FOXM1-Mediated Regulation of Reactive Oxygen Species and Radioresistance in Oral Squamous Cell Carcinoma Cells.

Radioresistance is a major obstacle to the successful treatment of oral squamous cell carcinoma (OSCC). To help overcome this issue, we have developed clinically relevant radioresistant (CRR) cell lines generated by irradiating parental cells over time, which are useful for OSCC research. In the present study, we conducted gene expression analysis using CRR cells and their parental lines to investigate the regulation of radioresistance in OSCC cells. Based on gene expression changes over time in CRR cells and parental lines subjected to irradiation, forkhead box M1 (FOXM1) was selected for further analysis in terms of its expression in OSCC cell lines, including CRR cell lines and clinical specimens. We suppressed or upregulated the expression of FOXM1 in OSCC cell lines, including CRR cell lines, and examined radiosensitivity, DNA damage, and cell viability under various conditions. The molecular network regulating radiotolerance was also investigated, especially the redox pathway, and the radiosensitizing effect of FOXM1 inhibitors was examined as a potential therapeutic application. We found that FOXM1 was not expressed in normal human keratinocytes but was expressed in several OSCC cell lines. The expression of FOXM1 was upregulated in CRR cells compared with that detected in the parental cell lines. In a xenograft model and clinical specimens, FOXM1 expression was upregulated in cells that survived irradiation. FOXM1-specific small interfering RNA (siRNA) treatment increased radiosensitivity, whereas FOXM1 overexpression decreased radiosensitivity, and DNA damage was altered significantly under both conditions, as well as the levels of redox-related molecules and reactive oxygen species production. Treatment with the FOXM1 inhibitor thiostrepton had a radiosensitizing effect and overcame radiotolerance in CRR cells. According to these results, the FOXM1-mediated regulation of reactive oxygen species could be a novel therapeutic target for the treatment of radioresistant OSCC; thus, treatment strategies targeting this axis might overcome radioresistance in this disease.

The antioxidative stress regulator Nrf2 potentiates radioresistance of oral squamous cell carcinoma accompanied with metabolic modulation.

Nuclear factor erythroid 2-related factor 2 (Nrf2), which regulates the expression of critical antioxidant proteins, was recently demonstrated to play a key role in cancer progression. Resistance to radiotherapy is a major obstacle in treating oral squamous cell carcinoma (OSCC). However, little is known about the association between Nrf2 and radioresistance in OSCC. Two OSCC cell lines (SAS and HSC-2) and their clinically relevant radioresistant (CRR) clones (SAS-R, HSC-2-R) were used. The effects of Nrf2 downregulation on radiosensitivity and the involvement of glycolysis in Nrf2-mediated radioresistance were evaluated. Immunohistochemistry of phosphorylated Nrf2 (p-Nrf2) was performed in 110 patients with OSCC who underwent preoperative chemoradiotherapy and surgery. Nrf2 was stably upregulated in CRR cells in vitro and in a mouse xenograft model. Moreover, elevated Nrf2 expression was associated with radioresistance. The enhancement of Nrf2-dependent glycolysis and glutathione synthesis was involved in the development of radioresistance. Additionally, p-Nrf2 expression was closely related to the pathological response to chemoradiotherapy, and its expression was predictive of prognosis in patients with advanced OSCC. Our results suggest that Nrf2 plays an important role in the radioresistance of OSCC accompanied by metabolic reprogramming. Targeting Nrf2 antioxidant pathway may represent a promising treatment strategy for highly malignant OSCC.

Circulating miRNA-1290 as a potential biomarker for response to chemoradiotherapy and prognosis of patients with advanced oral squamous cell carcinoma: A single-center retrospective study.

