Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

Microarray Analysis Of The Transcriptional Response To Carbon Ion Irradiation In Murine Tumors

Purpose/Objective(s):The purpose of this study was to identify molecular mechanism induced by carbon ion radiotherapy in order to provide information on potential targets for prediction of its effectiveness.Materials/Methods:Murine squamous cell carcinomas, NR-S1 (resistant to gamma-irradiation), and SCCVII (sensitive), were transplanted in hind legs of C3H/He male mice and established solid tumors (7.5-8.5 mm in diameter) were locally irradiated with carbon ion beam at 30Gy. Carbon-12 ions were accelerated by the Heavy Ion Medical Accelerator in Chiba or HIMAC synchrotron up to 290 MeV/u with a dose rate of approximately 3 Gy /min. Tumor growth delay (TGD) time, reduction rate of tumor, and recurrence rate of tumor were investigated as parameters of radiosensitivity of tumors. The mice were sacrificed and immediately dissected before irradiation and after different time points, such as 6,12,18h, 1, 3, 5, 7, 10, 15, 20 days after irradiation or recurrence for transcriptome assays and pathological investigation. Expression analyses were performed using single-color analysis microarrays consisting of 55k genes. Principal Compornent Analysis (PCA) was used to investigate similarity of comprehensive overview of the changes in gene expression between expression profiles of two tumors. Analysis of variance (ANOVA) was applied to the intensity of each tumor at different time point to evaluate significant differences.Results:TGD time of NR-S1 and SCCVII was 30 days and 56 days, reduction rate of NR-S1 and SCCVII was 40% and 100%, and recurrence rate of NR-S1 and SCCVII was 75% and 50%, respectively.PCA showed that all expression profiles of NR-S1 were identified as a group, while those of SCCVII were identified as another group. Recurred tumors showed different profiles from non-irradiation control tumors. We detected genes, whose expressions were significantly up-regulated or down regulated at each time point after carbon-irradiation (p value< 0.0001). At 6 hours after irradiation, fourteen genes, which were related with cell cycle regulation, were differentially expressed in both tumors. Eleven genes, which were related with inflammation or extracellular matrix, were up-regulated at 6 hours in both tumors, however, their expression changes on time-course were different. Pathological specimen showed duplet cells in both tumors 1 day after irradiation and continuous infiltration of inflammatory cells in SCCVII.Conclusions:Tumor growth assays revealed that two murine tumors, which have different radiosensitivity to gamma irradiation, kept their intrinsic radiosensitivity to carbon-ion irradiation. Transcriptional profiling of two tumors identified a number of carbon-ion irradiation response genes in murine tumors. We have also identified genes as being candidates for predictive markers of radiosensitivity to carbon-ion therapy.48th Annual Meeting of the American Society for Therapeutic Radiology and Oncolog

EFNA1, a radioresistant marker, detected in a murine tumor model by gamma and carbon ion irradiation

Using single-color oligo-microarrays, we analyzed the gene expression profiles of two murine squamous cell carcinomas: NR-S1 (radioresistant) and SCCVII (radiosensitive), after irradiation with 137-Cs gamma rays at doses of 30, 50 and 70 Gy or 290 MeV/u carbon ions at a dose of 30 Gy. Potential genes related to radiosensitivity were selected by comparing the expression values before and after irradiation (with both gamma rays and carbon ions) using a filter for at least 1.5-fold changes. Furthermore, candidate genes which had significantly different ratio values between the two tumors (P < 0.05) were detected by unpaired Student\u27s t-tests. Subsequent analysis by quantitative reverse-transcription polymerase chain reaction (RT-PCR) confirmed our microarray data. Protein expression and function were examined by immunohistochemical studies.Results: Four genes, Efna1, Sprr1a, Srgap3 and Xrra1, were selected as potential genes related to radioresistance after gamma and carbon ion irradiation. RT-PCR confirmed that Efna1 was induced in radioresistant NR-S1. Efna1, a proangiogenic factor, was expressed in the cytoplasm of tumor cells and significant increases in microvascular density were observed in the radioresistant NR-S1.Conclusions: We found that Efna1 may be a potential molecule related to radioresistance in murine tumors.The 14th European Cancer Conference, ESTRO26 meeting, European Society for therapeutic radiology and oncolog

The proangiogenic factor ephrin-A1 is up-regulated in radioresistant murine tumor by irradiation.

