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Patients with biliary tract cancer (BTC) were associated with poor prognosis and limited therapeutic options after first-line therapy currently. In this study, we sought to evaluate the feasibility and tolerability of sintilimab plus anlotinib as the second-line treatment for patients with advanced BTC. Eligible patients had histologically confirmed locally advanced unresectable or metastatic BTC and failed after the first-line treatment were recruited. The primary endpoint was overall survival (OS). Simultaneously, association between clinical outcomes and genomic profiling and gut microbiome were explored to identify the potential biomarkers for this regimen. Twenty patients were consecutively enrolled and received study therapy. The trail met its primary endpoint with a median OS of 12.3 months (95% CI: 10.1-14.5). Only four (20%) patients were observed of the grade 3 treatment-related adverse events (TRAEs) and no grade 4 or 5 TRAEs were detected. Mutation of AGO2 was correlated with a significantly longer OS. Abundance of Proteobacteria was associated with inferior clinical response. Therefore, sintilimab plus anlotinib demonstrated encouraging anti-tumor activity with a tolerable safety profile and deserved to be investigated in larger randomized trials for patients with advanced BTC subsequently.

577 Background: The incidence rate of biliary tract cancer (BTCs) is gradually increasing in china, while chemotherapy and immune checkpoint inhibitors monotherapy demonstrated limited efficacy for BTCs. New effective regimens are urgently needed. In this study, we explore anlotinib plus sintilimab as a chemo-free combination for second-line therapy. Previous results had been reported in 2021 ASCO GI (307 Poster Session). Updated results for longer follow-up and additional enrollment are presented here. Methods: BTC patients (pts) failed after the first-line treatment, aged 18-75 and an ECOG PS of 0-1 were recruited. Eligible pts received anlotinib (12mg, po, d1-14, q3w) and sintilimab (200mg, iv, d1, q3w) until disease progression, unacceptable toxicity or up to 2 years. The primary endpoint was Overall survival (OS). Secondary endpoints included Objective Response Rate, (ORR), Progression-Free Survival (PFS), Disease Control Rate (DCR) and safety. Results: At data cut-off date (Dec 30, 2021), a total of 29 pts were enrolled, of which 20 pts were available for efficacy assessment. The median treatment duration was 5.6 months (IQR: 2.5-10.0), and the median follow-up time was 12.5 months (IQR, 7.1-21.8). The median OS was 12.3 months (95% CI: 10.1-14.5).The median PFS was 6.5 months (95% CI: 4.2-8.8).The ORR was 30% (95% CI: 11.9%-54.3%) and DCR was 95% (95% CI: 75.1%-99.9%). Safety profile exhibited that the regimen was tolerable.The treatment-related adverse events (TRAEs) of any grade occurred in all patients (100%). The most common TRAEs were elevated GGT (75%), followed by hypertension (65%), elevated ALP (65%), elevated D-dimer (65%), elevated ALT (55%), anemia (50%), and elevated AST (50%). Grade 3 TRAEs occurred in 4 (20%) patients, including 2 (10%) hypertension, 1 (5%) elevated GGT, 1 (5%) elevated ALP, 1 (5%) elevated AST and 1 (5%) elevated ALT. No grade 4 or 5 TRAEs were observed. Conclusions: Anlotinib plus sintilimab demonstrated encouraging anti-tumor activity with a tolerable safety profile and deserve to be investigated in randomized trials for patients with advanced BTC. Clinical trial information: 1900022003 .

