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Purpose: MicroRNAs (miRNA) are involved in and are controlled by epigenetic regulation, and thereby form a reciprocal regulatory circuit. Using next-generation sequencing (NGS)–based miRNA profiling, this study aimed to discover esophageal squamous cell carcinoma (ESCC)–specific miRNAs and miRNA-related epigenetic modulations.Experimental Design: NGS-based miRNA profiles were generated for four pairs of ESCC tissues and adjacent normal tissues. In situ hybridization was used to assess miRNA expression and its correlation with prognosis. miRNA-related DNA methylations were identified using bisulfite genomic sequencing, and the role of DNA methyltransferase 1 (DNMT1) was investigated using RNA interference. miRNA targets were screened by mRNA sequencing, and functional validation was performed in vitro and in vivo.Results: NGS-based miRNA profiling identified 78 differentially expressed miRNAs in ESCC. Among them, microRNA126-3p (miR-126) was significantly downregulated, and its downregulation correlated with poor ESCC prognosis. Downregulation of miR-126 was due to promoter hypermethylation of its host gene, Egfl7. DNMT1 was aberrantly upregulated in ESCC and responsible for the hypermethylation of Egfl7. Intriguingly, DNMT1 was suppressed by overexpression of miR-126, indicating the existence of a regulatory feedback circuit. ADAM9 was identified as a key target of miR-126. Ectopic expression of miR-126 or silencing of ADAM9 reduced ESCC cell proliferation and migration by inhibiting epidermal growth factor receptor–AKT signaling.Conclusions: Our results indicate that miR-126 is a potential prognostic indicator for ESCC and suggest that a novel “DNMT1–miR-126 epigenetic circuit” is involved in ESCC progression. Consequently, miR-126–based epigenetic modulations may provide a basic rationale for new approaches to antitumor therapeutics. Clin Cancer Res; 21(4); 854–63. ©2014 AACR.

Supplementary Figure S3. Suppressive effect of miR-126 on ESCC cell migration and growth.

The file included six supplementary tables as following: Table S1.Clinicopathological Characteristics; Table S2. Differently expressed miRNAs between ECs and NMs; Table S3. Univariate and Multivariate analysis of factors associated with disease-specific survival; Table S4. A short list of functionally relevant genes significantly downredulated in 126-EC cells vs. NC-EC cells; Table S5. Primer sequences (5'-3'), Table S6. The primary antibodies for western blot and IHC.

This file contained the supplementary materials and methods including RNA extraction and quantitative real-time PCR, miRNA overexpression and RNA interference (RNAi), Cell proliferation and migration assays, Immunohistochemistry (IHC) and Western blotting, EGFR inhibitor (AG1478) and 5-aza-2'-deoxycytidine (5-aza-dC) treatments, ISH scoring, DNA methylation analysis, Target prediction, 3'-UTR vector preparation, and luciferase reporter assay, Tumor xenograft in nude mice, mRNA sequencing. As well as, legends of the six supplementary figures were included in this file.

Background: Wilms tumor gene 1 (WT-1 gene) is overexpressed in most patients with acute myeloid leukemia (AML) and provides the evidence for an indicator of minimal residual disease (MRD) monitoring, but further studies of the combined prognostic value of WT-1 gene are needed to be illustrated due to its relatively low specificity. The aim of the study is to explore the prognostic value of WT-1 gene combined with recurrent cytogenetic gene in AML. Methods: We dynamically examined the transcript expression of WT-1 gene in the bone marrow samples of adult patients with AML, and then validated the prognostic value of WT-1 gene with or without recurrent cytogenetic gene. Results: In AML, the transcript expression of WT-1 gene was closely related to leukemic tumor burden and could act as an accurate indicator of molecular MRD detection. Most patients with low level expression of WT-1 gene after induction and consolidation therapy were significantly associated with favorable relapse free survival (RFS) and overall survival (OS), but there were still 16.7% patients were relapsed and died of primary disease. However, when analyzing of WT-1 gene combined with recurrent cytogenetic gene, none of the patients with low level expression of WT-1 gene and negative of recurrent cytogenetic gene were relapsed and died in the median follow-up time of 19 months (range: 3-94months). Conclusion: WT-1 gene combined with recurrent cytogenetic gene is a more accurate indicator of MRD monitoring and prognosis evaluation in AML.

Long non-coding RNA HOTAIR has been reported to play a key role in regulating various biological processes in various cancers. However, the roles and mechanisms of HOTAIR in acute myeloid leukaemia (AML) are still unclear and need to be investigated. In this study, we induced differentiation of four AML cell lines by all-trans retinoic acid (ATRA) and found HOTAIR was significantly upregulated in the process. Chromatin immunoprecipitation (ChIP) assays indicated that C/EBPβ upregulated HOTAIR during ATRA induced differentiation in HL-60 cells. By gain- and loss-of-function analysis, we then observed that HOTAIR expression was positively correlated with ATRA-induced differentiation and negatively regulated G1 phase arrest in HL-60 cells. In addition, we found that HOTAIR promoted ATRA-induced differentiation via the regulation of the cell cycle regulator p21 via miR-17-5p. Moreover, we detected the expression of HOTAIR in 84 de novo AML patients, HOTAIR was found significantly downregulated in the AML patients compared to the iron deficiency anaemia (IDA) control group, negatively correlated with the platelet level in M2 patients. In all, our data suggest that HOTAIR may be subtype-specific in AML-M2 patients, also HOTAIR regulates AML differentiation by C/EBPBβ/HOTAIR/miR-17-5p/p21 pathway. The findings of the present study provide a novel insight into the mechanism of lncRNA-mediated differentiation and indicate that HOTAIR may be a promising therapeutic target for leukaemia, especially for AML with M2 type. Abbreviation: AML: acute myeloid leukaemia; APL: acute promyelocytic leukaemia; ATRA: all-trans retinoic acid; CCK8: cell Counting Kit-8; CDKs: cyclin-dependent kinases ; CeRNA: competing endogenous RNAs; ChIP: chromatin immunoprecipitation; CHX: cycloheximide; FAB: French–American–British; FCM: flow cytometry; HOTAIR: HOX transcript antisense RNA; IDA: iron-deficiency anemia; lncRNA: long non-coding RNA; 3′UTR: 3′untranslated region; MT: Mutation type; WT: Wild type; qRT-PCR: Quantitative real-time PCR