MicroRNAs are a class of small, endogenous, noncoding 18- to 24-nucleotide-long RNAs that can regulate multiple processes related to cancer progression. However, their clinical value in patients with oral squamous cell carcinoma has not yet been fully explored. Therefore, the aim of this study was to investigate the clinical significance of circulating microRNAs in oral squamous cell carcinoma patients. The expression levels of circulating miR-1246 and miR-1290 in healthy volunteers and oral squamous cell carcinoma patients were examined by quantitative real-time polymerase chain reaction. The expression levels of both microRNAs in the radioresistant oral squamous cell carcinoma cell line (SAS-R) and the parent cell line (SAS) and in the conditioned medium obtained from these cell lines were also examined by quantitative real-time polymerase chain reaction. In addition, the correlations between circulating microRNA status and various clinicopathological features in 55 oral squamous cell carcinoma patients with locally advanced oral squamous cell carcinoma who underwent surgery following 5-fluorouracil-based chemoradiotherapy were examined. The expression level of miR-1290 was significantly lower in the plasma of oral squamous cell carcinoma patients than in that of healthy volunteers (p < 0.01). The expression levels of microRNAs in the conditioned medium and in the cells varied from cell to cell. In the clinicopathological analyses, the frequency of patients with low miR-1290 levels was significantly higher among cases with lower pathological differentiation and among those with a poor pathological response for preoperative chemoradiotherapy (p = 0.030 each). Furthermore, Cox regression analysis based on the 5-year overall survival and disease-free survival revealed that miR-1290 status was a significant prognostic factor for patients with oral squamous cell carcinoma (hazard ratio = 0.169, p = 0.008, and hazard ratio = 0.186, p = 0.008, respectively). Circulating miR-1290 status could be a valuable biomarker for predicting the clinical response to chemoradiotherapy as well as overall survival in patients with oral squamous cell carcinoma.

FBXW7 expression affects the response to chemoradiotherapy and overall survival among patients with oral squamous cell carcinoma: A single-center retrospective study.

FBXW7 (F-box and WD repeat domain containing-7) is a tumor suppressor protein that regulates the degradation of various oncoproteins in several malignancies. However, limited information is available regarding FBXW7 expression in oral squamous cell carcinoma. Therefore, this study aimed to determine the clinical significance of FBXW7 expression in oral squamous cell carcinoma. The FBXW7 expression patterns in oral squamous cell carcinoma and adjacent normal tissues from 15 patients who underwent radical resection were evaluated using quantitative real-time polymerase chain reaction and immunohistochemical staining. In addition, immunohistochemistry was performed using paraffin-embedded sections from biopsy specimens obtained from 110 patients with oral squamous cell carcinoma who underwent surgery after 5 fluorouracil-based chemoradiotherapy. The associations of FBXW7 expression with various clinicopathological features and prognosis were evaluated in these patients. As a results, in the 15 matched samples, the FBXW7 expression was significantly decreased in the oral squamous cell carcinoma tissues compared to that in the adjacent normal tissues. In the clinicopathological analysis, compared to high protein expression, low FBXW7 expression was found to significantly associate with a poor histological response to preoperative chemoradiotherapy. Kaplan-Meier curve analysis revealed that low FBXW7 expression was significantly associated with a poor prognosis, and FBXW7 expression was found to be an independent predictor of overall survival in the multivariate analysis. Our results suggest that FBXW7 may function as a tumor suppressor protein in oral squamous cell carcinoma. In addition, FBXW7 could be a potential biomarker for predicting not only the clinical response to chemoradiotherapy but also overall survival in patients with oral squamous cell carcinoma.

IL-6 controls resistance to radiation by suppressing oxidative stress via the Nrf2-antioxidant pathway in oral squamous cell carcinoma.

