Spatial mapping of mitochondrial networks and bioenergetics in lung cancer.

Mitochondria are critical to the governance of metabolism and bioenergetics in cancer cells1. The mitochondria form highly organized networks, in which their outer and inner membrane structures define their bioenergetic capacity2,3. However, in vivo studies delineating the relationship between the structural organization of mitochondrial networks and their bioenergetic activity have been limited. Here we present an in vivo structural and functional analysis of mitochondrial networks and bioenergetic phenotypes in non-small cell lung cancer (NSCLC) using an integrated platform consisting of positron emission tomography imaging, respirometry and three-dimensional scanning block-face electron microscopy. The diverse bioenergetic phenotypes and metabolic dependencies we identified in NSCLC tumours align with distinct structural organization of mitochondrial networks present. Further, we discovered that mitochondrial networks are organized into distinct compartments within tumour cells. In tumours with high rates of oxidative phosphorylation (OXPHOSHI) and fatty acid oxidation, we identified peri-droplet mitochondrial networks wherein mitochondria contact and surround lipid droplets. By contrast, we discovered that in tumours with low rates of OXPHOS (OXPHOSLO), high glucose flux regulated perinuclear localization of mitochondria, structural remodelling of cristae and mitochondrial respiratory capacity. Our findings suggest that in NSCLC, mitochondrial networks are compartmentalized into distinct subpopulations that govern the bioenergetic capacity of tumours.

HPV Knowledge Retention and Concurrent Increase in Vaccination Rates 1.5 Years After a Novel HPV Workshop in Medical School.

Comprehensive education regarding human papillomavirus (HPV) pathogenesis, vaccination, and patient counseling are not routinely included in the medical school curriculum; consequently, student and provider knowledge, especially concerning head and neck pathology, remains low. The objective of this study was to demonstrate long-term retention of HPV knowledge and positive attitudes towards HPV vaccination after attending our novel HPV workshop, with a focus on knowledge of oropharyngeal cancer. A follow-up survey was administered to medical students 1.5 years after the initial completion of the workshop. HPV vaccination records from the student-led clinic were collected from the immunization information system. Awareness that HPV causes oropharyngeal cancer was present in 33% of medical students pre-curriculum; immediate and long-term post-curricular awareness of this association remained at 90% or higher (p < 0.0001). Comfort with HPV counseling, having enough information to recommend the vaccine, and knowledge of HPV malignancies, symptoms, transmission, and vaccination schedule remained persistently elevated over pre-curriculum scores (p < 0.05). Long-term knowledge scores were also higher than a control group of medical students at the same stage of training who had never participated in the workshop (p < 0.05). HPV vaccination rates at the medical school's student-run clinic also increased after the curriculum, from an average of 1.89 HPV vaccines given per clinic to 3.55 (p = 0.001). This study demonstrates that knowledge and positive attitudes were maintained 1.5 years after participating in this HPV curriculum during students' preclinical years of medical school. Additionally, an increase in HPV vaccination rates occurred at a student-led clinic, indicating a positive clinical impact on the curriculum.

Label-free, real-time detection of perineural invasion and cancer margins in a murine model of head and neck cancer surgery.

Surgical management of head and neck cancer requires a careful balance between complete resection of malignancy and preservation of function. Surgeons must also determine whether to resect important cranial nerves that harbor perineural invasion (PNI), as sacrificing nerves can result in significant morbidity including facial paralysis. Our group has previously reported that Dynamic Optical Contrast Imaging (DOCI), a novel non-invasive imaging system, can determine margins between malignant and healthy tissues. Herein, we use an in vivo murine model to demonstrate that DOCI can accurately identify cancer margins and perineural invasion, concordant with companion histology. Eight C3H/HeJ male mice were injected subcutaneously into the bilateral flanks with SCCVIISF, a murine head and neck cancer cell line. DOCI imaging was performed prior to resection to determine margins. Both tumor and margins were sent for histologic sectioning. After validating that DOCI can delineate HNSCC margins, we investigated whether DOCI can identify PNI. In six C3H/HeJ male mice, the left sciatic nerve was injected with PBS and the right with SCCVIISF. After DOCI imaging, the sciatic nerves were harvested for histologic analysis. All DOCI images were acquired intraoperatively and in real-time (10 s per channel), with an operatively relevant wide field of view. DOCI values distinguishing cancer from adjacent healthy tissue types were statistically significant (P < 0.05). DOCI imaging was also able to detect perineural invasion with 100% accuracy compared to control (P < 0.05). DOCI allows for intraoperative, real-time visualization of malignant and healthy tissue margins and perineural invasion to help guide tumor resection.

Immune Checkpoint Blockade Enhances Immune Activity of Therapeutic Lung Cancer Vaccine.

