Novel model integrating computed tomography-based image markers with genetic markers for discriminating radiation pneumonitis in patients with unresectable stage III non-small cell lung cancer receiving radiotherapy: a retrospective multi-center radiogenomics study.

BACKGROUND: Chemoradiotherapy is a critical treatment for patients with locally advanced and unresectable non-small cell lung cancer (NSCLC), and it is essential to identify high-risk patients as early as possible owing to the high incidence of radiation pneumonitis (RP). Increasing attention is being paid to the effects of endogenous factors for RP. This study aimed to investigate the value of computed tomography (CT)-based radiomics combined with genomics in analyzing the risk of grade ≥ 2 RP in unresectable stage III NSCLC. METHODS: In this retrospective multi-center observational study, 100 patients with unresectable stage III NSCLC who were treated with chemoradiotherapy were analyzed. Radiomics features of the entire lung were extracted from pre-radiotherapy CT images. The least absolute shrinkage and selection operator algorithm was used for optimal feature selection to calculate the Rad-score for predicting grade ≥ 2 RP. Genomic DNA was extracted from formalin-fixed paraffin-embedded pretreatment biopsy tissues. Univariate and multivariate logistic regression analyses were performed to identify predictors of RP for model development. The area under the receiver operating characteristic curve was used to evaluate the predictive capacity of the model. Statistical comparisons of the area under the curve values between different models were performed using the DeLong test. Calibration and decision curves were used to demonstrate discriminatory and clinical benefit ratios, respectively. RESULTS: The Rad-score was constructed from nine radiomic features to predict grade ≥ 2 RP. Multivariate analysis demonstrated that histology, Rad-score, and XRCC1 (rs25487) allele mutation were independent high-risk factors correlated with RP. The area under the curve of the integrated model combining clinical factors, radiomics, and genomics was significantly higher than that of any single model (0.827 versus 0.594, 0.738, or 0.641). Calibration and decision curve analyses confirmed the satisfactory clinical feasibility and utility of the nomogram. CONCLUSION: Histology, Rad-score, and XRCC1 (rs25487) allele mutation could predict grade ≥ 2 RP in patients with locally advanced unresectable NSCLC after chemoradiotherapy, and the integrated model combining clinical factors, radiomics, and genomics demonstrated the best predictive efficacy.

Radiation pneumonitis in relation to pulmonary function, dosimetric factors, TGFß1 expression, and quality of life in breast cancer patients receiving post-operative radiotherapy: a prospective 6-month follow-up study.

PURPOSE: To investigate development of radiation pneumonitis (RP) in relation to pulmonary function, dosimetric factors, and transforming growth factor beta-1 (TGFß1) expression in irradiated breast cancer patients. METHODS: A total of 49 breast cancer patients who received post-operative radiotherapy (RT) were evaluated in terms of pulmonary function tests (PFTs), quality of life (QoL), development of RP, dosimetric factors, cytokine levels, and lung high-resolution computed tomography (HRCT) before and after RT. ROC analysis was performed for performance of dosimetric factors in predicting RP, while frequencies of single nucleotide polymorphisms (SNPs) genotyped for TGFß1 (rs11466345 and rs1800470) were also evaluated. RESULTS: All cases with RP (10.2%) recovered clinically at the end of third post-RT month. PFT and HRCT parameters were similar before and after RT overall, as well as by RP and the radiation field subgroups. ROC analysis revealed the significant role of the ipsilateral V5 (cutoff value of 45.9%, p = 0.039), V10 (29.4%, p = 0.015), V20 (23%, p = 0.017), and MLD (1200 cGy, p = 0.030) in predicting RP. Higher post-RT TGFß1 levels (p = 0.037) were noted overall and in patients with RP. Patient and control groups were similar in terms of frequencies of SNPs genotyped for TGFß1 (rs11466345 and rs1800470). EORTC QLQ-C30 and QLQ-BR-23 scores were similar in patients with vs. without RP. CONCLUSION: Our findings revealed significant role of dosimetric factors including MLD, V20 as well as the low dose-volume metrics in predicting the risk of RP among breast cancer patients who received post-operative RT. Implementation of RT, extent of radiation field or the presence of RP had no significant impact on PFTs.

Genetic variations in DNA repair gene NEIL1 associated with radiation pneumonitis risk in lung cancer patients.

