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The emergence of immunotherapy has provided an option of treatment methods for bladder cancer (BC). However, the beneficiaries of immunotherapy are still limited to small‐scale patients, and immunotherapy‐related adverse events often occur. It is a major challenge for clinical work to study the immune subtypes of BC and the molecular mechanism of immune escape, and identify the immune responders accurately. Here, we explore the immune molecular subtypes of bladder cancer and potential escape mechanisms. First, we screened the expression profiles of 303 differentially expressed immune‐related genes in BC patients from the Cancer Genome Atlas (TCGA) database, and successfully identified 4 molecular subtypes of BC. By comparing the clinical characteristics, immune cells infiltration, the expression of checkpoint genes, human leukocyte antigen (HLA) genes, and gene mutation status of different subtypes, we identified different clinical and immunological characteristics of 4 subtypes. Among 4 subtypes, Cluster 2 met the general characteristics of immunotherapy responders and responded well to immunotherapy, while Cluster 4 had the highest expression of immune characteristics, and is similar to the immune environment of normal bladder tissue. Then, the weighted gene co‐expression network analysis (WGCNA) of immune‐related genes revealed that brown module was positively correlated with subtypes. Pathway enrichment analysis explored the major pathways associated with subtypes, which are also associated with immune escape mechanisms. Moreover, the decision tree model, which was constructed by the principle of random forest screening factors, was also validated in internal validation set and external validation set from the Gene Expression Omnibus (GEO) cohort (GSE133624), and could achieve accurate subtypes prediction for BC patients with high‐throughput sequencing. Taken together, we explored the immune molecular subtypes and their mechanisms of BC, and these results may provide guidance for the development of new BC immunotherapy strategies., We successfully identified 4 molecular subtypes of BC via TCGA database. Moreover, the establishment of decision tree model, by the principle of random forest screening factors, can achieve accurate subtype prediction for clinical BC patients with high‐throughput sequencing.

Background Cancer stem cells (CSCs) play an important role in tumor invasion and metastasis. CD44 is the most commonly used marker of CSCs, with the potential to act as a determinant against the invasion and migration of CSCs and as the key factor in epithelial–mesenchymal transition (EMT)-like changes that occur in colorectal cancer (CRC). Runt-related transcription factor-2 (RUNX2) is a mesenchymal stem marker for cancer that is involved in stem cell biology and tumorigenesis. However, whether RUNX2 is involved in CSC and in inducing EMT-like changes in CRC remains uncertain, warranting further investigation. Methods We evaluated the role of RUNX2 in the invasion and migration of CRC cells as a promoter of CD44-induced stem cell- and EMT-like modifications. For this purpose, western blotting was employed to analyze the expression of differential proteins in CRC cells. We conducted sphere formation, wound healing, and transwell assays to investigate the biological functions of RUNX2 in CRC cells. Cellular immunofluorescence and coimmunoprecipitation (co-IP) assays were performed to study the relationship between RUNX2 and BRG1. Real-time quantitative PCR (RT-qPCR) and immunohistochemistry (IHC) were performed to analyze the expressions of RUNX2, BRG1, and CD44 in the CRC tissues. Results We found that RUNX2 could markedly induce the CRC cell sphere-forming ability and EMT. Interestingly, the RUNX2-mediated EMT in CRC cell may be associated with the activation of CD44. Furthermore, RUNX2 was found to interact with BRG1 to promote the recruitment of RUNX2 to the CD44 promoter. Conclusions Our cumulative findings suggest that RUNX2 and BRG1 can form a compact complex to regulate the transcription and expression of CD44, which has possible involvement in the invasion and migration of CRC cells.

Muscle‐invasive bladder cancer (MIBC) is one of the common malignant tumors. Patients with MIBC still have high tumor recurrence and progression rates after surgery. Bioinformatics analysis of stromal infiltration‐related genes in the tumor microenvironment (TME) of MIBC patients was performed in this study to determine the major stromal cells types and biomarkers for their progression and poor prognosis. The ESTIMATE algorithm was applied to evaluate the stromal score and immune score of samples from MIBC patients in The Cancer Genome Atlas (TCGA) and found that stromal score was closely related to the clinical characteristics of the patients. The Gene Set Enrichment Analysis (GSEA) further revealed that stromal cells were involved in biological processes such as activation of leukocytes and positive regulation of cell migration during MIBC progression, as well as PI3K‐Akt, MAPK, and Rap1 signaling pathways. Five hub genes related to prognosis, including ACTA2, COL5A1, DCN, LUM, and PRRX1 were identified by the Weighted Gene Co‐Expression Network Analysis (WGCNA), Protein‐Protein Interaction (PPI), survival analysis, and Oncomine, Gene Expression Omnibus (GEO) database validation. Besides, we identified five stromal cell types associated with overall survival time, among which chondrocytes and fibroblasts were identified as the major stromal cell types through correlation analysis. Finally, the Receiver Operating Characteristic (ROC) curve and immunohistochemistry were used to verify the diagnostic value and expression of hub genes in different invasive tumors. In summary, we investigated the biological behavior of stromal cells in the TME of MIBC to promote tumor progression obtained hub genes associated with progression and poor prognosis and identified the main stromal cells types in the TME., Through our analysis, we found that stromal cells are closely related to the clinical prognosis of patients. Stromal cells are mainly involved in cell migration, leukocyte activation, and PI3K/AKT, MAPK, and Rap1 signaling pathways in tumor progression. In addition, we identified the hub genes of stromal cells promoting tumor progression based on bioinformatics analysis, and further identified chondrocytes and fibroblasts as the main types of 13 stromal cells.