BACKGROUND: Immune checkpoint blockade that downregulates T cell evasion for effective immunity has provided a renewed interest in therapeutic cancer vaccines. METHODS: Utilizing murine lung cancer models, we determined: tumor burden, TIL cytolysis, immunohistochemistry, flow cytometry, RNA Sequencing, CD4 T cells, CD8 T cells, CXCL9 chemokine, and CXCL10 chemokine neutralization to evaluate the efficacy of Programmed cell death protein 1 (PD-1) blockade combined with chemokine (C-C motif) ligand 21-dendritic cell tumor antigen (CCL21-DC tumor Ag) vaccine. RESULTS: Anti-PD1 combined with CCL21-DC tumor Ag vaccine eradicated 75% of 12-day established tumors (150 mm3) that was enhanced to 90% by administering CCL21-DC tumor Ag vaccine prior to combined therapy. The effect of combined therapy was blocked by CD4, CD8, CXCL9, and CXCL10 neutralizing antibodies. CONCLUSION: PD-1 blockade therapy plus CCL21-DC tumor Ag vaccine could be beneficial to lung cancer patients.

Oropharynx-directed ipsilateral irradiation for p16-positive squamous cell carcinoma involving the cervical lymph nodes of unknown primary origin.

BACKGROUND: The purpose of this study was to present our findings on the use of limited-field, oropharynx-directed ipsilateral irradiation for p16-positive squamous cell carcinoma of unknown primary origin. METHODS: Between April 2011 and January 2016, 25 patients with a histological diagnosis of p16-positive squamous cell carcinoma were selectively irradiated to the ipsilateral oropharynx and cervical neck for tumors of unknown primary origin. The dose to the oropharynx ranged from 54-60 Gy (median 60 Gy) in 30-33 fractions. Concurrent cisplatin-based chemotherapy was administered to 8 patients (32%). RESULTS: The actuarial 2-year estimates of locoregional control, progression-free survival, and overall survival were 91%, 87%, and 92%, respectively. One patient failed in the contralateral neck. There was no grade 3 + toxicity in either the acute or late setting. CONCLUSION: Oropharynx-directed, ipsilateral radiation results in disease control that compares favorably with historical controls treated by comprehensive mucosal and bilateral neck radiation.

Prognostic significance of p16 in squamous cell carcinoma of the larynx and hypopharynx.

PURPOSE: To evaluate the prognostic significance of p16 expression among patients with squamous cell carcinoma of the larynx (LSCC) and hypopharynx (HSCC). METHODS: The medical records of all patients with locally advanced, non-metastatic LSCC/HSCC were reviewed. p16INK4A (p16) protein expression was evaluated on pathological specimens by immunohistochemistry (IHC), and the Kaplan-Meier method was used to estimate overall survival (OS) and locoregional control (LRC). In select cases, p16 expression was correlated to high-risk and low-risk HPV genotypes using in situ hybridization (ISH). RESULTS: Thirty-one patients (23 LSCC; 8 HSCC) were identified. Seventeen (54.8%) patients were p16 negative; 14 (45.2%) were p16-positive. The primary treatment modality was radiation therapy for 22 (71.0%) patients and surgery for 9 (29.0%). Nineteen (61.3%) patients were evaluated for high-risk HPV and low-risk HPV genotypes by IHC, of whom 2 (10.5%) patients were positive for high-risk HPV and 1 (5.3%) was positive for low-risk HPV. For high-risk HPV, the positive predictive value (PPV), sensitivity, and specificity of p16 was 20.0%, 100%, and 52.9%. There was no significant difference in the 2-year actuarial rates of OS (91% vs. 64%, p=0.34) or LRC (51% vs. 46%, p=0.69) between the p16-positive and p-16 negative patients. CONCLUSION: In this small cohort of 31 LSCC and HSCC patients, p16 was not a significant predictive of either LRC or OS. Furthermore, p16 was poorly correlated with HPV genotyping as identified by ISH.

CCL21 Cancer Immunotherapy.

Cancer, a major health problem, affects 12 million people worldwide every year. With surgery and chemo-radiation the long term survival rate for the majority of cancer patients is dismal. Thus novel treatments are urgently needed. Immunotherapy, the harnessing of the immune system to destroy cancer cells is an attractive option with potential for long term anti-tumor benefit. Cytokines are biological response modifiers that stimulate anti-tumor immune responses. In this review, we discuss the anti-tumor efficacy of the chemotactic cytokine CCL21 and its pre-clinical and clinical application in cancer.

Combination Treatment with Apricoxib and IL-27 Enhances Inhibition of Epithelial-Mesenchymal Transition in Human Lung Cancer Cells through a STAT1 Dominant Pathway.

