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Circular RNAs (CircRNAs) are a group of noncoding RNAs that have essential function in the development and progression of various cancers. The expression pattern and function of circRNA in lung cancer is not fully understood. In the present study, we aimed to investigate the expression profiles and underlying mechanism of circRNA circ_POLA2 in lung cancer cell stemness. Circ_POLA2 was highly expressed in lung cancer tissues and predicted a poor prognosis in lung cancer patients. Knockdown of circ_POLA2 inhibited the stemness of lung cancer cells, which is evident by the decreased sphere-formation ability, ALDH1 activity, and stemness marker expression, but had no effects on cell viability. Mechanistically, circ_POLA2 functioned as a ceRNA by sponging miR-326. Furthermore, miR-326 negatively regulated G protein subunit beta 1 (GNB1) expression by targeting its 3'-UTR (untranslated region). Intriguingly, we found that GNB1 was overexpressed and associated with poor prognosis in lung cancer patients. Overexpression of GNB1 could antagonize the inhibitory effect of circ_POLA2 knockdown on lung cancer cell stemness. In conclusion, circ_POLA2 promotes lung cancer cell stemness and progression via regulating the miR-326/GNB1 axis, which might serve as a novel therapeutic target for lung cancer patients.

Cutaneous malignant melanoma (hereafter called melanoma) is one of the most aggressive cancers with increasing incidence and mortality rates worldwide. In this study, we performed a systematic investigation of the tumor microenvironmental and genetic factors associated with melanoma to identify prognostic biomarkers for melanoma. We calculated the immune and stromal scores of melanoma patients from the Cancer Genome Atlas (TCGA) using the ESTIMATE algorithm and found that they were closely associated with patients' prognosis. Then the differentially expressed genes were obtained based on the immune and stromal scores, and prognostic immune-related genes further identified. Functional analysis and the protein-protein interaction network further revealed that these genes enriched in many immune-related biological processes. In addition, the abundance of six infiltrating immune cells was analyzed using prognostic immune-related genes by TIMER algorithm. The unsupervised clustering analysis using immune-cell proportions revealed eight clusters with distinct survival patterns, suggesting that dendritic cells were most abundant in the microenvironment and CD8+ T cells and neutrophils were significantly related to patients' prognosis. Finally, we validated these genes in three independent cohorts from the Gene Expression Omnibus database. In conclusion, this study comprehensively analyzed the tumor microenvironment and identified prognostic immune-related biomarkers for melanoma.

4063 Background: Adding PD-1 inhibitors to neoadjuvant chemoradiotherapy have shown promising results in treatment of locally advanced esophageal squamous cell carcinoma (ESCC). However, there is a need to explore more effective and safe treatment doses and schedules. The aim of this single arm phase Ib trial is to determine the safety and efficacy of short course neoadjuvant chemo-radiotherapy plus toripalimab following by esophagectomy in patients with locally advanced resectable ESCC. Methods: Patients (pts) with histopathologically confirmed thoracic ESCC, diagnosed as clinical stage of cT3-4aN0M0/cT1-4aN+M0 per AJCC 8th and ECOG PS of 0-1 were eligible. Toripalimab (240mg) was given with paclitaxel (135 mg/m2) and carboplatin (AUC=5) on D1 every 3 weeks for two cycles. Short course neoadjuvant radiotherapy (3000 cGy in 12 fractions on 5 days per week) was administered from D3 to D18. Esophagectomy was scheduled 4 to 6 weeks after the completion of neoadjuvant therapy. Target total enrollment was 20 pts. The primary endpoint was treatment safety. Pre- and post-treatment specimens were collected for PD-L1 expression assay and gene expression profiles (GEPs) analysis using the nCounter platform. Results: From Jan 29, 2021 to Nov 3, 2021, 26 patients were screened and 23 met the inclusion criteria. The majority of them were diagnosed with clinical stage of cT3-T4 (95.6%), cN1-2 (62.5%), and cTNM III-IVA (78.2%). All 23 pts finished neoadjuvant radiotherapy, while two of them didn’t receive or received reduced dose of the second cycle chemotherapy or toripalimab due to toxicity. Notable grade 3 or higher AEs included leukopenia (n=21), neutropenia (n=21), anorexia (n=19) and nausea (n=18). No treatment related oesophagitis, pneumonia and gastrointestinal hemorrhage above Grade 3 were observed. 20 pts underwent surgery after a median of 7 weeks post neoadjuvant therapy. Reasons for not undergoing surgery were patients’ choice (n=2) and tuberculosis reactivation (n=1). 3 pts with Grade III surgical complications were observed during the perioperative period. 11/20 (55%) complete pathological responses (ypT0N0) were observed, 16/20 (80%) pts were major pathologic response (MPR) and the remaining 4 pts achieved partial response. Pre-treatment GEP analysis revealed significantly higher expression levels of CXCL10, CXCL11, OAS2, and lower level of CD209 and KLRB1 in pCR (n=6) than in Non-pCR group (n=4). Moreover, pCR group showed a trend of upregulated signatures of tumor infiltrating leukocytes, cytolytic activity, cytotoxic T cell, T cell markers and Teff score (P= 0.062) after treatment. Conclusions: Combining short course nCRT with toripalimab is feasible and effective in patients with locally advanced resectable ESCC, and might be a promising approach for neoadjuvant treatment. Clinical trial information: ChCTR2100045104.