The aim of the present study was to analyze the microarray data of human colorectal cancer (CRC) tissues and identify novel therapeutic targets for CRC. Microarray analysis from the GSE73360 and GSE84984 datasets was performed to identify novel long non-coding RNAs (lncRNAs) that were differentially expressed in human CRC tissues. Additionally, small interfering RNAs were used to deplete the expression of the indicated lncRNAs in cells. Colony-formation, wound-closure, and transwell assays were performed on CRC cells to assess their proliferation and migration capacities. Through microarray analysis, SCARNA9L, SLMO2-ATP5E and LOC100132062 were identified as differentially expressed lncRNAs in CRC tissues. The present study demonstrated that the ablation of SCARNA9L inhibited cell proliferation and arrested the cell cycle of SW480 and SW620 CRC cells. Additionally, depletion of SCARNA9L restrained the migration of CRC cells in vitro. Overall, the present study investigated the potential involvement of SCARNA9L in CRC and suggests SCARNA9L as a potential biomarker.

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer featured with high intra-tumoral heterogeneity and poor prognosis. To comprehensively delineate the PDAC intra-tumoral heterogeneity and the underlying mechanism for PDAC progression, we employed single-cell RNA-seq (scRNA-seq) to acquire the transcriptomic atlas of 57,530 individual pancreatic cells from primary PDAC tumors and control pancreases, and identified diverse malignant and stromal cell types, including two ductal subtypes with abnormal and malignant gene expression profiles respectively, in PDAC. We found that the heterogenous malignant subtype was composed of several subpopulations with differential proliferative and migratory potentials. Cell trajectory analysis revealed that components of multiple tumor-related pathways and transcription factors (TFs) were differentially expressed along PDAC progression. Furthermore, we found a subset of ductal cells with unique proliferative features were associated with an inactivation state in tumor-infiltrating T cells, providing novel markers for the prediction of antitumor immune response. Together, our findings provide a valuable resource for deciphering the intra-tumoral heterogeneity in PDAC and uncover a connection between tumor intrinsic transcriptional state and T cell activation, suggesting potential biomarkers for anticancer treatment such as targeted therapy and immunotherapy.

Background: To analyze perioperative conditions and long-term efficacy of open modified ureterosigmoidostomy urinary diversion (OMUUD) in patients with bladder cancer who underwent open radical cystectomy (ORC) and laparoscopic radical cystectomy (LRC).Methods: In this retrospective study, the clinical data of patients who underwent open and laparoscopic radical cystectomy plus open modified ureterosigmoidostomy urinary diversion in our hospital were collected from January 2011 to December 2019. In addition, perioperative data of 56 patients who underwent ORC and OMUUD were compared with those of 118 patients who underwent laparoscopic radical cystectomy (LRC) plus OMUUD. A long-term follow-up was performed to compare the overall survival (OS) and progression-free survival (PFS) rate between the two groups.Results: Results showed that there was no significant difference between ORC+OMUUD group and LRC+OMUUD group in terms of gender, age, body index, pre-operative ASA grade, history of transurethral resection of bladder tumor (TURBT) before surgery, tumor T stage, lymph node dissection range, pathological grade, and positive postoperative surgical margin. The mean operation time in the open group was shorter than that in the laparoscopic group （PConclusion: This study shows that the LRC group has long operation time, but less estimated intraoperative blood loss, short postoperative hospital stay, small trauma, and fast postoperative recovery compared to open surgery. Moreover, the incidence of complications at 30 - and 90-days postoperation, as well as the OS and PFS is not different between laparoscopy and open surgery.

