Purpose/Objective(s) Radiation-induced intestinal injuries (RIII) commonly occur during abdomin-pelvic cancer radiotherapy, but its pathogenesis is not well understood. Goblet cells (GCs) are specialized epithelial cells that secrete mucus to physically separate the host and its microbiota, thus preventing bacterial invasion and inflammation to maintain intestinal homeostasis. However, how goblet cells control the amount of mucus they secrete after exposure to ionizing radiation (IR) is unclear. Materials/Methods C57BL/6J and germ-free mice were treated with 13 Gy of whole abdominal irradiation (WAI), and the level of mucus secretion was evaluated by periodic acid–Schiff (PAS) staining and MUC2 fluorescence staining. Primary GCs were isolated from the small intestine of mice, and changes in endoplasmic reticulum stress (ERS) were tested via WB, ELISA, and scanning electron microscopy. The ERS inhibitor salubrinal (SAL) was used to detect changes in GCs mucus secretion after IR in vitro and in vivo. Through transcriptome sequencing, 16S rRNA sequencing and LC‒MS analysis, key genes and metabolites regulating ERS were identified. Results The PAS and MUC2 fluorescence staining results showed that IR significantly reduced GC and mucus secretion in each crypt (P<0.001), while the key proteins ERS, CHOP and Grp78 were significantly activated. Compared to those of the control group, the survival rate of the SAL-treated group was significantly greater (30%) (P<0.05), and the clinical score was improved after WAI. The histological and WB results showed that SAL reversed ERS in the intestine after IR and promoted mucus secretion in GCs, suggesting that ERS is involved in the mucus secretion process. Compared with C57BL/6J mice, germ-free mice irradiated at the same dose also experienced ERS in the intestine, but the amount of mucus remaining unchanged, indicating that the control of mucus secretion by ERS is regulated by the microbiota. 16S rRNA gene-based microbiota analysis and metabolomics analysis indicated that IR regulates the reprogramming of microbial metabolism, particularly the inhibition of tryptophan metabolism. The radioprotective effects of six tryptophan-related indole derivatives were screened at the level of mouse intestinal organoids, and it was found that indole-3-carboxaldehyde (I3A) had the best protective effect. Finally, we found that I3A inhibits ERS and requires the bacterial intracellular sensor Nod2 to promote excessive mucus secretion. Conclusion Radiation-induced ER-stress-mediated regulation of mucus secretion is microbiota dependent which promoted the development of radiation-induced intestinal injury and treatment with I3A could promote intestinal recovery by modulating ERS. Our findings provide new insights into the pathogenesis of radiation-induced intestinal injury.

Purpose/Objective(s) The high radiosensitivity of intestinal epithelium is the main limiting factor for the effect of radiotherapy for abdominal and pelvic malignant tumors. Unfortunately, there is no effective prevention or treatment strategy to mitigate radiation-induced intestinal injury (RIII). At present, studies show that direct DNA damage and oxidative stress are the two major pathogenesis of radiation-induced intestinal injury. Sestrin-2 (SESN2) is a conservative antioxidant protein. The expression of SESN2 will be up-regulated due to oxidative stress, which is a mechanism to compensate the cell damage by the way of inhibiting ROS and activating autophagy. The aim of this study is to provide a new target for the prevention and treatment of radiation-induced intestinal injury by studying the function and molecular mechanism of SESN2 in the process of radiation-induced intestinal injury repair. Materials/Methods Male C57BL/6J mice were exposed to 14Gy of whole abdominal irradiation (WAI) to establish a mouse model of radiation-induced intestinal injury. The levels of mRNA and m6A of genes in small intestine after irradiation were observed by RNA-seq, MeRIP-seq and MeRIP-PCR. Through a series of experiments such as the Cell Counting Kit 8, flow cytometry, γH2AX immunofluorescence and comet assays in vivo and in vitro, we studied the radiosensitivity of intestinal epithelium irradiated with the knockout or overexpression of SESN2 and detect of DNA damage and antioxidant reduction indexes in nucleus and mitochondria. Furthermore, the molecular mechanism of the interaction between SESN2 and m6A reader IGF2BP2 was analyzed by RNA pull-down, mass spectrometry analysis, RIP and dual luciferase reporter gene assays. Results The analysis of sequencing data revealed that SESN2 is regulated by m6A. The expression level of SESN2 in human intestinal epithelial cells is regulated by ionizing radiation. In vivo and in vitro experiments, its overexpression can promote radiation resistance. Through the detection of DNA damage and antioxidant reduction indexes, we found that SESN2 can regulate ROS production both in nucleus and mitochondria. Through RNA pull-down, mass spectrometry analysis, RIP, we found that SESN2 is regulated by m6A reader IGF2BP2 after ionizing radiation. And there is a decrease in survival rate and weight mice after inhibiting IGF2BP2 in vivo. Finally, we conducted the recovery experiment. Knockdown of SESN2 with overexpression of IGF2BP2 resulted in an increase in SESN2 mRNA, m6A, and protein levels and reducing DNA damage and production of ROS in HIEC-6 cells after irradiation, providing evidence for the regulation of SESN2 by IGF2BP2. Conclusion In conclusion, SESN2 depends on m6A modification of IGF2BP2 and further regulates the production of ROS in the nucleus and mitochondria, which may be a novel mechanism involved in the occurrence and development of RIII.