Background. The tumor volume of high-grade glioma (HGG) after surgery is usually determined by contrast-enhanced MRI (CE-MRI), but the clinical target volume remains controversial. Functional magnetic resonance imaging (multimodality MRI) techniques such as magnetic resonance perfusion-weighted imaging (PWI) and diffusion-tensor imaging (DTI) can make up for CE-MRI. This study explored the survival outcomes and failure patterns of patients with HGG by comparing the combination of multimodality MRI and CE-MRI imaging with CE-MRI alone. Methods. 102 patients with postoperative HGG between 2012 and 2016 were included. 50 were delineated based on multimodality MRI (PWI, DTI) and CE-MRI (enhanced T1), and the other 52 were delineated based on CE-MRI as control. Results. The median survival benefit was 6 months. The 2-year overall survival, progression-free survival, and local–regional control rates were 48% vs. 25%, 42% vs. 13.46%, and 40% vs. 13.46% for the multimodality MRI and CE-MRI cohorts, respectively. The two cohorts had similar rates of disease progression and recurrence but different proportions of failure patterns. The univariate analysis shows that characteristics of patients such as combined with epilepsy, the dose of radiotherapy, the selection of MRI were significant influence factors for 2-year overall survival. However, in multivariate analyses, only the selection of MRI was an independent significant predictor of overall survival. Conclusions. This study was the first to explore the clinical value of multimodality MRI in the delineation of radiotherapy target volume for HGG. The conclusions of the study have positive reference significance to the combination of multimodality MRI and CE-MRI in guiding the delineation of the radiotherapy target area for HGG patients.

Background Prognostic biomarker, which can inform the treatment outcome of adjuvant chemotherapy (ACT) after complete resection of early-stage non-small cell lung cancer (NSCLC), is urgently needed for the personalized treatment of these patients. Patients and methods The prognostic value of gene expression of the estrogen receptor (ER) on the effect of ACT in completely resected NSCLC was investigated in the present study. Two independent datasets from Gene Expression Omnibus (GEO) with a total of 309 patients were included in this study. The prognostic value of ER gene expression on ACT’s efficacy was evaluated by survival analysis and Cox hazards models. Results We found a consistent and significant prognostic value of ERβ (ESR2) expression for ACT’s efficacy in completely resected NSCLC in both of the two independent cohorts. After multivariate adjustment, a significant survival benefit of ACT was observed in patients with low expression of ESR2, with a hazard ratio (HR) of 0.19 (95%CI 0.05–0.82, p = 0.026) in the discovery cohort and an HR of 0.27 (95%CI 0.10–0.76, p = 0.012) in the validation group. No significant benefit of ACT in the subgroup of patients with high expression of ESR2 was observed, with an HR of 0.80 (95%CI 0.31–2.09, p = 0.644) in the discovery cohort and an HR of 1.05 (95%CI 0.48–2.29, p = 0.896) in the validation group. Conclusion A significant survival benefit from ACT was observed in patients with low ESR2 expression. No significant survival benefit was observed in patients with high ESR2 expression. Detection of ESR2 expression in NSCLC may help personalize its treatment after complete resection.