Background Bladder cancer is the most common malignancy in urinary system, but the therapeutic targets remain elusive. This study aims to reveal the relationship between AMIGO2 and proliferation, migration, drug-resistance and tumorigenicity of bladder cancer, and explore the potential molecular mechanisms. Methods The expression of AMIGO2 in human bladder cancer tissues is measured by qRT-PCR and immunohistochemistry (IHC). Stable AMIGO2 knockdown cell lines T24 and 5637 were established by lentivirus transfection. Cell viability assay (CCK-8 assay) was used to determine cell proliferation, flow cytometry analysis was utilized to detect cell cycle, and wound healing assay was proceeded to test migration ability of bladder cancer cells. Chemosensitivity to cisplatin was measured by CCK-8 assay. Xenograft mouse model was established for investigating the effect of AMIGO2 on tumor formation in vivo. The RNA Sequencing technology was used to explore differentially expressed genes (DEGs) between knockdown group and negative control group of T24. Bioinformatics analysis upon the results of RNA-Seq was proceeded to understand underlying mechanisms. Results AMIGO2 was upregulated in bladder cancer cells and tissues. Inhibited expression of AMIGO2 suppresses cell proliferation and migration, which might be mediated by cell cycle arrest in G1 phase. AMIGO2 could reduce chemoresistance to cisplatin in bladder cancer cells. Low AMIGO2 expression inhibited tumorigenicity of T24 in nude mice. 917 DEGs were identified by RNA-Sequencing technology and bioinformatics analysis. The DEGs were mainly enriched in cell-cell adhesion, ATP-binding cassette transporters (ABC transporters), PPAR signaling pathway and some other pathways. Among ten hub genes, four of them might be associated with the prognosis of bladder cancer patients. Conclusion AMIGO2 is overexpressed in bladder cancer cells and tissues and serves as an oncogene in bladder cancer. It also reduces chemoresistance to cisplatin. The process might be regulated by particular pathways including ABC transporters and PPAR signaling pathway. Four hub genes might be associated with prognosis of bladder cancer patients.

There is growing evidence that tumour neoantigens have important roles in generating spontaneous antitumour immune responses and predicting clinical responses to immunotherapies1,2. Despite the presence of numerous neoantigens in patients, complete tumour elimination is rare, owing to failures in mounting a sufficient and lasting antitumour immune response3,4. Here we show that durable neoantigen-specific immunity is regulated by mRNA N6-methyadenosine (m6A) methylation through the m6A-binding protein YTHDF15. In contrast to wild-type mice, Ythdf1-deficient mice show an elevated antigen-specific CD8+ T cell antitumour response. Loss of YTHDF1 in classical dendritic cells enhanced the cross-presentation of tumour antigens and the cross-priming of CD8+ T cells in vivo. Mechanistically, transcripts encoding lysosomal proteases are marked by m6A and recognized by YTHDF1. Binding of YTHDF1 to these transcripts increases the translation of lysosomal cathepsins in dendritic cells, and inhibition of cathepsins markedly enhances cross-presentation of wild-type dendritic cells. Furthermore, the therapeutic efficacy of PD-L1 checkpoint blockade is enhanced in Ythdf1-/- mice, implicating YTHDF1 as a potential therapeutic target in anticancer immunotherapy.

The emergence of the new coronavirus (nCoV-19) has brought global impact on human health, whilst the interaction between the virus and the host is the foundation of the disease. The viral genome codes a cluster of proteins, each with a unique function in the event of host invasion or viral development. Under current adverse situation, we employ virtual screening tools in searching for drugs and nature products which have been already deposited in the DrugBank in attempt to accelerate the drug discovery process. This study provides an initial evaluation of current drug candidates from various reports using our systemic in silico drug screening based on structures of viral proteins and human ACE2 receptor. Besides, we built an interactive online platform (https://shennongproject.com:11443/#/home) for browsing these results with the visual display of small molecule docked on its potential target protein, without installing any specialized structural software. With continuous maintenance and incorporation of data from laboratory works, it may serve not only as the assessment tool for the new drug discovery but also an educational website to meet general interest from the public.

The emergence of the new coronavirus (nCoV-19) has brought global impact on human health, whilst the interaction between the virus and the host is the foundation of the disease. The viral genome codes a cluster of proteins, each with a unique function in the event of host invasion or viral development. Under current adverse situation, we employ virtual screening tools in searching for drugs and nature products which have been already deposited in the DrugBank in attempt to accelerate the drug discovery process. This study provides an initial evaluation of current drug candidates from various reports using our systemic in silico drug screening based on structures of viral proteins and human ACE2 receptor. Besides, we built an interactive online platform (https://shennongproject.com:11443/#/home) for browsing these results with the visual display of small molecule docked on its potential target protein, without installing any specialized structural software. With continuous maintenance and incorporation of data from laboratory works, it may serve not only as the assessment tool for the new drug discovery but also an educational website to meet general interest from the public.

