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This study aimed to elucidate the effect of radiotherapy on expression of immune response-related genes in cervical cancer tissues.Tumor tissues were obtained from 16 patients with cervical cancer before initiation of radiotherapy and after treatment with 10 Gy X-rays, delivered in five fractions. Expression of 730 immune response-related genes was assessed using an nCounter PanCancer Immune Profiling Panel (NanoString Technologies. Seattle, WA, USA).Of the 730 genes examined, 41 showed significant changes (fold change of1.5 or0.66) in expression in post-radiotherapy samples (28 up-regulated and 13 down-regulated). Analysis of immune cell type-specific genes suggested predominant upregulation of those related to innate immunity postradiotherapy. Interestingly, cytotoxic T-lymphocyte-associated protein (CTLA4), a key negative regulator of T-cell activation, was marked down-regulated in 93.7% of patients, with an average fold-change of 2.0.To our knowledge, this study is the first to show down-regulation of CTLA4 in clinical cervical cancer tissues after treatment with radiotherapy.

This study aimed to establish a setup for ultra-high-dose-rate (FLASH) carbon-ion irradiation, and to conduct the first human cell experiments using FLASH carbon ions.A system for FLASH carbon-ion irradiation (1-3 Gy at 13 or 50 keV/μm) was developed. The growth and senescence of HFL1 lung fibroblasts were assessed by crystal violet staining assays and senescence-associated β-galactosidase staining, respectively. Survival of HSGc-C5 cancer cells was assessed by clonogenic assays.The dose rates of carbon ions ranged from 96-195 Gy/s, meeting the definition of FLASH. With both 13 and 50 keV/μm beams, no FLASH sparing effect was observed on the growth suppression and senescence of HFL1 cells, nor on the survival of HSGc-C5 cells.We successfully conducted the first human cell experiments with FLASH carbon ions. No FLASH effect was observed under the conditions examined.

To evaluate the usefulness of single-energy metal artifact reduction (SEMAR) for target delineation in brachytherapy for cervical cancer patients with metal hip implants.A series of four definitive brachytherapy sessions in the same patient was analyzed. At each brachytherapy session, the identical set of computed tomography images was subjected with or without SEMAR treatment. For both SEMAR-treated and -untreated sets, five radiation oncologists delineated the high-risk clinical target volume (HR-CTV), bladder, and rectum, for which the volume, Dice coefficient, and the dose volume parameters were compared between SEMAR-treated and -untreated datasets.The bladder volume was significantly greater in the SEMAR-treated datasets compared with the SEMAR-untreated datasets. Importantly, for the bladder, Dice coefficient among five radiation oncologists was significantly higher for the SEMAR-treated datasets compared with the SEMAR-untreated datasets. These effects of SEMAR treatment were not evident for HR-CTV and the rectum.These data indicate that SEMAR treatment contributes to improve delineation of the bladder in brachytherapy for cervical cancer patients with metal hip implants.

Purpose Tumor mutational burden (TMB) is a surrogate biomarker of neo-antigens and high TMB status is associated with favorable response to immune-checkpoint inhibitors (ICIs). This study aimed to elucidate the association between TMB and the outcome of definitive radiotherapy in patients with cervical cancer. Materials and methods TMB and treatment outcome were retrospectively analyzed in patients with newly diagnosed cervical cancer treated with definitive radiotherapy available with somatic mutation data of pre-treatment tumors obtained using a commercially available gene panel. Results The study enrolled 98 patients (median follow-up period, 61 months). The median TMB was 9.5 mutations per megabase (range, 3.0–35.5 mutations per megabase). After dichotomization based on this median value, the 5-year overall survival (OS) for TMB-high patients was significantly worse than that of TMB-low patients (61.1% vs. 82.2%). Multivariate analysis identified high TMB status as a significant prognostic factor for worse OS, along with advanced stage, para-aortic lymph node involvement, and absence of concurrent chemotherapy. Conclusion These data indicate that TMB is a potential prognostic factor for worse survival in patients with cervical cancer treated with definitive radiotherapy, thereby providing a rationale for treatment of TMB-high cervical cancers with a combination of ICIs plus radiotherapy. Secondary abstract This retrospective study of 98 patients demonstrates for the first time that tumor mutational burden (TMB) is an independent prognostic factor for worse overall survival of patients treated with definitive radiotherapy, providing a rationale for treatment of TMB-high cervical cancers with a combination of immune-checkpoint inhibitors plus radiotherapy.

BACKGROUND/AIM Carbon-ion radiotherapy has strong antitumor effects in X-ray-resistant tumors. However, the mechanisms underlying the strong antitumor effect, especially on metabolic alterations, are not fully elucidated. This study aimed to determine the effect of therapeutic carbon ions on metabolic alterations in cancer cells. MATERIALS AND METHODS Five human cancer cell lines were used in this study. The change in 333 metabolite levels in response to carbon ions was analyzed using gas chromatography-mass spectrometry. RESULTS Fifty-two metabolites were commonly detected in all cell lines. The levels of five metabolites significantly changed in four or more cell lines. Three of the five metabolites (i.e., 2-ketoglutaric acid, fumaric acid and 2-hydroxyglutaric acid) were associated with the TCA cycle. TCA cycle intermediates and the downstream oncometabolite 2-hydroxyglutaric acid were up-regulated by carbon ions. CONCLUSION We demonstrated for the first time that TCA cycle intermediates and 2-hydroxyglutaric acid are up-regulated after carbon-ion irradiation.

Ionizing radiation activates cytoprotective pathways in cancer cells. Fibroblast growth factor receptor (FGFR) is a key player in these pathways. Thus, FGFR signaling is a potential target to induce radiosensitization. LY2874455 is an orally administrable selective pan-FGFR inhibitor. However, the radiosensitizing effects of LY2874455 remain unclear. In this study, we addressed this issue by using radioresistant human cancer cell lines H1703 (FGFR1 mutant), A549 (FGFR1–4 wild-type), and H1299 (FGFR1–4 wild-type). At an X-ray dose corresponding to 50%-clonogenic survival as the endpoint, 100 nM LY2874455 increased the sensitivity of H1703, A549, and H1299 cells by 31%, 62%, and 53%, respectively. The combination of X-rays and LY2874455 led to a marked induction of mitotic catastrophe, a hallmark of radiation-induced cell death. Furthermore, combination treatment suppressed the growth of A549 xenografts to a significantly greater extent than either X-rays or the drug alone without noticeable toxicity. This is the first report to show the radiosensitizing effect of a selective pan-FGFR inhibitor. These data suggest the potential efficacy of LY2874455 as a radiosensitizer, warranting clinical validation.

Objective To elucidate tumor mutation profiles associated with outcomes of uterine cervical cancer (UCC) patients treated with definitive radiotherapy. Methods Ninety-eight patients with newly diagnosed and pathologically confirmed UCC (82 squamous cell carcinomas, 12 adenocarcinomas, and four adenosquamous carcinomas) who were treated with definitive radiotherapy were analyzed. DNA was extracted from pre-treatment tumor biopsy specimens. The exons of 409 cancer-related genes were sequenced using a next-generation sequencer. Genetic mutations were identified and analyzed for correlations with clinical outcome. Results Recurrent mutations were observed in PIK3CA (35.7%), ARID1A (25.5%), NOTCH1 (19.4%), FGFR3 (16.3%), FBXW7 (19.4%), TP53 (13.3%), EP300 (12.2%), and FGFR4 (10.2%). The prevalence of mutations in FGFR family genes (i.e., FGFR1–4) was almost as high (24.5%) as that in PIK3CA and ARID1A, both of which are well-studied drivers of UCC. Fifty-five percent (21 of 38) of the identified FGFR mutations were located in the FGFR protein tyrosine kinase domain. Five-year progression-free survival (PFS) rates for FGFR mutation-positive patients (n = 24) were significantly worse than those for FGFR mutation-negative patients (n = 74) (43.9% vs. 68.5%, respectively; P = 0.010). Multivariate analysis identified FGFR mutations as significant predictors of worse 5 year PFS (P = 0.005), independent of clinicopathological variables. Conclusions FGFR mutations are associated with worse PFS in UCC patients treated with definitive radiotherapy. These results warrant further validation in prospective studies.