While the pre-treatment status of cancer is generally correlated with outcome, little is known about microenvironmental change caused by anti-cancer treatment and how it may affect outcome. For example, treatment may lead to induction of gene expression that promotes resistance to therapy. In the present study, we attempted to find a gene that was both induced by irradiation and associated with radioresistance in tumors. Using single-color oligo-microarrays, we analyzed the gene expression profiles of two murine squamous cell carcinomas, NR-S1, which is highly radioresistant, and SCCVII, which is radiosensitive, after irradiation with 137-Cs gamma rays or carbon ions. Candidate genes were those differentially regulated between NR-S1 and SCCVII after any kind of irradiation. Four genes, Efna1 (Ephrin-A1), Sprr1a (small proline-rich protein 1A), Srgap3 (SLIT-ROBO Rho GTPase activating protein 3) and Xrra1 [RIKEN 2 days neonate thymus thymic cells (NOD) cDNA clone E430023D08 3\u27], were selected as candidate genes associated with radiotherapy-induced radioresistance. We focused on Efna1, which encodes a ligand for the Eph receptor tyrosine kinase known to be involved in the vascular endothelial growth factor (VEGF) pathway. We used immunohistochemical methods to detect expression of Ephrin-A1, VEGF, and the microvascular marker CD31 in radioresistant NR-S1 tumor cells. Ephrin-A1 was detected in the cytoplasm of NR-S1 tumor cells after irradiation, but not in SCCVII tumor cells. Irradiation of NR-S1 tumor cells also led to significant increases in microvascular density, and up-regulation of VEGF expression. Our results suggest that radiotherapy-induced changes in gene expression related with angiogenesis might also modulate microenvironment and influence responsiveness of tumors

Comparing the Japanese Version of the Ocular Surface Disease Index and Dry Eye-Related Quality-of-Life Score for Dry Eye Symptom Assessment

The aim of this study was to compare patient-reported symptoms of dry eye disease (DED) between the Japanese version of the Ocular Surface Disease Index (J-OSDI) and the Dry Eye-Related Quality-of-Life Score (DEQS). A total of 169 participants were enrolled between September 2017 and May 2018. Patients were administered the J-OSDI and DEQS questionnaires at their first (baseline) and follow-up visits to evaluate DED-related symptoms. The correlations between the J-OSDI total score and DEQS (Frequency and Degree) scores were evaluated using Pearson&rsquo;s correlation coefficient, and their clinical differences were assessed using the Bland&ndash;Altman analysis. At the baseline visit, the J-OSDI score and DEQS (Frequency and Degree) were significantly correlated (r = 0.855, r = 0.897, respectively). Moreover, a significant correlation was found between the J-OSDI score and DEQS (Frequency and Degree) at the follow-up visit (r = 0.852, r = 0.888, respectively). The Bland&ndash;Altman analysis revealed a difference (bias) of 4.18 units at the baseline and 4.08 units at the follow-up between the scores of the two questionnaires. The J-OSDI and DEQS were significantly correlated with negligible score differences, suggesting that the J-OSDI can be reliably used for Japanese patients, allowing for cross-country comparisons

Using the AllerSearch Smartphone App to Assess the Association Between Dry Eye and Hay Fever: mHealth-Based Cross-Sectional Study