BACKGROUND: The cyclooxygenase 2 (COX-2) pathway has been implicated in the molecular pathogenesis of many malignancies, including lung cancer. Apricoxib, a selective COX-2 inhibitor, has been described to inhibit epithelial-mesenchymal transition (EMT) in human malignancies. The mechanism by which apricoxib may alter the tumor microenvironment by affecting EMT through other important signaling pathways is poorly defined. IL-27 has been shown to have anti-tumor activity and our recent study showed that IL-27 inhibited EMT through a STAT1 dominant pathway. OBJECTIVE: The purpose of this study is to investigate the role of apricoxib combined with IL-27 in inhibiting lung carcinogenesis by modulation of EMT through STAT signaling. METHODS AND RESULTS: Western blot analysis revealed that IL-27 stimulation of human non-small cell lung cancer (NSCLC) cell lines results in STAT1 and STAT3 activation, decreased Snail protein and mesenchymal markers (N-cadherin and vimentin) and a concomitant increase in expression of epithelial markers (E-cadherin, ß-and γ-catenins), and inhibition of cell migration. The combination of apricoxib and IL-27 resulted in augmentation of STAT1 activation. However, IL-27 mediated STAT3 activation was decreased by the addition of apricoxib. STAT1 siRNA was used to determine the involvement of STAT1 pathway in the enhanced inhibition of EMT and cell migration by the combined IL-27 and apricoxib treatment. Pretreatment of cells with STAT1 siRNA inhibited the effect of combined IL-27 and apricoxib in the activation of STAT1 and STAT3. In addition, the augmented expression of epithelial markers, decreased expression mesenchymal markers, and inhibited cell migration by the combination treatment were also inhibited by STAT1 siRNA, suggesting that the STAT1 pathway is important in the enhanced effect from the combination treatment. CONCLUSION: Combined apricoxib and IL-27 has an enhanced effect in inhibition of epithelial-mesenchymal transition and cell migration in human lung cancer cells through a STAT1 dominant pathway.

Parotidectomy and neck dissection in the management of conjunctival melanoma: are they necessary?

OBJECTIVES/HYPOTHESIS: The objectives of this study were to review traditional techniques for the management of conjunctival melanoma and assess the need for parotidectomy and neck dissection in the management of conjunctival melanoma. STUDY DESIGN: Retrospective review. METHODS: This study was a retrospective review conducted in a tertiary academic medical center of patients diagnosed with conjunctival melanoma over a 20-year period RESULTS: There were 39 patients diagnosed with conjunctival melanoma identified from January 1990 to December 2010. Follow-up varied from 2 to 201 months (median, 25 months). Of the patients, 16 (41%) had local recurrences at the primary site, two (13%) of whom later presented with parotid disease. One patient with parotid recurrence had a subsequent neck dissection for confirmed metastatic spread. No patient in this series had metastatic cervical disease without initial spread to the parotid. The probability of disease-free survival at 1, 2, and 5 years was 77%, 68%, and 50%, respectively. The probability of parotid free progression at 1, 2, and 5 years was 100%, 96%, and 90%, respectively. CONCLUSIONS: Conjunctival melanoma is a rare malignancy traditionally managed with aggressive treatment to optimize local control. The role for staging parotidectomy with or without neck dissection has been heavily debated. Based on our review, parotidectomy only needs to be undertaken when high suspicion for metastatic spread is present, such as a palpable or radiographically evident mass. In addition, without documented parotid disease, neck dissection is not required.

Optimal timing of surgical intervention following adult laryngeal trauma.

OBJECTIVE: Laryngeal trauma is an infrequent diagnosis with a scarcity of published data. We aim to further define the factors associated with positive surgical outcomes of adult laryngeal trauma. STUDY DESIGN: Multi-institution database analysis. METHODS: Of the 1.9 million trauma cases from the National Trauma Database (NTDB), 564 adult trauma events were selected with ICD-9 codes specific to laryngeal trauma. RESULTS: Laryngeal trauma was seen predominately in white (61.5%), middle-aged (40.6 years), male (83.7%) patients experiencing blunt (70.7%) laryngeal injury with multiorgan system (92.2%) trauma. There was an overall 17.9% mortality rate. Within the 564 cases, 133 direct laryngoscopies, 185 tracheostomies, 53 laryngeal suturing, and 60 laryngeal fracture repairs were performed. In univariate negative binomial regression models, trauma severity (P ≤ .01), placement of tracheostomy (P lt; .01), and delayed tracheostomy placement (P = .04, .03, .048) were associated with increased ventilator dependence, intensive care unit (ICU) stay, and overall hospital admission duration. Multivariate regression models demonstrated significant associations between tracheostomy performed within 24 hours and shortened ICU stay (P = .03, ß = -.28, SE = 1.7) and overall hospital stay (P = .009, ß = -.23, SE = 3.1). CONCLUSIONS: The NTDB allows study of the largest laryngeal trauma cohort in modern literature. Although complexities arise in the treatment of laryngeal traumas, when indicated, surgical airway should be placed within 24 hours of presentation to improve the overall hospital course.