BACKGROUND: Radiation pneumonitis (RP) is a common side effect in lung cancer patients who received radiotherapy. Our previous study found genetic variations in DNA repair gene NEIL1 may be a predictor of RP in patients with esophageal cancer. So, we hypothesis genetic variations in NEIL1 gene could affect the risk of RP in lung cancer patients following radiotherapy. METHODS: Genetic variations rs4462560 G>C and rs7402844 C>G in NEIL1 gene were genotyped in 174 lung cancer patients received radio(chemo)therapy. Luciferase assay, real-time PCR and Western blot were used to access the effect of the variants on NEIL1 in HELF and HEF cell lines which were transfected with plasmids containing rs4462560 G>C and rs7402844 C>G. RESULTS: Patients with rs4462560 CC genotype had a lower risk of RP grade ≥2 than GG genotype. Compared with the CC genotype, rs7402844 GG genotype was associated with an increased RP grade ≥2 risk. What is more, rs4462560 G decreased the relative luciferase activity of NEIL1 gene promoter compared with the negative control in vitro, while rs4462560 C can increase the relative luciferase activity. The mRNA and protein level of the NEIL1 gene in rs4462560 G were lower than rs4462560 C. CONCLUSIONS: Genetic variants of NEIL1 are associated with RP risk through regulation of NEIL1 expression and serve as independent biomarkers for prediction of RP in patients treated with thoracic radiotherapy.

Identification of miRNA signatures associated with radiation-induced late lung injury in mice.

Acute radiation exposure of the thorax can lead to late serious, and even life-threatening, pulmonary and cardiac damage. Sporadic in nature, late complications tend to be difficult to predict, which prompted this investigation into identifying non-invasive, tissue-specific biomarkers for the early detection of late radiation injury. Levels of circulating microRNA (miRNA) were measured in C3H and C57Bl/6 mice after whole thorax irradiation at doses yielding approximately 70% mortality in 120 or 180 days, respectively (LD70/120 or 180). Within the first two weeks after exposure, weight gain slowed compared to sham treated mice along with a temporary drop in white blood cell counts. 52% of C3H (33 of 64) and 72% of C57Bl/6 (46 of 64) irradiated mice died due to late radiation injury. Lung and heart damage, as assessed by computed tomography (CT) and histology at 150 (C3H mice) and 180 (C57Bl/6 mice) days, correlated well with the appearance of a local, miRNA signature in the lung and heart tissue of irradiated animals, consistent with inherent differences in the C3H and C57Bl/6 strains in their propensity for developing radiation-induced pneumonitis or fibrosis, respectively. Radiation-induced changes in the circulating miRNA profile were most prominent within the first 30 days after exposure and included miRNA known to regulate inflammation and fibrosis. Importantly, early changes in plasma miRNA expression predicted survival with reasonable accuracy (88-92%). The miRNA signature that predicted survival in C3H mice, including miR-34a-5p, -100-5p, and -150-5p, were associated with pro-inflammatory NF-κB-mediated signaling pathways, whereas the signature identified in C57Bl/6 mice (miR-34b-3p, -96-5p, and -802-5p) was associated with TGF-ß/SMAD signaling. This study supports the hypothesis that plasma miRNA profiles could be used to identify individuals at high risk of organ-specific late radiation damage, with applications for radiation oncology clinical practice or in the context of a radiological incident.

A Likely Role for a Novel Cell Therapeutic Target of Transforming Growth Factor-ß1 on Radiation Pneumonitis in Lung and Nasopharyngeal Cancer Patients.

The association between the polymorphism of transforming growth factor (TGF)-ß1 and risk of radiation pneumonitis has been extensively investigated; however, conclusive results were unavailable. Eligible studies were identified from the database of Medline, Web of Science, EMBASE, and CNKI (China Knowledge Resource Integrated Database) up to September 2019. The odds ratio (OR) and 95% confidence interval (95% CI) were used to assess the strength of the relationship. The results showed that there were associations between TGF 869 T/C (rs1982073) and risks of radiation pneumonitis. Subgroup analyses showed that TGF 869 T/C was associated with risk of radiation pneumonitis in Caucasians (OR [95% CI]: 0.45 [0.31 to 0.67] for C carriers vs. TT). In addition, subgroup analyses also suggested that the C allele was associated with decreased risks of radiation pneumonitis among hospital-based case-control studies (0.56 [0.39 to 0.82] for C carriers vs. TT). Meanwhile, C allele was also suggested to be associated with decreased risk of radiation pneumonitis among PCC (0.60 [0.38 to 0.96] for C carriers vs. TT). Especially, C allele was also found to be associated with decreased risk of radiation pneumonitis from the participants with lung cancer (0.57 [0.37 to 0.90] for C carriers vs. TT). Our meta-analysis shows that T allele in TGF 869 T/C is significantly associated with the increased risk of radiation pneumonitis, especially for Caucasians, and for the participants with lung cancer.

