotic syndrome. Stent patency rate, clinical improvement and risk factors were evaluated during a six-months course after venoplasty.Results: One-hundred-sixty-four patients were included. Six-months primary, assisted primary and secondary patency rates were 98.86%, 100% and100% in the non-thrombotic-iliac-vein-lesions group and 88%, 93% and 96% in the post thrombotic syndrome groups (p-value = .005, p-value = .02, and p-value = .09, respectively). Pain, claudication and edema were the most common symptoms in both groups and significantly improved after six months. Early thrombosis in the PTS group was more common (9 vs. 1, P value = .007).Conclusion: Percutaneous stenting in patients with venous outflow obstruction is safe and effective with a high patency rate and significant decrease in clinical score in both post-thrombotic syndrome and non-thrombotic-iliac-vein lesions groups.

Rational genomic optimization of DNA detection for human papillomavirus type 16 in head and neck squamous cell carcinoma.

BACKGROUND: We aimed to use genomic data for optimizing polymerase chain reaction (PCR) primer/probe sets for detection of human papillomavirus (HPV)-16 in body fluids of patients with HPV-related head and neck squamous cell carcinoma (HPV-HNSCC). METHODS: We used genomic HPV-HNSCC sequencing data from a single institutional and a TCGA cohort. Optimized primer/probe sets were designed and tested for analytical performance in CaSki HPV-16 genome and confirmed in salivary rinse samples from patients with HPV-HNSCC. RESULTS: The highest read density was observed between E5 and L2 regions. The E1 region contained a region that was universally present. Among candidate PCR primer/probe sets created, six reliably detected 30 HPV-16 copy number. In a CLIA certified laboratory setting, the combination of two novel primer/probe with E7 sets improved performance in salivary rinse samples with a sensitivity of 96% and specificity of 100%. CONCLUSIONS: PCR-based detection of HPV-16 DNA in HPV-HNSCC can be improved using rational genomic design.

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.

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.

Characterization of Alternative Splicing Events in HPV-Negative Head and Neck Squamous Cell Carcinoma Identifies an Oncogenic DOCK5 Variant.

Purpose: Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers worldwide, and alternative splicing is considered to play important roles in tumor progression. Our study is designed to identify alternative splicing events (ASEs) in human papillomavirus (HPV)-negative HNSCC.Experimental Design: RNA sequencing data of 407 HPV-negative HNSCC and 38 normal samples were obtained from The Cancer Genome Atlas (TCGA), and splice junctions were discovered using MapSplice. Outlier analysis was used to identify significant splicing junctions between HPV-negative HNSCC and normal samples. To explore the functional role of the identified DOCK5 variant, we checked its expression with qRT-PCR in a separate primary tumor validation set and performed proliferation, migration, and invasion assays.Results: A total of 580 significant splicing events were identified in HPV-negative HNSCC, and the most common type of splicing events was an alternative start site (33.3%). The prevalence of a given individual ASE among the tumor cohort ranged from 9.8% and 64.4%. Within the 407 HPV-negative HNSCC samples in TCGA, the number of significant ASEs differentially expressed in each tumor ranged from 17 to 290. We identified a novel candidate oncogenic DOCK5 variant confirmed using qRT-PCR in a separate primary tumor validation set. Loss- and gain-of-function experiments indicated that DOCK5 variant promoted proliferation, migration, and invasion of HPV-negative HNSCC cells, and patients with higher expression of DOCK5 variant showed decreased overall survival.Conclusions: Analysis of ASEs in HPV-negative HNSCC identifies multiple alterations likely related to carcinogenesis, including an oncogenic DOCK5 variant. Clin Cancer Res; 24(20); 5123-32. ©2018 AACR.