Radiotherapy treatment strategies should be personalized based on the radiosensitivity of individual tumors. Clonogenic assays are the gold standard method for in vitro assessment of radiosensitivity. Reproducibility is the critical factor for scientific rigor; however, this is reduced by insufficient reporting of methodologies. In reality, the reporting standards of methodologies pertaining to clonogenic assays remain unclear. To address this, we performed a literature search and qualitative analysis of the reporting of methodologies pertaining to clonogenic assays. A comprehensive literature review identified 1672 papers that report the radiosensitivity of human cancer cells based on clonogenic assays. From the identified papers, important experimental parameters (i.e. number of biological replicates, technical replicates, radiation source and dose rate) were recorded and analyzed. We found that, among the studies, (i) 30.5% did not report biological or technical replicates; (ii) 47.0% did not use biological or technical replicates; (iii) 3.8% did not report the radiation source; and (iv) 32.3% did not report the dose rate. These data suggest that reporting of methodologies pertaining to clonogenic assays in a considerable number of previously published studies is insufficient, thereby threatening reproducibility. This highlights the need to raise awareness of standardization of the methodologies used to conduct clonogenic assays.

Background Nasopharyngeal cancer is endemic to Southeast Asia. However, there is limited clinical evidence of nasopharyngeal cancer in Indonesia, which has the largest population in Southeast Asia. Methods Patterns of care and treatment outcomes in 428 patients with newly-diagnosed and pathologically-confirmed nasopharyngeal cancer were retrospectively analyzed. Results Concurrent chemo-radiotherapy (CCRT) was the first-line treatment for stages I-IVB diseases. The 2-year overall survival (OS) of all patients were 100.0%, 100.0%, 93.8%, 86.2%, 82.9%, and 62.4% for stages I, II, III, IVA, IVB, and IVC, respectively. The 2-year OS of CCRT-treated patients were 100.0%, 100.0%, 92.6%, 82.4%, and 78.3% for stages I, II, III, IVA, and IVB, respectively. Conclusion The patterns of care and treatment outcomes were potentially consistent with world standards, needing future validation. This is the largest study of newly diagnosed nasopharyngeal cancer in Indonesia, a huge disease burden, providing an important basis for the clinical management of this disease.

Little information is available on the risk factors for nasolacrimal duct obstruction after radiotherapy for head and neck tumors. We investigated the incidence and predictive dosimetric parameters for nasolacrimal duct obstruction following carbon-ion radiotherapy for head and neck tumors.Twenty-eight patients with head and neck non-squamous cell carcinoma were analyzed in this single-institution prospective study. More than half of the tumors were located in the nasal cavity and maxillary sinus. Carbon-ion radiotherapy consisting of 57.6 or 64.0 Gy(relative biological effectiveness; RBE) in 16 fractions was administered. Nasolacrimal duct obstruction was recorded according to Common Terminology Criteria for Adverse Events version 4.0. Cutoff values were determined using receiver operating characteristic (ROC) curve analysis. VX indicates the volume irradiated with X Gy(RBE).The median follow-up period was 60.3 months. Incidences of Grade 1 and 2 nasolacrimal duct obstructions were 46% (13/28) and 7% (2/28), respectively; no Grade 3 or greater toxicities were recorded. Throughout the dose range, the volumes of the irradiated nasolacrimal ducts were significantly higher in the obstruction-positive patients than in the obstruction-negative patients (p 0.001 for V10, V20, V30, V40, V50, and V60). Cutoff values determined by the ROC curve analysis classified the obstruction-positive patients with an accuracy of96% over the entire range of V10-V60.The incidence and predictive dosimetric parameters for nasolacrimal duct obstruction after carbon-ion radiotherapy were demonstrated in a prospective cohort. These data should help optimize carbon-ion radiotherapy treatments for patients with head and neck tumors.

The metabolic dependencies of cancer cells have substantial potential to be exploited to improve the diagnosis and treatment of cancer. Creatine riboside (CR) is identified as a urinary metabolite associated with risk and prognosis in lung and liver cancer. However, the source of high CR levels in patients with cancer as well as their implications for the treatment of these aggressive cancers remain unclear. By integrating multiomics data on lung and liver cancer, we have shown that CR is a cancer cell-derived metabolite. Global metabolomics and gene expression analysis of human tumors and matched liquid biopsies, together with functional studies, revealed that dysregulation of the mitochondrial urea cycle and a nucleotide imbalance were associated with high CR levels and indicators of a poor prognosis. This metabolic phenotype was associated with reduced immune infiltration and supported rapid cancer cell proliferation that drove aggressive tumor growth. CRhi cancer cells were auxotrophic for arginine, revealing a metabolic vulnerability that may be exploited therapeutically. This highlights the potential of CR not only as a poor-prognosis biomarker but also as a companion biomarker to inform the administration of arginine-targeted therapies in precision medicine strategies to improve survival for patients with cancer.

The promising results of the PACIFIC study led to the approval of consolidation durvalumab for coverage by the National Health Insurance (NHI) in 2018 for patients with locally-advanced unresectable non-small cell lung carcinoma (NSCLC) treated with definitive concurrent chemoradiotherapy (CCRT). However, the effect of NHI coverage on the patterns of care for this population remains unclear. Here, we conducted a questionnaire-based survey to determine the patterns of care for patients with stage II-III NSCLC treated with definitive radiotherapy in 2017 (pre-durvalumab era) or in 2019 (post-durvalumab era). Data were obtained from 11 radiotherapy facilities in Gunma prefecture, which has a population of 1.94 million. We identified 80 and 83 patients with stage II-III NSCLC who received definitive radiotherapy in Gunma in 2017 and 2019, respectively. At a given facility, CCRT was the treatment of choice in a significantly greater proportion of patients in 2019 than in 2017 (66% ± 20% vs 51% ± 29%, P = 0.041). Intensity-modulated radiotherapy (IMRT) was more frequent in 2019 than in 2017 (24% vs 1.2%). Carboplatin plus paclitaxel was used for CCRT at higher rate in 2019 than in 2017 (73% vs 44%). Consolidation durvalumab was performed in 73% (40/55) of CCRT-treated patients in 2019, and the treatment was performed for the planned 12 months in 45% (18/40) of patients. These data indicate that NHI coverage of durvalumab might be a possible reason for choosing CCRT in patients with stage II-III NSCLC in the real-world setting.

Background and purpose Integrated analysis of existing radiosensitivity data obtained by the gold-standard clonogenic assay has the potential to improve our understanding of cancer cell radioresistance. However, extraction of radiosensitivity data from the literature is highly labor-intensive. To aid in this task, using deep convolutional neural networks (CNNs) and other computer technologies, we developed an analysis pipeline that extracts radiosensitivity data derived from clonogenic assays from the literature. Materials and methods Three classifiers (C1–3) were developed to identify publications containing radiosensitivity data derived from clonogenic assays. C1 uses Faster Regions CNN with Inception Resnet v2 (fRCNN-IRv2), VGG-16, and Optical Character Recognition (OCR) to identify publications that contain semi-logarithmic graphs showing radiosensitivity data derived from clonogenic assays. C2 uses fRCNN-IRv2 and OCR to identify publications that contain bar graphs showing radiosensitivity data derived from clonogenic assays. C3 is a program that identifies publications containing keywords related to radiosensitivity data derived from clonogenic assays. A program (iSF2) was developed using Mask RCNN and OCR to extract surviving fraction after 2-Gy irradiation (SF2) as assessed by clonogenic assays, presented in semi-logarithmic graphs. The efficacy of C1–3 and iSF2 was tested using seven datasets (1805 and 222 publications in total, respectively). Results C1–3 yielded sensitivity of 91.2% ± 3.4% and specificity of 90.7% ± 3.6%. iSF2 returned SF2 values that were within 2.9% ± 2.6% of the SF2 values determined by radiation oncologists. Conclusion Our analysis pipeline is potentially useful to acquire radiosensitivity data derived from clonogenic assays from the literature.