Histopathological examination (biopsy) is the “gold standard” for the diagnosis of colorectal cancer (CRC). However, biopsy is an invasive method, and due to the temporal and spatial heterogeneity of the tumor, a single biopsy cannot reveal the comprehensive biological characteristics and dynamic changes of the tumor. Therefore, there is a need for new biomarkers to improve CRC diagnosis and to monitor and treat CRC patients. Numerous studies have shown that “liquid biopsy” is a promising minimally invasive method for early CRC detection. A liquid biopsy mainly samples circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), microRNA (miRNA) and extracellular vesicles (EVs). CTCs are malignant cells that are shed from the primary tumors and/or metastases into the peripheral circulation. CTCs carry information on both primary tumors and metastases that can reflect dynamic changes in tumors in a timely manner. As a promising biomarker, CTCs can be used for early disease detection, treatment response and disease progression evaluation, disease mechanism elucidation, and therapeutic target identification for drug development. This review will discuss currently available technologies for plasma CTC isolation and detection, their utility in the management of CRC patients and future research directions.

Background: Spindle cell sarcoma (SCS) is a rare malignant tumor which relapses quickly and has poor prognosis.Case presentation: We report a case of SCS deriving from the left side of chestpossessed a novel unreported ALK fusion gene named NSF-ALK found by Next Generation Sequencing (NGS) technology. Despite receiving three surgical resections, local recurrence occurred rapidly and distant brain metastasis was eventually observed in this patient. The subsequent administration of crizotinib1, an oral anaplastic lymphoma kinase inhibitor, resulted in dramatic tumor shrinkage and the patient entered a remission period that has been ongoing for more than 10 months.Conclusions: In summary, this study is the first to describe a novel NSF-ALK fusion in spindle cell sarcoma which has promising efficacy by crizotinib treatment. More importantly, the case also shows that next generation sequencing provids more information of mutation to guide treatment in clinical decisions by presenting molecular changes.

Background: The main reason for esophageal squamous cell carcinoma (ESCC) treatment failure is metastasis. Little is known about the mechanisms involved in the metastasis of ESCC, and there is a lack of effective therapeutic targets. In our previous study, we found that patients with high levels of BC200 tended to have poor prognoses. Methods: First, we applied qRT-PCR to detect the expression level of BC200 in normal esophageal squamous epithelial cells and ESCC cells with different degrees of differentiation ability. Then, we changed BC200 expression by transfecting constructed lentiviruses that included BC200 shRNA (LV-BC200-shRNA, KD), negative control (CON053, NC), or BC200 gene (LV-BC200, BC200) to create BC200-deficient cell models in KYSE410 and KYSE70 cells and BC200 overexpression cell models in EC9706 cells and verified the transfection effect by qRT-PCR. Then, we examined cell migration by wound healing assay, invasion by Transwell assay, and proliferation by MTT assay and examined the metastasis ability in a xenograft mouse model. Gene expression profiling was performed to screen a panel of mRNAs following inhibition of BC200 expression. We then used ingenuity pathway analysis (IPA) to analyze the functions of the changed molecules and their interactions. The results from the microarray were validated by qRT-PCR and Western blotting. Results: In this study, we found that the expression of BC200 in poorly differentiated cell lines was significantly higher than that in well-differentiated cell lines. BC200 can significantly promote the migration and invasion but not the proliferation ability of ESCC cells in vitro and BC200 shRNA can significantly suppress tumor metastasis in vivo. Our genome-wide expression profile chip showed 406 differentially expressed genes, with 91 upregulated genes and 315 downregulated genes. The upstream regulator analysis showed that ATF4 was predicted to be strongly inhibited and 21 genes were consistently inhibited by this gene. Our qRT-PCR and Western blotting data also identified the reduced expression of ATF4 and some selected downstream genes, such as SNAIL2, GADD45A, and PSAT1, as a consequence of downregulating BC200 expression in ESCC. Conclusion: Our data showed that BC200 promoted the metastasis of ESCC cells and could regulate the expression of ATF4 and its downstream genes.

