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Supplementary Table 1: Radiosensitivity parameters of nasopharyngeal carcinoma cell lines

Supplementary Figure legends

Copper (Cu) is an essential trace element whose serum levels have been reported to act as an effective indicator of the efficacy of radiotherapy. However, little is known about the role of Cu in radiotherapy. In this study we aimed to determine this role and investigate the precise mechanism by which Cu or Cu-related proteins regulate the radiosensitivity of hepatocellular carcinoma (HCC).The expression and function of Cu and copper metabolism MURR1 domain 10 (COMMD10) were assessed via a Cu detection assay, immunostaining, real-time PCR, western blot, a radiation clonogenic assay and a 5-ethynyl-2'-deoxyuridine assay. Ferroptosis was determined by detecting glutathione, lipid peroxidation, malondialdehyde and ferrous ion (Fe) levels. The in vivo effects of Cu and COMMD10 were examined with Cu/Cu chelator treatment or lentivirus modification of COMMD10 expression in radiated mouse models.We identified a novel role of Cu in promoting the radioresistance of HCC cells. Ionizing radiation (IR) induced a reduction of COMMD10, which increased intracellular Cu and led to radioresistance of HCC. COMMD10 enhanced ferroptosis and radiosensitivity in vitro and in vivo. Mechanistically, low expression of COMMD10 induced by IR inhibited the ubiquitin degradation of HIF1α (by inducing Cu accumulation) and simultaneously impaired its combination with HIF1α, promoting HIF1α nuclear translocation and the transcription of ceruloplasmin (CP) and SLC7A11, which jointly inhibited ferroptosis in HCC cells. In addition, elevated CP promoted HIF1α expression by reducing Fe, forming a positive feedback loop.COMMD10 inhibits the HIF1α/CP loop to enhance ferroptosis and radiosensitivity by disrupting Cu-Fe homeostasis in HCC. This work provides new targets and treatment strategies for overcoming radioresistance in HCC.Radiotherapy benefits patients with unresectable or advanced hepatocellular carcinoma (HCC), but its effectiveness is hampered by radioresistance. Herein, we uncovered a novel role for copper in promoting the radioresistance of HCCs. This work has revealed new targets and potential treatment strategies that could be used to sensitize HCC to radiotherapy.

Supplemental Methods and References. Description of additional methods and procedures used in the study. Also includes Supplemental References.

Upregulation of matrix metalloproteinase MMP-14 (MT1-MMP) is associated with poor prognosis in cancer patients, but it is unclear how MMP-14 becomes elevated in tumors. Here, we show that miR-181a-5p is downregulated in aggressive human breast and colon cancers where its levels correlate inversely with MMP-14 expression. In clinical specimens, enhanced expression of MMP-14 was observed in cancer cells located at the invasive front of tumors where miR-181a-5p was downregulated relative to adjacent normal cells. Bioinformatics analyses defined a potential miR-181a-5p response element within the 3′-untranslated region of MMP-14 that was validated in reporter gene experiments. Ectopic miR-181a-5p reduced MMP-14 expression, whereas miR-181a-5p attenuation elevated MMP-14 expression. In support of a critical relationship between these two genes, miR-181a-5p–mediated reduction of MMP-14 levels was sufficient to decrease cancer cell migration, invasion, and activation of pro-MMP-2. Furthermore, this reduction in MMP-14 levels was sufficient to reduce in vivo invasion and angiogenesis in chick chorioallantoic membrane assays. Taken together, our results establish the regulation of MMP-14 in cancers by miR-181a-5p through a posttranscriptional mechanism, and they further suggest strategies to elevate miR-181a-5p to prevent cancer metastasis. Cancer Res; 75(13); 2674–85. ©2015 AACR.

Brain necrosis (RN) is a common radiotherapy sequela for brain metastases. Bevacizumab is identified as a therapeutic strategy for RN. This study aimed to study the clinical and radiobiological impacts on the efficacy of Bevacizumab in treating RN following stereotactic radiosurgery (SRS) for brain metastases.From April 2011 to November 2019, 40 patients diagnosed with RN after SRS for brain metastases were retrospectively analyzed. Patients were treated with Bevacizumab for RN and follow-up for 6 months using MR imaging at different timepoints. Linear regression was performed to evaluate the relationship between these variables.The median time course from the end of radiotherapy to the onset of RN was 11 months (range, 7-35 months). No significant difference was found in the edema volume between the chemotherapy group and non-chemotherapy group (P0.05). Patients received with SRS + WBRT exhibited relatively larger edema volumes post radiotherapy than those without WBRT (P0.05). Interestingly, the ratio of BED/GTV (Gy/cm3 ) correlated positively with the severity (time for half-reduction dose of corticosteroids) (r2 =0.13, P0.05), and negatively with the latency period (time course for development of radiation-induced brain necrosis) (r2 =0.21, P0.01). A new radiation doses volume index, BED × GTV (Gy·cm3 ), was proposed to facilitate the risk stratifications of patients for radiation-induced brain necrosis. Furthermore, no significant difference was found in alleviating brain edema between different regimens of Bevacizumab, i.e., 5 vs. 10 mg/kg, 2 vs.2 cycles (both P0.05).Bevacizumab is a feasible and favorable salvage treatment of BN after SRS for patients with BM. The efficacy is mainly manifested in radiological improvement and symptoms alleviation. The development of RN was found to be largely associated with radiation dose and gross tumor volume, and thus we proposed two new indexes, i.e., BED/GTV (Gy/cm3 ) for quantitative assessment of the severity and latency time, and BED × GTV (Gy·cm3 ) for risk stratifications for BN. A low dose with two cycles of Bevacizumab is recommended.