Impact of genetic variant of HIPK2 on the risk of severe radiation pneumonitis in lung cancer patients treated with radiation therapy.

BACKGROUND: Homeodomain-interacting protein kinase 2 (HIPK2) has increasingly drawn attention as recent researches demonstrated its unique role in the regulation of multiple fundamental processes such as apoptosis, proliferation and DNA damage repair. Most importantly, HIPK2 was shown to play regulatory role in inflammation and influence the phenotype and activity of fibroblasts. In this study, we aimed to evaluate the impact of HIPK2 gene variant on risk of radiation pneumonitis for patients with pulmonary malignancies. METHODS: 169 lung cancer patients with radiotherapy were included in our prospective study and genotyped by Sanger Sequence method. Multivariable Cox hazard analysis and multiple testing were applied to estimate the hazard ratio (HR) and 95% confidence intervals (CIs) of all factors possibly related to the risk of radiation pneumonitis (RP). RESULTS: Patients with Mean Lung Dose (MLD) ≥ 15Gy, Lung V20 ≥ 24% had higher risk of RP ≥ grade 2 compared with those counterparts (HR = 1.888, 95% CI: 1.186-3.004, P = 0.007; HR = 2.126, 95% CI: 1.338-3.378, P = 0.001, respectively). Importantly, CC genotype of HIPK2: rs2030712 were strongly related to an increased occurrence of RP ≥ grade 2 (HR = 2.146, 95% CI: 1.215-3.791, P = 0.009). CONCLUSION: HIPK2: rs2030712 was found to be significantly related to RP of grade ≥ 2 in our cohort, and may thus be one of the important predictors of severe RP before radiotherapy, if further validated in larger population. TRIAL REGISTRATION: Our study was prospective and observational. The research was registered in ClinicalTrials.gov database as NCT02490319.

Validation study of the association between genetic variant of IL4 and severe radiation pneumonitis in lung cancer patients treated with radiation therapy.

BACKGROUND AND PURPOSE: Recent researches demonstrated that single nucleotide polymorphisms (SNPs) of genes involving inflammation, DNA repair, etc. were associated with risk of radiation pneumonitis (RP). However, these studies were single-centered, from single ethnic origin, without validation from independent cohort studies from other populations. In order to identify clinical valuable SNPs for RP, in this study we selected 19 RP-related SNPs candidates previously published before 2016 for validation in our cohort. MATERIAL AND METHODS: 359 lung cancer patients with radiotherapy were included in our prospective study (NCT02490319). Peripheral blood samples from these patients were genotyped by MassArray and Sanger Sequence method. Multivariate Cox hazard and other analyses were applied to estimate the hazard ratio (HR) and 95% confidence intervals (CIs) of all factors possibly related to the risk of RP. RESULTS: Patients with elder age, MLD ≥15 Gy, V20 ≥24% had higher risk of RP ≥grade 3 compared with their counterparts (HR = 2.020, 95% CI: 1.045-3.906, P = 0.037; HR = 2.502, 95% CI: 1.346-4.652, P = 0.004; HR = 2.256, 95% CI: 1.191-4.272, P = 0.013, respectively). Moreover, patients receiving IMRT were associated with decreased incidence of RP (HR = 0.520, 95% CI: 0.280-0.963, P = 0.037). Importantly, CT + TT genotype of IL4: rs2243250 was strongly related to decreased risk of RP ≥grade 3 (HR = 0.195, 95% CI: 0.090-0.424, P = 0.000037, Pc = 0.0006). CONCLUSION: IL4: rs2243250 was validated to be significantly related to RP of grade ≥3 in our cohort. Our results further emphasized the prevalence and clinical value of IL4: rs2243250 on RP, and may thus be one of the important predictors of severe RP before radiotherapy.