Background Diffuse large B-cell lymphoma (DLBCL) is a highly heterogeneous malignancy with varied outcomes. However, the fundamental mechanisms remain to be fully defined. Aim We aimed to identify core differentially co-expressed hub genes and perturbed pathways relevant to the pathogenesis and prognosis of DLBCL. Methods We retrieved the raw gene expression profile and clinical information of GSE12453 from the Gene Expression Omnibus (GEO) database. We used integrated bioinformatics analysis to identify differentially co-expressed genes. The CIBERSORT analysis was also applied to predict tumor-infiltrating immune cells (TIICs) in the GSE12453 dataset. We performed survival and ssGSEA (single-sample Gene Set Enrichment Analysis) (for TIICs) analyses and validated the hub genes using GEPIA2 and an independent GSE31312 dataset. Results We identified 46 differentially co-expressed hub genes in the GSE12453 dataset. Gene expression levels and survival analysis found 15 differentially co-expressed core hub genes. The core genes prognostic values and expression levels were further validated in the GEPIA2 database and GSE31312 dataset to be reliable (p < 0.01). The core genes’ main KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichments were Ribosome and Coronavirus disease-COVID-19. High expressions of the 15 core hub genes had prognostic value in DLBCL. The core genes showed significant predictive accuracy in distinguishing DLBCL cases from non-tumor controls, with the area under the curve (AUC) ranging from 0.992 to 1.00. Finally, CIBERSORT analysis on GSE12453 revealed immune cells, including activated memory CD4+ T cells and M0, M1, and M2-macrophages as the infiltrates in the DLBCL microenvironment. Conclusion Our study found differentially co-expressed core hub genes and relevant pathways involved in ribosome and COVID-19 disease that may be potential targets for prognosis and novel therapeutic intervention in DLBCL.

Chimeric antigen receptor (CAR) T cell therapy improves the remission rate of refractory/relapsed B-acute lymphoblastic leukemia (R/R B-ALL) patients, but the relapse rate remains high. Recent studies suggest patients who underwent post-chimeric antigen receptor T cell therapy hematopoietic stem cell transplantation (post- HSCT) would achieve durable remission and better survival, but this remains controversial. To this end, we conducted a meta-analysis to assess the role of post-HSCT in R/R B-ALL. The Cochrane Library, Embase, and PubMed were used to identify relevant studies; the latest search update was on July 05, 2020. We used the Cochran Q test and I-squared statistics to test for heterogeneity among the studies analyzed. The fixed model and random model were used to combine results when appropriate. We performed all statistical analyses with Stata 12, and P < 0.05 was considered statistically significant. We included 18 studies with 758 patients in the meta-analysis. Our results indicated that post-HSCT was associated with lower relapse rate (RR: 0.40, 95% CI: 0.32-0.50, P = 0.000), better overall survival (HR: 0.37, 95% CI: 0.19-0.71, P = 0.003), better leukemia-free survival (HR: 0.20, 95% CI: 0.10-0.40, P = 0.000). However, post-HSCT did not influence OS in Caucasians, and CAR-T cells with CD28 co-stimulation factor bridged to HSCT did not influence OS. Post-HSCT decreased the relapse rate and improved the long-term survival of R/R B-ALL patients. R/R B-ALL patients would benefit from post-HSCT after CAR-T cell therapy.

Background: Hemophagocytic lymphohistiocytosis (HLH) a hyperinflammation disease and have high early mortality. The neutrophil-lymphocyte ratio (NLR) plays a prognostic role in various inflammation conditions. However, the role of NLR in HLH remains unknown.Results: 92 patients were collected from Sep 2014 to Dec 2019, the median age was 50 years (16-88) and 54 patients (58.6%) were male. 39 patients (42.3%) died during a 30-day follow-up. In addition, NLR was higher in nonsurvivor than survivor (P=0.005), higher NLR was correlated with older age (P=0.020), worse survival status (P=0.006), lower HB level (P=0.008), longer PT level (P=0.010), higher creatinine level (0.027) and higher IL-6 level (P=0.008)and in multivariate analysis, NLR (HR=2.508, 95%CI: 1.275-4.934), was indicated as an independent prognostic factor for early death. Conclusions: NLR was correlated with early death in HLH, suggest NLR could be used as a reference indicator for the monitoring and management of HLH.

Long non-coding RNAs (lncRNAs) have been demonstrated that they not only play important roles in tumorgenicity but also associate with cancer prognosis. Recently, highly up-regulated in liver cancer (HULC) is abnormally expressed in liver cancer and other cancers, and participated in cancers progression; however, it is unclear whether its expression is associated with prognosis. Here, we performed a meta-analysis and systematic review to evaluate the prognostic value and metastasis of HULC in various cancer patients. The meta-analysis was performed using a systematic search of PubMed, Web of Science, ScienceDirect and Wiley Online Library database to eligible studies. The pooled hazard ratios (HRs) with a 95% confidence interval (95% CI) were calculated to assess its prognosis and metastasis in human cancer. A total of 1134 patients from 11 studies were included. The results indicated that overexpression of HULC was associated with poor overall survival (OS) (HR = 1.89, 95% CI: 1.32-2.47). Furthermore, subgroup analysis showed that cancer type (digestive system cancer or non-digestive system cancers) and sample size (more or less than 100) significantly associated between HULC and OS. In addition, overexpression of HULC expression was significantly associated with metastasis in cancers (HR = 2.67, 95% CI: 0.94-4.39). The meta-analysis indicated that lncRNA HULC could serve as a new molecular marker for cancer prognosis and metastasis.