Myeloid-derived suppressor cells (MDSCs) are notable contributors to the immunosuppressive tumor microenvironment (TME) and are closely associated with tumor progression; in addition, MDSCs are present in most patients with cancer. However, the molecular mechanisms that regulate MDSCs in the etiopathogenesis of human tumor immunity remain unclear. The secreted alarmin high mobility group box 1 (HMGB1) is a proinflammatory factor and inducer of many inflammatory molecules during MDSC development. In this review, we detail the currently reported characteristics of MDSCs in tumor immune escape and the regulatory role of secreted HMGB1 in MDSC differentiation, proliferation, activity and survival. Notably, different posttranslational modifications of HMGB1 may have various effects on MDSCs, and these effects need further identification. Moreover, exosome-derived HMGB1 is speculated to exert a regulatory effect on MDSCs, but no report has confirmed this hypothesis. Therefore, the effects of HMGB1 on MDSCs need more research attention, and additional investigations should be conducted.

Chemodynamic therapy (CDT) has been critically challenged by insufficient H2O2 in cancer tissues and inefficient reactive oxygen species (ROS) production. Herein, we have reported the facile synthesis of an efficient ROS generator (GOx@Pd@ZIF-8) that exerts synergistic anticancer activity by blocking glucose metabolism and producing ROS. Glucose oxidase (GOx) and palladium (Pd) cube nanozymes were incorporated in zeolitic imidazolate framework-8 (ZIF-8) by biomimetic mineralization. Systematic characterization indicated the successful entrapment and embedding of GOx and Pd during ZIF-8 crystal growth. The GOx@Pd@ZIF-8 composite showed favorable catalytic glucose activity and stable ROS production. In vitro experiments showed that the GOx@Pd@ZIF-8 composite effectively inhibited cancer cell proliferation, invasion, and migration and promoted apoptosis through the ROS-mediated signaling pathway, which was further confirmed by bioinformatics analyses of RNA-seq data obtained from in vitro experiments. Furthermore, the GOx@Pd@ZIF-8 composite inhibited tumor growth with few to no side effects on other tissues in vivo. This work provides a novel antitumor strategy involving the construction of a stable and highly active ROS generator that shows promise for the treatment of solid cancers.

Background We retrieved different reports containing different genetic effects of A/B polymorphism within the Glutathione S-Transferase Mu 3 (GSTM3) intron 6 on the susceptibility to cancer in the overall population. Methods Herein, we first conducted a meta-analysis to comprehensively assess such a genetic relationship after collecting the available published evidence. STATA 12.0 software was used for the statistical analysis. Results By retrieving and screening database literature, a total of fifty-three eligible articles were finally selected. Even though no significant difference between overall cancer cases and negative controls was detected under the allelic, homozygotic, heterozygotic, dominant and recessive genetic model, we observed a reduced risk of head and neck cancer in cases, compared with controls, under the homozygotic model (BB vs. AA, P =0.047, OR=0.75) and recessive model (BB vs. AA+AB, P =0.045, OR=0.76), but not other models. Furthermore, a decreased risk of head and neck SCC cancer was detected under all the genetic models (allelic B vs. A, P =0.007, OR=0.78; BB vs. AA, P =0.006, OR=0.54; AB vs. AA, P =0.045, OR=0.82; AB+BB vs. AA, P =0.002, OR=0.77; BB vs. AA+AB, P =0.021, OR=0.63; carrier B vs. A, P =0.032, OR=0.83). Conclusion Our findings suggested that the GSTM3 intron 6 A/B polymorphism may confer the protective susceptibility to the squamous cell carcinoma of head and neck.

Background Long noncoding RNA (lncRNA) has been implicated in numerous tumors, including pancreatic cancer (PC). However, the precise cellular roles and molecular mechanisms of lncRNA DIO3OS on PC development remains to be fully clarified. Methods We performed the meta-analysis on PC samples and non-tumor samples retrieved from the TCGA database, and measured the levels of DIO3OS in PC cell lines and a normal pancreatic duct epithelial cell line HPDE6-C7. Cell proliferation was evaluated via CCK-8 assay. Cell invasion in vitro was investigated by transwell assay. The RNA immunoprecipitation assay and luciferase reporter assay was utilized to confirm the putative miR-122-binding site in DIO3OS. The effects of DIO3OS on PC progression were tested using in vivo subcutaneous xenografts. Results Our results showed that DIO3OS was highly expressed in human PC tissues and PC cell lines. DIO3OS exhibited oncogenic properties in stimulating PC cell proliferation and invasion in vitro and promoting cancer growth in vivo. Through online predictive tools and functional experiments, we found that DIO3OS could bind directly to microRNA-122 (miR-122) and inhibited its expression, which functioned as a tumor suppressor in PC cells. We also verified that ALDOA was the direct target of miR-122, and the tumor suppressive effects caused by DIO3OS knockdown or miR-122 overexpression could be rescued by re-expression of ALDOA in PC cells. Conclusions Overall, our study suggested that lncRNA DIO3OS promotes PC cell growth and invasion by competing for miR-122 to modulate the expression of ALDOA. These findings yield a better understanding of the potential mechanisms by which gain of DIO3OS expression accelerates PC progression.