12-Lipoxygenase is a Critical Mediator of Type II Pneumocyte Senescence, Macrophage Polarization and Pulmonary Fibrosis after Irradiation.

Radiation-induced pulmonary fibrosis (RIPF) is a chronic, progressive complication of therapeutic irradiation of the thorax. It has been suggested that senescence of type II pneumocytes (AECIIs), an alveolar stem cell, plays a role in the development of RIPF through loss of replicative reserve and via senescent AECII-driven release of proinflammatory and profibrotic cytokines. Within this context, we hypothesized that arachidonate 12-lipoxygenase (12-LOX) is a critical mediator of AECII senescence and RIPF. Treatment of wild-type AECIIs with 12S-hydroxyeicosateraenoic acid (12S-HETE), a downstream product of 12-LOX, was sufficient to induce senescence in a NADPH oxidase 4 (NOX4)-dependent manner. Mice deficient in 12-LOX exhibited reduced AECII senescence, pulmonary collagen accumulation and accumulation of alternatively activated (M2) macrophages after thoracic irradiation (5 × 6 Gy) compared to wild-type mice. Conditioned media from irradiated or 12S-HETE-treated primary pneumocytes contained elevated levels of IL-4 and IL-13 compared to untreated pneumocytes. Primary macrophages treated with conditioned media from irradiated AECII demonstrated preferential M2 type polarization when AECIIs were derived from wild-type mice compared to 12-LOX-deficient mice. Together, these data identified 12-LOX as a critical component of RIPF and a therapeutic target for radiation-induced lung injury.

Identification of molecular signatures involved in radiation-induced lung fibrosis.

In radiotherapy, radiation (IR)-induced lung fibrosis has severe and dose-limiting side effects. To elucidate the molecular effects of IR fibrosis, we examined the fibrosis process in irradiated mouse lung tissues. High focal IR (90 Gy) was exposed to a 3-mm volume of the left lung in C57BL6 mice. In the diffused irradiation, 20 Gy dose delivered with a 7-mm collimator almost covered the entire left lung. Histological examination for lung tissues of both irradiated and neighboring regions was done for 4 weeks after irradiation. Long-term effects (12 months) of 20Gy IR were compared on a diffuse region of the left lung and non-irradiated right lung. Fibrosis was initiated as early as 2 weeks after IR in the irradiated lung region and neighboring region. Upregulation of gtse1 in both 90Gy-irradiated and neighboring regions was observed. Upregulation of fgl1 in both 20Gy diffused irradiated and non-irradiated lungs was identified. When gtse1 or flg1 was knock-downed, TGFß or IR-induced epithelial-mesenchymal transition was inhibited, accompanied with the inhibition of cellular migration, suggesting fibrosis responsible genes. Immunofluorescence analysis using mouse fibrotic lung tissues suggested that fibrotic regions showed increased expressions of Gtse1 and Fgl1, indicating novel molecular signatures of gtse1and fgl1 for IR-induced lung fibrosis. Even though their molecular mechanisms and IR doses or irradiated volumes for lung fibrosis may be different, these genes may be novel targets for understanding IR-induced lung fibrosis and in treatment strategies. KEY MESSAGES: Upregulation of gtse1 by 90Gy focal irradiation and upregulation of fgl1 by 20Gy diffused irradiation are identified in mouse lung fibrosis model. Gtse1 and Fgl1 are involved in radiation or TGFß-induced epithelial-mesenchymal transition. Radiation-induced fibrotic regions of mouse lungs showed increased expressions of Gtse1 and Fgl1. Gtse1 and Fgl1 are suggested to be novel targets for radiation-induced lung fibrosis.

Regulation of miR-125a expression by rs12976445 single-nucleotide polymorphism is associated with radiotherapy-induced pneumonitis in lung carcinoma patients.