Purpose/Objective(s) There is currently no effective strategy available to mitigate radiation-induced intestinal injury (RIII). Because of its central role in the DNA damage response, p53 is often referred to as the 'guardian of the genome’. When activated in an oscillatory manner, it promotes repair of DNA damage, but sustained activation can lead to cell death. The aim of this study was to investigate the regulatory mechanism of p53 oscillatory activation in intestinal stem cells (ISCs) following radiation exposure. Additionally, a mathematical model of the protein regulatory network was used to predict the most effective method of drug delivery to promote p53 oscillatory activation. Materials/Methods Male C57BL/6J mice were exposed to 14Gy of whole abdominal irradiation (WAI). Immunofluorescence (IF) staining was used to observe the expression of p53 protein in crypts at various time points after WAI. To assess the mRNA and m6A levels of genes associated with the p53 pathway, we utilized RNA-seq, MeRIP-seq, and MeRIP-PCR. Additionally, we identified the murine double minute 2 (MDM2) N6-methyladenosine (m6A)-modified methylase through RNA-pulldown, mass spectrometry analysis, RIP, and dual luciferase reporter gene assays. A mathematical model of the p53 regulatory network was constructed, incorporating methyltransferase-like protein 3 (mettl3), and used to predict the effects of a mettl3 inhibitor (S-Adenosylhomocysteine, SAH) on p53 activation. To assess the impact of SAH, we measured the number of germinations, surface area, and survival rate of mouse intestinal crypt organoids. We also observed the survival and body weight. Furthermore, we performed immunofluorescence tests for intestinal stem cell markers and p53. Results After WAI, it was observed that P53 expression in mouse crypts occurred in an oscillatory pattern. Further analysis of sequencing data revealed that MDM2, the most important negative feedback regulator of p53, is regulated by m6A. This was confirmed through subsequent experiments, which demonstrated the binding of mettl3 to MDM2 mRNA. Knockdown of mettl3 resulted in a significant decrease in MDM2 mRNA, m6A, and protein levels in HIEC-6 cells after irradiation, providing evidence for the regulation of MDM2 by mettl3. Mathematical modeling of the p53 regulatory network showed that administering the appropriate dose of SAH resulted in an elevated level of p53 oscillatory activation, leading to an increase in the germination rate, surface area, and survival rate of irradiated organoids, as well as an improvement in the survival and body weight of mice. Furthermore, both organoid and mouse models showed an increase in the number of stem cells and a decrease in the amount of DNA damage. Conclusion Overall, the present study shows that IR-responsive mettl3 is involved in IR-induced DNA damage repair in ISCs, probably by regulating the p53 oscillatory activation via MDM2 m6A modification, which may be a novel mechanism involved in the occurrence and development of RIII.

Purpose/Objective(s) Acute radiation-induced intestinal injury (ARIII) is a common side effect of abdominal and pelvic radiotherapy, which can have negative effects on the prognosis and quality of life of cancer patients. This study aimed to evaluate the potential of supplementing Bifidobacterium longum BL21 (referred to as BL21) in preventing ARIII, and its influence on the compositions of intestinal microbiota and metabolites. Materials/Methods Acute radiation-induced intestinal injury (ARIII) is a common side effect of abdominal and pelvic radiotherapy, which can have negative effects on the prognosis and quality of life of cancer patients. This study aimed to evaluate the potential of supplementing Bifidobacterium longum BL21 (referred to as BL21) in preventing ARIII, and its influence on the compositions of intestinal microbiota and metabolites. Results From April 7, 2023, to October 31, 2023, a total of 44 patients were enrolled. The overall incidence of ARIII was 84.1%, with only 27.2% experiencing grade ≥2 ARIII, significantly lower than the reported 65% in historical control groups. No adverse events related to BL21 were reported in any patients. Regarding intestinal microbiota, the α-diversity of intestinal microbiota significantly decreased after radiotherapy and was associated with the severity of ARIII. The abundance of Lachnospiraceae significantly increased after radiotherapy (P=0.025). In patients with grade 0/1 ARIII, certain metabolites showed significant upregulation in stool samples at the end of radiotherapy. These included Indole-3-carboxylic acid (P=0.025), alanine (P=0.025), homocysteine (P=0.021), valine (P=0.0028), and phenylalanine (P=0.039). However, this difference was not observed in patients who developed ≥ grade 2 ARIII. Indole-3-carboxylic acid is a key intermediate product in the tryptophan metabolism pathway, while the others are precursor substances in the fatty acid metabolism pathway. Conclusion Prophylactic use of BL21 can prevent grade ≥2 ARIII, making it a safe, simple, and effective method in clinical practice. The potential mechanism of the preventive effect of BL21 may involve restructuring the intestinal flora to enhance beneficial bacteria and suppress harmful bacteria. Additionally, it may upregulate the tryptophan metabolic pathway and stimulate the production of short-chain fatty acids.