Background Multiple myeloma (MM), the second most hematological malignancy, has high incidence and remains incurable till now. The pathogenesis of MM is poorly understood. This study aimed to identify novel prognostic model for MM on gene expression profiles. Methods Gene expression datas of MM (GSE6477, GSE136337) were downloaded from Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) in GSE6477 between case samples and normal control samples were screened by the limma package. Meanwhile, enrichment analysis was conducted, and a protein-protein interaction (PPI) network of these DEGs was established by STRING and cytoscape software. Co-expression modules of genes were built by Weighted Correlation Network Analysis (WGCNA). Key genes were identified both from hub genes and the DEGs. Univariate and multivariate Cox congression were performed to screen independent prognostic genes to construct a predictive model. The predictive power of the model was evaluated by Kaplan–Meier curve and time-dependent receiver operating characteristic (ROC) curves. Finally, univariate and multivariate Cox regression analyse were used to investigate whether the prognostic model could be independent of other clinical parameters. Results GSE6477, including 101 case and 15 normal control, were screened as the datasets. A total of 178 DEGs were identified, including 59 up-regulated and 119 down-regulated genes. In WGCNA analysis, module black and module purple were the most relevant modules with cancer traits, and 92 hub genes in these two modules were selected for further analysis. Next, 47 genes were chosen both from the DEGs and hub genes as key genes. Three genes (LYVE1, RNASE1, and RNASE2) were finally screened by univariate and multivariate Cox regression analyses and used to construct a risk model. In addition, the three-gene prognostic model revealed independent and accurate prognostic capacity in relation to other clinical parameters for MM patients. Conclusion In summary, we identified and constructed a three-gene-based prognostic model that could be used to predict overall survival of MM patients.

The differential expression of soluble programmed death ligand 1 (sPD-L1) has been found in some cancers; however, the correlation between sPD-L1 expression and prognosis value in tumour is still unclear. Here, we conducted a meta-analysis and systematic review to assess the prognostic value of sPD-L1 in patients with cancer. Eligible studies were searched for in the databases including PubMed, Web of Science, ScienceDirect and Wiley Online Library database. The pooled hazard ratios (HRs) with a 95% confidence interval (95%CI) were calculated to assess the prognostic significance of sPD-L1 in human cancer. Eight studies and 1102 patients with cancer were included in the final analysis, and the combined analysis indicated that a higher level of sPD-L1 was associated with worse overall survival (OS) (HR = 1.60, 95%CI: 1.21-1.99). Furthermore, statistical significance was also observed in subgroup analysis stratified by the cancer type (haematological neoplasms or non-haematological neoplasms), sample size (more or less than 100), cut-off value of sPD-L1 (more or less than 6.51 ng/ml) and ethnicity (Asian or European). The meta-analysis indicates that circulating sPD-L1 changes may serve as a useful biomarker for cancer prognosis, and higher level of sPD-L1 may also be associated with poor outcomes in patients with cancer.

// Linhui Hu 1 , Manman Li 1 , Yangyang Ding 1 , Lianfang Pu 1 , Jun Liu 1 , Jingxin Xie 2 , Michael Cabanero 3 , Jingrong Li 4 , Ru Xiang 5 and Shudao Xiong 1 1 Department of Hematology/Hematological Lab, The Second Hospital of Anhui Medical University, Hefei, Anhui, People’s Republic of China 2 Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Fudan University, Shanghai, People’s Republic of China 3 University Health Network, University of Toronto, Ontario, Canada 4 Department of Emergency, The Second Hospital of Anhui Medical university, Hefei, Anhui, People’s Republic of China 5 School of Nursing, Anhui Medical University, Hefei, Anhui, People’s Republic of China Correspondence to: Shudao Xiong, email: // Keywords : red blood cell distribution width, prognosis, cancer, meta-analysis Received : August 15, 2016 Accepted : November 22, 2016 Published : December 02, 2016 Abstract Red blood cell distribution width (RDW), a parameter that used to differentiate the type of anemia for several decades, recent studies suggest it was a prognostic factor in various types of cancer patients. However, the prognostic value of RDW in cancer patients remains controversial. Here, we performed a meta-analysis and systematic review to evaluate the prognostic value of RDW in cancer patients. Relevant studies were picked out from the databases of Web of Science, Embase, Pubmed and Cochrane Library. A total of 16 papers with 4267 patients were included in this meta-analysis, and the combined results indicated that elevated RDW was associated with poor over survival (OS) (HR = 1.47, 95%CI:1.29-1.66), poor cancer-specific survival (CSS) (HR = 1.46, 95%CI:1.08-1.85), poor disease-free survival (DFS) (HR = 1.91, 95%CI:1.27-2.56), poor event-free survival (EFS) (HR = 2.98, 95%CI:0.57-5.39) and poor progress-free survival (PFS) (HR = 3.21, 95%CI:0.33-6.75) after treatment. Furthermore, the similar results were observed in subgroup analysis stratified by cancer type, cutoff value of RDW, sample size and ethnicity. In conclusion, this meta-analysis demonstrated that RDW may be a potential prognostic marker in patients with cancer, and high RDW may also be associated with poor outcomes.