While cancer immunotherapy has been remarkably successful in some malignancies, some cancers derive limited benefit from current immunotherapies. Here, we combined immune landscape signatures with hepatocellular carcinoma clinical and prognostic features to classify them into distinct subtypes. The immunogenomic profiles, stromal cell features and immune cell composition of the subtypes were then systematically analyzed. Two independent prognostic indexes were established based on 6 immune-related genes and 17 differentially expressed genes associated with stromal cell content. These indexes were significantly correlated with tumor mutation burden, deficient DNA mismatch repair and microsatellite instability. In addition, tumor-infiltrating lymphocytes, including activated NK cells, resting memory CD4 T-cells, eosinophils, and activated mast cells were significantly correlated with hepatocellular carcinoma survival. In conclusion, we have comprehensively described the immune landscape signatures and identified prognostic immune-associated biomarkers of hepatocellular carcinoma. Our findings highlight potential novel avenues for improving responses to immunotherapy.

BackgroundWe conducted this study to evaluate if a reduced cumulative dose of induction and concurrent cisplatin conferred similar favorable outcomes when compared to trial NPC-0501.MethodsNewly diagnosed nasopharyngeal carcinoma (NPC) with stage III-IVA were prospectively recruited from January 2015 to September 2019. Induction chemotherapy (IC) consisted of cisplatin 80mg/m2 on day 1 and capecitabine 1000mg/m2 twice daily from day 1 to 14 every 3 weeks for 3 cycles followed by concurrent chemoradiotherapy (CCRT) with 2 cycles of cisplatin 100mg/m2 given every 3 weeks. Tumor response was evaluated according to RECIST v1.1. Acute and late adverse events (AEs) were graded with CTCAE v4.0 and Late Radiation Morbidity Scoring of the RTOG, respectively.Results135 patients were recruited. At 16 weeks after CCRT, all 130 patients who completed the entire course of radiotherapy (RT) had a complete response upon final assessment. With a median follow-up of 36.2 months, 22 treatment failures and 8 deaths were observed. The 3-year progression-free survival, overall survival, locoregional recurrence-free survival, and distant recurrence-free survival were 83.7%, 94.1%, 94.1%, and 85.9%, respectively. Our survival data outcomes were similar to those reported in the cisplatin and capecitabine (PX) induction arm of the 0501 trial. 103 patients (76.3%) reported acute grade 3-4 AEs. Two patients (1.5%) had late grade 3-4 complications, numerically fewer than those reported in the NPC-0501 trial.ConclusionsInduction PX and concurrent cisplatin with a reduced cumulative cisplatin dose yield survival outcomes comparable to those reported in the NPC-0501 trial with excellent tolerability. Therefore, a reduced cumulative dose of cisplatin is a promising treatment scheme for nasopharyngeal carcinoma.

Background: Glioblastoma (GBM) is a common primary brain tumor with a high incidence in adults with malignant and fast-growing biological characteristics. In this study, we explore the immune-related prognosis markers of GBM at the mRNA level.Methods: The sequencing data and clinical information of GBM patients were downloaded from The Cancer Genome Atlas (TCGA) and the Chinese Glioma Genome Atlas (CGGA). The differentially expressed genes (DEGs) were calculated between normal tissues from the Genotype-Tissue Expression (GTEx) database and tumor tissues from TCGA and CGGA. We obtained immune genes from the ImmProt database. The intersection of DEGs and immune genes were defined as the immune-related differential genes (IR-DEGs), based on which, survival associated IR-DEGs were determined by multivariate Cox regression analysis. The survival risk score (SRS) was determined for each sample with the top 6 prognostic associated IR-DEGs. One-year, two-year, and three-year potential survival were predicted by the prognosis prediction model established by multivariate and univariate Cox regression. In addition, we performed CIBERSORT in GBM patients with samples from TCGA cohort; association analysis was performed with prognostic IR-DEGs and immune cells. Furthermore, the influence of prognostic IR-DEGs on the brain tumor microenvironment (TME) was validated in single-cell sequencing analysis.Results: We found 301 IR-DRGs in GBM primary tumor compared with normal tissue, and 19 of them could predict the overall survival (OS) more accurately in GBM patients. Six IR-DEGs (PLAUR, TNFSF14, CTSB, SOCS3, PTX3, and FCGR2B) were selected to construct the SRS, with which, GBM patients were divided into two different groups which combined with high and low risk. The SRS was found to be an independent prognostic factor for GBM and could predict GBM patients’ possible survival with an acceptable efficiency. Moreover, the expression of 6 IR-DEGs and their co-expressed IR-DEGs could influence TME and were associated with GBM prognosis.Conclusions: This study identified a potential immune prognostic signature of glioblastoma, which could enhance the prognosis prediction ability for GBM patients. The immune-related-genes in the TME could potentially benefit the immunotherapy development for GBM patients.