Objective Extranodal involvement represents a peculiar presentation of diffuse large B-cell lymphoma (DLBCL). Previous studies have suggested that older patients are more prone to extranodal involvement. This study retrospectively addressed the distribution, prognostic value and treatment options of extranodal involvement in young patients with DLBCL. Methods A total of 329 patients were enrolled according to the inclusion requirements. The effects of gender, extranodal involvement, age-adjusted international prognostic index (aaIPI), rituximab infusion and radiotherapy on patient outcomes were evaluated. Results Among these patients, 59% presented extranodal involvement in 16 anatomic sites. More than one instance was linked to many poorer clinical characteristics and poorer survival compared with either nodal disease or one instance. In patients with one extranodal lesion, multivariate analysis revealed that the site of extranodal involvement, but not the aaIPI or rituximab infusion, was independently related to the outcome, and radiotherapy had a negative influence on survival. Conclusions Extranodal involvement is common in younger patients and exhibits a ubiquitous distribution. The site of extranodal involvement is of strong prognostic significance. Radiotherapy for extranodal lesions does not improve patient outcomes.

// Hai-ying Wang 1 , Zhi-hua Yao 1 , Hong Tang 1 , Yan Zhao 1 , Shui-ling Jin 2 , Wen-ping Zhou 1 , Shu-na Yao 1 , Shu-jun Yang 1 , Yan-yan Liu 1 , Su-xia Luo 1 1 Department of Medical Oncology of Henan Cancer Hospital, Zhengzhou University Affiliated Cancer Hospital, Zhengzhou, Henan, China 2 Department of Internal Medicine, The Second Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China Correspondence to: Su-xia Luo, email: 470242877@qq.com Hai-ying Wang, email: luosxrm@163.com Keywords: advanced esophageal squamous cell carcinoma, Paclitaxel, S-1, cisplatin, palliative chemotherap Received: July 18, 2016 Accepted: November 14, 2016 Published: November 25, 2016 ABSTRACT Background: In advanced esophageal squamous cell carcinoma (ESCC), paclitaxel plus cisplatin are considered as active and tolerable. The current clinical study was conducted to retrospectively compare the efficacy and safety of first-line paclitaxel/S-1(PS) and paclitaxel/cisplatin(TP) regimens in advanced ESCC. Results: The overall response rate of PS was slightly, but not significantly, higher (25 patients, 46%) than that of TP (23 patients, 39%, P = 0.432). Median overall survival (OS) was similar for PS and TP (11.5 months vs. 10.4 months, p = 0.37). However PS had longer median progression-free survival than TP (PFS: 5.5 months vs5.0months, p = 0.04). When compared with PS, more grade 3 or 4 adverse events were recorded for TP, including leukopenia, neutropenia, anemia, anorexia and vomiting ( P < 0.05). No treatment-related deaths were recorded in either group. Patients and Methods: Between 2008 and 2014, all patients diagnosed with advanced ESCC and treated with paclitaxel/S-1 or paclitaxel/cisplatin at Cancer Hospital Affiliated to Zhengzhou University were analyzed retrospectively. One hundred and thirteen patients were included in this study. Disease control rates and progression-free survival (PFS) and overall survival (OS) were recorded. Survival analysis was calculated by using Kaplan–Meier method. Conclusions: The PS option improves PFS and its OS is similar to TP. Moreover, the PS regimen is an effective and safe first-line treatment for ESCC with less hematological and non-hematological toxicity.