BackgroundElevated red blood cell distribution width (RDW) and decreased platelet count (PLT) can be clinically relevant to the prognosis in cancer patients. However, their prognostic values in patients with diffuse large B-cell lymphoma (DLBCL) need to be further explored.MethodsHealthy donors (n = 130) and patients with DLBCL (n = 349) were included and evaluated retrospectively in this study. The prognostic influence of clinical and pathological factors including RDW and PLT on overall survival (OS) and progression-free survival (PFS) were studied by Kaplan-Meier curves. To evaluate the independent prognostic relevance of RDW and PLT, univariate and multivariate Cox proportional hazards regression models were applied. The adjusted IPI model was established based on the results of multivariate analysis, and verified by Harrell’s C statistical analysis.ResultsKaplan-Meier curves indicated that an elevated RDW value and thrombocytopenia are poor factors for OS (P P = 0.006) and PFS (P = 0.003,P P = 0.003) and decreased PLT count (HR =1.749, 95%CI = 1.010–3.028,P = 0.046) were both independent prognostic factors. The c-index of IPI and NCCN-IPI were increased when RDW level and PLT were supplemented in our cohort.ConclusionsOur study shows that elevated RDW level and decreased PLT are independent poor prognostic factors in newly diagnosed DLBCL patients. Adding RDW and PLT to the IPI score may improve its predictive ability, and the adjusted IPI may be more powerful in predicting the survival of DLBCL patients in the rituximab era.

Myeloid-derived suppressor cells (MDSCs) comprise of a population of cells, which suppress the innate and adaptive immune system via different mechanisms. MDSCs are accumulated under pathological conditions. The present study aimed to clarify the pathological role of MDSCs in systemic lupus erythematosus (SLE) patients. Consequently, the level of circulating M-MDSCs was significantly increased in newly diagnosed SLE patients as compared to healthy controls. An elevated level of M-MDSCs was positively correlated with the disease severity in SLE patients and an immunosuppressive role was exerted in an iNOS-dependent manner. The decrease in the number of M-MDSCs after therapy rendered them as an indicator for the efficacy of treatment. These results demonstrated that M-MDSCs participated in the pathological progress in SLE patients. Thus, MDSCs are attractive biomarkers and therapeutic targets for SLE patients.

Myeloid-derived suppressor cells (MDSCs) comprise of a population of cells, which suppress the innate and adaptive immune system via different mechanisms. MDSCs are accumulated under pathological conditions. The present study aimed to clarify the pathological role of MDSCs in systemic lupus erythematosus (SLE) patients. Consequently, the level of circulating M-MDSCs was significantly increased in newly diagnosed SLE patients as compared to healthy controls. An elevated level of M-MDSCs was positively correlated with the disease severity in SLE patients and an immunosuppressive role was exerted in an iNOS-dependent manner. The decrease in the number of M-MDSCs after therapy rendered them as an indicator for the efficacy of treatment. These results demonstrated that M-MDSCs participated in the pathological progress in SLE patients. Thus, MDSCs are attractive biomarkers and therapeutic targets for SLE patients.

Additional file 1: Figure S1. ROC curves analysis for RDW(a) and PLT(b) in the training set (N=200) of patients with DLBCL. Figure S2. Correlation between RDW, PLT and WBC, NLR, ALB, HB levels in all patients with DLBCL. Correlation between RDW and WBC(a), NLR(b), ALB(c) and HB(d) levels in all patients with DLBCL. (2)Correlation between PLT and WBC(e), NLR(f), ALB(g) and HB(h) levels in all patients with DLBCL. Figure S3. Correlation between HB and WBC(a), NLR(b) and ALB(c) levels in all patients with DLBCL. Figure S4. Survival curves according to RDW and PLT levels in the training, overall and testing set. (1)OS(a,c) and PFS(b,d) according to the RDW and PLT levels in the training set. (2)OS(e,g) and PFS(f,h) according to the RDW and PLT levels in the overall set. (3)OS(i,k) and PFS(j,l) according to the RDW and PLT levels in the testing set. Figure S5. Kaplan–Meier curves for OS and PFS comparing low (12 g/dL for women, >13 g/dL for men) Hb levels in the training(a,b), overall(c,d), testing set(e,f), CHOP cohort(g,h) and R-CHOP cohort(i,j). Figure S6. Survival curves according to RDW and PLT levels in CHOP cohort and the whole cohort with IPI score 3-5. (1) OS(a,c) and PFS(b,d) according to RDW levels and PLT counts in CHOP cohort with IPI score 3-5. (2) OS(e,g) and PFS(f,h) according to RDW levels and PLT counts in the whole cohort with IPI score 3-5. Table S1. Clinical characteristics of healthy donors and DLBCL patients. Table S2. Baseline clinical characteristics of patients with DLBCL. Table S3. Univariate analysis of clinicopathological parameters for the prediction of OS and PFS in CHOP cohort patients(n=175). Table S4. Univariate analysis of clinicopathological parameters for the prediction of OS and PFS in RCHOP cohort patients(n=174).

Background: In the hematology department, the availability of biomarkers for early detection of infection is difficult to obtain. The present study aimed to compare the diagnostic values of neutrophil CD64 Index, procalcitonin (PCT), interleukin-6 (IL-6) and C-reactive protein (CRP) and to determine whether the combined analysis of these biomarkers offer stronger predictive power in the diagnosis for the infection of febrile patients. Methods: Neutrophil CD64 Index, PCT, IL-6 and CRP levels were determined in 356 febrile patients in the hematology ward from May 2013 to May 2015. Sensitivity, specificity, positive and negative likelihood ratios, positive and negative predictive values, receiver operating characteristic (ROC) areas under the curve (AUC), and logistic regression analysis were determined to evaluate the diagnostic values of these biomarkers. Results: The levels of the four biomarkers were higher in the infection patients (p Conclusions: The neutrophil CD64 Index is useful for early identification of infections in febrile patients in the hematology department. The combined analysis of the CD64 Index, PCT and IL-6 could further improve its sensitivity.