Purpose/Objective(s) The intestine is vulnerable to structural and functional damage caused by exposure to radiation. Unfortunately, there is currently no effective prophylactic or therapeutic strategy available to mitigate radiation-induced intestinal injury (RIII). Deubiquitinating enzymes (DUBs) play a crucial role in repairing DNA breaks. Therefore, we conducted a new study on the pathogenesis of RIII by examining the role of DUBs, in order to identify potential directions for therapeutic or preventive measures in this area. Materials/Methods The effects of 14 Gy whole abdominal irradiation (WAI) on DUB levels in the intestine of C57BL/6J mice were investigated by RNA-seq analysis. In vivo and in vitro experiments were conducted using the ubiquitin-specific proteases 15 (USP15) inhibitor (USP15-IN-1). The impact of USP15 on the radiosensitization of HIEC-6 cells was observed. The survival and body weight of mice in each irradiated group were recorded, and the severity of radiation-induced intestinal injury (RIII) was evaluated through HE staining, immunohistochemistry (IHC), and the TUNEL method. USP15-bound proteins were identified and validated through mass spectrometry analysis. The role of USP15 in ataxia-telangiectasia mutated (ATM) deubiquitination and stability was determined by constructing a USP15 mutant (C298A). Finally, the potential reversal of USP15 knockdown’s promotional effects on radiosensitizing effect in HIEC-6 cells by ATM was investigated. Results USP15 is one of the top 20 highly expressed genes in the intestinal tissue of mice after exposure to 14Gy of WAI. Inhibition of USP15 resulted in increased radiosensitivity of HIEC-6 cells, as evidenced by a decrease in colony-forming ability and an increase in the formation of micronuclei and apoptotic cells, as well as an increase in 8-OHdG fluorescence intensity. Comet assay and γ-H2AX staining revealed more DNA damage in irradiated HIEC-6 cells treated with USP15-IN-1. In vivo, USP15 was found to modulate apoptosis and DNA damage in the small intestine of mice exposed to WBI. Mass spectrometry analysis showed that USP15 interacts with the protein kinase ATM, a key regulator of DNA double-strand break (DSB) signaling and stress response. By creating a mutant form of USP15(C298A), it was discovered that USP15 directly interacts with ATM, independent of its DUB activity, and can regulate the stability of ATM protein. Furthermore, USP15 was found to specifically disassemble K48-linked polyubiquitination of ATM but had no significant effect on monoubiquitination or other types of polyubiquitination. The radiosensitizing effect produced by knockdown of USP15 in HIEC-6 cells can be reversed by ATM. Conclusion Our findings demonstrate that USP15 plays a crucial role in repairing radiation damage in intestinal epithelial cells by counteracting ATM ubiquitination and degradation, which may be a novel mechanism involved in the occurrence and development of RIII.

In this phase II clinical trial, compared to historical controls, the prophylactic use of EGCG significantly reduced the incidence and severity of RIII in patients receiving pelvic RT. Therefore, EGCG has the potential to become a novel medical countermeasure for the prevention of RIII for pelvic cancer patients.

Purpose/Objective(s) The intestine is a highly radiosensitive tissue that is susceptible to structural and functional damage due to systemic as well as localized radiation exposure. Unfortunately, no effective prophylactic or therapeutic agents are available at present to manage radiation-induced intestinal injuries (RIII). Gut microbiome play pivotal roles in intestinal homeostasis. Several studies have demonstrated the potential of gut microbiome and metabolites as a therapeutic for RIII. Our results demonstrate that microbiota-derived Indole-3-carboxaldehyde (I3A) plays a pivotal role in promoting ISC-mediated epithelial development. Materials/Methods Male C57BL/6J mice were treated with 13Gy of Whole abdominal irradiation (WAI). Probiotics or I3A were gavage before (once a day for 30 days) WAI. The survival and body weight were recorded, while the severity of RIII was evaluated by HE staining, Immunohistochemistry (IHC) and TUNEL assay of gut tissues for each irradiated group. Meanwhile, stool samples were obtained 6h and 3.5d after irradiation. Gut microbiome were measured by 16S rRNA sequencing, and metabolites were detected by LC-MS analysis. Results Compared to the control, probiotics (Lactobacillus plantarum, Bifidobacterium longum, Lactobacillus paracasei) treatment significantly increased survival rates by 50% (P<0.05) and improved clinical scores of mice after WAI. HE staining of jejunum tissues showed that probiotics mitigated RIII, as reflected by the dramatic attenuation of crypt-villi architecture destruction. IHC results showed that probiotics treatment remarkably increased the Lgr5+ cells, Paneth cells, and Ki67+cells (P<0.001) per crypt, indicating that probiotics promoted proliferation and differentiation of ISC after WAI. The most highly enriched metabolites from the feces of probiotics treatment mice in the tryptophan metabolic pathway, with a threefold change seen in level of I3A. Specifically, I3A -treated groups showed survival rates of ∼20%%, with the protection persisting for >200 days after radiation. In addition, mice fed the I3A daily for 3 days before IR had significantly more Lgr5+, Paneth cells, Goblet cells, and Ki67+cells in the SI crypt than mice of control. Moreover, treatment with I3A resulted in enhanced numbers of Lgr5+ cells and Paneth cells and Wnt3 expression in SI organoids. Conclusion Collectively, our findings suggested that microbiota-derived I3A protects against RIII by promote the proliferation and differentiation of Lgr5+ intestinal stem cells via Wnt signaling pathway.