Programmed death-ligand 1 (PD-L1) is a key factor influencing cancer immunotherapy; however, the regulation of PD-L1 expression in cancer cells remains unclear, particularly regarding DNA damage, repair and its signalling. Herein, we demonstrate that oxidative DNA damage induced by exogenously applied hydrogen peroxide (H2O2) upregulates PD-L1 expression in cancer cells. Further, depletion of the base excision repair (BER) enzyme DNA glycosylase augments PD-L1 upregulation in response to H2O2. PD-L1 upregulation in BER-depleted cells requires ATR/Chk1 kinase activities, demonstrating that PD-L1 upregulation is mediated by DNA damage signalling. Further analysis of The Cancer Genome Atlas revealed that the expression of PD-L1 is negatively correlated with that of the BER/single-strand break repair (SSBR) and tumours with low BER/SSBR gene expression show high microsatellite instability and neoantigen production. Hence, these results suggest that PD-L1 expression is regulated in cancer cells via the DNA damage signalling and neoantigen–interferon-γ pathway under oxidative stress.

Background Adenoid cystic carcinoma (ACC) is a rare malignant tumor involving mostly the head and neck region, and frequently the salivary glands. The development of lung metastasis after treatment of the primary tumor is a common occurrence in ACC. Although lung metastases show a slow rate of growth, approximately 10% of patients die from distant metastases. The radioresistance of ACC limits the efficacy of conventional radiotherapy for lung metastases, and the optimal dose remains to be determined. Stereotactic body radiotherapy (SBRT) using CyberKnife can deliver a high dose to the lung tumor, while sparing the surrounding normal lung tissues, leading to favorable local control in non-squamous cell lung cancer and metastatic lung tumors. We report a case of lung metastases from ACC treated successfully with SBRT using CyberKnife. Case presentation A 76-year-old Japanese man with ACC who was treated with carbon ion radiotherapy for a primary oropharynx tumor presented with three metastatic lesions in the lung. The tumor masses were located in the right upper, right lower, and left lower lobes of the lungs. Surgical resection was not indicated because of the presence of multiple tumors. The patient underwent SBRT at 60 Gy in 10 sequential fractions for each tumor. The biologically effective dose based on an alpha/beta ratio of 2 Gy was 240 Gy per tumor. The percentage of the total lung volume irradiated with >20 Gy was 4.9%, 3.2%, and 2.6% for each tumor. The patient developed acute radiation pneumonitis during the initial therapy, which resolved at 6 months after the CyberKnife treatment. At 21 months after the first CyberKnife treatment, three tumors showed no signs of recurrence. No late toxicity was observed. Conclusions SBRT using CyberKnife is an effective and feasible approach to the management of multiple lung metastases of ACC.

Castration-resistant prostate cancer shows resistance to not only androgen deprivation therapy (ADT) but also X-ray therapy. On the other hand, carbon ion beams have a high biological effect and are used for various cancers showing resistance to X-ray therapy. The purposes of this study are to clarify the difference in the sensitivity of Castration-resistant prostate cancer to X-ray and carbon ion beams and to elucidate the mechanism. The androgen-insensitive prostate cancer cell line LNCaP-LA established by culturing the androgen-sensitive prostate cancer cell line LNCaP for 2 years in androgen-free medium was used for this study. First, colony formation assays were performed to investigate its sensitivity to X-ray and carbon ion beams. Next, DNA mutation analysis on 409 cancer-related genes and comprehensive transcriptome analysis (RNA-seq) were performed with a next-generation sequencer. Lethal dose 50 values of X-rays for LNCaP and LNCaP-LA were 1.4 Gy and 2.8 Gy, respectively (P

Programmed death ligand 1 (PD-L1) expression on the surface of cancer cells affects the efficacy of anti-PD-1/PD-L1 immune checkpoint therapy. However, the mechanism underlying PD-L1 expression in cancer cells is not fully understood, particularly after ionizing radiation (IR). Here, we examined the impact of high linear energy transfer (LET) carbon-ion irradiation on the expression of PD-L1 in human osteosarcoma U2OS cells. We found that the upregulation of PD-L1 expression after high LET carbon-ion irradiation was greater than that induced by X-rays at the same physical and relative biological effectiveness (RBE) dose, and that the upregulation of PD-L1 induced by high LET carbon-ion irradiation was predominantly dependent on ataxia telangiectasia and Rad3-related (ATR) kinase activity. Moreover, we showed that the downstream signaling, e.g. STAT1 phosphorylation and IRF1 expression, was upregulated to a greater extent after high LET carbon-ion irradiation than X-rays, and that IRF1 upregulation was also ATR dependent. Finally, to visualize PD-L1 molecules on the cell surface in 3D, we applied immunofluorescence-based super-resolution imaging. The three-dimensional structured illumination microscopy (3D-SIM) analyses revealed substantial increases in the number of presented PD-L1 molecules on the cell surface after high LET carbon-ion irradiation compared with X-ray irradiation.

Background Chromosomal inversions involving anaplastic lymphoma kinase (ALK) and echinoderm microtubule associated protein like 4 (EML4) generate a fusion protein EML4-ALK in non-small cell lung cancer (NSCLC). The understanding of EML4-ALK function can be improved by a functional study using normal human cells. Methods Here we for the first time conduct such study to examine the effects of EML4-ALK on cell proliferation, cellular senescence, DNA damage, gene expression profiles and transformed phenotypes. Results The lentiviral expression of EML4-ALK in mortal, normal human fibroblasts caused, through its constitutive ALK kinase activity, an early induction of cellular senescence with accumulated DNA damage, upregulation of p16INK4A and p21WAF1, and senescence-associated β-galactosidase (SA-β-gal) activity. In contrast, when EML4-ALK was expressed in normal human fibroblasts transduced with telomerase reverse transcriptase (hTERT), which is activated in the vast majority of NSCLC, the cells showed accelerated proliferation and acquired anchorage-independent growth ability in soft-agar medium, without accumulated DNA damage, chromosome aberration, nor p53 mutation. EML4-ALK induced the phosphorylation of STAT3 in both mortal and hTERT-transduced cells, but RNA sequencing analysis suggested that the different signaling pathways contributed to the different phenotypic outcomes in these cells. While EML4-ALK also induced anchorage-independent growth in hTERT-immortalized human bronchial epithelial cells in vitro, the expression of EML4-ALK alone did not cause detectable in vivo tumorigenicity in immunodeficient mice. Conclusions Our data indicate that the expression of hTERT is critical for EML4-ALK to manifest its in vitro transforming activity in human cells. This study provides the isogenic pairs of human cells with and without EML4-ALK expression.

Background This study aimed to explain the dynamics of prostate-specific antigen (PSA) levels in patients with prostate cancer who were treated with carbon ion radiotherapy (CIRT) and neoadjuvant androgen-deprivation therapy (ADT). Methods Eighty-five patients with intermediate-risk prostate cancer who received CIRT and neoadjuvant ADT from December 2015 to December 2017 were analyzed in the present study. The total dose of CIRT was set at 51.6 Gy (relative biological effectiveness) delivered in 12 fractions over 3 weeks. The PSA bounce was defined as a ≥0.4 ng/ml increase of PSA levels from the nadir, followed by any decrease. PSA failure was defined using the Phoenix criteria. Results The median patient age was 68 (range, 48–81) years. The median follow-up duration was 33 (range, 20–48) months. The clinical T stage was T1c, T2a, and T2b in 27, 44, and 14 patients, respectively. The Gleason score was 6 in 3 patients and 7 in 82 patients. The median pretreatment PSA level was 7.37 (range, 3.33–19.0) ng/ml. All patients received neoadjuvant ADT for a median of 6 (range, 2–117) months. PSA bounces were observed in 39 patients (45.9%), occurring a median of 12 (range, 6–30) months after CIRT. PSA failure was observed in eight patients (9.4%), occurring a median of 21 (range, 15–33) months after CIRT. The 3-year PSA failure-free survival rate was 88.5%. No clinical recurrence was observed during the follow-up period. Younger age and lower T stage were significant predictors of PSA bounce. Younger age was a significant predictor of PSA failure. Conclusions In this study, we identified the significant predictors of the occurrence of PSA bounce and failure. Further follow-up is needed to reveal the clinical significance of PSA dynamics.