Acute myeloid leukemia (AML) is the most common hematological malignancy in adults, but the etiology of it remains poorly understood. IL-35 is a recently described cytokine composed of an IL-12 subunit p35 and an IL-27 subunit Epstein-Barr virus induced gene 3 (EBI3), and has an immunosuppressive effect on inflammation through induction of regulatory T cells (Tregs) and suppression of Th1 and Th17. Recently, we have illustrated that concentrations of IL-35 in peripheral blood are up-regulated in newly diagnosed (ND) AML patients. However, whether IL-35 in bone marrow is increased in AML patients is not clear. In this study, we examined IL-35 in bone marrow by various methods including RT-PCR, ELISA, FCM and IHC, and found that IL-35 levels are also increased significantly in bone marrow of adult AML patients. Furthermore, we investigated that concentrations of bone marrow IL-35 in ND group were higher than that in complete remission (CR) group and control group, but there was no significant difference compared to that in relapse group. In conclusion, IL-35 was elevated in bone marrow of adult AML patients and this increase was correlated with the clinical stages of malignancy, suggesting that IL-35 is involved in pathogenesis of AML.

Tumor immune escape mechanism mediated by CD4+CD25+regulatory T cells (Tregs) is a key factor in the pathogenesis of acute myeloid leukemia (AML). IL-35, as a novel inhibitory cytokine, is produced by Tregs specially and regulates functions of Tregs in murine. However, IL-35 expression of Tregs in human is still disputed, and its role in AML is yet to be elucidated. In this study, we found that IL-35 was expressed highly in peripheral blood plasma of adult patients with AML and significantly correlated with the clinical stages of malignancy. Tregs-derived from adult AML patients produced IL-35 in a stimulation-dependent manner. IL-35 promoted AML blasts immune escape by expanding Tregs and inhibiting CD4+CD25-effector T cells (Teffs). Furthermore, IL-35 directly promoted the proliferation of AML blasts and reduced the apoptosis of AML blasts. Together, our study demonstrates that IL-35-derived from Tregs promotes the growth of adult AML blasts, suggesting that IL-35 has an important role in the pathogenesis of AML.

Increased platelet heterogeneity has been reported in myeloproliferative neoplasms (MPN) with thrombocytosis. However, whether abnormal thrombopoiesis occurs in patients with chronic myeloid leukemia (CML) who have normal platelet counts, remains unclear. In order to explore this question, 25 patients with CML with normal platelet counts (CML-N), 40 patients with CML with elevated platelet counts (CML-E) and 33 healthy adults were recruited. The association of platelet count with mean platelet volume (MPV), platelet large cell ratio (P-LCR) and platelet distribution width (PDW) was examined. Bone marrow smears were also reviewed to assess the proliferation and abnormal lobation of megakaryocytes. The results showed that the two CML groups exhibited higher MPV, P-LCR and PDW values than those of the controls (P

The Philadelphia (Ph; BCR-ABL) chromosome originates from a translocation event between chromosomes 9 and 22, and results in the BCR-ABL fusion gene. In chronic myelogenous leukemia (CML), the BCR-ABL gene is mainly coded for by a major breakpoint cluster region (M-bcr, e13a2 and e14a2). However, in some patients, BCR-ABL genes are encoded by a minor (m)-bcr, e1a2, and a micro (µ)-bcr region, e19a2. These transcripts revealed a different clinical course. The present study described a CML patient whose cytogenetics and FISH analyses of bone marrow revealed a karyotype of 46, XY t(9,22) (q34;q11), while the commercial kits of quantitative PCR (qPCR) failed to detect the BCR-ABL fusion gene. Further multiplex Reverse transcription-PCR (RT-PCR) and sequencing analyses identified a rare e14a3 (b3a3) fusion transcript.

microRNAs are an abundant class of short endogenous non-coding RNAs that function as important regulators of multiple target genes and participate in diverse biological roles in carcinogenesis. However, the role of miR-7 in lung cancer remains unclear and requires further elucidation. In the present study, we found a reduction of miR-7 expression in Lewis lung cancer (3LL) cells originating from mice by real-time RT-PCR. Restoration of miR-7 inhibited 3LL cell proliferation, induced cell apoptosis in vitro and reduced tumorigenicity in vivo. We further confirmed that miR-7 downregulated the expression of both epidermal growth factor receptor (EGFR) and murine leukemia viral oncogene homologue-1 (RAF-1) oncogenes by real-time PCR and western blot analysis. Furthermore, inhibition of EGFR showed similar effects to miR-7 enforcement in 3LL cells. Taken together, these findings revealed that miR-7 acts as an antitumor miRNA in 3LL by targeting and suppressing the expression of both EGFR and RAF-1 oncogenes. This study may provide a rationale for the use of miR-7 in lung cancer target therapy.

Paclitaxel (PTX) is the front-line chemotherapeutic agent against human non-small cell lung cancer (NSCLC). However, its therapeutic efficacy is restricted by the increasing frequency of chemotherapeutic resistance in NSCLC. Accumulating evidence has shown the potential role of microRNAs (miRNAs) in the chemotherapeutic sensitivity of cancer cells. Previously it was reported that microRNA-7 (miR-7) acts as an important tumor suppressor in NSCLC. Therefore, the present study was conducted to determine the regulatory role of miR-7 in PTX chemotherapy for NSCLC. Four NSCLC cell lines were used to analyze the correlation of the PTX-sensitivity and endogenoaus miR-7 expression. miR-7 expression was up- and downregulated using miR-7 mimics and inhibitors respectively, and the role of miR-7 in sensitizing NSCLC cells to PTX was assessed by cell viability and apoptosis assays. The molecular mechanism of PTX sensitivity was determined by quantitative polymerase chain reaction and western blotting. It was found that the sensitivity of NSCLC cells to PTX was dependent on endogenous miR-7. Upregulation of miR-7 enhanced the PTX-sensitivity of NSCLC cells by suppressing cell proliferation and promoting cell apoptosis, while the inhibition of miR-7 abrogated the antiproliferative proapoptotic effects of PTX. Pretreatment of miR-7 mimics enhanced the PTX-mediated downregulation of epidermal growth factor receptor (EGFR) in NSCLC cells. These results have identified miR-7 as a potential EGFR-targeting sensitizer in PTX therapy. These data may facilitate the development of novel chemotherapeutic approaches for NSCLC.