Purpose/Objective(s)Statistically, a disease is considered curable when the mortality rate of survivors returns to the same level as that of the general population. We aimed to assess the curability of extranodal nasal-type NK/T-cell lymphoma (ENKTCL) in the modern treatment era.Materials/MethodsThe data of 1995 patients treated between 2000 and 2016 and registered in China Lymphoma Collaborative Group (CLCG) database were analyzed. We estimated cure fractions, median survival times, and the time point of cure using a non-mixture cure model.ResultsThe relative survival curves attained plateau for the entire cohort and most subsets, indicating that the notion of cure was robust. The overall cure fraction was 72.1%. Median survival was 1.10 years in uncured patients. The cure time was 4.5 years, i.e., beyond this time, mortality in ENKTCL patients was equivalent to that in the general population. In multivariate analysis, cure probability was associated with stage, performance status, primary tumor invasion, and lactate dehydrogenase level. Elderly patients (>60 years) had cure fraction similar to that of younger patients. Five-year overall survival rate correlated well with the cure fraction across risk-stratified groups. Thus, statistical cure is possible in ENKTCL patients receiving modern treatment strategies.ConclusionOverall probability of cure is good, though it is affected by presence of risk factors. These findings provide the rationale for treatment and follow-up of patients with different cure probabilities.

Dysbiosis of both gut microbiota and metabolome develops in patients with RIII. Gut bacteria Erysipelatoclostridium and its related metabolite ptilosteroid A may collaboratively predict grade 2 RIII, and could be used as a prediction model.

Seneparib (previously known as IMP4297) is a novel oral PARP inhibitor which is 20-fold more potent than olaparib in vivo and showing strong antitumor activity in preclinical studies. This phase I study of senaparib is to evaluate PK, safety/tolerability and preliminary antitumor activity in patients with advanced solid tumours. The primary objective was to evaluate the safety profile of senaparib given orally QD, including the identification of the maximum tolerated dose and the recommended phase II dose (RP2D). Dose escalation was guided by a modified Fibonacci sequence, starting at 2 mg/day, with a traditional 3+3 design. Dose expansion cohort was planned to start from the dose level in which efficacy was observed. As of Feb 29, 2020, 54 patients, including 34 BRCA mutation carriers, had been enrolled at 2-120 mg dose level. No DLT was observed. The most frequent treatment-related adverse events (TRAE) were anemia (48%), followed by leukopenia (41%), thrombocytopenia (26%), neutropenia (22%), nausea (22%) and fatigue (22%). Most of them were grade 1 or 2 in severity. Seven (13%) patients interrupted and 3 (6%) patients discontinued treatment due to AEs. Among 41 pts with at least one follow-up RECIST 1.1 assessment, 6 germline BRCA+ patients, 1 somatic BRCA+ patient and 2 BRCA- patients had PR, including one long-lasting PR over 9 months (BRCA+ ovarian cancer, 60mg). The overall ORR and DCR was 22% and 61% respectively. In 17 BRCA+ ovarian cancer patients, the ORR is 24% and DCR is 82%. An alternative dosing regimen of 50mg bid is ongoing to compare PK characteristic between QD and BID dosing. We further confirmed senaparib is well-tolerated, with mild to moderate haematologic toxicity as the most frequent TRAEs and showed encouraging signs of clinical activity. The 100 mg orally QD was selected as the RP2D based on safety, pharmacokinetics and clinical activity.

PARP inhibitors are promising anti-cancer agents with proven clinical activity based on mechanism of synthetic lethality. Senaparib (previously known as IMP4297) is a novel highly potent and selective oral PARP1/2 inhibitor with strong antitumor activity in preclinical studies. This first in human study investigated the tolerability, safety, PK, and preliminary antitumor activity of senaparib in Australia. Adults with advanced, refractory solid tumours received senaparib orally QD, starting at 2mg. Dose escalation used a traditional 3+3 design and a modified Fibonacci sequence with 3-6 patients per cohort. DLT was evaluated in the first cycle. Dose expansion cohort enrolled patients with BRCA mutated (BRCA+) advanced solid tumors. As of Feb 25, 2020, 39 patients were enrolled in 10 dose levels (2 to 150mg). No DLTs were observed. The most frequent treatment emergent adverse events (TEAE) were headache (25.6%), fatigue (25.6%), constipation (17.9%), diarrhea (15.4%), nausea (12.8%), vomiting (12.8%) and anemia (10.3%). Treatment-related adverse events (TRAE) were observed in 8 (21%) patients starting from 40mg dose group. The most frequent TRAEs were nausea (8%), thrombocytopenia (5%) and fatigue (5%). An event of grade 4 thrombopenia in 80mg was the only serious TRAE. Four (10%) patients interrupted and 6 (15%) patients discontinued therapy due to AEs. The overall ORR and DCR was 15% and 85% respectively. In 8 evaluable ovarian cancer patients, ORR was 38% and DCR was 75%. A prolonged (> 20 months) PR response was observed in one BRCA+ ovarian cancer patient and a > 50% decrease of PSA for 11 months was observed in one BRCA- prostate cancer patients. The plasma exposure increased proportionally with doses ranging from 2mg to 80mg and became nonlinear ranging from 80mg to150mg cohorts. Senaparib demonstrated encouraging clinical benefit and a favorable tolerability profile in patients with advanced solid tumour. The 100 mg orally QD was selected as the RP2D in Australia based on safety, pharmacokinetics and clinical activity. Clinical trial information: NCT03507543.