Photons, such as X- or γ-rays, induce DNA damage (distributed throughout the nucleus) as a result of low-density energy deposition. In contrast, particle irradiation with high linear energy transfer (LET) deposits high-density energy along the particle track. High-LET heavy-ion irradiation generates a greater number and more complex critical chromosomal aberrations, such as dicentrics and translocations, compared with X-ray or γ irradiation. In addition, the formation of >1000 bp deletions, which is rarely observed after X-ray irradiation, has been identified following high-LET heavy-ion irradiation. Previously, these chromosomal aberrations have been thought to be the result of misrepair of complex DNA lesions, defined as DNA damage through DNA double-strand breaks (DSBs) and single-strand breaks as well as base damage within 1–2 helical turns (

Programmed cell death-1 (PD-1) and its ligand (programmed death-ligand 1, PD-L1) are key factors that regulate a cytotoxic immune reaction. Anti-PD-1 therapy provides significant clinical benefits for patients with cancer, even those with advanced-stage cancer. We have recently demonstrated that DNA damage signaling from DNA double-strand breaks (DSBs) promotes PD-L1 upregulation in cancer cells. In the present study, we aimed to investigate PD-L1 expression in primary normal human dermal fibroblasts (NHDFs) in response to DSBs. We demonstrated that PD-L1 expression in NHDFs is not upregulated after ionizing radiation (IR). In addition, interferon (IFN) regulatory factor 1 (IRF1) and signal transducer and activator of transcription 1 (STAT1) phosphorylation do not respond in NHDFs after IR. In contrast, IFNγ treatment upregulates PD-L1 and IRF1 expressions and STAT1 phosphorylation. The nonresponsiveness was also observed after treatment with other DNA-damaging agents, such as camptothecin and etoposide. Treatment with a histone deacetylase inhibitor (HDACi), which causes chromatin relaxation and restores gene silencing, upregulates PD-L1 without exogenous DNA damage; however, IR-dependent upregulation is not observed in NHDFs treated with HDACi. Taken together, our data suggest that DNA-damage signaling is insufficient for upregulating PD-L1 in NHDFs.

Radiotherapy is a definitive treatment for early-stage cervical cancer; however, a subset of this disease recurs locally, necessitating establishment of predictive biomarkers and treatment strategies. To address this issue, we performed gene panel-based sequencing of 18 stage IB cervical cancers treated with definitive radiotherapy, including two cases of local recurrence, followed by in vitro and in silico analyses. Simultaneous mutations in KRAS and SMAD4 (KRASmt/SMAD4mt) were detected only in a local recurrence case, indicating potential association of this mutation signature with radioresistance. In isogenic cell-based experiments, a combination of activating KRAS mutation and SMAD4 deficiency led to X-ray resistance, whereas either of these factors alone did not. Analysis of genomic data from 55,308 cancers showed a significant trend toward co-occurrence of mutations in KRAS and SMAD4. Gene Set Enrichment Analysis of the Cancer Cell Line Encyclopedia dataset suggested upregulation of the pathways involved in epithelial mesenchymal transition and inflammatory responses in KRASmt/SMAD4mt cancer cells. Notably, irradiation with therapeutic carbon ions led to robust killing of X-ray-resistant KRASmt/SMAD4mt cancer cells. These data indicate that the KRASmt/SMAD4mt signature is a potential predictor of radioresistance, and that carbon ion radiotherapy is a potential option to treat early-stage cervical cancers with the KRASmt/SMAD4mt signature.

Despite multidisciplinary treatment for patients with advanced gastric cancer, their prognosis remains poor. Therefore, the development of novel therapeutic strategies is urgently needed, and immunotherapy utilizing anti-programmed death 1/-programmed death ligand-1 mAb is an attractive approach. However, as there is limited information on how programmed death ligand-1 is upregulated on tumor cells within the tumor microenvironment, we examined the mechanism of programmed death ligand-1 regulation with a particular focus on interferon gamma in an in vitro setting and in clinical samples. Our in vitro findings showed that interferon gamma upregulated programmed death ligand-1 expression on solid tumor cells through the JAK-signal transducer and activator of transcription pathway, and impaired the cytotoxicity of tumor antigen-specific CTL against tumor cells. Following treatment of cells with anti-programmed death ligand-1 mAb after interferon gamma-pre-treatment, the reduced anti-tumor CTL activity by interferon gamma reached a higher level than the non-treatment control targets. In contrast, programmed death ligand-1 expression on tumor cells also significantly correlated with epithelial-mesenchymal transition phenotype in a panel of solid tumor cells. In clinical gastric cancer samples, tumor membrane programmed death ligand-1 expression significantly positively correlated with the presence of CD8-positive T cells in the stroma and interferon gamma expression in the tumor. The results suggest that gastric cancer patients with high CD8-positive T-cell infiltration may be more responsive to anti-programmed death 1/-programmed death ligand-1 mAb therapy.

Immunotherapy is expected to be promising as a next generation cancer therapy. Immunoreceptors are often activated constitutively in cancer cells, however, such levels of ligand expression are not effectively recognized by the native immune system due to tumor microenvironmental adaptation. Studies have demonstrated that natural-killer group 2, member D (NKG2D), a major activating immunoreceptor, responds to DNA damage. The upregulation of major histocompatibility complex class I-related chain A and B (MICA/B) (members of NKG2D ligands) expression after DNA damage is associated with NK cell-mediated killing of cancer cells. However, the regulation of DNA damage-induced MICA/B expression has not been fully elucidated in the context of the types of cancer cell lines. In the present study, we found that MICA/B expression varied between cancer cell lines after DNA damage. Screening in terms of chromatin remodeling identified that inhibitors related to chromatin relaxation via post-translational modification on histone H3K9, i.e. HDAC, Suv39 or G9a inhibition, restored DNA damage-dependent MICA/B expression in insensitive cells. In addition, we revealed that the restored MICA/B expression was dependent on ATR as well as E2F1, a transcription factor. We further revealed that low-dose treatment of an HDAC inhibitor was sufficient to restore MICA/B expression in insensitive cells. Finally, we demonstrated that HDAC inhibition restored DNA damage-dependent cytotoxic NK activity against insensitive cells. Thus, the present study revealed that DNA damage-dependent MICA/B expression in insensitive cancer cells can be restored by chromatin relaxation via the HDAC/Suv39/G9a pathway. Collectively, manipulation of chromatin status by therapeutic cancer drugs may potentiate the antitumor effect by enhancing immune activation following radiotherapy and DNA damage-associated chemotherapy.

Herein, we investigate the long-term clinical outcomes for cervical cancer patients treated with in-room computed tomography–based brachytherapy. Eighty patients with Stage IB1–IVA cervical cancer, who had undergone treatment with combined 3D high-dose rate brachytherapy and conformal radiotherapy between October 2008 and May 2011, were retrospectively analyzed. External beam radiotherapy (50 Gy) with central shielding after 20–40 Gy was performed for each patient. Cisplatin-based chemotherapy was administered concurrently to advanced-stage patients aged ≤75 years. Brachytherapy was delivered in four fractions of 6 Gy per week. In-room computed tomography imaging with applicator insertion was performed for treatment planning. Information from physical examinations at diagnosis, and brachytherapy and magnetic resonance imaging at diagnosis and just before the first brachytherapy session, were referred to for contouring of the high-risk clinical target volume. The median follow-up duration was 60 months. The 5-year local control, pelvic progression-free survival and overall survival rates were 94%, 90% and 86%, respectively. No significant differences in 5-year local control rates were observed between Stage I, Stage II and Stage III–IVA patients. Conversely, a significant difference in the 5-year overall survival rate was observed between Stage II and III–IVA patients (97% vs 72%; P = 0.006). One patient developed Grade 3 late bladder toxicity. No other Grade 3 or higher late toxicities were reported in the rectum or bladder. In conclusion, excellent local control rates were achieved with minimal late toxicities in the rectum or bladder, irrespective of clinical stage.