There is no consensus in clinical practice on the optimal bladder volume during brachytherapy. The present study aimed to assess the effect of bladder volume on radiation dose to organs at risk and tumor in cervical cancer during image-guided adaptive brachytherapy and clinical outcome. The retrospective study included patients treated at University of Hong Kong-Shenzhen Hospital between January 2015 and July 2019. Patients with International Federation of Gynecology and Obstetrics (2009) stage IB1-IVB (retroperitoneal lymph nodes metastasis only) cervical cancer treated by external beam radiotherapy with concurrent cisplatin followed by brachytherapy were assessed. A total of 421 brachytherapy insertions were analyzed. Every 83 and 90 cm3 (cc) increase in bladder volume led to an incremental raise of 1 Gy in bladder wall minimum dose received by the most irradiated 1 and 2 cc volumes (D1 and D2cc) of the bladder wall, respectively. An increase in bladder volume was associated with increased D1 and D2cc of bladder (both P89.6 Gy (42.1 months vs. not reached, P=0.001). Patients with grade 2 acute urinary toxicity had significantly higher cumulative bladder wall D2cc than those with acute urinary toxicity

Background The Copper Metabolism MURR1 (COMM) domain family has been reported to play important roles in tumorigenesis. As a prototype for the COMMD family, the expression pattern and biological function of COMMD6 in human tumours remain unknown. Methods COMMD6 expression in BALB/c mice and human tissues was examined using real-time PCR and immunohistochemistry. Kaplan–Meier analysis was applied to evaluate the prognosis of COMMD6 in tumours. Competing endogenous RNA (ceRNA) and transcriptional regulation network were constructed based on differentially expressed mRNAs, microRNAs and long non-coding RNAs from the cancer genome atlas database. GO and KEGG enrichment analysis were used to explore the bioinformatics implication. Results COMMD6 expression was widely observed in BALB/c mice and human tissues, which predicted prognosis of cancer patients. Furthermore, we shed light on the underlying tumour promoting role and mechanism of COMMD6 by constructing a TEX41-miR-340-COMMD6 ceRNA network in head and neck squamous cell carcinoma and miR-218-CDX1-COMMD6 transcriptional network in cholangiocarcinoma. In addition, COMMD6 may modulate the ubiquitination and degradation of NF-κB subunits and regulate ribonucleoprotein and spliceosome complex biogenesis in tumours. Conclusions This study may help to elucidate the functions and mechanisms of COMMD6 in human tumours, providing a potential biomarker for tumour prevention and therapy.

Upregulation of histone methyltransferase SET domain bifurcated 1 (SETDB1) is associated with poor prognosis in cancer patients. However, the mechanism of oncogenicity of SETDB1 in cancer is hitherto unknown. Here, we show that SETDB1 is upregulated in human colorectal cancer (CRC) where its level correlates with poor clinical outcome. Ectopic SETDB1 promotes CRC cell proliferation, whereas SETDB1 attenuation inhibits this process. Flow cytometry reveals that SETDB1 promotes proliferation by driving the CRC cell cycle from G0/G1 phase to S phase. Mechanistically, SETDB1 binds directly to the STAT1 promoter region resulting in increased STAT1 expression. Functional characterization reveals that STAT1-CCND1/CDK6 axis is a downstream effector of SETDB1-mediated CRC cell proliferation. Furthermore, SETDB1 upregulation is sufficient to accelerate in vivo proliferation in xenograft animal model. Taken together, our results provide insight into the upregulation of SETDB1 within CRC and can lead to novel treatment strategies targeting this cell proliferation-promoting gene.

Background Aberrant expression of microRNA-664 was involved in tumor growth and metastasis of various cancers. The specific role of miR-664 in cutaneous squamous cell carcinoma (cSCC) is yet to be elucidated. Objective The present study aimed to investigate the molecular mechanisms underpinning of cSCC development and provide translational insights for future therapeutics. Methods Human cSCC specimens were used to determine the miR-664 by in situhybridization and IRF2 by immunohistochemistry. To study the potential mechanisms in tumorigenesis, three cSCC cell lines including HSC-5, HSC-1 and A431 as well as BALB/C mouse tumor model was utilized. Results We found that miR-664 was remarkably high in cSCC patient specimens and cSCC cell lines. Overexpression of miR-664 promotes tumorigenic behaviors such as increased cell proliferation, migration and invasion capacities in vitro and enhanced tumorigenicity in xenograft mouse model. Our data further identified IRF2 as a direct downstream target of miR-664. Knockdown of IRF2 reverses pro-tumorigenesis phenotype of miR-664; whereas IRF2 over-expression inhibits miR-664 tumorigenesis in cSCC. Together, it revealed miR-664 functions as an oncogene in cSCC via suppression of IRF2. Conclusion Our data demonstrates that aberrant expression of miR-664 plays a critical role in carcinogenesis of cSCC. The discovery of novel targets such as miR-664 and IRF2 will facilitate future development of therapeutic interventions.