BACKGROUND: In promoting tumour malignancy IL-6 signalling is considered to have an important role. However, the biological roles of IL-6 on radiosensitivity in oral squamous cell carcinoma (OSCC) remain largely unclear. The objective of this study is to determine the effects and molecular mechanisms of IL-6 on radiosensitivity in OSCC. METHODS: Two OSCC cell lines, and OSCC tissue samples with radioresistant cells were used. We examined the effects of IL-6, or tocilizumab, a humanised anti-human IL-6 receptor antibody, or both on radiosensitivity and DNA damage after X-ray irradiation in vitro. In addition, we investigated the involvement of the Nrf2-antioxidant pathway in IL-6-mediated radioresistant mechanisms using OSCC cell lines and tissues. RESULTS: Increased levels of IL-6 suppressed radiation-induced cell death, and the blockade of IL-6 signalling by tocilizumab sensitised tumour cells to radiation. The radioresistant effect of IL-6 was associated with decreased DNA damage after radiation. We also found that IL-6 promotes the activation of not only the downstream molecule STAT3 but also the Nrf2-antioxidant pathway, leading to a significant decrease in oxidative stress by upregulating Mn-SOD. CONCLUSIONS: These results indicate that the blockade of IL-6 signalling combined with conventional radiotherapy could augment the treatment response and survival rate in patients with radioresistant OSCC.

[Potential uncertainty about image registration in thoracic image-guided radiotherapy].

PURPOSE: Although image-guided radiotherapy (IGRT) is widely used to determine and correct daily setup errors, the additional interpretation for image registration would provide another error. We evaluated the uncertainty in image registration in IGRT. METHOD: The subjects consisted of 12 consecutive patients treated with IGRT for thoracic esophageal cancer. Two radiation therapists had consensually achieved daily 3D registration between planning computed tomography (CT) and cone beam CT (CBCT). The original data sets of image registration in all fractions except for boost irradiations with a change in the isocenter positions were selected for evaluation. There were 20 to 32 data sets for each patient: a total of 318 data sets. To evaluate daily setup errors, the mean 3D displacement vector was calculated for each patient. To assess the reproducibility of image registration, two other radiation therapists reviewed the data sets and recorded geometric differences as uncertainty in the image registration. RESULTS: The mean 3D displacement vector for each patient ranged from 4.9 to 15.5 mm for setup errors and 0.7 to 2.2 mm for uncertainty in image registration. There was a positive correlation between the 3D vectors for setup error and uncertainty in image registration (r = 0.487, p = 0.016). CONCLUSION: Although IGRT can correct the setup errors, potential uncertainty exists in image registration. The setup error would disturb the image registration in IGRT.

[Development of a quality assurance phantom for brachytherapy: the feasibility of daily check with the phantom].

PURPOSE: We developed a quality assurance (QA) phantom to enable easy confirmation of radiation source output measurements of a high dose rate (192)Ir intracavitary brachytherapy unit in gynecology. The purpose of this study was to evaluate the feasibility of daily checks using the QA phantom. METHODS AND MATERIALS: The QA phantom was designed with tough water phantoms to hold a Farmer-type ionization chamber, with semiconductor detectors used as in vivo dosimeters to measure rectal dose, and three transfer tubes for gynecology. To test the reliability of our QA phantom for the detection of abnormalities in source output or semiconductor detectors, we applied different doses. RESULTS: Variations due to different settings of the QA phantom were within 2%. The temporal variations were less than 2% and 5% in the Farmer-type ionization chamber and semiconductor detectors, respectively. Interobserver variations were below 3%. CONCLUSIONS: With tolerance levels of 2% and 5% for a Farmer-type ionization chamber and semiconductor detectors, respectively, a QA phantom is potentially useful for easily detecting abnormalities by applying daily checks of the brachytherapy unit.

Added value of high-b-value (b = 3000 s/mm2) diffusion-weighted imaging at 3 T in relation to fluid-attenuated inversion recovery images for the evaluation of cortical lesions in inflammatory brain diseases.