Purpose/Objective(s) Intracranial metastasis failed to standard systematic treatment is common in advanced non-small cell lung cancer (NSCLC), contributing significantly to morbidity and mortality. Post Hoc analysis of a phase III randomized control trial (ALTER0303) has demonstrated that anlotinib can benefit patients with advanced NSCLC with brain metastases (BM) and is highly potent in the management of intracranial lesions. Here we report results of a comparative analysis of survival in patients with NSCLC with BM receiving whole brain radiotherapy (WBRT) with and without anlotinib, for whom intracranial disease had progressed or developed after at least one lines of prior systematic therapy. Materials/Methods From 2019 to 2020, a total of 96 patients of NSCLC in our institution with BM failed to at least one lines of systematic treatment met inclusion criteria for the study. Patients were treated with concurrent anlotinib and WBRT followed by anlotinib consolidation (an-WBRT group) or WBRT with other regimens at the discretion of the treating physician (WBRT group) at intracranial progression. Overall survival (OS) and intracranial progression-free survival (iPFS) were measured from the date of brain progression. Results The median lines of prior therapy are both 2 for an-WBRT and WBRT groups. The median OS for the an-WBRT (n = 26) and WBRT (n = 70) cohorts was 12.8 and 14.3 months, respectively (P = 0.82). The an-WBRT group has a longer iPFS than WBRT group (HR, 0.33; 95% CI, 0.21–0.65), with median iPFS of 8.1 months and 4.0 months respectively. Conclusion This retrospective analysis demonstrated that the combination of anlotinib and WBRT is associated with better iPFS than WBRT in NSCLC patients with intracranial metastasis failed to standard systematic treatment. Randomized, prospective studies are needed to further validate these findings. Author Disclosure C. Kong: None. X. Zhu: None. M. Jiang: None. X. Song: None. P. Qian: None. J. Zhu: None. J. Xu: None. X. He: None.

Esophageal squamous cell cancer is a highly aggressive cancer with a dismal 5-year survival rate. CD47 is a cell transmembrane protein that is involved in cell apoptosis, proliferation, adhesion, migration, and antigen presentation in the immune system. By interacting with signal regulatory protein-α expressed in antigen-presenting cells (APCs), CD47 acts as an antiphagocytic mechanism to inhibit APC-dependent antigen presentation. Overexpression of CD47 was found in various types of cancer. However, its role in esophageal squamous cell cancer is not yet clear. Anti-CD47 is an antagonist of CD47 signaling pathways by competing with its ligand. In the current study, we investigated the effects of anti-CD47 treatment on the antitumor immune response in an esophageal squamous cell cancer preclinical model. We found that anti-CD47 treatment enhanced proinflammatory responses and increased CD8+ T-cell infiltration in tumor tissue in the animal model. T cells in anti-CD47-treated tumors showed higher PD-1 and CTLA-4 expression, indicating T-cell activation and the rationale of combining anti-CD47 with anti-PD-1 and CLTA-4. The combinatory treatment showed the best antitumor response, implying a novel treatment strategy. The effects of anti-CD47 depended on dendritic cell function. In patient samples, expression of CD47 was negatively correlated with CD8+ T-cell infiltration in esophageal squamous cell cancer patients. Taken together, CD47 might be a novel target to enhance anti-PD-1 and CLTA-4 efficacy in esophageal squamous cell cancer.

It has been demonstrated that microRNAs (miRNAs) serve important roles in various biological processes, such as tumorigenesis. In the present study, the role of miR‑30a‑3p in the pathogenesis of esophageal carcinoma (EC) was investigated. Reverse transcription‑quantitative polymerase chain reaction was performed to determine the levels of miR‑30a‑3p expression in EC tissues and cell lines. Then, the effects of miR‑30a‑3p on the migration, invasion and radiosensitivity of EC cells were investigated using scratch‑wound, Transwell and radiosensitivity assays, respectively. A dual‑luciferase reporter assay was performed to determine potential interactions between miR‑30a‑3p and the 3'‑untranslated region (3'‑UTR) of insulin‑like growth factor 1 receptor (IGF‑1R). The results demonstrated that the levels of miR‑30a‑3p expression in EC tissues and cell lines were significantly decreased compared with those in paired healthy tissues and a human esophageal epithelial cell line. Upregulation of miR‑30a‑3p expression significantly suppressed migration, invasion and epithelial‑mesenchymal transition (EMT), and enhanced radiosensitivity in EC cells. Analysis of luciferase activity demonstrated that miR‑30a‑3p interacted with the 3'‑UTR of IGF‑1R, and knockdown of IGF‑1R induced similar effects on the migration, invasion, EMT and radiosensitivity of EC cells. The results indicated that miR‑30a‑3p suppressed metastasis and enhanced the radiosensitivity of EC cells via downregulation IGF‑1R, suggesting that miR‑30a‑3p may be a potential therapeutic target in the treatment of EC.

