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Background: Cumulative data have shown that microRNAs (miRNA) are involved in the etiology and prognosis of colorectal cancer (CRC). Genetic polymorphisms in pre-miRNA genes may influence the biogenesis and functions of their host miRNAs. However, whether these polymorphisms are associated with CRC prognosis remains unknown.Methods: We analyzed the effects of seven single-nucleotide polymorphisms (SNP) in pre-miRNA genes on the prognosis of a Chinese population with 408 CRC patients with surgically-resected adenocarcinoma.Results: Two SNPs were identified to be significantly associated with recurrence-free survival and overall survival of the patients. The most significant SNP was rs6505162 in pre-miR-423. Compared with the homozygous wild-type genotype, the variant-containing genotypes of this SNP were significantly associated with both the overall survival (HR = 2.12, 95% CI = 1.34–3.34, P = 0.001) and the recurrence-free survival (HR = 1.59, 95% CI = 1.08–2.36, P = 0.019). Another SNP, rs4919510 in pre-miR-608, was also associated with altered recurrence-free survival (HR = 0.61, 95% CI = 0.41–0.92, P = 0.017). These effects were evident only in patients receiving chemotherapy but not in those without chemotherapy. In addition, the combined analysis of the two SNPs conferred a 2.84-fold (95% CI = 1.50–5.37, P = 0.001) increased risk of recurrence and/or death. Similarly, this effect was only prominent in those receiving chemotherapy (P < 0.001) but not in those without chemotherapy (P = 0.999).Conclusions: Our data suggest that genetic polymorphisms in pre-miRNA genes may impact CRC prognosis especially in patients receiving chemotherapy, a finding that warrants further independent validation.Impact: This is one of the first studies showing a prognostic role of pre-miRNA gene SNPs in CRC. Cancer Epidemiol Biomarkers Prev; 21(1); 217–27. ©2011 AACR.

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Circulating cell‐free DNA (cfDNA) fragmentomics, which encompasses the measurement of cfDNA length and short nucleotide motifs at the ends of cfDNA molecules, is an emerging field for cancer diagnosis. The utilization of cfDNA fragmentomics for the diagnosis of patients with hepatocellular carcinoma (HCC) caused by hepatitis B virus (HBV) is currently limited. In this study, we utilized whole‐genome sequencing data of cfDNA in samples from patients with HCC (n = 197) and HBV (n = 187) to analyze the association of fragment size selection (, Circulating cell‐free DNA (cfDNA) fragmentomics, encompassing the measurement of cfDNA length and short nucleotide motifs at cfDNA ends, is an emerging field in cancer diagnostics. In this study, we utilized whole‐genome sequencing data of cfDNA in patients with hepatocellular carcinoma (HCC). We observed that representative abnormal copy number variation (CNV) alterations and shorter fragment size (

Background Mitochondrial dynamics homeostasis is important for cell metabolism, growth, proliferation, and immune responses. The critical GTPase for mitochondrial fission, Drp1 is frequently upregulated in many cancers and is closely implicated in tumorigenesis. However, the underling mechanism by Drp1 influence tumor progression is largely unknown, especially in esophageal squamous cell carcinoma (ESCC). Methods Immunohistochemistry was used to examine Drp1 and LC3B expression in tissues of ESCC patients. Autophagic vesicles were investigated by transmission electron microscopy. Fluorescent LC3B puncta and mitochondrial nucleoid were observed by microscope and confocal microscopy. Mitochondrial function was evaluated by mitochondrial membrane potential, ROS and ATP measurement. Xenograft nude mice model was performed in BALB/c nude mice to confirm the role of Drp1 on ESCC progression. Results We found that Drp1 high expression is correlated with poor overall survival of ESCC patients. Drp1 overexpression promotes cell proliferation and xenograft ESCC tumor growth by triggering autophagy. Furthermore, we demonstrated that Drp1 overexpression disturbs mitochondrial function and subsequent induces mitochondrial DNA (mtDNA) released into the cytosol, which is recognized as cytosolic mtDNA stress. Mechanistically, cytosolic mtDNA stress activates the cGAS-STING pathway and facilitates autophagy, which promotes ESCC cancer growth. Moreover, mtDNA digestion with DNase I and autophagy inhibition with chloroquine attenuates the cGAS-STING pathway activation and ESCC cancer growth. Conclusions Our finding reveals that Drp1 overexpression induces mitochondrial dysfunction and cytosolic mitochondrial DNA stress, which subsequently activates the cGAS-STING pathway, triggers autophagy and promotes ESCC progression.