Supplemental Digital Content is available in the text, Background and objective: Krüppel-like factor 8 (KLF8), a transcription factor, belongs to the KLF8 family. Currently, studies have shown that KLF8 is highly expressed in some tumors. However, the prognostic value and metastasis of KLF8 in cancers remain unclear. For the first time, we conducted meta-analysis to explore the relationship between KLF8 expression with prognosis and metastasis in various carcinomas patients. Methods: Web of Science, PubMed, Embase, and Cochrane Library were systematically searched for eligible articles. Pooled hazard ratios (HRs) and their 95% confidence intervals (95% CIs) were calculated to evaluate the prognostic value and metastasis of KLF8 expression in human cancer patients. Results: The result revealed that highly expression level of KLF8 was significantly associated with poor overall survival (OS) (HR = 1.56, 95% CI: 1.26–1.87). Meanwhile, this significant correlation was also observed in subgroup analysis stratified by cancer types, source of HR, sample size, follow-up (months). In addition, highly expression of KLF8 was also closely associated with metastasis (HR = 1.37, 95% CI: 0.57–2.17) and tumor node metastasis stage (HR = 1.58, 95% CI: 0.90–2.25) in carcinomas. Conclusion: In summary, our meta-analysis indicates that overexpression of KLF8 may be associated with poor prognosis and higher incidence of metastasis in various carcinomas, and KLF8 may be used as a prognostic and metastatic indicator in human cancers.

Background: MicroRNA-7 (miR-7) has been reported to be a tumour suppressor gene. However, whether it has a role in the growth of non-small-cell lung cancer (NSCLC) and what is its target involved in the tumour growth is still under investigation. Methods: NSCLC tissue sample, NSCLC cell lines and tissue microarray were investigated in this study. Total RNA, miRNA and protein were used for RT-PCR and western blot analysis. Immunohistochemistry was performed in tissues microarray. Cell culture and intervention experiments were performed in vitro and in vivo. Bioinformatics prediction, western blot and luciferase assay were identified the target of miR-7. Results: In this study, we found that the expression of miR-7 was significantly downregulated not only in NSCLC cell lines, but also in human NSCLC tissues compared with the matched adjacent tissues. Restoration of its expression through miR-7 mimics in A549 and H1299 NSCLC cells inhibited cell proliferation, colony formation, and cell-cycle progression in vitro. More importantly, the tumorigenicity in nude mice was reduced after administration of miR-7 in vivo. In advance, through bioinformatic analysis, luciferase assay and western blot, we identified a novel target of miR-7, PA28gamma (a proteasome activator) to be enrolled in the regulation with tumour. PA28gamma mRNA and protein levels are markedly upregulated in NSCLC cell lines and tumour samples, exhibiting a strong inverse relation with that of miR-7. In addition, knockdown of PA28gamma induced similar effects as overexpression of miR-7 in NSCLC cells. Furthermore, miR-7 overexpression or silencing of PA28gamma reduced the cyclinD1 expression at mRNA and protein level in NSCLC cell lines. Conclusion: All these findings strongly imply that the overexpression of PA28gamma resulted from miR-7 downexpression in NSCLC has an important role in promoting cancer cell progress and consequently results in NSCLC growth. Thus, strategies targeting PA28gamma and/or miR-7 may become promising molecular therapies in NSCLC treatment.

Cytokine-induced killer (CIK) cells are usually generated from peripheral blood mononuclear cells with the stimulation of IL-2 in vitro. Unlike the conventional IL-2-stimulated CIK cells (IL-2-CIK cells), we investigated the characteristics and potential mechanism of IL-15-stimulated CIK cells (IL-15-CIK cells) in this study. Compared with IL-2-CIK cells, the percentage of CD3CD56 cells was significantly increased in IL-15-CIK cells, but the expression of regulatory T (Treg) cells and IL-35 was significantly decreased in IL-15-CIK cells. Meanwhile, the in vitro cytotoxicity against human myeloid leukemia cells K562 of IL-15-CIK cells was significantly augmented compared with IL-2-CIK cells. These data suggest that IL-15 may improve the cytotoxicity of CIK cells against leukemia cells by upregulating CD3CD56 cells and downregulating Treg cells and IL-35.

Glucocorticoids (GCs) are among the most widely used and effective therapies for many chronic inflammatory diseases. Although attempts have been made to identify important protein-coding genes and pathways involved in the anti-inflammatory effect of GCs, knowledge of genomic aberrations associated with noncoding genes, such as micro-RNAs (miRNAs), and their contributions is relatively limited. In this study, a systematic screening of the miRNA expression profile by microarray showed that GCs inhibited the expression of miR-155 in lipopolysaccharide (LPS)-induced macrophage inflammatory responses. Overexpression of miR-155 markedly reversed the suppressive action of GCs, whereas inhibition of miR-155 exhibited an effect similar to that of GCs on LPS-treated RAW264.7 cells, indicating miR-155 to be a functional regulator in the anti-inflammatory effect of GCs. Furthermore, GCs inhibited miR-155 expression in a GC receptor- and NF-κB-dependent manner. Bioinformatics analysis and luciferase assay revealed that the NF-κB binding site located in the promoter region of the B-cell integration cluster was important in mediating the GC-driven suppression of miR-155 in response to LPS stimulation. In addition, the combination of treatment with GCs and inhibition of miR-155 enhanced the anti-inflammatory effect of GCs on LPS-stimulated RAW264.7 cells. Therefore, we identify miR-155 to be a novel target through which GCs exert their anti-inflammatory effect on the LPS-induced macrophage inflammatory response. These findings may provide a basic rationale for new approaches in the effort to develop anti-inflammatory therapeutics.