e15265 Background: Piwi-interacting RNAs (piRNAs) are a novel type of small noncoding RNAs which have important biological functions in malignant diseases. However, little is known about the clinical value of piRNAs in esophageal squamous cell carcinoma (ESCC). Hence, we demonstrated that two representative piRNAs, piR-36026 and piR-651, to dig out their clinical value in ESCC. Methods: Quantitative real-time PCR was employed to detect the levels of piR-36026 and piR-651 expression in ESCC tissues. Furthermore, the correlation between them and ESCC patients' clinicopathological characteristics were explored. Receiver operating characteristic (ROC) curves were constructed for measuring significances of piR-36026 and piR-651 for ESCC diagnosis. Results: Both of piR-36026 and piR-651 were prominently increased in ESCC tissues compared with normal. Additionally, piR-36026 was related with ESCC patients' diameter (P = 0.013), TNM stage (P = 0.027), tumor differentiation (P = 0.008) and CEA (P = 0.013); and piR-651 was associated with diameter (P = 0.005), lymph node metastasis (P = 0.002), tumor differentiation (P = 0.008) and SCC (P = 0.002). The area under the ROC curve (AUC) of piR-36026 was up to 0.8634 (95% confidence interval: 0.8171-0.9089, P < 0.05). Similarly, the AUC of piR-651 was 0.8525 (95% confidence interval: 0.7804-0.9247, P < 0.05). Conclusions: These results demonstrated that both piR-36026 and piR-651 may play a crucial role in ESCC oncogenesis; and they may be diagnostic biomarker for ESCC.

Objective To evaluate cell-free DNA (cfDNA) in plasma as a promising biomarker for lymphoma, altered levels of cfDNA and its association with clinical parameters are investigated in patients suffered from lymphomas. Methods Peripheral blood specimens were collected from 60 patients with lymphoma during initial diagnosis and those of another 107 patients with lymphoma during treated stage were also collected, 93 healthy volunteers were selected as control group. Quantitative PCR was used to detect cfDNA level in each group, cfDNA level in different groups was analyzed to understand its relationship with lymphoma patients' clinical features. After correlation analysis between cfDNA and clinical characteristics, Receiver operator characteristic curve was performed to analyze sensitivity and specificity of cfDNA and LDH . Results cfDNA concentration and integrity in initial stage of lymphoma patients were significantly higher than those in treated stage, and cfDNA concentration in treated phase was significantly higher than cfDNA concentration in control group. There was no significant difference in cfDNA integrity at treated stage compared with control group. There was no significant correlation between patient's age, gender, extranodal invasion and lymphoma pathological type and cfDNA concentration and integrity; In contrast, there was a significant correlation between ECOG score, LDH content, Ann Arbor stage, IPI, B-symptoms , Ki-67 expression and radiotherapy and cfDNA concentration and integrity, both at the time of initial diagnosis and treated stage. cfDNA concentration detection is an optimal diagnostic indicator, followed by cfDNA integrity detection, the sensitivity and specificity of both are superior to the traditional LDH detection. Conclusion cfDNA level is significantly increased in lymphomas patient plasma and may help lymphoma screening. cfDNA level may serve as a potential indicator of lymphomas treatment efficacy.