BACKGROUND: The expression of miR-125 is regulated by an single-nucleotide polymorphism (SNP), rs12976445, which may be involved in the risk of pneumonitis among non-small-cell lung carcinoma patients undergoing the radiotherapy. We investigated this hypothesis via clinical data analysis and in vitro experiments. METHODS: An online microRNA (miRNA) database (www.mirdb.org) and luciferase reporter assays were used to confirm the role of transforming growth factor-ß1 (TGFB1) as a target gene of miR-125a. Quantification by real-time PCR and Western blot analysis were used to measure the expression of miRNA-125a and TGFB1 among different groups (carrying CC, CT, and TT genotypes of rs12976445) or cells transfected with a scramble control, miR-125a mimics, TGFB1 siRNA, or miR-125a inhibitors. RESULTS: We evaluated 699 NSCLC patients and found that the patients carrying the TT or CT genotype of rs12976445 had a higher risk of radiotherapy-induced pneumonitis. Computational analysis and luciferase assays validated that TGFB1 is a target gene of miR-125a. The expression level of miR-125a mRNA was significantly downregulated in the CT and TT groups, while the expression levels of TGFB1 and SMAD2 were significantly upregulated in the CC group. The expression of TGFB1 and SMAD2 was regulated by miR-125a in A549 cells. CONCLUSION: The rs12976445 SNP in miR-125a is associated with the risk of pneumonitis after in lung cancer patients undergoing the radiotherapy by regulating the expression of miR-125a and TGFB1.

Recurrent DNA damage is associated with persistent injury in progressive radiation-induced pulmonary fibrosis.

PURPOSE: Radiation-induced lung injuries (RILI), namely radiation pneumonitis and/or fibrosis, are dose-limiting outcomes following treatment for thoracic cancers. As part of a search for mitigation targets, we sought to determine if persistent DNA damage is a characteristic of this progressive injury. METHODS: C57BL/6J female mice were sacrificed at 24 h, 1, 4, 12, 16, 24 and 32 weeks following a single dose of 12.5 Gy thorax only gamma radiation; their lungs were compared to age-matched unirradiated animals. Tissues were examined for DNA double-strand breaks (DSBs) (γ-H2A.X and p53bp1), cellular senescence (senescence-associated beta-galactosidase and p21) and oxidative stress (malondialdehyde). RESULTS: Data revealed consistently higher numbers of DSBs compared to age-matched controls, with increases in γ-H2A.X positivity beyond 24 h post-exposure, particularly during the pathological phases, suggesting periods of recurrent DNA damage. Additional intermittent increases in both cellular senescence and oxidative stress also appeared to coincide with pneumonitis and fibrosis. CONCLUSIONS: These novel, long-term data indicate (a) increased and persistent levels of DSBs, oxidative stress and cellular senescence may serve as bioindicators of RILI, and (b) prevention of genotoxicity, via mitigation of free radical production, continues to be a potential strategy for the prevention of pulmonary radiation injury.

A microRNA­125a variant, which affects its mature processing, increases the risk of radiation­induced pneumonitis in patients with non­small­cell lung cancer.

The present study aimed to investigate the role of microRNA (miR)­125a in the development of pneumonitis inpatients with non­small­cell lung cancer that received radiotherapy. In addition, the study aimed to determine how the miR­125a affects its target, transforming growth factor ß (TGFß). Bioinformatics tools were used to identify a potential miR­125a binding site in the 3'untranslated region of TGFß, which was subsequently confirmed using a dual­luciferase reporter system. In addition, tissue samples were collected from patients with lung cancer and genotyped as CC (n=36), CT (n=28) or TT (n=6). The expression levels of miR­125a and TGFß in these samples were determined, and CC genotype samples demonstrated upregulated miR­125a expression, and downregulated TGFß protein and mRNA expression compared with samples carrying the minor allele, T. To further investigate the association between the rs12976445 polymorphism and the risk of pneumonitis in patients with lung cancer that received radiotherapy, 534 lung cancer patients diagnosed with pneumonitis and 489lung cancer patients without pneumonitis were recruited. rs12976445 was shown to be significantly associated with the risk of pneumonitis. In conclusion, the rs12976445 polymorphism increased expression levels of TGFß by decreasing the expression of miR­125a, and therefore may be associated with the development of pneumonitis in patients with lung cancer that receive radiotherapy.

Potentially Functional Variants of ATG16L2 Predict Radiation Pneumonitis and Outcomes in Patients with Non-Small Cell Lung Cancer after Definitive Radiotherapy.