Ribosomes are important cellular components that maintain cellular homeostasis through overall protein synthesis. The nucleolus is a prominent subnuclear structure that contains ribosomal DNA (rDNA) encoding ribosomal RNA (rRNA), an essential component of ribosomes. Despite the significant role of the rDNA‑rRNA‑ribosome axis in cellular homeostasis, the stability of rDNA in the context of the DNA damage response has not been fully investigated. In the present study, the number and morphological changes of nucleolin, a marker of the nucleolus, were examined following ionizing radiation (IR) in order to investigate the impact of DNA damage on nucleolar stability. An increase in the number of nucleoli per cell was found in HCT116 and U2OS cells following IR. Interestingly, the IR‑dependent increase in nucleolar fragmentation was enhanced by p53 deficiency. In addition, the morphological analysis revealed several distinct types of nucleolar fragmentation following IR. The pattern of nucleolar morphology differed between HCT116 and U2OS cells, and the p53 deficiency altered the pattern of nucleolar morphology. Finally, a significant decrease in rRNA synthesis was observed in HCT116 p53‑/‑ cells following IR, suggesting that severe nucleolar fragmentation downregulates rRNA transcription. The findings of the present study suggest that p53 plays a key role in protecting the transcriptional activity of rDNA in response to DNA damage.

Radiotherapy is an essential component of cancer therapy. Carbon ion radiotherapy (CIRT) promises to improve outcomes compared with standard of care in many cancers. Nevertheless, clinicians often observe in-field recurrence after CIRT. This indicates the presence of a subset of cancers that harbor intrinsic resistance to CIRT. Thus, the development of methods to identify and sensitize CIRT-resistant cancers is needed. To address this issue, we analyzed a unique donor-matched pair of clinical specimens: a treatment-naïve tumor, and the tumor that recurred locally after CIRT in the same patient. Exon sequencing of 409 cancer-related genes identified enrichment of somatic mutations in FGFR3 and FGFR4 in the recurrent tumor compared with the treatment-naïve tumor, indicating a pivotal role for FGFR signaling in cancer cell survival through CIRT. Inhibition of FGFR using the clinically available pan-FGFR inhibitor LY2874455 sensitized multiple cancer cell lines to carbon ions at 3 Gy (RBE: relative biological effectiveness), the daily dose prescribed to the patient. The sensitizer enhancement ratio was 1.66 ± 0.17, 1.27 ± 0.09, and 1.20 ± 0.18 in A549, H1299, and H1703 cells, respectively. Our data indicate the potential usefulness of the analytical pipeline employed in this pilot study to identify targetable mutations associated with resistance to CIRT, and of LY21874455 as a sensitizer for CIRT-resistant cancers. The results warrant validation in larger cohorts.

Photon radiation therapy is a major curative treatment for cancer. However, the lack of robust predictive biomarkers for radiosensitivity precludes personalized radiation therapy. Clonogenic assays are the gold standard method for measuring the radiosensitivity of cancer cells. Although a large number of publications describe the use of clonogenic assays to measure cancer cell radiosensitivity, the robustness of results from different studies is unclear. To address this, we conducted a comprehensive detailed literature search of 256 common cancer cell lines and identified the eight cell lines most-frequently examined for photon sensitivity using clonogenic assays. Survival endpoints and experimental parameters from all 620 relevant experiments were compiled and analyzed. We found that the coefficients of variation for SF2 (surviving fraction after 2 Gy irradiation) and for D10 (dose that yields a surviving fraction of 10%) were below 30% for all cell lines, indicating that SF2 and D10 have acceptable inter-assay precision. These data support further analysis of published data on clonogenic assays using SF2 and D10 as survival endpoints, which facilitates robust identification of biological profiles representative of cancer cell sensitivity to photons.

In the era of precision medicine, radiotherapy strategies should be determined based on genetic profiles that predict tumor radiosensitivity. Accordingly, pre-clinical research aimed at discovering clinically applicable genetic profiles is needed. However, how a given genetic profile affects cancer cell radiosensitivity is unclear. To address this issue, we performed a pilot in vitro study by utilizing EGFR mutational status as a model for genetic profile. Clonogenic assays of EGFR mutant (n = 6) and wild-type (n = 9) non-small cell lung carcinoma (NSCLC) cell lines were performed independently by two oncologists. Clonogenic survival parameters SF2, SF4, SF6, SF8, mean inactivation dose (MID), D10, D50, α, and β were obtained using the linear quadratic model. The differences in the clonogenic survival parameters between the EGFR mutant and wild-type cell lines were assessed using the Mann–Whitney U test. As a result, for both datasets, the p values for SF2, SF4, D50, α, and α/β were below 0.05, and those for SF2 were lowest. These data indicate that a genetic profile of NSCLC cell lines might be predictive for their radiation response; i.e., EGFR mutant cell lines might be more sensitive to low dose- and low fraction sized-irradiation.

Background/aim The aim of this study was to evaluate the efficacy and safety of hypofractionated intensity-modulated radiotherapy (IMRT) for intermediate- and high-risk prostate cancer. Patients and methods Seventy-five consecutive patients with intermediate- and high-risk prostate cancer treated with IMRT (63 Gy/21 fractions/7 weeks) between 2010 and 2013 were retrospectively analyzed. PSA relapse and adverse events were determined based on the Phoenix criteria and the Common Terminology Criteria for Adverse Events v4.0, respectively. Results The 5-year PSA relapse-free rate, clinical relapse-free rate, and overall survival rate for all patients was 92.1%, 95.1%, and 92.9%, respectively. The incidence of late grade 2 gastrointestinal- and genitourinary-toxicity at 5 years was 1.3% and 17.1%, respectively. No grade 3 or greater toxicities were observed. Conclusion These data indicate that hypofractionated IMRT (63 Gy in a total of 21 fractions with 3 fractions per week) is effective and safe for intermediate- and high-risk prostate cancer.

Creatine riboside (CR) is a novel metabolite of cancer metabolism. It is a urinary diagnostic biomarker of lung and liver cancer risk and prognosis. The level of CR is highly positive correlated in tumor and urine indicating that it is derived from human lung and liver cancers. A precise and sensitive ultra-pressure liquid chromatography-tandem mass spectrometry (UPLC-ESI-MS/MS) method was developed and validated for simultaneous quantification of the noninvasive biomarker CR, along with creatinine riboside (CNR), and their precursors creatine and creatinine, utilizing the labeled internal standard creatine riboside-(13)C,(15)N(2) (CR-(13)C,(15)N(2)). Chromatography was carried out on a hydrophilic interaction chromatography column under a gradient mobile phase condition. MRM transitions were monitored for CR (264.1 > 132.1, m/z), CNR (246.1 > 113.9, m/z), creatine (132.0 > 72.0, m/z), creatinine (114.0 > 85.8, m/z) and CR-(13)C,(15)N(2) (267.1 > 134.9, m/z) with a 11.0 min run time in the positive mode ionization. The calibration plot of the method was linear over the concentration range of 4.50–10,000 nM. Method validation was performed according to regulatory guidelines established for sensitivity, selectivity, calibration curve, stability at different storage conditions, reinjection reproducibility, ruggedness with acceptable accuracy, and precision. This assay was applied for the quantification of CR along with CNR, creatine and creatinine in a subset of urine and serum samples from the National Cancer Institute – Maryland (NCI-MD) cohort population controls and lung cancer cases. It can be standardized and used in multiple laboratories for cancer diagnosis and determining the efficacy of cancer therapy and monitoring cancer recurrence.

This study aimed to elucidate the contribution of T cell-mediated antitumor immunity in the antitumor effect of local hyperthermia (LH).C57BL/6J mice were injected with the mouse lymphoma cell line, E.G7-OVA, in the right femur on day 0. LH was induced by immersing the right femur in a water bath at 42 °C for 60 min on day 7, followed by administration of anti-CD8 monoclonal antibodies (mAb) or anti-CTLA-4 mAb (days 8, 11, and 14). The effect of LH on tumor growth (TG) was assessed by measuring the duration until tumor volume reached 1000 mmTG with and without LH treatment was 9.0 ± 9.6 and 7.0 ± 1.6 days, respectively. TG was significantly slower with LH treatment (p = .01). The therapeutic effect of LH was mitigated by addition of anti-CD8 mAb (p .05 for both TG and survival) compared with the untreated (control) group. Furthermore, addition of anti-CTLA-4 mAb did not significantly affect the therapeutic effect of LH. The ELISpot assay showed that the number of spots in the LH group (276.3 ± 14.5) was significantly greater than in the control group (59.0 ± 4.5, p .001).CD8-positive T cell-mediated antitumor immunity significantly contributes to the antitumor effect of LH.