OBJECTIVE: The purpose of this study was to determine how the gray-to-white matter contrast in healthy subjects changes on high-b-value diffusion-weighted imaging (DWI) acquired at 3 T and evaluate whether high-b-value DWI at 3 T is useful for the detection of cortical lesions in inflammatory brain diseases. METHODS: Ten healthy volunteers underwent DWI at b = 1000, 2000, 3000, 4000, and 5000 s/mm(2) on a 3-T MRI unit. On DW images, 1 radiologist performed region-of-interest measurements of the signal intensity of 8 gray matter structures. The gray-to-white matter contrast ratio (GWCR) was calculated. Ten patients with inflammatory cortical lesions were also included. All patients underwent conventional MRI and DWI at b = 1000 and 3000 s/mm(2). Using a 4-point grading system, 2 radiologists independently assessed the presence of additional information on DW images compared with fluid-attenuated inversion recovery images. Interobserver agreement was assessed by κ statistics. RESULTS: In the healthy subjects, the b value increased as the GWCR decreased in all evaluated gray matter structures. On DW images acquired at b = 3000 s/mm(2), mean GWCR was less than 1.0 in 7 of 8 structures. For both reviewers, DWI at b = 3000 s/mm(2) yielded significantly more additional information than did DWI at b = 1000 s/mm(2) (P < 0.05). Interobserver agreement for DWI at b = 1000 s/mm(2) and b = 3000 s/mm(2) was fair (κ = 0.35) and excellent (κ = 1.0), respectively. CONCLUSIONS: At 3-T DWI, the gray-to-white matter contrast in most gray matter structures reverses at b = 3000 s/mm. In the evaluation of cortical lesions in patients with inflammatory brain diseases, 3-T DWI at b = 3000 s/mm was more useful than b = 1000 s/mm(2).

Can MRI-derived factors predict the survival in glioblastoma patients treated with postoperative chemoradiation therapy?

BACKGROUND: Advanced diagnostic and therapeutic developments may yield novel prognostic factors in patients with glioblastoma multiforme (GBM). PURPOSE: To validate the predictive values of pretreatment quantitative diffusion-weighted (DW) magnetic resonance imaging (MRI) and MRI performed within 72 h after surgery in patients with GBM. MATERIAL AND METHODS: Between January 2000 and September 2009, 138 patients with GBM underwent postoperative chemoradiation therapy (chemo-RT) and longitudinal MRI before surgery, in the early postoperative period, and at 1-month intervals thereafter. The role of the patient age, Karnofsky performance scale (KPS) score, minimum apparent diffusion coefficient (ADC) on pretreatment DW-MRI, and gross residual tumor on early postoperative MRI were assessed by factor analysis of overall survival (OS). Survival curves were calculated using the Kaplan-Meier method; the multivariate Cox's proportional hazards model was used to adjust for the influence of prognostic factors. Radiation Therapy Oncology Group-recursive partitioning analysis (RTOG-RPA) criteria were used to validate the predictive value of the MRI-derived factors. RESULTS: Substantial independent prognostic factors were the KPS score (hazard ratio [HR], 1.812), minimum ADC (HR, 2.365), and gross residual tumor (HR, 1.777). Based on MRI-derived factors, we assigned the patients to different prognostic groups in the RTOG-RPA classification and grouped them according to the level of risk, i.e. a high-risk group with low minimum ADCs (<0.93 × 10(-3) mm(2)/s) with gross residual tumor and a low-risk group with high minimum ADCs (≥0.93 × 10(-3) mm(2)/s) without gross residual tumor; the other patients were assigned to the intermediate-risk group. Median OS for the low-, intermediate-, and high-risk groups were 28.2, 14.7, and 10.8 months, respectively (P < 0.001). CONCLUSION: The minimum ADC on pretreatment DW-MRI and gross residual tumor on early postoperative MRI can predict the survival in GBM patients treated with postoperative chemo-RT.

Linear ubiquitination-induced necrotic tumor remodeling elicits immune evasion.