Background There have been no reliable scientific studies examining whether the interval between induction chemotherapy (IC) and initiating radiotherapy is associated with poor outcomes of nasopharyngeal carcinoma (NPC). Patients and methods In this retrospective study, we included a total of 239 local advanced NPC patients who underwent concurrent chemoradiotherapy and IC. Based on the interval between IC and intensity-modulated radiation therapy (IMRT), the patients were classified into three groups as follows: Group A (≤7 vs >7 days), Group B (≤14 vs >14 days), and Group C (≤ 21 vs >21 days). Univariate and multivariate regression analyses were performed to determine the prognostic factors of survival outcomes. The differences between the two groups were compared by the log-rank test. Results The median IC-IMRT interval was 9 days (range, 1-76 days). The median follow-up time was 40 months (range, 4-58 months). The IC-IMRT interval including Group A, Group B, and Group C was not significantly associated with overall survival (OS), distant metastasis-free survival (DMFS), locoregional relapse-free survival (LRFS), or disease-free survival (DFS). Multivariate analysis showed that the tumor stage was the independent significant predictor for OS, DMFS, LRFS, and DFS. But it appears that there was a trend toward improvement in the outcome of ≤7 days group in OS from the Kaplan-Meier curves. Conclusion It is also feasible to postpone radiotherapy for 1-3 weeks if patients were unable to receive treatment immediately due to chemotherapy complications such as bone marrow suppression. However, we suggest that patients should start IMRT as soon as possible after IC.

COMMD10, a member of COMMD protein, has been proved to target p65 NF-kappaB (nuclear factor-kappaB) subunit and reduce its nuclear translocation, thereby leading to the inactivation of NF-kappaB pathway and suppression of colorectal cancer invasion and metastasis. The aim of this study is to explore its expression pattern and tissue distribution in human normal tissues and other tumor tissues and to investigate the relevant mechanism. We firstly provided the expression profile and histological distribution of COMMD10 in various BALB/c mice tissues and identified the biological distribution of COMMD10 in different kinds of human normal and tumor tissues. We verified the expression profile of COMMD10 using TCGA database. The interacting genes of COMMD10 were predicted by using STRING using. Finally, we performed database, and the microRNAs targeting COMMD10 were predicted using miRDB, miRWalk, TargetScan and microRNA. GO and KEGG pathway analyses were performed to predict the biological function of COMMD10 and its interacting genes. mRNA expression of COMMD10 showed the highest level in the lung and spleen, and the lowest level in the heart and brain. Immunohistochemistry detection revealed that COMMD10 was expressed in different tissues with different degrees and was was located mainly in the cytoplasm. Subsequently, we showed that COMMD10 displayed various degrees of expression in different human normal tissues that mainly located in cytoplasm, while COMMD10 of liver cells resided in both nucleus and cytoplasm. All the tumor tissues except breast small cell carcinoma, breast phyllodes tumor, lung adenocarcinoma, thymoma, cervical cancer and bladder urothelial carcinoma showed that COMMD10 was positive staining in cytoplasm. Kaplan-Meier plotter indicated that renal clear cell carcinoma patients with increased expression level of COMMD10 exhibited longer survival. STRING database revealed that COMMD10 had 41 interacting genes, and data from 4 different databases indicated that hsa-miR-590-3p may be the potential regulator of COMMD10. GO analysis demonstrated that COMMD10 and its interacting genes were mainly enriched in Cullin-RING ubiquitin ligase complexes, binding and transport of copper ions, the transport and steady-state maintenance of copper ions, transcription, translation and transport of proteins, and negatively regulate the activity of NF-kappaB transcription factors. KEGG pathway showed that COMMD10 and its interacting genes were mainly involved in renal cell carcinoma, HIF-1 signaling pathways, ubiquitination-mediated proteolysis, endocytosis and mineral absorption. COMMD10 may play a tumor suppressive role in renal clear cell carcinoma through the miR-590-3p-COMMD10-Cul2-RBX1-NF-κB/HIF/NRF2 pathway and regulate the chemotherapy resistance of various tumor cells to cisplatin.