Anti-tumor immunity modulates the local effects of radiation therapy. High mobility group box 1 (HMGB1) plays a pivotal role in activating antigen-specific T-cell responses. Here, we examined the relationship between linear energy transfer (LET) and HMGB1 release. We assessed the proportions of KYSE-70, HeLa and SiHa cells surviving after carbon-ion (C-ion) beam irradiation with different LET values, using a clonogenic assay. The D10, the dose at which 10% of cells survived, was calculated using a linear-quadratic model. HMGB1 levels in the culture supernatants of C-ion beam-irradiated tumor cells were assessed by enzyme-linked immunosorbent assay. The D10 doses for 13 keV/μm of C-ion irradiation in KYSE-70, HeLa and SiHa cells were 2.8, 3.9 and 4.1 Gy, respectively, whereas those for 70 keV/μm C-ion irradiation were 1.4, 1.9 and 2.3 Gy, respectively. We found that 70 keV/μm of C-ion irradiation significantly increased HMGB1 levels in the culture supernatants of all cell lines 72 h after irradiation compared with non-irradiated controls. Furthermore, 70 keV/μm of C-ion irradiation significantly increased HMGB1 levels in the culture supernatants of all cell lines 72 h after irradiation compared with 13 keV/μm. The results suggest that HMGB1 release from several cancer cell lines increases with increased LET.

Carbon-ion radiotherapy (CIRT) holds promise in the treatment of glioblastoma, an aggressive X-ray-resistant brain tumor. However, since glioblastoma cells show a highly invasive nature, carbon-ion (C-ion) irradiation of normal tissues surrounding the tumor is inevitable. Recent studies have revealed the existence of neural stem cells in the adult brain. Therefore, the damaging effect of C-ion beams on the neural stem cells has to be carefully considered in the treatment planning of CIRT. Here, we investigated the growth and death mode of human neural stem cells (hNSCs) and glioblastoma A172 cells after X-ray or C-ion beam irradiation. The X-ray dose resulting in a 50% growth rate (D(50)) was 0.8 Gy in hNSCs and 3.0 Gy in A172 cells, while the D(50) for C-ion beams was 0.4 Gy in hNSCs and 1.6 Gy in A172 cells; the relative biological effectiveness value of C-ion beams was 2.0 in hNSCs and 1.9 in A172 cells. Importantly, both X-rays and C-ion beams preferentially induced apoptosis, not necrosis, in hNSCs; however, radiation-induced apoptosis was less evident in A172 cells. The apoptosis-susceptible nature of the irradiated hNSCs was associated with prolonged upregulation of phosphorylated p53, whereas the apoptosis-resistant nature of A172 cells was associated with a high basal level of nuclear factor kappa B expression. Taken together, these data indicate that apoptosis is the major cell death pathway in hNSCs after irradiation. The high sensitivity of hNSCs to C-ion beams underscores the importance of careful target volume delineation in the treatment planning of CIRT for glioblastoma.

X-ray radiotherapy activates tumor antigen-specific T-cell responses, and increases in the serum levels of high mobility group box 1 (HMGB1) induced by X-ray irradiation play a pivotal role in activating anti-tumor immunity. Here, we examined whether carbon-ion beams, as well as X-rays, can induce HMGB1 release from human cancer cell lines. The study examined five human cancer cell lines: TE2, KYSE70, A549, NCI-H460 and WiDr. The proportion of cells surviving X- or carbon-ion beam irradiation was assessed in a clonogenic assay. The D10, the dose at which 10% of cells survive, was calculated using a linear-quadratic model. HMGB1 levels in the culture supernatants were assessed by an ELISA. The D10 dose for X-rays in TE2, KYSE70, A549, NCI-H460 and WiDr cells was 2.1, 6.7, 8.0, 4.8 and 7.1 Gy, respectively, whereas that for carbon-ion beams was 0.9, 2.5, 2.7, 1.8 and 3.5 Gy, respectively. X-rays and carbon-ion beams significantly increased HMGB1 levels in the culture supernatants of A549, NCI-H460 and WiDr cells at 72 h post-irradiation with a D10 dose. Furthermore, irradiation with X-rays or carbon-ion beams significantly increased HMGB1 levels in the culture supernatants of all five cell lines at 96 h post-irradiation. There was no significant difference in the amount of HMGB1 induced by X-rays and carbon-ion beams at any time-point (except at 96 h for NCI-H460 cells); thus we conclude that comparable levels of HMGB1 were detected after irradiation with iso-survival doses of X-rays and carbon-ion beams.

The present study investigated the ability of carboplatin and paclitaxel to sensitize human non-small-cell lung cancer (NSCLC) cells to carbon-ion beam irradiation. NSCLC H460 cells treated with carboplatin or paclitaxel were irradiated with X-rays or carbon-ion beams, and radiosensitivity was evaluated by clonogenic survival assay. Cell proliferation was determined by counting the number of viable cells using Trypan blue. Apoptosis and senescence were evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining and senescence-associated β-galactosidase (SA-β-gal) staining, respectively. The expression of cleaved caspase-3, Bax, p53 and p21 was analyzed by western blotting. Clonogenic survival assays demonstrated a synergistic radiosensitizing effect of carboplatin and paclitaxel with carbon-ion beams; the sensitizer enhancement ratios (SERs) at the dose giving a 10% survival fraction (D10) were 1.21 and 1.22, respectively. Similarly, carboplatin and paclitaxel showed a radiosensitizing effect with X-rays; the SERs were 1.41 and 1.29, respectively. Cell proliferation assays validated the radiosensitizing effect of carboplatin and paclitaxel with both carbon-ion beam and X-ray irradiation. Carboplatin and paclitaxel treatment combined with carbon-ion beams increased TUNEL-positive cells and the expression of cleaved caspase-3 and Bax, indicating the enhancement of apoptosis. The combined treatment also increased SA-β-gal-positive cells and the expression of p53 and p21, indicating the enhancement of senescence. In summary, carboplatin and paclitaxel radiosensitized H460 cells to carbon-ion beam irradiation by enhancing irradiation-induced apoptosis and senescence.

Accumulating evidence suggests that exogenous cellular stress induces PD-L1 upregulation in cancer. A DNA double-strand break (DSB) is the most critical type of genotoxic stress, but the involvement of DSB repair in PD-L1 expression has not been investigated. Here we show that PD-L1 expression in cancer cells is upregulated in response to DSBs. This upregulation requires ATM/ATR/Chk1 kinases. Using an siRNA library targeting DSB repair genes, we discover that BRCA2 depletion enhances Chk1-dependent PD-L1 upregulation after X-rays or PARP inhibition. In addition, we show that Ku70/80 depletion substantially enhances PD-L1 upregulation after X-rays. The upregulation by Ku80 depletion requires Chk1 activation following DNA end-resection by Exonuclease 1. DSBs activate STAT1 and STAT3 signalling, and IRF1 is required for DSB-dependent PD-L1 upregulation. Thus, our findings reveal the involvement of DSB repair in PD-L1 expression and provide mechanistic insight into how PD-L1 expression is regulated after DSBs., PD-L1 is upregulated in many cancers due to exogenous cellular stress. Here the authors show that PD-L1 is upregulated in response to DNA double strand breaks via STAT and IRF1 signalling.