BackgroundDry eye (DE) and hay fever (HF) show synergistic exacerbation of each other’s pathology through inflammatory pathways. ObjectiveThis study aimed to investigate the association between DE and HF comorbidity and the related risk factors. MethodsA cross-sectional observational study was conducted using crowdsourced multidimensional data from individuals who downloaded the AllerSearch smartphone app in Japan between February 2018 and May 2020. AllerSearch collected the demographics, medical history, lifestyle and residential information, HF status, DE symptoms, and HF-related quality of life. HF symptoms were evaluated using the nasal symptom score (0-15 points) and nonnasal symptom score (0-12 points). HF was defined by the participants’ responses to the questionnaire as HF, non-HF, or unknown. Symptomatic DE was defined as an Ocular Surface Disease Index total score (0-100 points), with a threshold score of 13 points. HF-related quality of life was assessed using the Japanese Allergic Conjunctival Disease Standard Quality of Life Questionnaire (0-68 points). We conducted a multivariable linear regression analysis to examine the association between the severity of DE and HF symptoms. We subsequently conducted a multivariable logistic regression analysis to identify the factors associated with symptomatic DE (vs nonsymptomatic DE) among individuals with HF. Dimension reduction via Uniform Manifold Approximation and Projection stratified the comorbid DE and HF symptoms. The symptom profiles in each cluster were identified using hierarchical heat maps. ResultsThis study included 11,284 participants, classified into experiencing HF (9041 participants), non-HF (720 participants), and unknown (1523 participants) groups. The prevalence of symptomatic DE among individuals with HF was 49.99% (4429/9041). Severe DE symptoms were significantly associated with severe HF symptoms: coefficient 1.33 (95% CI 1.10-1.57; P<.001) for mild DE, coefficient 2.16 (95% CI 1.84-2.48; P<.001) for moderate DE, and coefficient 3.80 (95% CI 3.50-4.11; P<.001) for severe DE. The risk factors for comorbid symptomatic DE among individuals with HF were identified as female sex; lower BMI; medicated hypertension; history of hematologic, collagen, heart, liver, respiratory, or atopic disease; tomato allergy; current and previous mental illness; pet ownership; living room and bedrooms furnished with materials other than hardwood, carpet, tatami, and vinyl; discontinuation of contact lens use during the HF season; current contact lens use; smoking habits; and sleep duration of <6 hours per day. Uniform Manifold Approximation and Projection stratified the heterogeneous comorbid DE and HF symptoms into 14 clusters. In the hierarchical heat map, cluster 9 was comorbid with the most severe HF and DE symptoms, and cluster 1 showed severe HF symptoms with minimal DE-related symptoms. ConclusionsThis crowdsourced study suggested a significant association between severe DE and HF symptoms. Detecting DE among individuals with HF could allow effective prevention and interventions through concurrent treatment for ocular surface management along with HF treatment

Diagnostic Ability of a Smartphone App for Dry Eye Disease: Protocol for a Multicenter, Open-Label, Prospective, and Cross-sectional Study.

BACKGROUND: Dry eye disease (DED) is one of the most common ocular surface diseases. Numerous patients with DED remain undiagnosed and inadequately treated, experiencing various subjective symptoms and a decrease in quality of life and work productivity. A mobile health smartphone app, namely, the DEA01, has been developed as a noninvasive, noncontact, and remote screening device, in the context of an ongoing paradigm shift in the health care system, to facilitate a diagnosis of DED. OBJECTIVE: This study aimed to evaluate the capabilities of the DEA01 smartphone app to facilitate a DED diagnosis. METHODS: In this multicenter, open-label, prospective, and cross-sectional study, the test method will involve using the DEA01 smartphone app to collect and evaluate DED symptoms, based on the Japanese version of the Ocular Surface Disease Index (J-OSDI), and to measure the maximum blink interval (MBI). The standard method will then involve a paper-based J-OSDI evaluation of subjective symptoms of DED and tear film breakup time (TFBUT) measurement in an in-person encounter. We will allocate 220 patients to DED and non-DED groups, based on the standard method. The primary outcome will be the sensitivity and specificity of the DED diagnosis according to the test method. Secondary outcomes will be the validity and reliability of the test method. The concordance rate, positive and negative predictive values, and the likelihood ratio between the test and standard methods will be assessed. The area under the curve of the test method will be evaluated using a receiver operating characteristic curve. The internal consistency of the app-based J-OSDI and the correlation between the app-based J-OSDI and paper-based J-OSDI will be assessed. A DED diagnosis cutoff value for the app-based MBI will be determined using a receiver operating characteristic curve. The app-based MBI will be assessed to determine a correlation between a slit lamp-based MBI and TFBUT. Adverse events and DEA01 failure data will be collected. Operability and usability will be assessed using a 5-point Likert scale questionnaire. RESULTS: Patient enrollment will start in February 2023 and end in July 2023. The findings will be analyzed in August 2023, and the results will be reported from March 2024 onward. CONCLUSIONS: This study may have implications in identifying a noninvasive, noncontact route to facilitate a diagnosis of DED. The DEA01 may enable a comprehensive diagnostic evaluation within a telemedicine setting and facilitate early intervention for undiagnosed patients with DED confronting health care access barriers. TRIAL REGISTRATION: Japan Registry of Clinical Trials jRCTs032220524; https://jrct.niph.go.jp/latest-detail/jRCTs032220524. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): PRR1-10.2196/45218