Introduction Acute myeloid leukemia (AML) is an aggressive disease with genetic and phenotypic heterogeneity that results in a highly variable response to standard chemotherapy. Azacitidine (AZA) is a hypomethylating agent (HMA) and has been investigated in combination with intensive chemotherapy as an epigenetic primer to sensitize leukemic cells to treatment. In a phase 1 trial, this regimen was safe and well-tolerated with overall response rate (CR+CRi) of 61% and complete remission rate of 41% (Cahill et al, Blood Adv 2020). Predictive biomarkers for response to this treatment strategy have not yet been identified. Since 5-hydroxymethylcytosine (5hmC) is an epigenetic biomarker in cancer, we hypothesized that Nano-5hmC-Seal sequencing technology may serve as a novel approach to identifying 5hmC profiles predictive of treatment response to epigenetic priming. Methods We performed RNA-seq gene expression and Nano-5hmC-Seal DNA profiling from peripheral blood/bone marrow samples of patients with high-risk AML to identify potential 5hmC profile biomarkers and gene expression changes (Figure 1A). Patients (n=46) were treated in a 3+3 dose-escalation scheme of AZA (37.5 mg/m 2, 50 mg/m 2, or 75 mg/m 2) on days 1-5 followed by high-dose cytarabine (3000 mg/m 2) and mitoxantrone (30 mg/m 2) (AZA-HiDAC-Mito) on day 6 and day 10 in a phase 1 trial previously reported (Cahill et al, Blood Adv 2020). We compared pre-treatment RNA-seq gene expression and 5hmC DNA profiles between responders (CR+CRi) and non-responders, as well as between pre-treatment and after 5 days of AZA for individual patients. We used an XGBoost machine learning model in Python based on a training set of patients to develop a 5hmC gene signature to predict response to AZA-HiDAC-Mito in an independent test set of patients. We compared continuous variables with two-tailed Student's t-test and used the Kaplan-Meier method with log-rank test for survival analysis. Results Thirty-three patients (72%) had adequate RNA samples for RNA-seq gene expression analysis. Eighteen responded to treatment (CR +CRi) and were enriched with gene expression patterns involved in cell-cell interaction and activation of cell cycle, while non-responders (n=15) had a higher expression of leukemic stem cell (LSC) signatures. There was no difference in gene expression profile when comparing pre-treatment samples to day 5 samples after AZA exposure. From the 5hmC profiling [n=40 (87%) patients with adequate samples], increased 5hmC in LSC genes was associated with treatment resistance to AZA-HiDAC-Mito (p=0.044). The number of differentially hydroxy-methylated genes (DhMGs) increased with higher doses of AZA exposure suggesting a dose-dependent epigenetic effect from AZA. Patients with a greater number of DhMGs following 5 days of AZA treatment had improved survival (p=0.015) (Figure 1B). Using the 5hmC-based XGBoost machine learning model comparing 5hmC profiles between responders to non-responders from a training set of patients (n=22), we developed an 11-gene 5hmC pre-treatment signature (including SKP1, WNT8A, CYP2E1, and NBPF9) to predict treatment response. The model was highly effective in predicting response to therapy, with an area under the curve (AUC) of 0.86 in an independent test set of patients (n=18) treated with AZA-HiDAC-Mito (Figure 1C). Conclusion In patients with AML treated with AZA-HiDAC-Mito, a pre-treatment LSC gene expression signature enriched with 5hmC was associated with treatment resistance. More DhMGs at day 5 appear to be a dose-dependent epigenetic effect that is induced by AZA and is associated with longer survival despite the absence of an immediate change in gene expression levels. An 11-gene 5hmC pre-treatment signature may be a predictive biomarker for AZA-HiDAC-Mito therapy and other HMA-based approaches. These findings warrant validation in a larger prospective trial. Figure 1 Figure 1. Disclosures Zhang: Bristol-Myers Squibb: Current Employment. Stock: Pfizer: Consultancy, Honoraria, Research Funding; amgen: Honoraria; agios: Honoraria; jazz: Honoraria; kura: Honoraria; kite: Honoraria; morphosys: Honoraria; servier: Honoraria; syndax: Consultancy, Honoraria; Pluristeem: Consultancy, Honoraria. Odenike: Celgene, Incyte, AstraZeneca, Astex, NS Pharma, AbbVie, Gilead, Janssen, Oncotherapy, Agios, CTI/Baxalta, Aprea: Research Funding; AbbVie, Celgene, Impact Biomedicines, Novartis, Taiho Oncology, Takeda: Consultancy. He: Epican Genetech: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company.

Tumour-associated macrophages (TAMs) are associated with both the progression and poor prognosis of a variety of solid tumours. This study aimed to investigate and clarify the tumour-promoting role of CXCL8 secreted by TAMs in the urothelial carcinoma microenvironment of the bladder. Immunohistochemistry (n = 55) was used to detect Chemokine (C-X-C motif) ligand 8 (CXCL8), CD163 (a TAM marker), Matrixmetalloproteinase-9 (MMP-9), vascular endothelial growth factor (VEGF), and E-cadherin in cancerous and adjacent tissues of bladder cancer patients. TAMs-like PBM (peripheral blood mononuclear)-derived macrophages were developed using in vitro experiments. T24, 5637, and UM-UC-3 were treated with conditioned medium (CM) for the experimental intervention group, without CM for the blank control group, and with CM and an anti-CXCL8 neutralizing antibody for the experimental control group, respectively. The immunohistochemical study showed that the expression of CXCL8 was significantly upregulated as the number of infiltrating TAMs increased in the tumour tissues. A high expression of CXCL8 significantly correlated with an increase in the expression of MMP-9 and VEGF and a decrease in expression of E-cadherin in the microenvironment. This revealed that TAM-derived CXCL8 is highly associated with bladder cancer migration, invasion, and angiogenesis. The concentration of CXCL8 was significantly higher in CM collected from TAM-like PBM-derived macrophages than that from THP-1 cells. In subsequent in vitro experiments, we found that CM derived from TAM-like PBM-derived macrophages can also increase the migration rate, invasiveness, and pro-angiogenic properties of tumour cells. Additionally, the effect of CXCL8 was significantly diminished by the addition of an anti-CXCL8 neutralizing antibody to CM. The infiltration of TAMs in the tumour microenvironment leads to the elevation of CXCL8, which in turn promotes the secretion of MMP-9, VEGF, and E-cadherin by bladder cancer cells. This alters the migration, invasion, and pro-angiogenic capacity of bladder cancer cells and accelerates cancer progression.