The gut is highly radio-sensitive, and the early triage of affected populations by biodosimeters is essential for the effective treatment of radiation induced intestinal injury (RIII) after radiological events. Gut microbiome plays pivotal roles in intestinal homeostasis. Due to the reproducibility, convenience, stability and non-invasive access, several studies have demonstrated the potential of gut microbiome and metabolites as biomarkers for the early diagnosis of intestinal diseases. Here, we tested the feasibility of gut microbiome and metabolites as biodosimeters for the early triage of RIII in rodent. Male C57BL/6 mice were given total body irradiation (TBI) with a single dose of 0, 4, 8 and 12Gy, respectively. The survival and body weight were recorded, while the severity of RIII was evaluated by HE staining, Immunohistochemistry (IHC) and TUNEL assay of gut tissues for each irradiated group. Meanwhile, stool samples were obtained 6h and 3.5d after irradiation. Gut microbiomes were measured by 16S rRNA sequencing, and metabolites were detected by LC -MS analysis. Compared to the control group, the survival rates and body weights significantly decreased in a dose-dependent way after TBI. The villus lengths, number of crypts and Ki-67 positive cells decreased, while the TUNEL positive cells of intestinal tissues increased in a dose-dependent manner, indicating severer RIII with higher TBI doses. For the gut microbiome, at genus level the relative abundance of Alistipes (1.3%, 6%, 9%), Lachnospiraceae_NK4A136_group (1.4%, 0.8%, 0.3%), Marvinbryantia (0.2%, 0.4%, 0.6%), Parabacteroides (1%, 3%, 6%) and Parasutterella (1%, 2%, 3%) exerted dose-dependent changes 3.5d after TBI (P<0.05). As for the gut metabolites, the expressions of Erysopine, Roquefortine, Harderoporphyrin, PHENYL-gamma-Aminobutyric Acid, 3-(3,4-dihydroxyphenyl)-N-[2-(4-hydroxyphenyl)ethyl] propanimidic acid, 3alpha,7beta,12alpha-Trihydroxy-6-oxo-5alpha-cholan-24-oic Acid and Nalbuphine showed dose- and time-dependent changes 6h and 3.5d after TBI (P<0.05). The data from this study could be used to develop a multi-biodosimeters panel based on gut microbiome and metabolites that could be used for dose assessment and early triage of RIII after radiological events.

Esophageal carcinoma (EC) is one of the deadliest cancers worldwide, and is the sixth leading cause of death from malignancy. Radio-resistance remains the major obstacle to improve the prognosis of EC. Epigenetics including acetylation and N6-methyladenosine (m6A) play pivotal roles in tumor radio-resistance. Therefore, the aim of this study is to investigate the biological effect and underline mechanisms of acetyltransferase Tip60 in the radio-resistance of EC. TCGA data was used to determine the genetic alterations of Tip60 in EC, and Kaplan-Meier analysis was performed to determine the clinical impact of Tip60 on EC survival. Then, colony formation assay was used to measure the radio-sensitization after Tip60 knockdown in EC cell line model. Next, the impact of Tip60 knockdown on global m6A level was detected by a m6A quantification kit. Meanwhile, the expression of key m6A modification enzymes was measured by western blot and qRT-PCR. Finally, mRNA sequencing (mRNA-seq) and m6A immunoprecipitation sequencing (meRIP-seq) were conducted to investigate the downstream targets. In a TCGA dataset which contains 186 EC cases, Tip60 genetically alters in 11% cases (most are upregulation), and Kaplan-Meier survival analysis demonstrated poorer median overall survival of Tip60 altered cases than normal cases (12.4 months versus 31.2 months, P = 0.02). Knockdown of Tip60 by shRNA exerted radio-sensitization effect, and decreased both global m6A level and m6A methyltransferase METTL16 expression in EC cells. To understand the regulatory role of Tip60 in comprehensive gene expression, mRNA-seq identified 369 genes that were differentially expressed by at least 2-fold after Tip60 knockdown, which primarily involved in p53 pathway and PI3K-AKT pathway. To screen out genes that were differentially expressed by METTL16-mediated m6A methylation, meRIP-seq and mRNA-seq data were jointly analyzed, and a total of 4 genes that decreased at m6A level while increased at mRNA level at least 2-fold after Tip60 knockdown were selected, including MAP1A, FBXO2, AXL and DDX58. Tip60 inhibition radio-sensitizes EC via m6A methyltransferase METTL16, and supports the clinical development of Tip60 inhibitors for the radio-sensitization of EC.