307 Background: Gemcitabine combined with cisplatin (GP) has been established as the standard first-line treatment in patients with unresectable biliary tract cancers (BTC), a heterogeneous group of rare and aggressive malignancies arising from the epithelium of the biliary duct system. Nevertheless, there has been no approved standard of care for second-line treatment. In this study, we explored the efficacy and safety of the combination of anlotinib and sintlimab as a novel second-line regimen in BTC. Methods: In this phase II trial, patients with advanced BCT, who experienced progression after first-line chemotherapy, received anlotinib (12 mg, PO, D1-14, Q3W) combined with sintlimab (200 mg, iv, D1, Q3W) until disease progression or unacceptable toxicity. The primary endpoint was OS, and the secondary endpoints included ORR, PFS, DCR, and safety. The exploratory endpoint was the correlation of clinical outcomes with biomarkers. Results: From Aug 2019 to Sep 2020, 17 patients were enrolled. 9(52.9%) were men, and the median age was 59(43-69) years. Nine patients were diagnosed with ICC, 2 with ECC, and 6 with GBC. 15 patients were assessable for response. At a median follow-up of 8.76 months (95%CI: 3.60-13.94 months), the primary endpoint OS has not been reached, the median PFS was 6.50 months (95%CI: 3.60-9.40 months). ORR was 40.00%, and DCR was 86.67%. The incidence of treatment-related adverse events (TRAEs) was 70.60%, and the most common grade 1-2 AEs were hypertension (70.60%), diarrhea (17.65%), and hypothyroidism (17.65%). Conclusions: Anlotinib plus sintlimab demonstrates significant clinical activity and acceptable toxicity in patients with advanced BTC who failed first-line chemotherapy. Clinical trial information: ChiCTR1900022003.

Background Thymosin alpha-1 (TA) has been reported to inhibit tumor growth as an immunomodulator. However, its mechanism of action in immunosuppressive cells is unclear. The purpose of this study was to investigate whether TA can reshape the immune microenvironment by inhibiting the function of myeloid-derived suppressor cells (MDSCs) in non-small cell lung carcinoma (NSCLC). Methods The effects of TA on peripheral blood monocytic MDSCs (M-MDSCs) in patients with NSCLC and on the apoptosis and migration of M-MDSCs were studied. A mouse subcutaneous xenograft tumor model was constructed, and the effect of TA on M-MDSC migration was evaluated. Quantitative real-time PCR, Western blotting, flow cytometry and immunohistochemistry were used to examine the mechanism by which TA affects M-MDSCs. Results TA not only promoted the apoptosis of M-MDSCs by reducing the Bcl-2/BAX ratio but also and more importantly inhibited the migration of MDSCs to the tumor microenvironment by suppressing the production of vascular endothelial growth factor (VEGF) through the downregulation of hypoxia-inducible factor (HIF)-1α in tumor cells. Conclusions TA may have a novel antitumor effect mediated by decreasing M-MDSC accumulation in the tumor microenvironment through reduced VEGF production.