Tumor-elicited inflammation confers tumorigenic properties, including cell death resistance, proliferation, or immune evasion. To focus on inflammatory signaling in tumors, we investigated linear ubiquitination, which enhances the nuclear factor-κB signaling pathway and prevents extrinsic programmed cell death under inflammatory environments. Here, we showed that linear ubiquitination was augmented especially in tumor cells around a necrotic core. Linear ubiquitination allowed melanomas to tolerate the hostile tumor microenvironment and to extend a necrosis-containing morphology. Loss of linear ubiquitination resulted in few necrotic lesions and growth regression, further leading to repression of innate anti-PD-1 therapy resistance signatures in melanoma as well as activation of interferon responses and antigen presentation that promote immune-mediated tumor eradication. Collectively, linear ubiquitination promotes tumor-specific tissue remodeling and the ensuing immune evasion.

Impacts of dosing and drug withdrawal period on tacrolimus-based triple therapy in a non-human primate renal transplantation model.

Non-human primate (NHP) renal transplantation models are widely used vivo models for researching new immunosuppressive therapies including allograft tolerance strategies. To enroll animals into a tolerance study, an immunosuppressive regimen that efficiently establishes stable renal function in NHPs is needed. Here, we assessed the effect of triple therapy comprising 2.0 mg/kg tacrolimus, mycophenolate mofetil and a steroid and its success rate for achieving stable renal function. In addition, to predict the pathophysiological consequences of withdrawing immunosuppressants, an indispensable process after induction of tolerance, we also assessed changes in the stable renal state maintained by triple therapy after drug withdrawal. Six cynomolgus monkeys were used. The median survival time was >176 days over the dosing period and 45 days after drug withdrawal. The triple therapy successfully induced stable graft function without calcineurin inhibitor nephrotoxicity in three of six recipients, although adopting trough-dependent tacrolimus dose adjustment rather than a preset dose regimen could improve on the present strategy. Further, drug withdrawal led to deterioration of renal function, de novo donor specific antibody production and increased the memory/naïve T cell ratio within two weeks post drug withdrawal. We expect that these findings contribute to establish one of the choices for animal model for evaluating future tolerance therapy for renal transplantation.

Laterality on FDG-PET/CT in clinically node-negative early-stage oral squamous cell carcinoma: a retrospective analysis of patients with late neck metastasis.

This study aimed to assess the role of preoperative 18F-fluorodeoxyglucose (FDG)-positron emission tomography (PET)/computed tomography (CT) for predicting late neck metastasis in clinically node-negative (cN0) early-stage oral squamous cell carcinoma (OSCC). We retrospectively investigated the standardized uptake value (SUV) parameters in patients with late neck metastasis based on the neck node level. The study population consisted of 16 patients with cT1N0 or cT2N0 oral SCC who were evaluated with dual-phase FDG-PET/CT and were treated with local resection of the primary tumor and watchful waiting for neck management. The SUV at each level was measured on the early and delayed images, and the laterality of the SUV was calculated. The laterality on the delayed images significantly differed between positive and negative pairs at the levels Ib (p = 0.002) and IIb (p = 0.013); a cut-off value of 1.4 yielded a true-positive rate of 50% and a false-positive rate of 6%. The laterality of FDG-uptake should be used to stratify the risk for nodal-level metastasis.

Longitudinal changes over 2 years in parotid glands of patients treated with preoperative 30-Gy irradiation for oral cancer.