INTRODUCTION: Autophagy not only plays an important role in the progression of cancer but is also involved in tissue inflammatory response. However, few published studies have investigated associations between functional genetic variants of autophagy-related genes and radiation pneumonitis (RP) as well as clinical outcomes in patients with NSCLC after definitive radiotherapy. METHODS: We genotyped nine potentially functional single-nucleotide polymorphisms (SNPs) in four autophagy-related genes (autophagy related 2B gene [ATG2B], autophagy related 10 gene [ATG10], autophagy related 12 gene [ATG12], and autophagy related 16 like 2 gene [ATG16L2]) in 393 North American patients with NSCLC treated by definitive radiotherapy and assessed their associations with RP, local recurrence-free survival (LRFS), progression-free survival (PFS), and overall survival (OS) in multivariable Cox proportional hazard regression analyses. RESULTS: We found that patients with the ATG16L2 rs10898880 CC variant genotype had a better LRFS, PFS, and OS (adjusted hazard ratio = 0.59, 0.64, and 0.64; 95% confidence interval: 0.45-0.79, 0.48-0.84, and 0.48-0.86; p = 0.0004, 0.002, and 0.003, respectively), but a greater risk for development of severe RP (adjusted hazard ratio = 1.80, 95% confidence interval: 1.04-3.12, p = 0.037) than did patients with AA/AC genotypes. Further functional analyses suggested that the ATG16L2 rs10898880 C variant allele modulated expression of the ATG16L2 gene. CONCLUSION: This is the first report that one potentially functional SNP rs10898880 in ATG16L2 may be a predictor of RP, LRFS, PFS, and OS in patients with NSCLC after definitive radiotherapy. Additional larger, prospective studies are needed to confirm these findings.

Association between ATM gene polymorphisms, lung cancer susceptibility and radiation-induced pneumonitis: a meta-analysis.

BACKGROUND: Previous studies have suggested that DNA double-strand break (DSB) repair is an important protective pathway after damage. The ataxia telangiectasia mutated (ATM) gene plays an important role in the DNA DSB repair pathway. DNA damage is a major cytotoxic effect that can be caused by radiation, and the ability to repair DNA after damage varies among different tissues. Impaired DNA repair pathways are associated with high sensitivity to radiation exposure. Hence, ATM gene polymorphisms are thought to influence the risk of cancer and radiation-induced pneumonitis (RP) risk in cancer patients treated with radiotherapy. However, the results of previous studies are inconsistent. We therefore conducted this comprehensive meta-analysis. METHODS: A systematic literature search was performed in the PubMed, Embase, China National Knowledge Internet (CNKI) and Wanfang databases to identify studies that investigated the association between the ATM gene polymorphisms and both lung cancer and RP radiotherapy-treated lung cancer (the last search was conducted on Dec.10, 2015). The odds ratio (OR) and 95% confidence interval (CI) were used to investigate the strength of these relationships. Funnel plots and Begg's and Egger's tests were conducted to assess the publication bias. All analyses were performed in STATA 13.0 software. RESULTS: Ten eligible case-control studies (4731 cases and 5142 controls) on lung cancer susceptibility and four (192 cases and 772 controls) on RP risk were included. The results of the overall and subgroup analyses indicated that in the ATM gene, the rs189037 (-111G > A, -4519G > A), rs664677 (44831C > T, 49238C > T) and rs664143 (131,717 T > G) polymorphisms were significantly associated with lung cancer susceptibility (OR = 1.21, 95% CI = 1.04-1.39, P = 0.01; OR = 1.26, 95% CI = 1.06-1.49, P = 0.01; OR = 1.43, 95% CI = 1.15-1.78, P < 0.01). Additionally, the rs189037 variant was significantly associated with RP risk (OR = 1.74, 95% CI = 1.02-2.97, P = 0.04). No publication bias was found in the funnel plots, Begg's tests or Egger's tests. CONCLUSIONS: The results indicate that the ATM rs189037, rs664677 and rs664143 gene polymorphisms are risk factors for lung cancer, while the ATM rs189037 variant was significantly associated with RP risk.

Polymorphisms in BMP2/BMP4, with estimates of mean lung dose, predict radiation pneumonitis among patients receiving definitive radiotherapy for non-small cell lung cancer.