Trichosanthin (TCS), extracted from the Chinese medicinal herb Trichosanthes kirilowi, has shown promise for the inhibition of tumor growth. However, its immunomodulatory effect on tumor-host interaction remains unknown. In this study, we focused on the effect of TCS on murine anti-tumor immune response in the 3LL Lewis lung carcinoma tumor model and explored the possible molecular pathways involved. In addition to inhibiting cell proliferation and inducing apoptosis in the 3LL tumor, TCS retarded tumor growth and prolonged mouse survival more significantly in C57BL/6 immunocompetent mice than in nude mice. This reflected the fact that the host immune system was involved in tumor eradication. Using FACS analysis, we found that TCS increased the percentage of effector T cells, particularly Interferon-gamma (IFN-γ) producing CD4(+) and CD8(+) T cells from tumor-bearing mice. TCS also promoted the vigorous proliferation of antigen-specific effector T cells, markedly increased Th1 cytokine secretion and elicited more memory T cells in tumor-bearing mice, consequently enhancing the anti-tumor response and inducing immune protection. Furthermore, we found that TCS upregulated the expression of tumor suppressor in lung cancer 1 (TSLC1) in 3LL tumor cells and the expression of its ligand, class I-restricted T cell-associated molecule (CRTAM), in effector T cells. Blocking TSLC1 expression with small interfering RNA (siRNA) significantly eliminated the effects of TCS on the proliferation and cytokine secretion of effector T cells, suggesting that TCS enhances anti-tumor immune response at least partially by boosting the interaction between TSLC1 and CRTAM. Collectively, our data demonstrate that TCS not only affects tumor cells directly, but also enhances anti-tumor immunity via the interaction between TSLC1 and CRTAM. These findings may lead to the development of a novel approach for tumor regression.

Paclitaxel has become one of the most effective and widely used chemotherapeutic agents over the past decades. Although it has shown promise to selectively deplete regulatory T (Treg) cells in our previous study, the underlying molecular mechanism remains to be further elucidated. The present study focused on the effect of paclitaxel on Treg cells in 3LL Lewis tumor model and explored the possible molecular pathways involved in this process. We found that paclitaxel significantly decreased the percentage of Treg cells in CD4(+) cells and impaired their suppressive functions, but effector T (Teff) cells remained unaffected. Compared with Teff cells, Treg cells exhibited a high sensitivity to paclitaxel-mediated apoptosis in vitro. Interestingly, though paclitaxel has been characterized as a mitotic inhibitor, tubulin was not involved in the selective function of paclitaxel. Treg cells exposed to paclitaxel displayed downregulation of Bcl-2 and upregulation of Bax. Blocking the Bcl-2 pathway eliminated the difference between Treg and Teff cells responding to paclitaxel. These results suggest that Bcl-2 rather than tubulin contributes to the distinctive effect of paclitaxel on Treg cells. Therefore, we here identify a molecular pathway through which paclitaxel selectively ablates Treg cells.

Low-dose total body irradiation (LTBI) is used in the treatment of some cancers mainly for immune enhancement rather than cell killing. However, the mechanism underlying LTBI remains unknown. In this study, by analyzing the immune patterns of lymphocytes, we found that the percentage and absolute number of CD4(+)CD25(+)Foxp3(+) regulatory T cells are markedly decreased in naive mice following treatment with LTBI. On the contrary, the CD4(+)CD44(+)/CD8(+)CD44(+) effect or-memory T cells are greatly increased. Importantly, naive mice treated with dendritic cell-gp 100 tumor vaccines under LTBI induced an enhancement of antigen-specific proliferation and cytotoxicity as well as interferon-gamma (IFN-gamma) secretion against F10 melanoma tumor challenge, compared to treatment with either the tumor vaccine or LTBI alone. Consequently, the treatment resulted in a reduced tumor burden and prolonged mouse survival. Our data demonstrate that LTBI's enhancement of antitumor immunity was mainly associated with selectively decreasing the proportion and number of T regulatory cells,implying the potential application of the combination of LTBI and a tumor vaccine in antitumor therapy.

We retrospectively evaluated the association between the expression of CD56 and CD117 on neoplastic plasma cells and patients prognosis in 50 newly diagnosed multiple myeloma (MM) patients. The overall survival (OS) was measured and Cox proportional hazards model was used to evaluate CD56 and CD117 as possible prognostic factors for OS. CD56+ and CD117+ were detected in 74% and 32% multiple myeloma cases, respectively. In Kaplan-Meier analysis, CD56 and CD117 expression demonstrated potential prognostic impacts and were associated with longer OS (CD56: p=0.004, CD117: p=0.022), absence of both of them showed significantly shorter OS (p=0.046 compared to CD56+/CD117+ group, p=0.014 compared to CD56-/CD117+ or CD56+/CD117- group). Multivariate analysis showed that CD56 was independently prognostic of longer OS (p=0.012). After induction chemotherapy, overall response rates (ORR) was higher in CD56-positive group compared to CD56-negative group (70.6% versus 30.0%, p=0.024), however, there was no difference between CD117-positive and CD117-negative group (69.2% versus 58.1%, p=0.448). This study demonstrated the prognostic value of CD56 and CD117 in patients with newly diagnosed MM patients. Absence of both of them was associated with the poorest prognosis.

Paraplegia following spinal injury is a rare complication subsequent to the administration of intrathecal chemotherapy; however, it is also one of the rare clinical features of central nervous system leukemia (CNSL). Distinguishing between the two is extremely important. The present study reports the case of a 46-year-old man who was diagnosed with acute lymphoblastic leukemia and subsequently achieved remission in the blood and bone marrow following the initial course of chemotherapy. However, the patient developed a sudden onset of paraplegia and urinary retention due to spinal cord infiltration of leukemia cells following the administration of intrathecal methotrexate and cytarabine. The paraplegia was initially reversible. However, a few weeks later, the patient developed irreversible paraplegia due to a complication of the intrathecal administration of chemotherapy (methotrexate and cytarabine arabinoside). The patient gave up further treatment in May 2013 and succumbed to the disease in June 2013.