Background The skin is considered a critical organ at risk in carbon ion radiotherapy (CIRT) for locally advanced malignant bone and soft tissue tumors (MBSTs). The predictive factors for acute skin reactions after CIRT have not been investigated. The present study aimed to identify these factors and evaluate the correlation between the severity of acute skin reactions and skin dose parameters. Methods CIRT with total doses of 64.0–70.4 Gy (relative biological effectiveness [RBE]) was administered to 22 patients with MBSTs. The skin-tumor distance (STD), maximum skin total dose (Dmax), and area of the skin receiving a total dose of X Gy (RBE) were evaluated. Results All patients developed acute skin reactions after CIRT, including Grades 1 and 2 dermatitis in 15 (71%) and 6 (29%) patients, respectively. There was a significant difference in the STD between the two groups (P = 0.007), and the cut-off value of STD for predicting Grade 2 acute skin reactions was 11 mm. There was a significant difference in Dmax between the groups (P

Research on ionizing radiation (IR)-induced clonogenic cell death is important for understanding the effect of IR on malignant tumors and normal tissues. Here, we describe a quick and cost-effective one-step assay for simultaneously assessing the major modes of clonogenic cell death induced by IR, i.e., apoptosis, mitotic catastrophe, and cellular senescence. In this method, cells grown on a cover slip are irradiated with X-rays and stained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI). Using fluorescence microscopy, apoptosis, mitotic catastrophe, and cellular senescence are identified based on the characteristic morphologies of the DAPI-stained nuclei. Apoptosis is determined by the presence of apoptotic bodies (i.e., condensed and fragmented nuclei). Mitotic catastrophe is determined by the presence of nuclei that exhibit two or more distinct lobes and micronuclei. Cellular senescence is determined by the presence of senescence-associated heterochromatic foci (i.e., nuclear DNA containing 30-50 bright, dense foci). This approach allows the experimenter to easily screen for clonogenic cell death modes using various cell lines, treatment settings, and/or time points, with the goal of elucidating the mechanisms of cell death in the target cells and conditions of interest.

In cancer therapy today, carbon ion radiotherapy is used mainly as monotherapy, whereas cisplatin is used concomitantly with X-ray radiotherapy. The effectiveness of concomitant carbon ions and cisplatin is unclear. To obtain the information on the mechanisms potentially shared between carbon ions or X-rays and cisplatin, we assessed the correlation of sensitivity to the single treatments. In 20 human cancer cell lines, sensitivity to X-rays strongly correlated with sensitivity to cisplatin, indicating the presence of potentially shared target mechanisms. Interestingly, the correlation of sensitivity to carbon ions and cisplatin was much weaker than that of sensitivity to X-rays and cisplatin, indicating the presence of potentially different target mechanisms between carbon ions and cisplatin. Assessment of clonogenic cell death by 4′,6-diamidino-2-phenylindole dihydrochloride staining showed that mitotic catastrophe was more efficiently induced by carbon ions than by the same physical dose of X-rays, while apoptosis and senescence were not. These data indicate that the correlation of sensitivity to carbon ions and cisplatin is weaker than that of sensitivity to X-rays and cisplatin, which are helpful as biological basis to understand the potentially shared mechanism among these treatments. Further investigation is mandatory to elucidate the clinical efficacy of carbon ions and cisplatin combination.

Summary: BRCA1 promotes homologous recombination (HR) by activating DNA-end resection. By contrast, 53BP1 forms a barrier that inhibits DNA-end resection. Here, we show that BRCA1 promotes DNA-end resection by relieving the 53BP1-dependent barrier. We show that 53BP1 is phosphorylated by ATM in S/G2 phase, promoting RIF1 recruitment, which inhibits resection. 53BP1 is promptly dephosphorylated and RIF1 released, despite remaining unrepaired DNA double-strand breaks (DSBs). When resection is impaired by CtIP/MRE11 endonuclease inhibition, 53BP1 phosphorylation and RIF1 are sustained due to ongoing ATM signaling. BRCA1 depletion also sustains 53BP1 phosphorylation and RIF1 recruitment. We identify the phosphatase PP4C as having a major role in 53BP1 dephosphorylation and RIF1 release. BRCA1 or PP4C depletion impairs 53BP1 repositioning, EXO1 recruitment, and HR progression. 53BP1 or RIF1 depletion restores resection, RAD51 loading, and HR in PP4C-depleted cells. Our findings suggest that BRCA1 promotes PP4C-dependent 53BP1 dephosphorylation and RIF1 release, directing repair toward HR. : Following induction of DNA double-strand break, a pro-end-joining environment is created in G2 by transient 53BP1 phosphorylation and RIF1 recruitment. Here, Isono et al. show that, if timely repair does not ensue, BRCA1 promotes 53BP1 dephosphorylation and RIF1 release, favoring repair by homologous recombination. Keywords: ATM, DNA-end resection, BRCA1, 53BP1, RIF1, PP4C, NHEJ, HR

This study aimed to investigate the effect of carbon ion (C-ion) irradiation on cell motility through the ras homolog gene family member (Rho) signaling pathway in the human lung adenocarcinoma cell line A549. Cell motility was assessed by a wound-healing assay, and the formation of cell protrusions was evaluated by F-actin staining. Cell viability was examined by the WST-1 assay. The expression of myosin light chain 2 (MLC2) and the phosphorylation of MLC2 at Ser19 (P-MLC2-S19) were analyzed by Western blot. At 48 h after irradiation, the wound-healing assay demonstrated that migration was significantly greater in cells irradiated with C-ion (2 or 8 Gy) than in unirradiated cells. Similarly, F-actin staining showed that the formation of protrusions was significantly increased in cells irradiated with C-ion (2 or 8 Gy) compared with unirradiated cells. The observed increase in cell motility due to C-ion irradiation was similar to that observed due to X-ray irradiation. Western-blot analysis showed that C-ion irradiation (8 Gy) increased P-MLC2-S19 expression compared with in unirradiated controls, while total MLC2 expression was unchanged. Exposure to a non-toxic concentration of Y-27632, a specific inhibitor of Rho-associated coiled-coil-forming protein kinase (ROCK), reduced the expression of P-MLC2-S19 after C-ion irradiation (8 Gy), resulting in a significant reduction in migration. These data suggest that C-ion irradiation increases cell motility in A549 cells via the Rho signaling pathway and that ROCK inhibition reduces that effect.

Chromatin-regulating proteins represent a large class of novel targets for cancer therapy. In the context of radiotherapy, acetylation and deacetylation of histones by histone acetyltransferases (HATs) and histone deacetylases (HDACs) play important roles in the repair of DNA double-strand breaks generated by ionizing irradiation, and are therefore attractive targets for radiosensitization. Small-molecule inhibitors of HATs (garcinol, anacardic acid and curcumin) and HDACs (vorinostat, sodium butyrate and valproic acid) have been shown to sensitize cancer cells to ionizing irradiation in preclinical models, and some of these molecules are being tested in clinical trials, either alone or in combination with radiotherapy. Meanwhile, recent large-scale genome analyses have identified frequent mutations in genes encoding chromatin-regulating proteins, especially in those encoding subunits of the SWI/SNF chromatin-remodeling complex, in various human cancers. These observations have driven researchers toward development of targeted therapies against cancers carrying these mutations. DOT1L inhibition in MLL-rearranged leukemia, EZH2 inhibition in EZH2-mutant or MLL-rearranged hematologic malignancies and SNF5-deficient tumors, BRD4 inhibition in various hematologic malignancies, and BRM inhibition in BRG1-deficient tumors have demonstrated promising anti-tumor effects in preclinical models, and these strategies are currently awaiting clinical application. Overall, the data collected so far suggest that targeting chromatin-regulating proteins is a promising strategy for tomorrow's cancer therapy, including radiotherapy and molecularly targeted chemotherapy.

To compare the incidence and degree of hematological toxicity between innovator and generic cisplatin formulations, decreases in white blood cell (WBC) count (leukopenia) and platelet counts (thrombocytopenia) were retrospectively examined, using the Common Toxicity Criteria for Adverse Events ver. 4.0, in patients with uterine cervical cancer treated with concurrent chemoradiotherapy using innovator (innovator group, n = 22) or generic (generic group, n = 22) cisplatin formulations. There were no significant differences in patient characteristics except in the technique of external irradiation; larger numbers of patients in the innovator and generic groups were irradiated using the parallel-opposed two-field technique and the four-field box technique, respectively (P = 0.00012), which is in line with the historical progress of external beam radiation therapy. The numbers of patients showing Grade 1, 2, 3 and 4 leukopenia were 1 (4.5%), 14 (64%), 7 (32%) and 0 (0.0%) in the innovator group, and 1 (4.5%), 6 (27%), 13 (59%) and 2 (9.0%) in the generic group, respectively. The number of patients showing Grade 3–4 leukopenia was significantly greater in the generic group than in the innovator group (P = 0.034). There was no significant relationship between the incidence of Grade 3–4 leukopenia and the technique of external irradiation. There were no significant differences in the incidence and degree of thrombocytopenia between the two groups. These results indicate the possibility that the generic cisplatin formulation may have a different toxicity profile compared to the innovator formulation in terms of the incidence of leukopenia.