OBJECTIVE: To evaluate longitudinal changes in parotid volumes and saliva production over 2 years after 30 Gy irradiation. METHODS: We retrospectively evaluated 15 assessable patients treated for advanced oral cancer. Eligibility criteria were a pathologic diagnosis of squamous cell carcinoma, preoperative radiation therapy with a total dose of 30 Gy delivered in 15 fractions, and the availability of longitudinal data of morphological assessments by computed tomography and functional assessments with the Saxon test spanning 2 years after radiation therapy. In the Saxon test, saliva production was measured by weighing a folded sterile gauze pad before and after chewing; the low-normal value is 2 g/2 min. Repeated-measures analysis of variance with Bonferroni adjustment for multiple comparisons was used to determine the longitudinal changes. RESULTS: The normalized ipsilateral parotid volumes 2 weeks and 6-, 12- and 24 months after radiation therapy were found to be 72.5, 63.7, 66.9 and 78.1%, respectively; the normalized contralateral volumes were 69.8, 64.6, 72.2 and 82.0%, respectively. The bilateral parotid volumes were significantly decreased after radiation therapy (P < 0.01). The nadir appeared at 6 months post-radiation therapy and the volumes substantially recuperated 24 months after radiation therapy (P < 0.01). Mean saliva production before radiation therapy was 3.7 g; the longitudinal changes after radiation therapy were 31.3, 38.0, 43.3 and 69.6%, respectively. Substantial recuperation of saliva production was observed 24 months after radiation therapy (P = 0.01). CONCLUSIONS: Although parotid volumes and saliva production were decreased after 30 Gy irradiation, we observed the recuperation of morphological and functional changes in the parotid glands 2 years after radiation therapy.

Oncolytic vaccinia virus induces a novel phenotype of CD8+ effector T cells characterized by high ICOS expression.

Characterization of the intratumoral immune status is important for developing immunotherapies and evaluating their antitumor effectiveness. CD8+ T cells are one of the most important cell types that directly and indirectly contribute to antitumor efficacy by releasing cytolytic molecules and inflammatory cytokines in the tumor microenvironment. Previously, we engineered a tumor-selective oncolytic vaccinia virus that encodes interleukin-7 (IL-7) and IL-12 and demonstrated its usefulness as an agent for in situ vaccination against tumors, with data showing that antitumor efficacy was reliant upon CD8+ T cells recruited by viral treatment. Here, we investigated the phenotypic changes in intratumoral CD8+ T cells caused by this oncolytic virus and found increased expression of inducible co-stimulator (ICOS) in PD-1-CD8+ T cells. Unlike previously reported ICOS+CD8+ T cells, a subset of ICOS+PD-1-CD8+ T cells showed effector function characterized by granzyme B expression. ICOS expression was induced by the backbone virus, which did not encode any immune transgenes and was independent of upregulation of the type I interferon pathway. Not only did we identify a novel effector cell subset characterized by ICOS expression, but our findings also shed light on a potential unknown aspect of the mechanism of oncolytic vaccinia virotherapy.

Can MRI-derived depth of invasion predict nodal recurrence in oral tongue cancer?

OBJECTIVES: To evaluate the prognostic value of preoperative radiological findings for nodal recurrence in clinically node-negative (cN0) patients with oral tongue squamous cell carcinoma (SCC). METHODS: The study population consisted of 52 patients with cT1-2N0 oral tongue SCC classified according to the 7th edition of the Union for International Cancer Control (UICC) staging system. The subjects had undergone preoperative radiological examinations, including magnetic resonance imaging (MRI) and 18F-fluorodeoxyglucose (FDG)-positron emission tomography (PET)/computed tomography. All patients were treated with local resection and watchful waiting for neck management. Using an unpaired t test, Pearson's chi-squared test, and the Kaplan-Meier method, the MRI-derived depth of invasion (DOI), the standardized uptake value (SUV) on FDG-PET, and the T stage according to the 7th and 8th UICC were assessed as prognostic factors. RESULTS: The MRI-derived DOI was recorded as ≤ 5 mm in 24 patients and > 5 mm in 28 patients. During the follow-up period, nine patients exhibited nodal recurrence, with the MRI-derived DOI being significantly higher in patients with positive than in those with negative (p = 0.011). The SUV was not significant. Five-year cumulative nodal recurrence probabilities were 4.5% for patients with an MRI-derived DOI ≤ 5 mm, while it was 32.1% for > 5 mm (p = 0.013). Although the T classifications were not significant, none of our patients whose T stage according to the 8th UICC was T1 suffered nodal recurrence. CONCLUSIONS: MRI-derived DOI can predict nodal recurrence, while preoperative information may assist in treatment planning for oral tongue SCC.