Highlights • Determining normal tissue tolerance of organs at risk is of utmost importance for the success of carbon ion radiotherapy (CIRT). • This manuscript is the first report on experimental RBE for development of lung fibrosis assessed by quantitative CT after fractionated CIRT. • We further propose RBE estimation by integrating RBEmax and α/βL based modified high-LET BED model. • This model was found to well predict RBE but is sensitive to the uncertainties of α/βL estimates from the reference photon irradiation. • Our findings will contribute to a more precise knowledge of RBE as well as the fractionated dose effects of carbon ion therapy on normal lung tissue., Background and purposes Carbon ion radiotherapy (CIRT) with raster scanning technology is a promising treatment for lung cancer and thoracic malignancies. Determining normal tissue tolerance of organs at risk is of utmost importance for the success of CIRT. Here we report the relative biological effectiveness (RBE) of CIRT as a function of dose and fractionation for development of pulmonary fibrosis using well established fibrosis index (FI) model. Materials and Methods Dose series of fractionated clinical quality CIRT versus conventional photon irradiation to the whole thorax were compared in C57BL6 mice. Quantitative assessment of pulmonary fibrosis was performed by applying the FI to computed tomography (CT) data acquired 24-weeks post irradiation. RBE was calculated as the ratio of photon to CIRT dose required for the same level of FI. Further RBE predictions were performed using the derived equation from high-linear energy transfer biologically effective dose (high-LET BED) model. Results The averaged lung fibrosis RBE of 5-fraction CIRT schedule was determined as 2.75 ± 0.55. The RBE estimate at the half maximum effective dose (RBEED50) was estimated at 2.82 for clinically relevant fractional sizes of 1–6 Gy. At the same dose range, an RBE value of 2.81 ± 0.40 was predicted by the high-LET BED model. The converted biologically effective dose (BED) of CIRT for induction of half maximum FI (BEDED50) was identified to be 58.12 Gy3.95. In accordance, an estimated RBE of 2.88 was obtained at the BEDED50 level. The LQ model radiosensitivity parameters for 5-fraction was obtained as αH = 0.3030 ± 0.0037 Gy−1 and βH = 0.0056 ± 0.0007 Gy−2. Conclusion This is the first report of RBE estimation for CIRT with the endpoint of pulmonary fibrosis in-vivo. We proposed in present study a novel way to mathematically modeling RBE by integrating RBEmax and α/βL based on conventional high-LET BED conception. This model well predicted RBE in the clinically relevant dose range but is sensitive to the uncertainties of α/β estimates from the reference photon irradiation (α/βL). These findings will assist to eliminate current uncertainties in prediction of CIRT induced normal tissue complications and builds a solid foundation for development of more accurate in-vivo data driven RBE estimates.

Background Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality worldwide. Currently, there is limited knowledge of dysregulation of cellular proliferation and apoptosis that contribute to the malignant phenotype in HCC. Copper metabolism gene MURR1 domain 10 (COMMD10) is initially identified as a suppressor gene in the pathogenesis of HCC in our observations. Here we aimed to explore its function and prognostic value in the progression of HCC. Methods Functional experiments were performed to explore the role of COMMD10 in HCC. The molecular mechanisms of COMMD10 were determined by luciferase assay, immunofluorescence, and immunoprecipitation. The nomogram was based on a retrospective and multicenter study of 516 patients who were pathologically diagnosed with HCC from three Chinese hospitals. The predictive accuracy and discriminative ability of the nomogram were determined by a C‐index and calibration curve and were compared with COMMD10 and the Barcelona Clinic Liver Cancer (BCLC) staging system. The primary endpoint was overall survival (OS). Results COMMD10 expression was significantly lower in HCC than that in normal liver tissues. In vitro and in vivo experiments revealed that COMMD10 suppressed cell proliferation and induced apoptosis in HCC. Mechanistically, COMMD10 inhibits TNFα mediated ubiquitination of IκBα and p65 nuclear translocation through the combination of COMMD10‐N terminal to the Rel homology domain of p65, which inhibited NF‐κB activity and increased expression of cleaved caspase9/3 in HCC. Clinically, COMMD10 stratifies early‐stage HCC patients into two risk groups with significantly different OS. Additionally, the nomogram based on COMMD10 and BCLC stage yielded more accuracy than BCLC stage alone for predicting OS of HCC patients in three cohorts. Conclusions COMMD10 suppresses proliferation and promotes apoptosis by inhibiting NF‐κB signaling and values up BCLC staging in predicting OS, which provides evidence for the identification of potential therapeutic targets and the accurate prediction of prognosis for patients with HCC., 1. COMMD10 stabilizes IκBα protein by inhibiting ubiquitination and degradation of IκBα. 2. COMMD10 interacts with the Rel homology domain of p65 and suppresses the nuclear translocation of p65. 3. COMMD10/NF‐κB axis promotes apoptosis by modulating Bcl‐2/Bax/caspase‐9/3 pathway in hepatocellular carcinoma.

