Radiation-Induced Microvascular Injury as a Mechanism of Salivary Gland Hypofunction and Potential Target for Radioprotectors

Radiation therapy is commonly used to treat patients with head and neck squamous cell carcinoma (HNSCC). One of the major side effects of radiotherapy is injury to the salivary glands (SG), which is thought to be mediated by microvascular dysfunction leading to permanent xerostomia. The goal of this study was to elucidate the mechanism of radiation-induced microvasculature damage and its impact on SG function. We measured bovine aortic endothelial cell (BAEC) apoptosis and ceramide production in response to 5 Gy irradiation, either alone or with reactive oxygen species (ROS) scavengers. We then investigated the effect of a single 15 Gy radiation dose on murine SG function. BAECs exposed to 5 Gy underwent apoptosis with increased ceramide production, both prevented by ROS scavengers. Among the 15 Gy irradiated mice, there was considerable weight loss, alopecia and SG hypofunction manifested by reduced saliva production and lower lysozyme levels. All of these effects, except for the lysozyme levels, were prevented by pretreatment with ROS scavengers. Microvessel density was significantly lower in the SG of irradiated mice compared to the control group, and this effect was significantly attenuated by pretreatment with Tempol. This study demonstrates that radiation-induced SG hypofunction is to a large extent mediated by microvascular dysfunction involving ceramide and ROS generation. These findings strongly suggest that ROS scavengers may serve as potential radioprotectors of SG function in patients undergoing radiotherapy for HNSCC

Stem cell-like T cell depletion in the recurrent head and neck cancer immune microenvironment

ABSTRACTHuman papilloma virus (HPV)-related oncogenesis in head and neck cancer establishes a local microenvironment rich with immune cells, however the composition of the microenvironment in recurrent disease following definitive treatment is poorly understood. Here, we investigate the composition and spatial relationships between tumor and immune cells in recurrent head and neck cancer following curative intent chemoradiotherapy. Multiplexed immunofluorescence with 12 unique markers, through two multiplex immunofluorescent panels, was performed to evaluate 27 tumor samples including 18 pre-treatment primary and 9 paired recurrent tumors. Tumor and immune cell populations were phenotyped and quantified using a previously validated semi-automated digital pathology platform for cell segmentation. Spatial analysis was conducted by evaluating immune cells within the tumor, peri-tumoral stroma, and distant stroma. Initial tumors in patients with subsequent recurrence were found to be enriched in tumor associated macrophages and displayed an immune excluded spatial distribution. Recurrent tumors after chemoradiation were hypo-inflamed, with a statistically significant reduction in the recently identified stem-like TCF1+ CD8 T-cells, which normally function to maintain HPV-specific immune responses in the setting of chronic antigen exposure. Our findings indicate that the tumor microenvironment of recurrent HPV-related head and neck cancers displays a reduction in stem-like T cells, consistent with an immune microenvironment with a reduced ability to mount T-cell-driven anti-tumor immune responses

Oral Microbiome in Nonsmoker Patients with Oral Cavity Squamous Cell Carcinoma, Defined by Metagenomic Shotgun Sequencing

Objectives: Smoking is the commonest cause of oral cavity squamous cell carcinoma (OC-SCC), but the etiology of OC-SCC in nonsmokers is unknown. Our primary goal was to use metagenomic shotgun sequencing (MSS) to define the taxonomic composition and functional potential of oral metagenome in nonsmokers with OC-SCC. Methods: We conducted a case–control study with 42 OC-SCC case and 45 control nonsmokers. MSS was performed on DNA extracted from mouthwash samples. Taxonomic analysis and pathway analysis were done using MetaPhlAn2 and HUMAnN2, respectively. Statistical difference was determined using the Mann–Whitney test controlling false discovery rate. Results: There was no significant difference in age, sex, race, or alcohol consumption between OC-SCC and control patients. There was a significant difference in beta diversity between OC-SCC and controls. At the phylum level, Bacteroidetes and Synergistetes were overly represented in OC-SCC while Actinobacteria and Firmicutes were overly represented in controls. At the genus level, Fusobacterium was overly represented in OC-SCC compared with controls, while Corynebacterium, Streptococcus, Actinomyces, Cryptobacterium, and Selenomonas were overly represented in controls. Bacterial pathway analysis identified overrepresentation in OC-SCC of pathways related to metabolism of flavin, biotin, thiamin, heme, sugars, fatty acids, peptidoglycans, and tRNA and overrepresentation of nucleotides and essential amino acids in controls. Conclusions: The oral microbiome in nonsmoker patients with OC-SCC is significantly different from that of nonsmoker control patients in taxonomic compositions and functional potentials. Our study’s MSS findings matched with previous 16S-based methods in taxonomic differentiation but varied greatly in functional differentiation of microbiomes in OC-SCC and controls

Concurrent Chemoradiotherapy With Cisplatin Versus Cetuximab for Squamous Cell Carcinoma of the Head and Neck

ObjectivesWe previously reported inferior outcomes for locally advanced head and neck cancer treated with cetuximab (C225) versus cisplatin (CDDP). We now examine if this difference persists when accounting for HPV status and update outcomes on the entire cohort.Materials and methodsFrom 3/106 to 4/1/08, 174 locally advanced head and neck cancer patients received definitive treatment with RT and CDDP (n=125) or RT and C225 (n=49). Of these, 62 patients had tissue available for HPV analysis.ResultsThe median follow-up was 47 months. The 3-year loco-regional failure, disease-free survival, and overall survival for CDDP versus C225 were 5.7% versus 40.2% (P<0.0001), 85.1% versus 35.4% (P<0.0001), and 90.0% versus 56.6% (P<0.0001), respectively. In the subset with tissue, there was no difference in rates of HPV or p16 positivity between the 2 groups. In this subset, the 3-year loco-regional failure, disease-free survival, and overall survival for CDDP versus C225 were 5.3% versus 32.0% (P=0.01), 86.8% versus 43.2% (P=0.002), and 86.7% versus 76.9% (P=0.09), respectively. Multivariate analysis continued to show a benefit for CDDP.ConclusionsWith longer follow-up and the inclusion of HPV and p16 status for about one third of patients where tissue was available, we continued to find superior outcomes with concurrent CDDP versus C225