Background and Objectives: An accurate delineation of the primary clinical target volume (CTVp) in esophageal squamous cell carcinoma (ESCC) significantly affects the outcomes of radiotherapy. However, when basing the CTVp on the primary gross tumor volume, there are no consistent guidelines for the size of the margin. We compared preoperative 18F-fluorodeoxyglucose (FDG) PET/CT images and large slices of resected pathological ESCC specimens for evidence and prediction of subclinical lesions. We also investigated associations between the maximum standardized uptake value (SUVmax), metabolic tumor volumes (MTVs), and lesions to improve estimates of the CTVp. Methods:55 patients underwent FDG PET/CT before surgery, and the SUVmax and MTVs were determined. To ensure that the in situ distances between the primary and secondary tumors were preserved, the esophageal specimens collected during radical surgery were processed to minimize shrinkage, and subclinical lesions were characterized by pathological examination. A 2-dimensional logistic regression model was used to assess the associations between clinicopathological features and microscopic spread of the lesions. Results: Subclinical lesions in pathological specimens were characterized as direct invasion, multicentric occurrence lesions, intra-mural metastasis, vascular invasion, and perineural invasion in 56.4, 40.0, 30.9, 21.8, and 18.2% of patients, respectively. The mean distances of the subclinical lesions from the primary tumor were 0.79 ± 1.28 cm and 0.87 ± 1.00 cm in the cranial and caudal directions, respectively. Together the SUVmax and MTV values could predict the presence of subclinical lesions that were not detectable in PET/CT images. Conclusions: To cover 94.5% of ESCC subclinical lesions in the CTVp, a 3-cm margin along the cranial-caudal axis should be added to the primary gross tumor volume as defined by FDG-PET/CT, as well as a cutoff SUVmax value of 2.5. Although preoperative FDG PET/CT images may not reveal lesions directly, the SUVmax and MTV measurements together could predict their presence.

Summary Tumor-associated macrophages (TAMs) can dampen the antitumor activity of T cells, yet the underlying mechanism remains incompletely understood. Here, we show that C1q+ TAMs are regulated by an RNA N6-methyladenosine (m6A) program and modulate tumor-infiltrating CD8+ T cells by expressing multiple immunomodulatory ligands. Macrophage-specific knockout of an m6A methyltransferase Mettl14 drives CD8+ T cell differentiation along a dysfunctional trajectory, impairing CD8+ T cells to eliminate tumors. Mettl14-deficient C1q+ TAMs show a decreased m6A abundance on and a higher level of transcripts of Ebi3, a cytokine subunit. In addition, neutralization of EBI3 leads to reinvigoration of dysfunctional CD8+ T cells and overcomes immunosuppressive impact in mice. We show that the METTL14-m6A levels are negatively correlated with dysfunctional T cell levels in patients with colorectal cancer, supporting the clinical relevance of this regulatory pathway. Thus, our study demonstrates how an m6A methyltransferase in TAMs promotes CD8+ T cell dysfunction and tumor progression.

Summary The ever-increasing understanding of the complexity of factors and regulatory layers that contribute to immune evasion facilitates the development of immunotherapies. However, the diversity of malignant tumors limits many known mechanisms in specific genetic and epigenetic contexts, manifesting the need to discover general driver genes. Here, we have identified the m6A demethylase FTO as an essential epitranscriptomic regulator utilized by tumors to escape immune surveillance through regulation of glycolytic metabolism. We show that FTO-mediated m6A demethylation in tumor cells elevates the transcription factors c-Jun, JunB, and C/EBPβ, which allows the rewiring of glycolytic metabolism. Fto knockdown impairs the glycolytic activity of tumor cells, which restores the function of CD8+ T cells, thereby inhibiting tumor growth. Furthermore, we developed a small-molecule compound, Dac51, that can inhibit the activity of FTO, block FTO-mediated immune evasion, and synergize with checkpoint blockade for better tumor control, suggesting reprogramming RNA epitranscriptome as a potential strategy for immunotherapy.