Extracellular vesicles derived from radioresistant oral squamous cell carcinoma cells contribute to the acquisition of radioresistance via the miR-503-3p-BAK axis.

Despite advancements in treatments, oral squamous cell carcinoma (OSCC) has not significantly improved in prognosis or survival rate primarily due to the presence of treatment-resistant OSCC. The intercellular communication between tumour cells is a molecular mechanism involved in acquiring OSCC treatment resistance. Extracellular vesicles (EVs) and encapsulated miRNAs are important mediators of intercellular communication. Here, we focused on EVs released from clinically relevant radioresistant (CRR) OSCC cells. Additionally, we evaluated the correlation between miRNA expression in the serum samples of patients who showed resistance to radiotherapy and in EVs released from CRR OSCC cells. We found that EVs released from CRR OSCC cells conferred radioresistance to radiosensitive OSCC cells via miR-503-3p contained in EVs. This miR-503-3p inhibited BAK and impaired the caspase cascade to suppress radiation-induced apoptosis. Furthermore, OSCC cells with BAK knockdown had increased radioresistance. Additionally, the expression of circulating miR-503-3p in patients with OSCC was correlated with a poor treatment response and prognosis of radiotherapy. Our results provide new insights into the relationship between EVs and the radioresistance of OSCC and suggest that the miR-503-3p-BAK axis may be a therapeutic target and that circulating miR-503-3p is a useful prognostic biomarker in the radiotherapy of OSCC.

Radiation-induced parotid gland changes in oral cancer patients: correlation between parotid volume and saliva production.

OBJECTIVE: To evaluate whether saliva production reflects the parotid volume during the course of radiation therapy (RT) in patients with head-and-neck cancer. METHODS: Twenty patients with advanced oral squamous cell carcinomas, who were treated with preoperative chemo-RT, underwent morphological assessment with CT or MRI and functional assessment with the Saxon test. For the Saxon test, saliva production was measured by weighing a gauze pad before and 2 min after chewing without swallowing; the low-normal value is 2 g. Saliva production and parotid volumes before and 2 weeks after RT were compared with the paired t-test, the Spearman rank correlation test and the Fisher exact test. RESULTS: After 30 Gy irradiation, mean saliva production was decreased from 4.2 to 1.0 g (P < 0.01); the reduction in saliva production ranged from 1.7 to 5.4 g (mean 3.2 g). The mean parotid volume was decreased from 68.2 to 47.9 cm(3) (P < 0.01); the post-RT:pre-RT parotid volume ratio ranged from 54% to 85% (mean 71%). Although the initial parotid ;volume was correlated with initial saliva production (r = 0.47, P = 0.04), no significant correlation was noted after RT (r = 0.08, P = 0.71), and there were considerable individual variations. The parotid volume ratio was inversely correlated with the saliva-reduction amount (r = - 0.79, P < 0.01). CONCLUSIONS: There was a correlation between decreased parotid gland volume and decreased saliva production in patients with head-and-neck cancer undergoing RT. Parotid volume reduction may predict parotid gland function.

Intratumoral expression of IL-7 and IL-12 using an oncolytic virus increases systemic sensitivity to immune checkpoint blockade.

