p53 deficiency augments nucleolar instability after ionizing irradiation

学位記番号：医博甲179

Dosimetric Impact of Voluntary Deep Inspiration Breath Hold (DIBH) in Mediastinal Hodgkin Lymphomas: A Comparative Evaluation of Three Different Intensity Modulated Radiation Therapy (IMRT) Delivery Methods Using Voluntary DIBH and Free Breathing Techniques

Hodgkin lymphomas are radiosensitive and curable tumors that often involve the mediastinum. However, the application of radiation therapy to the mediastinum is associated with late effects including cardiac and pulmonary toxicities and secondary cancers. The adoption of conformal IMRT and deep inspiration breath- hold (DIBH) can reduce the dose to healthy normal tissues (lungs, heart and breast). We compared the dosimetry of organs at risk (OARs) using different IMRT techniques for two breathing conditions, i.e., deep inspiration breath hold (DIBH) and free breathing. Twenty-three patients with early-stage mediastinal Hodgkin lymphomas were accrued in the prospective study. The patients were given treatment plans which utilized full arc volumetric modulated arc therapy (F-VMAT), Butterfly VMAT (B-VMAT), and fixed field IMRT (FF-IMRT) techniques for both DIBH and free breathing methods, respectively. All the plans were optimized to deliver 95% of the prescription dose which was 25.2 Gy to 95% of the PTV volume. The mean dose and standard error of the mean for each OAR, conformity index (CI), and homogeneity index (HI) for the target using the three planning techniques were calculated and compared using Student’s t-test for parametric data and Wilcoxon signed-rank test for non-parametric data. The HI and CI of the target was not compromised using the DIBH technique for mediastinal lymphomas. The mean values of CI and HI for both DIBH and FB were comparable. The mean heart doses were reduced by 2.1 Gy, 2.54 Gy, and 2.38 Gy in DIBH compared to FB for the F-VMAT, B-VMAT, and IMRT techniques, respectively. There was a significant reduction in V5Gy, V10Gy, and V15Gy to the heart (p p = 0.004). DIBH results in lower heart, lung, and breast doses than free breathing in mediastinal Hodgkin Lymphoma. Among the different IMRT techniques, FF-IMRT, B-VMAT, and F-VMAT showed similar PTV coverage, with similar conformity and homogeneity indices. However, the time taken for FF-IMRT was much longer than for the F-VMAT and B-VMAT techniques for both breathing methods. B-VMAT and F-VMAT emerged as the optimal planning techniques able to achieve the best target coverage and lower doses to the OARs, with less time required to deliver the prescribed dose

RNF8 promotes high linear energy transfer carbon-ion-induced DNA double-stranded break repair in serum-starved human cells

The cell-killing effect of radiotherapy largely depends on unrepaired DNA double-stranded breaks (DSBs) or lethal chromosome aberrations induced by DSBs. Thus, the capability of DSB repair status is critically important in cancer cell-killing effect after ionizing radiation. Here, we investigate the involvement of the DNA damage signaling factors ataxia telangiectasia mutated (ATM) and ring finger protein (RNF) 8 and RNF168 in quiescent G0/G1 cells, which occupy the majority population in tumors, after high-linear-energy-transfer (LET) carbon-ion irradiation. Interestingly, ATM inhibition caused a substantial DSB repair defect after high-LET carbon-ion irradiation. Similarly, RNF8 or RNF168 depletion causes a substantial DSB repair defect. ATM inhibition in RNF8-depleted cells did not have an additive effect, suggesting that ATM and RNF8 function in the same pathway. Importantly, we found that RNF8 RING mutant show similar DSB repair defect, suggesting the requirement of ubiquitin ligase activity in this repair pathway. RNF8 FHA domain, which is required for the interaction with MDC1, is also required for DSB repair in this axis. Furthermore, depletion of p53 binding protein 1 (53BP1), which is an important downstream factor in RNF8-dependent DSB repair, is also required for this repair. Importantly, either ATM inhibition or RNF8 depletion increased the frequency of chromosomal breaks, but reduced dicentric chromosome formation, demonstrating that ATM/RNF8 is required for the rejoining of DSB ends for dicentric chromosome formation. Finally, we show that RNF8 depletion augmented radiosensitivity after high-LET carbon-ion irradiation. Taken together, these results suggest that the inhibition of RNF8 activity or its downstream pathway may augment the efficacy of high-LET carbon-ion therapy

Expression of non‑homologous end joining factor, Ku80, is negatively correlated with PD‑L1 expression in cancer cells after X‑ray irradiation