DNA 5hmC biomarkers to predict the outcomes to the of azacitidine prior to hi-dose cytarabine and mitoxantrone chemotherapy for patients with high-risk AML. Azacitidine (AZA), a DNA methyltransferase inhibitor and epigenetic modulator, has been combined with chemotherapy to treat high-risk AML patients for better clinical outcomes. However, the complete remission (CR) rate and prognosis remains unsatisfactory. Here, we proposed that the use of AZA for high-risk AML patients helped to sensitize leukemia cells to chemotherapy. We analyzed 45 high-risk AML patients who had received AZA treatment followed by High-dose cytarabine and mitoxantrone (AZA-HiDAC-Mito) therapy. We performed RNA-Seq and DNA 5hmC-Seq on available RNA/DNA samples. By comparing differential expressed genes between patients responded to therapy (CR or CRi, responder) and those did not (non-responder), we found that patients with better response had lower expression on ephrin receptor gene and AR pathway related genes. Using XGBoost machine learning model, we developed an 11-gene-5hmC signature to predict treatment outcomes, and it achieved 87.76% sensitivity and 87.10% specificity (AUC=0.911). The signature was also capable of distinguishing which patients would have better response to HiDAC-Mito only treatment, indicating that the signature had a potential to serve as a general predictor for intensive chemotherapy. Our findings show that DNA 5hmC patterns could be used as the prognostic biomarker for AZA-HiDAC-Mito therapy, which could guide further biomarker-based clinical trials. Citation Format: Dali HAN, Wei Zhang. Identification of DNA 5hmC decision signature biomarkers for azacitidine prior to hi-dose cytarabine and mitoxantrone chemotherapy in patients with high-risk AML [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-178.

Haimin Lin,1,2,* Dali Han,1,* Guobin Fu,3 Chengxin Liu,1 Lili Wang,4 Shumei Han,5 Bo Liu,5 Jinming Yu1 1Department of Radiation Oncology, Shandong Cancer Hospital Affiliated to Shandong University, Jinan, Shandong 250117, China; 2School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Sciences, Jinan, Shandong 250200, China; 3Department of Medical Oncology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, China; 4Department of Bone and Soft Tumor, Shandong Cancer Hospital Affiliated to Shandong University, Jinan, Shandong 250117, China; 5Department of Medical Oncology, Shandong Cancer Hospital Affiliated to Shandong University, Jinan, Shandong250117,China *These authors contributed equally to this work Purpose: The aim of this study was to assess the efficacy and safety of concurrent apatinib and docetaxel therapy vs apatinib monotherapy as third- or subsequent-line treatment for advanced gastric adenocarcinoma (GAC).Methods: Patients, who had received apatinib with or without docetaxel as third or more line therapy for advanced GAC, were retrospectively reviewed. Propensity score matching (PSM) analysis was performed to minimize the potential confounding bias. Kaplan–Meier curve and log-rank test were used to analyze the survival. Prognostic factors were estimated by Cox regression. Adverse events (AEs) were evaluated using CTCAE 4.0.Results: Thirty-four patients received concurrent therapy, whereas 31 received monotherapy. The median progression-free survival (PFS) and overall survival (OS) in monotherapy and con-therapy groups were 2.5 and 4 months (P=0.002), 3.3 and 6 months (P=0.004), respectively. After PSM, the median PFS and OS in the con-therapy group were also superior to the monotherapy group (P=0.004 and P=0.017). Cox regression suggested that Eastern Cooperative Oncology Group performance status (ECOG PS; HR =2.437, 95% CI: 1.349–4.404, P=0.003), CA199 (HR =1.001, 95% CI: 1.000–1.002, P=0.016), and treatment options (HR =0.388, 95% CI: 0.222–0.679, P=0.001) had significant effects on OS. Grade 3/4 toxicities in the monotherapy and con-therapy groups were as follows: leukopenia (0% vs 8.8%), neutropenia (3.2% vs 2.9%), anemia (9.8% vs 8.8%), thrombocytopenia (6.4% vs 2.9%), proteinuria (3.2% vs 2.9%), aminotransferase (0% vs 11.8%), hyperbilirubinemia (9.8% vs 5.9%), hypertension (9.8% vs 5.9%), hand–foot syndrome (3.2% vs 8.8%), nausea and vomiting (0% vs 11.8%), diarrhea (0% vs 5.9%), and fatigue (6.5% vs 2.9%).Conclusion: Patients with advanced GAC benefit more from concurrent apatinib and docetaxel therapy than apatinib monotherapy. Keywords: propensity score matching, progression-free survival, overall survival

Copper is a transition metal that plays critical roles in many life processes. Controlling the cellular concentration and trafficking of copper offers a route to disrupt these processes. Here we report small molecules that inhibit the human copper-trafficking proteins Atox1 and CCS, and so provide a selective approach to disrupt cellular copper transport. The knockdown of Atox1 and CCS or their inhibition leads to a significantly reduced proliferation of cancer cells, but not of normal cells, as well as to attenuated tumour growth in mouse models. We show that blocking copper trafficking induces cellular oxidative stress and reduces levels of cellular ATP. The reduced level of ATP results in activation of the AMP-activated protein kinase that leads to reduced lipogenesis. Both effects contribute to the inhibition of cancer cell proliferation. Our results establish copper chaperones as new targets for future developments in anticancer therapies.