Single nucleotide polymorphisms (SNPs) in TGFß1 can predict the risk of radiation pneumonitis (RP) in patients with non-small cell lung cancer (NSCLC) after definitive radiotherapy. Here we investigated whether SNPs in TGFß superfamily members BMP2 and BMP4 are associated with RP in such patients. In total, we retrospectively analyzed 663 patients given ≥ 60 Gy for NSCLC. We randomly assigned 323 patients to the training cohort and 340 patients to the validation cohort. Potentially functional and tagging SNPs of BMP2 (rs170986, rs1979855, rs1980499, rs235768, rs3178250) and BMP4 (rs17563, rs4898820, rs762642) were genotyped. The median of mean lung dose (MLD) was 17.9 Gy (range, 0.15-32.74 Gy). Higher MLD was strongly associated with increased risk of grade ≥ 2 RP (hazard ratio [HR]=2.191, 95% confidence interval [CI] = 1.680-2.856, P < 0.001) and grade ≥ 3 RP (HR = 4.253, 95% CI = 2.493-7.257, P < 0.001). In multivariate analyses, BMP2 rs235768 AT/TT was associated with higher risk of grade ≥ 2 RP (HR = 1.866, 95% CI = 1.221-2.820, P = 0.004 vs. AA) both in training cohort and validation cohort. Similar results were observed for BMP2 rs1980499. BMP2 rs3178250 CT/TT was associated with lower risk of grade ≥ 3 RP (HR = 0.406, 95% CI = 0.175-0.942, P = 0.036 vs. CC) in the pooled analysis. Adding the rs235768 and rs1980499 SNPs to a model comprising age, performance status, and MLD raised the Harrell's C for predicting grade ≥ 2 RP from 0.6117 to 0.6235 (P = 0.0105). SNPs in BMP2 can predict grade ≥ 2 or 3 RP after radiotherapy for NSCLC and improve the predictive power of MLD model. Validation is underway through an ongoing prospective trial.

Polydatin alleviated radiation-induced lung injury through activation of Sirt3 and inhibition of epithelial-mesenchymal transition.

Radiation-induced lung injury (RILI) is one of the most common and fatal complications of thoracic radiotherapy. It is characterized with two main features including early radiation pneumonitis and fibrosis in later phase. This study was to investigate the potential radioprotective effects of polydatin (PD), which was shown to exert anti-inflammation and anti-oxidative capacities in other diseases. In this study, we demonstrated that PD-mitigated acute inflammation and late fibrosis caused by irradiation. PD treatment inhibited TGF-ß1-Smad3 signalling pathway and epithelial-mesenchymal transition. Moreover, radiation-induced imbalance of Th1/Th2 was also alleviated by PD treatment. Besides its free radical scavenging capacity, PD induced a huge increase of Sirt3 in culture cells and lung tissues. The level of Nrf2 and PGC1α in lung tissues was also elevated. In conclusion, our data showed that PD attenuated radiation-induced lung injury through inhibiting epithelial-mesenchymal transition and increased the expression of Sirt3, suggesting PD as a novel potential radioprotector for RILI.

Involvement of p38-ßTrCP-Tristetraprolin-TNFα axis in radiation pneumonitis.

Early release of tumor necrosis factor-alpha (TNF-α) during radiotherapy of thoracic cancers plays an important role in radiation pneumonitis, whose inhibition may provide lung radioprotection. We previously reported radiation inactivates Tristetraprolin (TTP), a negative regulator of TNF-α synthesis, which correlated with increased TNF-α release. However, the molecular events involved in radiation-induced TTP inactivation remain unclear. To determine if eliminating Ttp in mice resulted in a phenotypic response to radiation, Ttp-null mice lungs were exposed to a single dose of 15 Gy, and TNF-α release and lung inflammation were analyzed at different time points post-irradiation. Ttp-/- mice with elevated (9.5±0.6 fold) basal TNF-α showed further increase (12.2±0.9 fold, p<0.02) in TNF-α release and acute lung inflammation within a week post-irradiation. Further studies using mouse lung macrophage (MH-S), human lung fibroblast (MRC-5), and exogenous human TTP overexpressing U2OS and HEK293 cells upon irradiation (a single dose of 4 Gy) promoted p38-mediated TTP phosphorylation at the serine 186 position, which primed it to be recognized by an ubiquitin ligase (E3), beta transducing repeat containing protein (ß-TrCP), to promote polyubiquitination-mediated proteasomal degradation. Consequently, a serine 186 to alanine (SA) mutant of TTP was resistant to radiation-induced degradation. Similarly, either a p38 kinase inhibitor (SB203580), or siRNA-mediated ß-TrCP knockdown, or overexpression of dominant negative Cullin1 mutants protected TTP from radiation-induced degradation. Consequently, SB203580 pretreatment blocked radiation-induced TNF-α release and radioprotected macrophages. Together, these data establish the involvement of the p38-ßTrCP-TTP-TNFα signaling axis in radiation-induced lung inflammation and identified p38 inhibition as a possible lung radioprotection strategy.