Background: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide with a high recurrence rate. Although immune-checkpoint treatment provides survival benefits for patients with HCC, the mortality of HCC is still high. Therefore, it is necessary to understand the molecular mechanism, immune microenvironment and related prognostic factors of HCC. Methods: HCC patients with both RNAseq and miRNA expression data and their clinical information were downloaded from the NCI's Genomic Data Commons. Gene expression data were analyzed to identify differentially expressed genes (DEGs). The results were further used to construct the competing endogenous RNA (ceRNA) networks related to HCC. Meanwhile, the proportions of 22 immune cell types in HCC were estimated by using CIBERSORT (https://cibersort.stanford.edu). Subsequently, two nomograms based on the expression value of ceRNAs and the percentage of immune cells were constructed to predict the prognosis of HCC patients, respectively. The calibration and time series ROC curves were used to evaluate these models. Finally, the correlation between ceRNAs and immune cells were explored to reveal the underlying molecular mechanism. Results: Gene expression data and clinical information of 367 primary HCC and 50 adjacent normal samples were downloaded from the TCGA database. These data were used to identify DEGs. A total of 12 lncRNAs, 71 mRNAs and 39 miRNAs were identified to be in ceRNA networks according to starBase v2.0 database. The results showed that stages of cancer, the expression values of CBX2, MAFG, DNM3, BUB1, ARL5B and other 23 genes were important predictors. The AUCs of the constructed nomogram in predicting the 3- and 5-year OS were 0.79 and 0.81 respectively. Stages of cancer, proportions of resting memory CD4+ T cell, M0 macrophages and resting mast cells constructed another nomogram with AUC of 0.79 and 0.81 in predicting the 3- and 5-year OS. CIBERSORT results showed that the proportions of regulatory T cells, CD8+ T cells, follicular helper T cells and M0 macrophages were slightly higher while the proportions of M2 macrophages, monocytes and activated mast cells were slightly lower in HCC than in adjacent normal samples. Integration analysis of CIBERSORT results, ceRNAs and gene set enrichment analysis of immunologic signatures identified BUB1, E2F8, H2AFZ, JPT1, LPCAT1, MAFG and PSMC3IP to be positively related with regulatory T cells, follicular helper T cells, activated memory CD4+ T cells and M0 macrophages. This is consistent with current knowledge that immune activity is suppressed in certain types of cancers. However, the underlying mechanism warrant further exploration. Conclusions: In this study, two nomograms were constructed to predict the prognosis of HCC patients, which might be useful in predicting the prognosis of HCC patients. The results suggest that the immune suppress effect of HCC may be related to BUB1, E2F8, H2AFZ, JPT1, LPCAT1, MAFG and PSMC3IP genes. These genes may serve as potential therapeutic targets. Acknowledgments: This work was supported by the National Science and Technology Major Project of China (2018ZX10302205). Citation Format: Jianxiang Shi, Chi Cui, Yaru Duan, Hua Ye, Peng Wang, Jitian Li, Shuiling Jin, Liping Dai, Jianying Zhang. Construction of ce-RNA networks and comprehensive analysis with tumor infiltrating immune cells in hepatocellular carcinoma [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 35.

e16712 Background: Gemcitabine combined with cisplatin (GP) is currently used as a standard first-line chemotherapy regimen for aBTCs, However, the median overall survival (mOS) is only about 11.7 months, and there is no standard treatment option for patients who failed GP therapy. In this study, we aimed to investigate the efficacy and safety of anlotinib with cediranib as a second-line treatment for patients with aBTCs. Methods: A monocenter single-arm phase II study was conducted at the First Affiliated Hospital of Zhengzhou University. Patients with measurable aBTCs and progressed on GP were enrolled in this study. Patients received cediranib 200mg, on day1 + anlotinib 12mg on day1-14, Q3W until disease progression, intolerance of toxicity, investigator/patient decision to withdraw or other reasons specified in the protocol. Response (RECIST1.1) was assessed every 8 weeks. Plasma, stool and tumor tissues were collected for exploratory analyses. The primary endpoint was overall survival (OS). Secondary endpoints included progression-free survival (PFS), objective response rate (ORR), disease control rate (DCR) and drug safety. Results: We planned to include 20 patients. So far 9 patients were enrolled in this study, 66.7% (6/9) were men, 33.3% were women. The median age was 56y (43y-61y). The primary sites of the tumor were intrahepatic biliary (66.7%, 6/9) and gallbladder (33.3%, 3/9). At data cutoff (Dec 14, 2019), the median duration of follow-up was 2.5mos (range, 1.2 to 4) and all of the patients were still under treatment. 8 patients have undergone at least one efficacy evaluation, of which 2 (25%) PR, 5 SD (62.5%), DCR was 87.5% ((7/8). An SD patient had a long-term intermittent fever, which considered to be tumor fever, the body temperature returned to normal after 1 cycle of treatment. 1 patient was considered to be undefined because, at the first evaluation, the liver lesions were reduced however the lymph nodes in the retroperitoneum were enlarged. The median PFS and OS not yet reached. Grade 3-4 treatment-related adverse events (TRAEs) occurred in 11.1% (1/9) of patients. TRAEs led to discontinuation in 1 patient (grade 3 hypertension). TRAEs led to dose reduction of anlotinib in 2 patients. No TRAEs were fatal. Conclusions: The primary result showed that the combination of anlotinib and cediranib was well tolerated and demonstrates encouraging efficacy than historical control in second-line treatment for aBTC. Updated data, including safety, efficacy, and biomarkers will be presented. Clinical trial information: ChiCTR1900022003 .