Non-functional parathyroid carcinoma is an exceedingly rare disease with 31 reported cases since 1909. Because of the scarce number of cases of non-functional parathyroid carcinoma, there are no evidence-based recommendations for its optimal treatment. Surgery, including en bloc resection of the carcinoma, ipsilateral thyroid lobe and isthmus together with a neck dissection only in case of lymph node involvement, is the main treatment for non-functioning parathyroid carcinoma. The patient usually has a poorer prognosis because of detection at advanced stages, the relative ineffectiveness of adjuvant treatment modalities and the lack of adequate parameters for clinical follow-up. In this report, we present a case of non-functional parathyroid carcinoma at our institution, and we review the previous literature to discuss the latest advances in the diagnosis and treatment of this rare disease.

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer featured with high intra-tumoral heterogeneity and poor prognosis. To comprehensively delineate the PDAC intra-tumoral heterogeneity and the underlying mechanism for PDAC progression, we employed single-cell RNA-seq (scRNA-seq) to acquire the transcriptomic atlas of 57,530 individual pancreatic cells from primary PDAC tumors and control pancreases, and identified diverse malignant and stromal cell types, including two ductal subtypes with abnormal and malignant gene expression profiles respectively, in PDAC. We found that the heterogenous malignant subtype was composed of several subpopulations with differential proliferative and migratory potentials. Cell trajectory analysis revealed that components of multiple tumor-related pathways and transcription factors (TFs) were differentially expressed along PDAC progression. Furthermore, we found a subset of ductal cells with unique proliferative features were associated with an inactivation state in tumor-infiltrating T cells, providing novel markers for the prediction of antitumor immune response. Together, our findings provide a valuable resource for deciphering the intra-tumoral heterogeneity in PDAC and uncover a connection between tumor intrinsic transcriptional state and T cell activation, suggesting potential biomarkers for anticancer treatment such as targeted therapy and immunotherapy.

The primary aim of this study was to use serial 18F-3′-deoxy-3′-fluorothymidine (FLT) PET/CT to measure tumor cell proliferation during radiotherapy of squamous cell carcinoma (SCC) of the esophagus. Methods: Twenty-one patients with inoperable locally advanced SCC of the esophagus underwent serial 18F-FLT PET/CT during radiotherapy. Each patient received a pretreatment scan, followed by 1–3 scans after delivery of 2, 6, 10, 20, 30, 40, 50, or 60 Gy to the tumor. Results: Among the 19 patients who completed radiotherapy without interruption, parameters reflecting 18F-FLT uptake in the tumor (i.e., maximum tumor standardized uptake value [SUVmax] and proliferation target volume) decreased steadily. All patients demonstrated an almost complete absence of proliferating esophageal tumor after 30 Gy and a complete absence after 40 Gy. In the 2 patients whose radiotherapy course was interrupted, 18F-FLT uptake in the tumor was greater after the interruption than before the interruption. Marked early reduction of 18F-FLT uptake in irradiated bone marrow was observed in all patients, even after only 2 Gy. All showed a complete absence of proliferating marrow in irradiated regions after 10 Gy. Both patients who underwent scans after completing the entire radiotherapy course showed no tumor uptake on 18F-FLT PET/CT but high uptake on 18F-FDG PET/CT. Pathologic examination of these regions revealed inflammatory infiltrates but no residual tumor. Conclusion:18F-FLT uptake can be used to monitor the biologic response of esophageal SCC and normal tissue to radiotherapy. Increased uptake of 18F-FLT after treatment interruptions may reflect accelerated repopulation. 18F-FLT PET/CT may have an advantage over 18F-FDG PET/CT in differentiating inflammation from tumor.

Accurate delineation of clinical target volume (CTV) is critical in the effective management of squamous cell carcinoma (SCC) of esophagus using radiation therapy. Accurate delineation may improve the probability of local control and reduce the risk of complications. However, there are no consistent standards on the proper size of the margins added to the gross tumor volume (GTV). Different institutions and radiation oncologists have discordant opinions. In this paper, we review pathological and clinical outcomes to determine the most appropriate CTV for squamous cell carcinomas (SCC) of esophagus. The CTV for esophageal carcinoma should ensure that all subclinical lesions are encompassed regardless of the physical distance. The most precise method for delineating a reasonable CTV is to combine advanced imaging techniques, such as PET/CT and EUS, which allows the detection and prediction of subclinical lesions based on tumor characteristics such as the pathological type, differentiation, T disease, length and lymph node status.

N(6)-methyladenosine (m(6)A) is the most prevalent and reversible internal modification in mammalian messenger and noncoding RNAs. We report here that human methyltransferase-like 14 (METTL14) catalyzes m(6)A RNA methylation. Together with METTL3, the only previously known m(6)A methyltransferase, these two proteins form a stable heterodimer core complex of METTL3-METTL14 that functions in cellular m(6)A deposition on mammalian nuclear RNAs. WTAP, a mammalian splicing factor, can interact with this complex and affect this methylation.