This study aimed to evaluate the clinical features, prognostic factors, and survival outcomes for patients with intracranial nongerminomatous germ cell tumors (NGGCTs), with a particular focus on treatment toxicity for long-term survivors.Intracranial NGGCTs treated with platinum-based chemotherapy and craniospinal irradiation (CSI) in our institution were retrospectively analyzed. Hematological complications following sequential chemoradiotherapy as well as height and weight in childhood survivors were evaluated. Plasma growth hormone (GH) concentrations prior to and after radiotherapy were obtained for the comparisons.A total of 111 intracranial NGGCTs were included. The 3‑year overall survival (OS) and event-free survival (EFS) rates were 83.5% ± 3.9% and 71.0% ± 4.8%, respectively. A combined treatment modality consisting of ≥ 4 cycles of platinum-based chemotherapy and CSI was associated with an improved OS (P = 0.003) and EFS (P 0.001). Thrombocytopenia of any grade occurred in 35.4% (34/96) of patients, and the threshold age for an increased risk of thrombocytopenia was 14 years (area under the curve AUC = 0.752, P 0.0001) as derived from receiver operating characteristic (ROC) analysis. Growth impediment was found in 8 of 56 (14%) patients. The age for receiving radiotherapy was found to inversely correlate with height development, revealing a cut-off age of 11.5 years for risking growth impairment (AUC = 0.806, P = 0.004). Consistently, a significant decline in plasma growth hormone after radiotherapy was observed in patients ≤ 11.5 years (P 0.01) but not patients 11.5 years. (P 0.05).Our study suggested that a combined treatment modality with at least four cycles of chemotherapy and CSI was safe and effective for patients with intracranial NGGCTs. Radiotherapy should be used with caution for patients 11.5 years due to growth impairment.

Background and purpose: To introduce a standard linac with true attachment free, rapid arc and dose-drop scheme for positioning related dose deviation control based total body irradiation(TBI).Materials/Methods: One eight years old girl diagnosed with acute lymphocytic leukemia underwent TBI in 2020. Target volumes and organs at risk were contoured after CT simulation. Total sixteen ARC and four AP-PA from five isocenters were designed. A dose-drop scheme on both sides of adjacent region were performed to reduce positioning-related dose deviation. A series of quality assurance before radiotherapy and real-time dose monitoring during radiotherapy were carried out.Results: The average on board imaging (OBI) time of per fraction was 40.3 min, the average beam on time of per fraction was 37.2 min, the average time to change from head first to feet first position was 18.4 min. The average mean lung dose was 9.89 Gy, the maximum lens dose was 7.60 Gy, the mean PTV_total dose was 12.17Gy, 98.23% PTV_total volume was covered by 90% of the prescription dose. The maximum dose (Dmax) of PTV_total was 13.65Gy. Dmean and V10.8 of PTV_total are only slightly different(0.49% - 1.89% and 0.26% - 1.04% respectively) even with an error of 5 - 20mm longitudinal misalignments. Gamma passing rate(3mm/ 3% Gamma criteria) are between 93.5% and 100%. Real-time dose monitoring showed an overall deviation of -3.9%±5.51%.Conclusions: Standard linac, rapid arc and dose-drop for positioning-related dose deviation control based total body irradiation is feasible, accurate, and reliable. It is worthy of clinical application.

Carbohydrate sulfotransferase 4 (CHST4) plays an important role in lymphocyte homing and is abnormally expressed in several cancer types; however, its precise function in tumor development and progression is unknown. Here we confirm that CHST4 is aberrantly expressed in various tumor subtypes. In particular, we found that CHST4 expression was downregulated in hepatitis B virus-related hepatocellular carcinoma (HBV-HCC) tumors compared to paired normal tissue. We also showed that CHST4 overexpression inhibited the proliferation and metastasis of HCC cells in vitro. Clinically, CHST4 was identified as an independent prognostic factor for HBV-HCC patients. We further illuminated the anti-tumor role and mechanism of CHST4 in HBV-HCC by constructing a FENDRR–miR-10b-5p–CHST4 competing endogenous RNA network. We found that downregulation of CHST4 expression may promote HBV expression and regulate ribonucleoprotein complex biogenesis to promote malignant behaviors in HBV-HCC. CHST4 may also recruit CD4+ T cells, macrophages, dendritic cells, and neutrophils into the tumor microenvironment to inhibit the progression of HBV-HCC. Overall, our findings suggest that CHST4 acts as a tumor suppressor in HCC-HBV and represents a potential diagnostic and therapeutic target.

Background and purpose Leptomeningeal metastasis (LM) is a rare but detrimental complication in patients with non-small cell lung cancer (NSCLC). Although whole brain radiotherapy (WBRT) is used to eliminating cancer cells or microscopic foci, it is becoming less favorable due to the concerns over neurocognitive toxicity. This study aimed to re-evaluate the role of WBRT in the setting of modern targeted therapy. Materials and methods From December 2014 to March 2019, 80 NSCLC patients with cytologically and/or radiologically proven LM diagnosis were retrospectively analyzed. Results The median OS (mOS) after diagnosis of LM was 8.0 (95%CI: 4.4 to 11.6) months, and the one-year OS was 39.4%. The mOS for EGFR-mutated LM patients was 12.6 (3.0 to 22.2) months versus only 4.1 (2.8 to 5.4) for patients with wild-type EGFR (P P = 0.041). No survival benefits were found in EGFR-mutated patients who received WBRT (P = 0.490). In contrast, mOS was significantly prolonged in wild-type EGFR patients with WBRT versus non-WBRT (mOS: 8.0 vs. 2.1, P = 0.002). Multivariate analysis indicated that WBRT (P = 0.025) and younger age (P = 0.048) were independent prognostic factors that predicted prolonged survival for wild-type EGFR LM patients from NSCLC. Conclusion Our study demonstrated that WBRT has clear survival advantages for patients with wild-type EGFR, and molecular biological stratification of LM patients for WBRT is highly recommended.