The aim of this study was to correlate matrix metalloproteinase-2 and matrix metalloproteinase-9 expression with the clinicopathological features and outcome of patients with early gastric cancer and to clinically elucidate more information on the role of matrix metalloproteinase-2 and matrix metalloproteinase-9 protein overexpression with regard to lymph node metastasis of early gastric cancer. The levels of matrix metalloproteinase-2 and matrix metalloproteinase-9 protein expression were assessed by immunohistochemistry. An association was observed between matrix metalloproteinase-2, matrix metalloproteinase-9, and matrix metalloproteinase-2/matrix metalloproteinase-9 overexpression and clinicopathological factors, such as ulceration and lymph node metastasis. Furthermore, matrix metalloproteinase-9 and matrix metalloproteinase-2/matrix metalloproteinase-9 overexpression both were strongly correlated with histological grade. In addition, matrix metalloproteinase-2/matrix metalloproteinase-9 overexpression correlated with deep invasion. Multivariate Cox regression analysis revealed that matrix metalloproteinase-2 and matrix metalloproteinase-9 expression were both independent factors of overall survival in patients with early gastric cancer. In novelty, we found that matrix metalloproteinase-2/matrix metalloproteinase-9 overexpression was an independent indicator of lymph node metastasis in early gastric cancer which will be helpful in clinic to select the appropriate treatment of these patients.

More than 60% of myeloid dysplasia syndrome (MDS) contains mutations in genes encoding for splicing factors such as SF3B1, U2AF, SRSF2 and ZRSR2. Mutations in SF3B1 are associated with 80% cases of refractory anemia with ring sideroblast (RARS), a subtype of MDS. SF3B1K700E is the most frequently mutated site among mutations on SF3B1. Yet the molecular mechanisms on how mutations of splicing factors lead to defective erythropoiesis are not clear. SF3B1K700E mutant binds to an RNA binding protein, RBM15, stronger than the wild type SF3B1 protein in co-immunoprecipitation assays. In addition, K700E mutant alters the RNA splicing of transcription factors TAL1 and GATA1. Via alternative RNA splicing, a novel short TAL1 transcript variant (TAL1s) is generated. Enhanced interaction between SF3B1 and RBM15 promotes the production of full-length TAL1 (TAL1fl) mRNA, while reduction of RBM15 protein level via PRMT1-mediated degradation pathway changes TAL1s/TAL1fl ratio in favor of TAL1s. TAL1s contains the helix-loop-helix DNA binding domain but not the N terminal region upstream of the DNA binding domain. The TAL1s protein loses its interaction with ETO2, which represses early erythropoiesis. In this vein, overexpression of TAL1s stimulates the transcription of β-hemoglobin in human leukemia K562 cells and promotes erythroid differentiation of human cord blood CD34+ cells cultured in erythropoietin-containing medium. Therefore, mutations of SF3B1 may block erythropoiesis via dysregulation of alternative RNA splicing of transcription factor TAL1, and targeting PRMT1 may alleviate the anemic symptoms in MDS patients.