The growing importance of antitumour immunity by cancer immunotherapy has prompted studies on radiotherapy‑induced immune response. Previous studies have indicated that programmed cell death‑1 ligand (PD‑L1) expression is regulated by DNA damage signalling. However, PD‑L1 up‑regulation after radiotherapy has not been fully investigated at the clinical level, particularly in the context of expression of DNA repair factors. The present study examined the correlation of mRNA expression between PD‑L1 and non‑homologous end joining (NHEJ) factors using The Cancer Genome Atlas database analysis. Among NHEJ factors, Ku80 mRNA expression was negatively correlated with PD‑L1 mRNA expression levels in several types of cancer (colon adenocarcinoma, breast invasive carcinoma, skin cutaneous melanoma, lung adenocarcinoma, head and neck squamous cell carcinoma, uterine corpus endometrial carcinoma, cervical squamous cell carcinoma and endocervical adenocarcinoma). To verify the negative correlation in clinical samples, the present study analysed whether Ku80 expression levels affected PD‑L1 up‑regulation after radiotherapy using cervical squamous cell carcinoma samples. Quantitative evaluation using software analysis of immunohistochemically stained slides revealed that patients with low Ku80 positivity in biopsy specimens demonstrated increased PD‑L1 expression levels after 10 Gy irradiation (Spearman\u27s rank correlation coefficient=‑0.274; P=0.017). Furthermore, PD‑L1 induction levels in tumour cells after 10 Gy of irradiation were significantly inversely correlated with Ku80 expression levels (Spearman\u27s rank correlation coefficient=‑0.379; P<0.001). The present study also confirmed that short interfering RNA‑mediated Ku80 depletion was associated with greater X‑ray‑induced PD‑L1 up‑regulation in HeLa cells. These results indicated that radiotherapy could enhance PD‑L1 induction in tumour cells with low Ku80 expression in a clinical setting. Furthermore, these data highlighted Ku80 as a potential predictive biomarker for immune checkpoint therapy combined with radiotherapy

DNA double-strand break repair pathway regulates PD-L1 expression in cancer cells

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

Analysis of radiotherapy‑induced alteration of CD8+ T cells and PD‑L1 expression in patients with uterine cervical squamous cell carcinoma

Abstract. Radiotherapy induces an immune response in the cancer microenvironment that may influence clinical outcome. The present study aimed to analyse the alteration of CD8+ T‑cell infiltration and programmed death‑ligand 1 (PD‑L1) expression following radiotherapy in clinical samples from patients with uterine cervical squamous cell carcinoma. Additionally, the current study sought to analyse the association between these immune responses and clinical outcomes. A total of 75 patients who received either definitive chemoradiotherapy or radiotherapy were retrospectively analyzed. CD8+ T‑cell infiltration and PD‑L1 expression were determined by immunohistochemistry using biopsy specimens before radiotherapy (pre‑RT) and after 10 Gy radiotherapy (post‑10 Gy). The PD‑L1+ rate was significantly increased from 5% (4/75) pre‑RT to 52% (39/75) post‑10 Gy (P<0.01). Despite this increase in the PD‑L1+ rate post‑10 Gy, there was no significant association between both pre‑RT and post‑10 Gy and overall survival (OS), locoregional control (LC) and progression‑free survival (PFS). On the other hand, the CD8+ T‑cell infiltration density was significantly decreased for all patients (median, 23.1% pre‑RT vs. 16.9% post‑10 Gy; P=0.038); however, this tended to increase in patients treated with radiotherapy alone (median, 17.7% pre‑RT vs. 24.0% post‑10 Gy; P=0.400). Notably, patients with high CD8+ T‑cell infiltration either pre‑RT or post‑10 Gy exhibited positive associations with OS, LC and PFS. Thus, the present analysis suggested that CD8+ T‑cell infiltration may be a prognostic biomarker for patients with cervical cancer receiving radiotherapy. Furthermore, immune checkpoint inhibitors may be effective in patients who have received radiotherapy, since radiotherapy upregulated PD‑L1 expression in cervical cancer specimens

FGFR Signaling as a Candidate Therapeutic Target for Cancers Resistant to Carbon Ion Radiotherapy

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-na&iuml;ve 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-na&iuml;ve 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 &plusmn; 0.17, 1.27 &plusmn; 0.09, and 1.20 &plusmn; 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

FGFR Signaling as a Candidate Therapeutic Target for Cancers Resistant to Carbon Ion Radiotherapy.

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 and 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