Intestine is one of the most radio-sensitive tissues, and radiation induced intestinal injury (RIII) remains one of the most prevalent dose-limiting toxicities in radiotherapy for pelvic and abdominal cancers. The failure of synthetic compounds for efficient prevention or treatment of RIII led to growing interest in the study of natural derivatives from nontoxic plants. Epigallocatechin-3-gallate (EGCG), the major polyphenol in green tea, is a free radical scavenger and potent antioxidant agent, and has been widely studied in cancer chemoprevention. However, whether EGCG has the therapeutic effect on RIII remains unknown. Male C57BL/6J mice were treated with 9Gy of total body irradiation (TBI). 25mg/kg of EGCG were intraperitoneally injected before (once a day for 5 days) and 30minutes after TBI. Then, the survival rate and body weight were recorded. Meanwhile, jejunum tissues were collected and the severity of RIII was measured by HE staining, immunohistochemistry (IHC), immunofluorescence (IF) and TUNEL. Next, human intestinal epithelial cells (HIEC) were treated with EGCG and irradiated. Then, colony formation assay, flow cytometry, IF were conducted, and the level of reactive oxygen species (ROS) was assessed. Further, the expression of pivotal anti-oxidative response regulator nuclear factor erythroid 2 related factor 2 (Nrf2) was detected by IF and western blotting. Compared to the control, EGCG treatment significantly improved the survival time (from 6.1days to 10.4days, P<0.05) and increased the body weights of mice after TBI. HE staining of jejunum tissues showed that EGCG mitigated RIII, as reflected by the dramatic attenuation of crypt-villi architecture destruction. IHC results showed that EGCG treatment remarkably increased the Ki67-positive (from 15.7 to 30.1, P<0.001) and Lgr5-positive (from1.5 to 3.0, P<0.001) cells per crypt, indicating that EGCG promoted proliferation and survival of intestinal stem cells after TBI. Meanwhile, in EGCG treatment group the number of TUNEL-positive cells (from 4.9 to 1.0, P<0.001), γH2AX foci (from 2.8 to 0.4, P<0.001) and 8-OHdG-positive cells (from 3.5 to 0.6, P<0.001) significantly decreased per crypt, indicating that EGCG inhibited radiation-induced apoptosis and DNA damage in intestine. Consistently, the EGCG treatment remarkably improved the colony forming ability, while decreased apoptosis ratio, γH2AX foci and 8-OHdG foci of irradiated HIEC cells. For the mechanism study, EGCG treatment diminished the radiation-induced production of ROS, and promoted the nuclear translocation of key anti-oxidative response regulator Nrf2 of irradiated HIEC cells, indicating that EGCG scavenged radiation-induced ROS, and subsequently attenuated ROS-regulated intestinal oxidative DNA damage. Collectively, our findings suggested that EGCG protects against RIII by reducing ROS-induced DNA damage, and might be a novel therapeutic option for the clinical treatment of RIII.

Anaplastic thyroid carcinoma (ATC) is an aggressive cancer with extremely poor prognosis due to resistant to conventional therapies including radiotherapy. Our previous study demonstrated that elevated lysine acetyltransferase 5 (KAT5) expression in ATC is associated with poorer survival. Here, we sought to investigate the biological role and underline molecular mechanism of KAT5 in the radio-resistance of ATC. Human ATC cells 8505C and CAL-62, along with normal thyroid cell Nthy-ori 3-1 were cultured. KAT5 expressions were detected by western blot and qPCR. Then, radio-sensitivity after KAT5 knockdown or inhibition (by KAT5 specific inhibitor NU9056) was measured by colony formation assay, and DNA damage was quantified by γH2AX expression. For the mechanism study, Oncomine data and western blot were used to determine c-Myc expression in ATC. Finally, microRNA sequencing, qPCR and dual-luciferase reporter assay were conducted to identify the downstream of c-Myc. Endogenous KAT5 protein and mRNA levels were much higher in ATC cells than in normal thyroid cell. Inhibition of KAT5, by using both genetic (siRNA) and pharmacologic (NU9056) methods, exerted radio-sensitization effect and increased DNA damage in ATC cells, but showed no impact on the radio-sensitivity of normal thyroid cell. Our previous study found that KAT5-mediated acetylation stabilizes c-Myc protein via the inhibition of ubiquitin-proteasome system. Oncomine dataset revealed that c-Myc expression was most elevated in ATC than in other type of thyroid cancers. At cellular level, inhibition of KAT5 significantly shortened the half-life of c-Myc protein. MicroRNA sequencing showed that knockdown of KAT5 decreased microRNA-210 (miR-210) level. Consistently, qPCR showed that inhibition of KAT5 decreased miR-210 level. Finally, forced c-Myc expression remarkably enhanced the luciferase activity of miR-210 promotor, indicating that KAT5 up-regulates miR-210 expression at transcriptional level through c-Myc. Our results indicated that abnormally high expression of KAT5 leads to c-Myc stabilization, subsequently miR-210 over-expression, and results in ATC radio-resistance. This study may provide us with a promising strategy by targeting KAT5 to increase the efficacy of radiotherapy for ATC.

An all in one nano-system with active-targeting, enzyme-triggered deshielding and positive-charge characteristics was fabricated for chemo/photo-combination therapy to allow efficient tumor targeting, cellular internalization and lysosomal escape. The deshielding of NPs was induced by enzyme triggered degradation of the NP shell, and consequently exposure of the positively charged core accelerates escape of NPs from the lysosome to exert anticancer effects with high efficiency.