Protein arginine methyltransferase 1 (PRMT1) serves pivotal roles in various cellular processes. However, its role in megakaryocytic differentiation has not been clearly reported. The aim of the present study was to explore the role of the PRMT-RNA binding motif protein 15 (RBM15) axis in human MK differentiation and the feasibility of targeting PRMT1 for leukemia treatment. In the present study, PRMT1 was overexpressed and the RBM15 protein was knocked down in human umbilical cord blood cluster of differentiation (CD)34+ cells and the cells were then cultured in megakaryocytic differentiation medium. Flow cytometry was used to analyze CD41 and CD42 double-positive cells, as well as the protein expression levels of PRMT1 and RBM15. The results demonstrated that human cord blood CD34+ cells differentiate into mature MKs in high thrombopoitin medium, as demonstrated by CD41 and CD42 expression. Overexpression of PRMT1 in human umbilical cord blood CD34+ cells blocked the maturation of megakaryocytic cells. Knockdown of RBM15 by short hairpin RNA produced less mature MKs. PRMT1 inhibitor rescued PRMT1-blocked megakaryocytic differentiation. These results provide evidence for a novel role of PRMT1 in the negative regulation of megakaryocytic differentiation. PRMT1 may be a therapeutic target for leukemia treatment.

The isoforms of key transcription factors in hematopoiesis such as TAL1, GATA1 and RUNX1 are generated through alternative RNA splicing regulated by the PRMT1-RBM15 axis (Zhang et al. 2015). The functions of short isoforms of GATA1 (GATA1s) and RUNX1 (RUNX1a) are well characterized, yet it is unknown how the short isoform of TAL1 (TAL1s) regulates hematopoiesis. In this presentation, we report that the short isoform of TAL1, i.e. TAL1s, is generated via alternative RNA splicing as detected by isoform specific real-time PCR reactions using RNA isolated from leukemia cell lines and primary human cord blood cells. RBM15, an RNA binding protein, which is involved in chromosome translocation to produce RBM15-MKL1 fusion protein in acute megakaryocytic leukemia, regulates the alternative RNA splicing of TAL1. RBM15 promotes the production of full-length TAL1 mRNA, while reduction of RBM15 protein level via PRMT1-mediated degradation pathway favors the production of TAL1s. RBM15 directly binds to intronic regions on TAL1 pre-mRNA. Binding of RBM15 is responsible for recruiting SF3B1-associated RNA splicing complex. Given that PRMT1 senses the hypoxia status of hematopoietic cells, the changing of TAL1s/TAL1fl ratio by PRMT1 activity may be an adaptive response of hematopoietic cells to hypoxia status. The short form TAL1s still contains the helix-loop-helix DNA binding domain but not the N terminal regions upstream of the DNA binding domain. Thus, the TAL1s may act as a dominant negative mutant of the full-length TAL1fl to block TAL1fl-regulated transcription. We demonstrated that overexpression of TAL1s not the full-length TAL1promotes the erythroid differentiation of K562 cells. Although TAL1 gene is required for both erythroid and megakaryocyte differentiation at early stage of hematopoiesis, TAL1s does not promote megakaryocyte differentiation. Therefore, fine-tuning the TAL1 isoforms by the PRMT1-RBM15 axis determine the cell fate of a MEP progenitor cell. Using immunoprecipitation assays and mass spectrometry analysis, we identified proteins specifically associated with the N terminal region of TAL1. How unique TAL1s-associated transcriptional regulatory complex plays in erythroid differentiation will be discussed in the presentation in comparison with the Tal1fl-asociated protein complex. In summary, our findings stratify another new layer of regulation by PRMT1, which relays extracellular signals (such as hypoxia signal) to transcriptional regulatory program. Given that PRMT1 is often constitutively highly expressed in leukemia cells, how overproduction of short form TAL1 interferes with normal hematopoiesis may help to explain the molecular mechanisms of many hematological malignancies associated with dysregulation of TAL1 expression. Disclosures No relevant conflicts of interest to declare.