Inhibition of Bcl-2/xl With ABT-263 Selectively Kills Senescent Type II Pneumocytes and Reverses Persistent Pulmonary Fibrosis Induced by Ionizing Radiation in Mice.

PURPOSE: Ionizing radiation (IR)-induced pulmonary fibrosis (PF) is an irreversible and severe late effect of thoracic radiation therapy. The goal of this study was to determine whether clearance of senescent cells with ABT-263, a senolytic drug that can selectively kill senescent cells, can reverse PF. METHODS AND MATERIALS: C57BL/6J mice were exposed to a single dose of 17 Gy on the right side of the thorax. Sixteen weeks after IR, they were treated with 2 cycles of vehicle or ABT-263 (50 mg/kg per day for 5 days per cycle) by gavage. The effects of ABT-263 on IR-induced increases in senescent cells; elevation in the expression of selective inflammatory cytokines, matrix metalloproteinases, and tissue inhibitors of matrix metalloproteinases; and the severity of the tissue injury and fibrosis in the irradiated lungs were evaluated 3 weeks after the last treatment, in comparison with the changes observed in the irradiated lungs before treatment or after vehicle treatment. RESULTS: At 16 weeks after exposure of C57BL/6 mice to a single dose of 17 Gy, thoracic irradiation resulted in persistent PF associated with a significant increase in senescent cells. Treatment of the irradiated mice with ABT-263 after persistent PF had developed reduced senescent cells and reversed the disease. CONCLUSIONS: To our knowledge, this is the first study to demonstrate that PF can be reversed by a senolytic drug such as ABT-263 after it becomes a progressive disease. Therefore, ABT-263 has the potential to be developed as a new treatment for PF.

Standardized Herbal Formula PM014 Inhibits Radiation-Induced Pulmonary Inflammation in Mice.

Radiation therapy is widely used for thoracic cancers. However, it occasionally causes radiation-induced lung injuries, including pneumonitis and fibrosis. Chung-Sang-Bo-Ha-Tang (CSBHT) has been traditionally used to treat chronic pulmonary disease in Korea. PM014, a modified herbal formula derived from CSBHT, contains medicinal herbs of seven species. In our previous studies, PM014 exhibited anti-inflammatory effects in a chronic obstructive pulmonary disease model. In this study, we have evaluated the effects of PM014 on radiation-induced lung inflammation. Mice in the treatment group were orally administered PM014 six times for 2 weeks. Effects of PM014 on radiation pneumonitis were evaluated based on histological findings and differential cell count in bronchoalveolar lavage fluid. PM014 treatment significantly inhibited immune cell recruitment and collagen deposition in lung tissue. Normal lung volume, evaluated by radiological analysis, in PM014-treated mice was higher compared to that in irradiated control mice. PM014-treated mice exhibited significant changes in inspiratory capacity, compliance and tissue damping and elastance. Additionally, PM014 treatment resulted in the downregulation of inflammatory cytokines, chemokines, and fibrosis-related genes and a reduction in the transforming growth factor-ß1-positive cell population in lung tissue. Thus, PM014 is a potent therapeutic agent for radiation-induced lung fibrosis and inflammation.

Genetic variants in the plasminogen activator inhibitor-1 gene are associated with an increased risk of radiation pneumonitis in lung cancer patients.

Plasminogen activator inhibitor-1 (PAI-1) plays a crucial role in the process of lung injury, although its association with radiation pneumonitis (RP) is unclear. We hypothesized that genetic variants in PAI-1 may influence the risk of RP. In this study, 169 lung cancer patients were genotyped for six single-nucleotide polymorphisms in PAI-1 using the Sequenom MassARRAY system. The risk of RP was evaluated by Cox proportional hazards analyses. The cumulative RP probabilities by genotype were assessed using Kaplan-Meier analyses. Univariate and multivariate analyses revealed that PAI-1:rs7242 GT/GG was correlated with an increased occurrence of grade ≥3 RP (crude hazard ratio = 3.331; 95% confidence interval, 1.168-9.497; P = 0.024). Our results indicated that PAI-1:rs7242 in the 3'-untranslated region of PAI-1 can be a predictor of grade ≥3 RP before radiotherapy.