Background: Risk of colorectal cancer (CRC) is defined by genetic predisposition and environmental factors that often co-occur and interact, resulting in diversiform biological reactions. The present study attempted to investigate transcriptome alteration and adaptation associated with CRC progression. Methods: The study consisted of patients who presented at Memorial Sloan-Kettering Cancer Center, Guangzhou, China with a colonic neoplasm in 1992-2004. Microarray GSE41258 of the study was acquired from Gene Expression Omnibus and 253 included microarrays were categorized by groups of normal colon, early primary tumor, lymph node metastases primary tumor, advanced primary tumor and distant metastases. Short Time-series Expression Miner (STEM) was applied to discover tumor grade-dependent gene expression patterns. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were carried out to explore functional enrichment of differential expression genes (DEGs). Results: Overall, 2870 significant DEGs were screened out on all groups. Six significant grade-dependent gene expression patterns were statistically significant. DEGs in all significant patterns were mainly assembled in GO terms of metastases and deterioration of tumor, epithelial proteins and cytokines, and protein binding and bridging. DEGs in profile 0 down-regulated with higher tumor grade, prominently enriched in KEGG pathways of metabolism. Conclusion: Besides many well-known colorectal cancer-related pathways, DEGs of profiles especially those down-regulated with CRC progression, clustered in various metabolic pathways including starch and sucrose metabolism, fatty acid metabolism, nitrogen metabolism, as well as xenobiotics biotransformation that link to tumorigenesis, demonstrating the impairment of physiological metabolic pathways in the context of tumor progression. These results gave a high potential for therapeutic strategies.

Supplemental Material, Glioma_M1_Suppl_V8_upload for Increased M1 Macrophages Infiltration Correlated With Poor Survival Outcomes and Radiation Response in Gliomas by Zhaoming Zhou, Lei Wen, Mingyao Lai, Changguo Shan, Jian Wang, Rong Wang, Hainan Li, Longhua Chen, Linbo Cai, Meijuan Zhou and Cheng Zhou in Dose-Response

Background and purpose: Radioresistance is one of the main obstacles limiting the therapeutic efficacy of chemoradiotherapy (CRT) and favorable patient prognoses, and the molecular mechanisms underlying this type of resistance remain unclear. The purpose of this study was to identify characteristic genes involved in chemoradiotherapy resistance in nasopharyngeal carcinoma (NPC). Materials and methods: Clinicopathological data of 185 patients with NPC treated at Nanfang Hospital of Southern Medical University between January 2013 and December 2014 were retrospectively analyzed. SPSS statistical software was used to analyze the clinicopathological data related to radiotherapy efficacy. Three patients who achieved complete remission and three with disease progression after CRT were selected. Differentially expressed genes (DEGs) were screened via mRNA microarray analysis of primary diagnostic endoscopy specimens. Results: The peripheral blood leukocyte count, platelet count and EBV-DNA copy number in NPC patients who were resistant to radiotherapy were higher than those in NPC patients who were sensitive to radiotherapy. The RobustRankAggreg (RRA) analysis method identified 392 DEGs, and the 66 most closely related genes among the DEGs were identified from the PPI network. Conclusion: The results of this study indicate that screening for DEGs and pathways in NPC using integrated in silico analyses can help identify a series of genetic and clinical signatures for NPC patients treated with neoadjuvant chemotherapy followed by concurrent chemoradiotherapy. Funding Statement: This work was supported by the Clinical Research Startup Program of Southern Medical University by High-level University Construction Funding of Guangdong Provincial Department of Education (LC2019ZD008); the Clinical Research Startup Program of Nanfang Hospital (2018CR021);the National Natural Science Foundation of China (Grant Nos. 81602685 and 81672992); the Science and Technology Projects in Guangdong Province (Grant Nos. 2018-1201-SF-0019 ); the Health & Medical Collaborative Innovation Project of Guangzhou City, China (201803040003); and the Natural Science Foundation of Guangdong Province (Grant No. 2017A030313486); the Natural Science Foundation of Fujian Province (Grant No. 2017J01245). Declaration of Interests: We declare no competing interests. Ethics Approval Statement: The protocol was approved by the ethics committee of Nanfang Hospital and implemented in accordance with the principles of the Declaration of Helsinki. All participants provided informed consent.