Background To validate a gradient-based segmentation method for gross tumor volume(GTV) delineation on 8F-fluorothymidine (FLT)positron emission tomography (PET)/ computer tomography (CT) in esophageal squamous cell cancer through pathologic specimen, in comparison with standardized uptake values (SUV) threshold-based methods and CT. The corresponding impact of this GTV delineation method on treatment planning was evaluated. Methods and materials Ten patients with esophageal squamous cell cancer were enrolled. Before radical surgery, all patients underwent FLT-PET/CT. GTVs were delineated by using four methods. GTVGRAD, GTV1.4 and GTV30%max were segmented on FLT PET using a gradient-based method, a fixed threshold of 1.4 SUV and 30% of SUVmax, respectively. GTVCT was based on CT data alone. The maximum longitudinal tumor length of each segmented GTV was compared with the measured tumor length of the pathologic gross tumor length (LPath). GTVGRAD, GTV1.4 and GTV30%max were compared with GTVCT by overlap index. Two radiotherapy plannings (planGRAD) and (planCT) were designed for each patient based on GTVGRAD and GTVCT. The dose-volume parameters for target volume and normal tissues, CI and HI of planGRAD and planCT were compared. Results The mean ± standard deviation of LPath was 6.47 ± 2.70 cm. The mean ± standard deviation of LGRAD,L1.4, L30%max and LCT were 6.22 ± 2.61, 6.23 ± 2.80, 5.95 ± 2.50,7.17 ± 2.28 cm, respectively. The Pearson correlation coefficients between LPath and each segmentation method were 0.989, 0.920, 0.920 and 0.862, respectively. The overlap indices of GTVGRAD, GTV1.4, GTV30%max when compared with GTVCT were 0.75 ± 0.12, 0.71 ± 0.12, 0.57 ± 0.10, respectively. The V5, V10, V20, V30 and mean dose of total-lung,V30 and mean dose of heart of planGRAD were significantly lower than planCT. Conclusions The gradient-based method provided the closest estimation of target length. The radiotherapy plannings based on the gradient-based segmentation method reduced the irradiated volume of lung, heart in comparison to CT.

Summary Inhibition of cytosolic DNA sensing represents a strategy that tumor cells use for immune evasion, but the underlying mechanisms are unclear. Here we have shown that CD47-signal regulatory protein α (SIRPα) axis dictates the fate of ingested DNA in DCs for immune evasion. Although macrophages were more potent in uptaking tumor DNA, increase of DNA sensing by blocking the interaction of SIRPα with CD47 preferentially occurred in dendritic cells (DCs) but not in macrophages. Mechanistically, CD47 blockade enabled the activation of NADPH oxidase NOX2 in DCs, which in turn inhibited phagosomal acidification and reduced the degradation of tumor mitochondrial DNA (mtDNA) in DCs. mtDNA was recognized by cyclic-GMP-AMP synthase (cGAS) in the DC cytosol, contributing to type I interferon (IFN) production and antitumor adaptive immunity. Thus, our findings have demonstrated how tumor cells inhibit innate sensing in DCs and suggested that the CD47-SIRPα axis is critical for DC-driven antitumor immunity.

The primary aim of this prospective study was to investigate the value of (99m)Tc-methoxyisobutylisonitrile (MIBI) single photon emission computed tomography (SPECT) gated myocardial perfusion imaging (GMPI) in the detection of radiation-induced heart disease (RIHD) as early as during radiotherapy (RT) for oesophageal cancer (EC). The second aim was to analyse the correlation between cardiac toxicity and the dose-volume factors.The (99m)Tc-MIBI SPECT GMPI was performed both pre-RT and during RT (40Gray). The results of the SPECT were quantitatively analysed with QGS/QPS software and read by two experienced nuclear medicine physicians. The correlation between the changes in the SPECT parameters and the RT dosimetric data was analysed.Eighteen patients with locally advanced EC were enrolled in the study. Compared with the baseline, the imaging during RT showed not only significant decreases in the wall motion (WM) (1/20 segments), wall thickening (WT) (2/20 segments), end-diastolic perfusion (EDP) (5/20 segments) and end-systolic perfusion (ESP) (8/20 segments) (p0.05) but also a significant increase in the heart rate (74.63±7.79 vs 81.49±9.90, p=0.036). New myocardial perfusion defects were observed in 8 of the 18 patients. The V37-V40 was significantly higher (p0.05) in the patients with the new perfusion defects during RT than in the patients who did not exhibit these defects.Radiotherapy for EC induces cardiac damage from an early stage. (99m)Tc-MIBI SPECT GMPI can detect the occurrence of cardiac impairment during RT. The WM, WT, EDP and ESP may be valuable as early indicators of RIHD. The percentage of the heart volume that receives a high dose is an important factor that is correlated with RIHD.