The immune status of the tumor microenvironment is a key indicator in determining the antitumor effectiveness of immunotherapies. Data support the role of activation and expansion of tumor-infiltrating lymphocytes (TILs) in increasing the benefit of immunotherapies in patients with solid tumors. We found that intratumoral injection of a tumor-selective oncolytic vaccinia virus encoding interleukin-7 (IL-7) and IL-12 into tumor-bearing immunocompetent mice activated the inflammatory immune status of previously poorly immunogenic tumors and resulted in complete tumor regression, even in distant tumor deposits. Mice achieving complete tumor regression resisted rechallenge with the same tumor cells, suggesting establishment of long-term tumor-specific immune memory. Combining this virotherapy with anti-programmed cell death-1 (PD-1) or anti-cytotoxic T lymphocyte antigen 4 (CTLA4) antibody further increased the antitumor activity as compared to virotherapy alone, in tumor models unresponsive to either of the checkpoint inhibitor monotherapies. These findings suggest that administration of an oncolytic vaccinia virus carrying genes encoding for IL-7 and IL-12 has antitumor activity in both directly injected and distant noninjected tumors through immune status changes rendering tumors sensitive to immune checkpoint blockade. The benefit of intratumoral IL-7 and IL-12 expression was also observed in humanized mice bearing human cancer cells. These data support further investigation in patients with non-inflamed solid tumors.

Onodera's prognostic nutritional index correlates with tumor immune environment and survival in patients with oral squamous cell carcinoma undergoing chemoradiotherapy.

Pretreatment nutritional and immunological status is useful for predicting survival outcomes for various types of malignant tumors. Our objective was to determine the impact of the pretreatment Onodera's prognostic nutritional index (OPNI) on outcomes of patients who underwent definitive chemoradiotherapy for advanced oral squamous cell carcinoma (OSCC). We reviewed 47 patients treated for OSCC with definitive chemoradiotherapy (CRT) at our institution between January 2004 and December 2011. We determined the OPNI according to the following formula: 10 × serum albumin (g/dL) + 0.005 × total lymphocyte count (per µL). We determined the optimum OPNI cut-off through a receiver operating characteristic analysis. We analyzed the associations between OPNI status and various clinicopathological features and evaluated the effects of OPNI on the prognosis. We examined the relationships between OPNI and systemic inflammatory response parameters and analyzed intratumoral CD8+ T cells and their correlation with OPNI. The optimum OPNI cut-off was 42.7. A Kaplan-Meier curve analysis revealed that low OPNI was significantly associated with poor overall survival and cause-specific survival. The multivariate analysis revealed that low OPNI was independently correlated with poor 5 year overall survival and cause-specific survival. OPNI was significantly correlated with systemic inflammatory response parameters. Intratumoral CD8+ T cell counts in primary tumors were significantly lower for low OPNI than for high OPNI. The present data demonstrate that pretreatment OPNI is a valuable independent prognostic indicator of overall and cause-specific survival in advanced OSCC following definitive CRT. OPNI might reflect the tumor immune microenvironment characterization in OSCC.

Enhanced Expression of IGFBP-3 Reduces Radiosensitivity and Is Associated with Poor Prognosis in Oral Squamous Cell Carcinoma.

Insulin-like growth factor (IGF) binding protein-3 (IGFBP-3) modulates various cell functions through IGF-dependent or independent mechanisms. However, its biological roles in the radiosensitivity of oral squamous cell carcinoma (OSCC) remain largely unknown. The purpose of this study was to determine the clinical significance and molecular mechanisms of the association between IGFBP-3 and OSCC radiosensitivity. We performed an immunohistochemical analysis of IGFBP-3 in 52 OSCC specimens from patients treated with preoperative chemoradiotherapy and surgery (phase II study). Associations between IGFBP-3 expression and clinicopathological features were also evaluated. In addition, we examined the effects of IGFBP-3 on post-X-ray irradiation radiosensitivity and DNA damage in vitro. High IGFBP-3 expression was significantly correlated with poor chemoradiotherapy responses and prognosis. With IGFBP-3 knockdown, irradiated OSCC cells exhibited significantly higher radiosensitivity compared with that of control cells. Moreover, IGFBP-3 depletion in OSCC cells reduced phosphorylation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which is required for DNA double-strand break repair during non-homologous end joining. These findings indicate that IGFBP-3 may have a significant role in regulating DNA repair and is be a potential biomarker for predicting clinical response to radiotherapy and prognosis in OSCC.