Background: Immunotherapy has been proven to be effective in gastric cancer (GC) patients. But the efficacy of immunotherapy for HER2 positive GC has not been defined. Tumor mutation burden (TMB) has been considered as predictive biomarkers of immunotherapy. However, the association between HER2 alterations and TMB in GC is still unclear. We therefore analyzed the associated between HER2 alterations and TMB in Chinese patients with GC. Methods: Genomic profiling of DNA from fresh or FFPE tumor tissue of 163 Chinese GC was performed using next-generation sequencing on 381 cancer associated genes in 3D Medicines laboratory. Whole-exome sequencing and Genomic Identification of Significant Targets in Cancer (GISTIC) data of 435 GC from The Cancer Genome Altas (TCGA) were also analyzed to evaluate the association between HER2 alterations with TMB. TMB was defined as number of somatic non-synonymous mutations in coding region. HER2 amplification in TCGA was defined as "2" derived from the copy-number analysis algorithms GISTIC. Results: HER2 alterations including amplification, missense and fusion were present in 11.0% (18/163) of Chinese cohort and 17.0% (74/435) of TCGA cohort. 5.5% (9/163) of Chinese cohort and 14.3% (62/435) of TCGA cohort harbored HER2 amplification. Higher TMB level was observed in patients carrying any HER2 alterations (P = 0.004) in Chinese cohort, but the association was not found in TCGA cohort (P = 0.379). Prognosis analysis was also performed on patients in TCGA cohort. HER2 status was not an independent risk factors affecting disease free survival (HR, 1.007; 95%CI 0.60-1.69; P = 0.979) or overall survival (HR, 0.828; 95%CI 0.56-1.24; P = 0.357). Higher TMB level was associated with significantly better disease free survival (median, 43.04 vs. 25.53 months; HR, 0.685; 95%CI 0.47-1.00; P = 0.048) and a borderline improvement in overall survival (median, 42.51 vs. 25.69 months; HR, 0.75; 95%CI 0.56-1.02; P = 0.070). Conclusions: HER2 alterations are associated with higher TMB in Chinese GC patients, but not in patients from TCGA. Chinese GC patients may exhibit distinct characteristics. Further studies are warranted to evaluate the efficacy of immunotherapy in GC patients with HER2 alterations. Legal entity responsible for the study: Yanhong Gu. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.

Background: Tumor mutational burden (TMB) is associated with genome instability and immunogenicity, which has been reported to play an important role in predicting the efficacy of immune checkpoint inhibitors. However, the relationship of TMB and prognosis in solid tumors is not yet fully understood. In this study, we aimed to explore the association between TMB and prognosis across pan-cancers.Table: 57PDThe associations of TMB and OS across pan-cancerstumorfull namesTMB-median (interquartile)ALLStage I-IIStage III-IVNHR(95% CI)PNHR(95% CI)PNHR(95% CI)PBLCABladder Urothelial Carcinoma151(85-269)4080.51(0.33-0.78)<0.0011311.07(0.45-2.55)0.892750.41(0.24-0.68)<0.001BRCABreast invasive carcinoma39(26-69)9701.48(0.87-2.54)0.157200.98(0.49-1.94)0.952294.46(1.57-12.69)<0.001CHOLCholangiocarcinoma32(26-44)411.37(0.35-5.32)0.65282.33(0.44-12.49)0.32(-)(-)(-)COADColon adenocarcinoma111(82-167)3781.43(0.79-2.58)0.242023.24(0.93-11.37)0.071651.57(0.7-3.5)0.27ESCAEsophageal carcinoma100(77-144)1831.84(0.98-3.48)0.06973.01(1.18-7.68)0.02631.07(0.36-3.14)0.90GBMGlioblastoma multiforme47(37-64)3851.01(0.73-1.42)0.93(-)(-)(-)(-)(-)(-)HNSCHead and Neck squamous cell carcinoma92(60-140)5061.65(1.11-2.45)0.01963.94(1.34-11.59)0.013411.11(0.69-1.78)0.66KIRCKidney renal clear cell carcinoma48(35-63)3333.72(1.79-7.75)<0.0012228.23(1.9-35.6)<0.0011091.82(0.75-4.38)0.18KIRPKidney renal papillary cell carcinoma54(35-73)2770.39(0.13-1.15)0.091871.17(0.19-7.04)0.8663(-)(-)LGGBrain Lower Grade Glioma25(18-34)5015.03(2.87-8.8)<0.001(-)(-)(-)(-)(-)(-)LIHCLiver hepatocellular carcinoma77(55-107)3571.5(0.88-2.53)0.132501.87(0.87-3.98)0.11861.36(0.61-3.05)0.45LUADLung adenocarcinoma171(74-338)5010.83(0.54-1.28)0.393900.89(0.52-1.52)0.671040.69(0.33-1.46)0.33LUSCLung squamous cell carcinoma205(148-297)4830.75(0.52-1.09)0.133900.7(0.46-1.06)0.09890.92(0.38-2.21)0.84OVOvarian serous cystadenocarcinoma79(53-130)4290.73(0.52-1.03)0.08(-)(-)(-)(-)(-)(-)PAADPancreatic adenocarcinoma35(25-46)1731.62(0.88-3.02)0.12(-)(-)(-)(-)(-)(-)READRectum adenocarcinoma95(74-127)1270.68(0.21-2.27)0.53611.22(0.11-13.47)0.87580.6(0.1-3.72)0.59SARCSarcoma40(27-54)2351.25(0.63-2.49)0.52(-)(-)(-)(-)(-)(-)SKCMSkin Cutaneous Melanoma226(62-415)1030.45(0.15-1.3)0.14670.14(0.01-1.32)0.09311.51(0.36-6.32)0.58STADStomach adenocarcinoma106(66-216)4090.52(0.34-0.8)<0.0011780.45(0.2-0.98)0.052140.53(0.31-0.92)0.02THCAThyroid carcinoma9(6-13)4886.92(0.85-56.28)0.07325(-)(-)1611.34(0.16-11.19)0.79UCECUterine Corpus Endometrial Carcinoma89(47-562)5270.27(0.13-0.57)<0.001(-)(-)(-)(-)(-)(-)UVMUveal Melanoma12(9-15)682.5(0.45-13.88)0.2937(-)(-)302.4(0.19-30.98)0.50 Open table in a new tab Methods: Whole-exome sequencing from 7882 solid tumors, spanning 22 cancer types from The Cancer Genome Atlas were analyzed. TMB was defined by total non-silent somatic mutation counts in coding region. Patients were classified into four groups based on the quartiles of TMB in each tumor. All Hazard ratios (HR) were reported as the risk ratio of the top 25% to the bottom 25% group. Results: Among 22 tumors, the level of TMB of top five tumors (median, interquartile, sample size) were: SKCM (226, 62-415, 103), LUSC (205, 148-297, 483), LUAD (171, 74-338, 501), BLCA (151, 85-269, 408), COAD (111, 82-167, 378). In all patient analyses of each tumor, higher TMB was associated with longer overall survival (OS) in BLCA, STAD and UCEC, and was associated with shorter OS in HNSC, KIRC and LGG. In stage I-II of each tumor, higher TMB was significantly associated with prolonged OS in STAD, but with shorter OS in ESCA, HNSC and KIRC. In stage III-IV of each tumor, higher TMB was significantly associated with prolonged OS in BLCA and STAD, but with shorter OS in BRCA. In 5 tumors (including BLCA, HNSC, KIRC, LUSC and THCA), patients with higher pathological stage had significantly higher TMB (P < 0.05). However, only in COAD and READ, patients with higher stage had significantly lower TMB (P < 0.05). Conclusions: Higher TMB played different roles in various tumors, which may be attribute to different driver mutations or other factors. Further analyses of underlying mechanism were underway to confirm and explore the potential prognosis prediction of TMB across pan-cancers. Legal entity responsible for the study: Feng Qiu. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.