Autophagy inhibition is crucial for the improvement of the efficacy of radiotherapy in cancer. The aim of the present study was to determine the potential therapeutic value of autophagy and its correlation with mitochondria in human esophageal carcinoma cells following treatment with ionizing radiation (IR). Autophagy in Eca-109 cells was induced under poor nutrient conditions. The formation of autophagic vacuoles was monitored using electron microscopy. In addition, cell apoptosis after IR and mitochondrial membrane potential (MMP) were analyzed by flow cytometry. LC3, beclin-1, cytochrome c and apoptosis-related proteins were assayed by western blotting. A nude mouse xenograft model was also employed to verify the biological effects and mechanisms underlying autophagy in vivo. The formed autophagic vesicles and increased LC3 II/LC3 I ratio indicated marked induction of autophagy by Earle's balanced salt solution (EBSS) in Eca-109 cells. 3-Methyladenine or LY294002 significantly antagonized EBSS-induced autophagy and increased apoptosis of irradiated cells, suggesting that autophagy inhibition conferred radiosensitivity in vitro. Notably, IR induced prominent release of cytochrome c and Bax activation, and decreased Bcl-2 and MMP expression in Eca-109 cells under poor nutrient conditions. Of note, these changes were more prominent following pretreatment with autophagy inhibitors. In vivo, IR treatment mildly delayed tumor growth, but the radiotherapeutic effect was improved significantly by abolishing autophagy. Furthermore, mitochondrial signaling was investigated in the Eca-109 xenograft nude mice model, and the results were consistent with the in vitro study. Therefore, the mitochondrial pathway may be associated with improvement of radiosensitivity in Eca-109 cells.

We investigated the incidence of temporal lobe injury (TLI) in 132 nasopharyngeal carcinoma (NPC) patients who had undergone intensity-modulated radiotherapy (IMRT) in our hospital between March 2005 and November 2009; and identified significant dosimetric predictors of TLI development. Contrast-enhanced lesions or cysts in the temporal lobes, as detected by magnetic resonance imaging (MRI), were regarded as radiation-induced TLIs. We used the least absolute shrinkage and selection operator (LASSO) method to select Dmax (the maximum point dose) and the D1cc (the top dose delivered to a 1-mL volume) from 15 dose-volume-histogram-associated and four clinically relevant candidate factors; the Dmax and the D1cc were the most significant predictors of TLI development. We drew dose-response curves for Dmax and D1cc. The tolerance dose (TD) for the 5% and 50% probabilities of TLI development were 69.0 ± 1.6 and 82.1 ± 2.4 Gy for Dmax and 62.8 ± 2.2 and 80.9 ± 3.4 Gy for D1cc, respectively. The incidence of TLI in NPC patients after IMRT was higher than expected because the therapeutic window is narrow. High-quality longitudinal studies are needed to gain further insight into the complex spatiotemporal effects of non-uniform irradiation on TLI development in NPC patients.

Background N-stage is related to distant metastasis in nasopharyngeal carcinoma (NPC) patients. The purpose of this study was to evaluate the efficacy and toxicity of different nedaplatin-based chemotherapy regimens in advanced N2-3 stage NPC patients treated with intensity modulated radiation therapy (IMRT). Patients and methods Between April 2005 and December 2009, a total of 128 patients with N2-3 advanced NPC were retrospectively analyzed. Patients were treated with IMRT concurrent with 2 cycles of chemotherapy consisting of either nedaplatin plus paclitaxel (NP group, n = 67) or nedaplatin plus fluorouracil and paclitaxel (NFP group, n = 61). Two to four cycles of adjuvant chemotherapy were then administered every 21 days following concurrent chemoradiotherapy. Results With a median follow-up of 60 months, the 5-year overall survival (OS), progression-free survival (PFS), local-regional recurrence-free survival (LRRFS), and distant metastasis-free survival (DMFS) for all patients were 81.4%, 71.5%, 87.8% and 82.0%, respectively. No significant difference in PFS (66.6% vs. 76.7%, P = 0.212) and LRRFS rates (89.0% vs. 86.3%, P = 0.664) was observed between the NP and NFP groups. The 5-year OS (75.4% vs. 88.5%, P = 0.046) and DMFS (75.1% vs. 89.0%, P = 0.042) rate were superior in the NFP group compared with the NP group. The NFP group had a higher incidence of grade 3-4 acute toxicities including bone marrow suppression (leukopenia: χ2 = 3.935, P = 0.047; anemia: χ2 = 9.760, P = 0.002; thrombocytopenia: χ2 = 8.821, P = 0.003), and both liver and renal dysfunction (χ2 = 5.206, P = 0.023) compared with the NP group. Late toxicities were moderate and no difference was observed between the two groups. Conclusion IMRT concurrent with nedaplatin-based chemotherapy is an advocated regimen for patients with advanced N2-3 stage NPC. Patients with advanced N2-3 stage may be better candidates for the NFP regimen although this regimen was associated with a high acute toxicity rate.