Carbon ion radiotherapy shows great potential as a cure for X-ray-resistant tumors. Basic research suggests that the strong cell-killing effect induced by carbon ions is based on their ability to cause complex DNA double-strand breaks (DSBs). However, evidence supporting the formation of complex DSBs in actual patients is lacking. Here, we used advanced high-resolution microscopy with deconvolution to show that complex DSBs are formed in a human tumor clinically treated with carbon ion radiotherapy, but not in a tumor treated with X-ray radiotherapy. Furthermore, analysis using a physics model suggested that the complexity of radiotherapy-induced DSBs is related to linear energy transfer, which is much higher for carbon ion beams than for X-rays. Visualization of complex DSBs in clinical specimens will help us to understand the anti-tumor effects of carbon ion radiotherapy.

Chromosomal translocations arise from misrejoining of DNA double strand breaks (DSBs) between loci located on two chromosomes. One current model suggests that spatial proximity of potential chromosomal translocation partners influences translocation probability. Ionizing radiation (IR) is a potent inducer of translocations. Accumulating evidence demonstrates that particle irradiation more frequently causes translocations compared with X-ray irradiation. This observation has led to the hypothesis that the high frequency of translocations after particle irradiation may be due to the formation of DSBs at chromosome boundaries along the particle track, because such DSBs can be misrejoined between distinct chromosomes. In this study, we simultaneously visualized the site of IR-induced DSBs and chromosome position by combining Immunofluorescence and fluorescence in situ hybridization. Importantly, the frequency of γH2AX foci at the chromosome boundary of chromosome 1 after carbon-ion irradiation was4-fold higher than that after X-ray irradiation. This observation is consistent with the idea that particle irradiation generates DSBs at the boundaries of two chromosomes along the track. Further, we showed that resolution of γH2AX foci at chromosome boundaries is prevented by inhibition of DNA-PKcs activity, indicating that the DSB repair is NHEJ-dependent. Finally, we found that γH2AX foci at chromosome boundaries after carbon-ion irradiation contain DSBs undergoing DNA-end resection, which promotes repair utilizing microhomology mediated end-joining during translocation. Taken together, our study suggests that the frequency of DSB formation at chromosome boundaries is associated with the incidence of chromosomal translocations, supporting the notion that the spatial proximity between breaks is an important factor in translocation formation. © 2016 Wiley Periodicals, Inc.

Uterine cervical cancer/Radiotherapy/Small bowel/Pelvic insufficiency fracture/Common iliac lymph node region. The purpose of this study was to compare dose red uction to the small bowel and sacral bone by twofield and four-field techniques when the common iliac lymph node region is excluded from the radiation field in external beam radiotherapy of uterine cervical cancer. Thirteen patients with cervical cancer were entered into the study. Conventional treatment plans based on bony landmarks were made with parallelopposed two-field technique (C2F) and four-field box technique (C4F). Modified C2F (M2F) and C4F (M4F) plans of excluding the common iliac lymph node region from the conventional radiation fields were created in reference to the bifurcations of pelvic arteries in computed tomography images. For each patient, the dose volume histograms for the small bowel and sacral bone resulting from the C2F, C4F, M2F, and M4F plans were compared. The volumes were obtained at 10 levels of prescribed dose, at increments of 10%, from 5 Gy to 50 Gy. By sparing both small bowel and sacral bone, the M2F and M4F plans were s ignificantly better than the C2F and C4F plans at any dose level (p < 0.05), respectively. In addition, the M4F plan was significantly better than the M2F plan in sparing both small bowel at 10–50% of the prescribed dose (p < 0.05) and sacral bone at 40–100% of the prescribed dose (p < 0.05). The present study suggests that modified treatment planning could be useful for selected patients for reducing small bowel complications and insufficiency fracture after radiotherapy.

Carbon-ion radiotherapy (CIRT) holds promise to treat inoperable locally-advanced non-small cell lung carcinoma (NSCLC), a disease poorly controlled by standard chemoradiotherapy using X-rays. Since CIRT is an extremely limited medical resource, selection of NSCLC patients likely to benefit from it is important; however, biological predictors of response to CIRT are ill-defined. The present study investigated the association between the mutational status of EGFR and KRAS, driver genes frequently mutated in NSCLC and the relative biological effectiveness (RBE) of carbon-ion beams over X-rays. The assessment of 15 NSCLC lines of different EGFR/KRAS mutational status and that of isogenic NSCLC lines expressing wild-type or mutant EGFR revealed that EGFR-mutant NSCLC cells, but not KRAS-mutant cells, show low RBE. This was attributable to (i) the high X-ray sensitivity of EGFR-mutant cells, since EGFR mutation is associated with a defect in non-homologous end joining, a major pathway for DNA double-strand break (DSB) repair and (ii) the strong cell-killing effect of carbon-ion beams due to poor repair of carbon-ion beam-induced DSBs regardless of EGFR mutation status. These data highlight the potential of EGFR mutation status as a predictor of response to CIRT, i.e., CIRT may show a high therapeutic index in EGFR mutation-negative NSCLC.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, and it is associated with poor survival. The standard therapy for newly-diagnosed GBM is radiotherapy with concurrent temozolomide following maximal surgical resection. To improve the outcome of these patients, combinations of the standard therapy plus molecular-targeted agents have been tested in clinical trials. However, the addition of gefitinib to the standard therapy did not appear to improve clinical outcome, and the standard therapy plus bevacizumab showed no improvement in overall survival, although a 4-month improvement in progression-free survival (PFS) was observed. Phase II data have indicated the potential efficacy of talampanel combined with the standard therapy for patients with newly-diagnosed GBM, and these findings are awaiting validation in phase III trials. In addition, phase II trials have demonstrated that adjuvant immunotherapy is effective and tolerable for treatment of patients with GBM. In this article, we discuss topics in chemotherapy, molecular-targeted therapy, and immunotherapy for patients with newly-diagnosed GBM.

The mammalian target of rapamycin (mTOR) correlates with cell survival under hypoxia and regulates hypoxia-inducible factor-1α (HIF-1α), a key protein in hypoxia-related events. However, the role of mTOR in radio-resistance has not been fully investigated. Therefore, the effect of mTOR on the radio-resistance of cancer cells under hypoxia was evaluated using the mTOR inhibitor temsirolimus. Clonogenic survival was examined in the A549 human lung adenocarcinoma cell line under normoxia or hypoxia, with or without temsirolimus. An oxygen enhancement ratio (OER) was calculated using the D(10) values, the doses giving 10% survival. Western blotting was performed to investigate the effect of temsirolimus on mTOR and the HIF-1α pathway under normoxia and hypoxia. A549 cells showed a radio-resistance of 5.1 and 14.2 Gy, as indicated by D(10) values under normoxia and hypoxia, respectively; the OER was 2.8. The cell survival rates under hypoxia and with temsirolimus remarkably decreased compared with those under normoxia. The D(10) values of the cells under normoxia and hypoxia were 4.8 and 5.4 Gy, respectively (OER = 1.1). mTOR expression was suppressed by temsirolimus under both normoxia and hypoxia. HIF-1α expression decreased under hypoxia in the presence of temsirolimus. These results suggest that temsirolimus can overcome the radio-resistance induced by hypoxia. When the fact that mTOR acts upstream of HIF-1α is considered, our data suggest that the restoration of radiation sensitivity by temsirolimus under hypoxia may be associated with the suppression of the HIF-1α pathway. Temsirolimus could therefore be used as a hypoxic cell radio-sensitizer.