Aim Locally advanced rectal cancer (LARC) is frequently treated with neoadjuvant chemoradiotherapy (NACRT) to reduce the risk of local recurrence and improve survival. Tumour response to NACRT is variable and may influence the prognosis after subsequent surgery. This study compared the prognostic values of tumour regression grade (TRG) and neoadjuvant pathological (ypTNM) downstaging in patients with Stage II and III rectal cancer treated with NACRT followed by curative surgery.Method This study included 185 patients with LARC treated with long-course radiotherapy (45 Gy in 25 fractions) plus 5-fluorouracil over 5 weeks between 2005 and 2013. We used multivariate analysis to assess the relationship of Dworak's five-tier TRG, ypTNM stage and ypTNM downstaging with clinicopathological factors, 5-year disease-free survival (DFS) and 5-year overall survival (OS).Results Total regression (TRG4), good regression (TRG3), moderate regression (TRG2), minor regression (TRG1) and no regression (TRG0) were seen in 38 (20.6%), 65 (35.2%), 43 (23.2%), 28 (15.1%) and 11 (5.9%) patients, respectively. TNM downstaging following NACRT occurred in 109 (58.9%) patients. The 5-year DFS rates after NACRT for TRG0, TRG1, TRG2, TRG3 and TRG4 were 0%, 58.5%, 66.4%, 80.4% and 82.6%, respectively (P < 0.001). The ypTNM stage correlated with 5-year DFS (P = 0.004) but not 5-year OS (P = 0.075). Multivariate analysis demonstrated that TRG was related to both DFS and OS (P < 0.001).Conclusion TRG measured on a five-tier system was better than ypTNM stage for predicting outcome in patients with LARC treated with NACRT and surgery.

Background: Late adverse events lead to concern on long-term efficacy of drueluting stents (DES).The clear mechanism of late in-stent restenosis (ISR) remains unclear.Sustained hypersensitivity reaction played an important role in the formation of restenosis.Eosinophil cationic protein (ECP) was a sensitive marker of eosinophil activation.Previous studies have demonstrated that higher ECP levels were associated with late adverse events after DES implantation.Purpose: We aimed to investigate the relationship between serum ECP levels and late ISR.Methods: We enrolled 202 patients who underwent angiography follow up beyond 1 year after DES implantation in our hospital.Patients were divided into two groups (ISR, n=100 and non-ISR, n=102) according to the findings at follow-up angiography.Serum ECP levels were detected using sandwich ELISA methods and compared between groups.In subgroup analysis, we divided patients into DP-DES and BP-DES groups according to their implanted stent type.Results: Serum ECP levels were significantly higher in the ISR group than in the non-ISR group [20.6 (6.7, 45.6) vs. 10.5 (3.6, 25.3) μg/mL; p=0.003].Multivariate regression revealed that ECP levels, procedure age were independent risk factors for late ISR formation.In sub-group analysis, Serum ECP levels were significantly higher in patients with DP-DES in ISR group compared to non-ISR group [24.9 (8.4,59.1) vs. 10.5 (3.1, 22.4) μg/mL, p=0.002], while no difference was found in patients with BP-DES.Conclusion: Serum ECP levels were higher in patients with late ISR.ECP levels were only significantly higher in ISR patients after DP-DES implantation compared to non-ISR patients.