Purpose: MicroRNAs (miRNA) are involved in and are controlled by epigenetic regulation, and thereby form a reciprocal regulatory circuit. Using next-generation sequencing (NGS)–based miRNA profiling, this study aimed to discover esophageal squamous cell carcinoma (ESCC)–specific miRNAs and miRNA-related epigenetic modulations. Experimental Design: NGS-based miRNA profiles were generated for four pairs of ESCC tissues and adjacent normal tissues. In situ hybridization was used to assess miRNA expression and its correlation with prognosis. miRNA-related DNA methylations were identified using bisulfite genomic sequencing, and the role of DNA methyltransferase 1 (DNMT1) was investigated using RNA interference. miRNA targets were screened by mRNA sequencing, and functional validation was performed in vitro and in vivo. Results: NGS-based miRNA profiling identified 78 differentially expressed miRNAs in ESCC. Among them, microRNA126-3p (miR-126) was significantly downregulated, and its downregulation correlated with poor ESCC prognosis. Downregulation of miR-126 was due to promoter hypermethylation of its host gene, Egfl7. DNMT1 was aberrantly upregulated in ESCC and responsible for the hypermethylation of Egfl7. Intriguingly, DNMT1 was suppressed by overexpression of miR-126, indicating the existence of a regulatory feedback circuit. ADAM9 was identified as a key target of miR-126. Ectopic expression of miR-126 or silencing of ADAM9 reduced ESCC cell proliferation and migration by inhibiting epidermal growth factor receptor–AKT signaling. Conclusions: Our results indicate that miR-126 is a potential prognostic indicator for ESCC and suggest that a novel “DNMT1–miR-126 epigenetic circuit” is involved in ESCC progression. Consequently, miR-126–based epigenetic modulations may provide a basic rationale for new approaches to antitumor therapeutics. Clin Cancer Res; 21(4); 854–63. ©2014 AACR.

Tumor immune escape mechanism mediated by CD4+CD25+regulatory T cells (Tregs) is a key factor in the pathogenesis of acute myeloid leukemia (AML). IL-35, as a novel inhibitory cytokine, is produced by Tregs specially and regulates functions of Tregs in murine. However, IL-35 expression of Tregs in human is still disputed, and its role in AML is yet to be elucidated. In this study, we found that IL-35 was expressed highly in peripheral blood plasma of adult patients with AML and significantly correlated with the clinical stages of malignancy. Tregs-derived from adult AML patients produced IL-35 in a stimulation-dependent manner. IL-35 promoted AML blasts immune escape by expanding Tregs and inhibiting CD4+CD25-effector T cells (Teffs). Furthermore, IL-35 directly promoted the proliferation of AML blasts and reduced the apoptosis of AML blasts. Together, our study demonstrates that IL-35-derived from Tregs promotes the growth of adult AML blasts, suggesting that IL-35 has an important role in the pathogenesis of AML.

Myeloid-derived suppressor cells (MDSCs) are heterogeneous, immature, myeloid progenitor cells, which suppress immune responses against tumors. CD14(+)HLA-DR(-/low) monocytic MDSCs (M-MDSC) are increased in patients suffering from multiple myeloma (MM). However, the frequency and function of M-MDSCs with the relationship between the tumor development and outcome of therapy in MM remain unclear. In this study, we analyzed the changes in M-MDSCs in newly diagnosed, relapsed and remission MM patients. In addition, we also assessed the response of M-MDSCs in MM patients treated with a bortezomib-based therapy as well as the impact of bortezomib on the modulation of M-MDSCs in vitro. The levels of M-MDSCs in newly diagnosed and relapsed MM patients were significantly increased compared with those in remission MM patients and healthy donors. Moreover, the levels of M-MDSCs were shown to correlate with tumor progression. The decrease in M-MDSCs after proteasome inhibitory therapy suggested that M-MDSCs could be considered as an indicator for the efficacy of therapy. Finally, we found the plasma from newly diagnosed MM patients, and MM cells were able to induce the accumulation of M-MDSCs in vitro. These results indicated that M-MDSCs could be considered as a prognostic predictor and an important cell type contributing to immune suppressive microenvironment in MM patients. Treatments targeting for M-MDSCs may improve therapeutic outcomes for MM patients.

Regulatory T cells (Tregs) play an essential role in sustaining self-tolerance and immune homeostasis. Despite many studies on the correlation between Tregs accumulation and age, or malignancies, the related mechanism hasn't been well explored. To find out the mechanism of Tregs accumulation in aged urinary bladder cancer, we examined the novel cellular senesence gene SENEX and relevant apoptosis gene mRNA expression in sorted CD4+CD25(hi) Tregs from aged UBC donors, evaluated serum cytokine profiles related to tumor immunopathology, and further explored the relationship between SENEX expression, apoptosis gene expression and cytokine secretion. After having silenced down SENEX gene expression with RNA interference, we also evaluated the cellular apoptosis of Tregs sorted from aged UBC patients in response to H₂O₂-mediated stress. Our data indicated that upregulated SENEX mRNA expression in Tregs of aged UBC patients was correlated with pro-apoptotic gene expression and cytokine concentration. Silencing SENEX gene expression increased cellular apoptosis and pro-apoptotic gene expression of Tregs, in response to H₂O₂-mediated stress. Upregulated SENEX mRNA expression together with decreased pro-apoptotic gene expression and disturbances in cytokines synthesis may contribute to the Tregs proliferation and promote tumorigenesis and metastasis. Overall, upregulation of cellular senescence gene SENEX, was associated to regulatory T cells accumulation in aged urinary bladder cancer. Our study provides a new insight into understanding of peripheral Tregs accumulation in aged malignancies.