Background: In this study, we aim to identify the molecular markers associated with the nasopharyngeal carcinoma (NPC) prognosis using Weighted Gene Co-Expression Network Analysis (WGCNA) and DNA methylation analysis. Methods: GEO accession viewer was utilized to download three NPC mRNA microarray data from GEO. Limma package was used for the differential analysis of three databases (i.e. GSE12452, GSE53819 and GSE52068). On the basis of miRNA-mRNA interaction in the starBase (v3.0, http://starbase.sysu.edu.cn), we then screened the miRNA that were interacted with the genes in the modules. The biomarkers associated with the pathogenesis and prognosis of NPC were screened using WGCNA and DNA methylation analysis. Results: WGCNA identified two gene modules (the Brown module and Magenta module) that were extremely associated with NPC. Survival analysis of 26 hub genes was based on the Kaplan Meier Plotter to depict the survival curvature using the TCGA data. In total, 109 interacted pairs of mRNA-miRNA-lncRNA regulatory network were established. Prognostic evaluation was conducted to the hub genes using the head and neck cancer data in the TCGA database. The data indicated that the overall survival was significantly correlated with the expression of C22orf15, KIF6, EPC1, STAP2, OLR1 and WDR38. Conclusions: WGCNA and DNA methylation analysis were helpful for the screening of genes associated with the pathogenesis and prognosis of NPC. We screened 6 hub genes may be closely related to the prognosis of NPC patients. Funding Statement: This work was supported by the Natural Science Foundation of Fujian Province (Grant No. 2017J01245); the Clinical Research Startup Program of Southern Medical University by High-level University Construction Funding of Guangdong Provincial Department of Education (Grant No. LC2019ZD008); the Clinical Research Startup Program of Nanfang Hospital (Grant No. 2018CR021); the National Natural Science Foundation of China (Grant Nos. 81602685 and 81672992); the Science and Technology Projects in Guangdong Province (Grant Nos. 2018-1201-SF-0019); the Health & Medical Collaborative Innovation Project of Guangzhou City, China (Grant No. 201803040003); and the Natural Science Foundation of Guangdong Province (Grant No. 2017A030313486). Declaration of Interests: None. Ethics Approval Statement: The study was approved by the Ethics Committee of Nanfang Hospital, Southern Medical University and written informed consent was obtained from the patients.

Zinc finger protein 307 (ZNF307) is a new Krüppel-associated box zinc-finger protein gene and a member of the zinc-finger family of transcriptional factors. Notably, the role of ZNF307 and its underlying mechanisms involved in hepatocarcinogenesis are poorly investigated. In the present study, we found that the expression of ZNF307 was significantly downregulated in hepatocellular carcinoma (HCC) tissues, compared with that in adjacent non-tumor tissues. In vitro studies further demonstrated that ectopic expression of ZNF307 in HCC cell lines Bel7402 and HCCLM3 significantly reduced cell proliferation, migration and invasive ability. Concordantly, knockdown of ZNF307 increased cell proliferation, migration and invasive ability of HCC cell lines MHCC97L and QGY7701. In vivo functional studies showed that upregulation of ZNF307 expression in Bel7402 cells led to a suppression of tumorigenicity in mice, while knockdown of ZNF307 in MHCC97L cells resulted in reverse. effects. Importantly, flow cytometric analysis showed that ZNF307 overexpression increased the incidence of apoptosis, while ZNF307 knockdown decreased the incidence of apoptosis. Consistently, key regulators in apoptosis, such as caspase-3, BAX and BCL-2 were also regulated by ZNF307. Therefore, our results indicate that ZNF307 may serve as a tumor suppressor and inhibits cell proliferation of HCC via inducing apoptosis.

Activation of antiapoptotic genes has been indicated as one of the factors that contributes to hepatitis B virus (HBV) infection-induced liver cancer. The cellular inhibitor of apoptosis protein 2 (cIAP2), a member of the IAP family, is upregulated in various types of cancer and serves as a potential treatment target. However, to the best of our knowledge, the importance of cIAP2 in HBV-induced liver cancer has not been investigated. In the present study, cIAP2 expression in liver cells in response to HBV infection and the underlying mechanism involved was investigated. Western blot analysis of clinical liver samples showed that higher cIAP2 expression was detected in HBV-positive non-cancerous tissue compared with that in HBV-negative non-cancerous tissue, and the expression was further increased in HBV-positive liver cancer tissue. Reverse transcription-quantitative PCR and western blot experiments performed on two liver cell lines also confirmed that cIAP2 expression was increased upon HBV infection at both the mRNA and protein levels. Promoter analysis revealed that HBV could activate cIAP2 promoter in an infection dose-dependent manner, and this activation involved a NF-κB-binding site in the cIAP2 promoter. Further analysis demonstrated that HBV enhanced NF-κB phosphorylation and nuclear translocation via the PI3K/AKT signaling pathway, leading to the binding and activation of cIAP2 promoter. The present data demonstrates that HBV-infection induces cIAP2 expression in the liver by activation of the PI3K/AKT/NF-κB signaling pathway through promoting the binding of NF-κB to cIAP2 promoter, which may lead to carcinogenesis. The findings from the present study provide more information for understanding HBV-induced liver cancer and also offer a potential target for treatment or diagnosis of this disease.