Background Mitochondrial dynamics homeostasis is important for cell metabolism, growth, proliferation, and immune responses. The critical GTPase for mitochondrial fission, Drp1 is frequently upregulated in many cancers and is closely implicated in tumorigenesis. However, the mechanism underling Drp1 to influence tumor progression is largely unknown, especially in esophageal squamous cell carcinoma (ESCC). Methods Immunohistochemistry was used to examine Drp1 and LC3B expression in tissues of ESCC patients. Autophagic vesicles were investigated by transmission electron microscopy. Fluorescent LC3B puncta and mitochondrial nucleoid were observed by fluorescent and confocal microscopy. Mitochondrial function was evaluated by mitochondrial membrane potential, ROS and ATP levels. Xenograft tumor model was performed in BALB/c nude mice to analyze the role of Drp1 on ESCC progression. Results We found that Drp1 high expression is correlated with poor overall survival of ESCC patients. Drp1 overexpression promotes cell proliferation and xenograft ESCC tumor growth by triggering autophagy. Furthermore, we demonstrated that Drp1 overexpression disturbs mitochondrial function and subsequent induces mitochondrial DNA (mtDNA) released into the cytosol thereby inducing cytosolic mtDNA stress. Mechanistically, cytosolic mtDNA activates the cGAS-STING pathway and facilitates autophagy, which promotes ESCC cancer growth. Moreover, mtDNA digestion with DNase I and autophagy inhibition with chloroquine attenuates the cGAS-STING pathway activation and ESCC cancer growth. Conclusions Our finding reveals that Drp1 overexpression induces mitochondrial dysfunction and cytosolic mtDNA stress, which subsequently activates the cGAS-STING pathway, triggers autophagy and promotes ESCC progression.

H1 histamine receptor (H1HR) belongs to the family of rhodopsin-like G-protein-coupled receptors. Recent studies have shown that H1HR expression is increased in several types of cancer. However, its functional roles in tumor progression remain largely unknown, especially in hepatocellular carcinoma (HCC). We found that H1HR is frequently unregulated in HCC, which is significantly associated with both recurrence-free survival and overall survival in HCC patients. Functional experiments revealed that H1HR promoted both the growth and metastasis of HCC cells by inducing cell cycle progression, formation of lamellipodia, production of matrix metalloproteinase 2, and suppression of cell apoptosis. Activation of cyclic adenosine monophosphate-dependent protein kinase A was found to be involved in H1HR-mediated HCC cell growth and metastasis. In addition, we found that overexpression of H1HR was mainly due to the downregulation of miR-940 in HCC cells. Moreover, the H1HR inhibitor terfenadine significantly suppressed tumor growth and metastasis in an HCC xenograft nude mice model. Our findings demonstrate that H1HR plays a critical role in the growth and metastasis of HCC cells, which provides experimental evidence supporting H1HR as a potential drug target for the treatment of HCC.

Tumor-associated macrophages (TAMs) contribute to hepatocellular carcinoma (HCC) progression. However, the molecular mechanism underlying the infiltration of TAMs into HCC microenvironment is largely unclear. Recent studies have reported that alteration of mitochondrial nucleoid structures induces mitochondrial DNA (mtDNA) release into the cytosol, which is recognized as mtDNA stress, and consequently regulates innate immunity. Here we aimed to investigate whether mitochondrial fission induces mtDNA stress and then promotes TAM infiltration and HCC progression. Confocal microscopy and real-time PCR were used to detect cytosolic mtDNA content in HCC cells. The relationship between the expression of mitochondrial fission key regulator dynamin-related protein 1 (Drp1) and the percentage of CD163 (a marker of TAMs)-positive cells was investigated in HCC tissues using immunohistochemistry. Finally, the effect of Drp1 overexpression in HCC cells on recruitment and polarization of TAMs was investigated. Our data showed that increased Drp1 expression was positively correlated with the infiltration of TAMs into HCC tissues. Drp1-mediated mitochondrial fission induced the cytosolic mtDNA stress to enhance the CCL2 secretion from HCC cells by TLR9-mediated NF-κB signaling pathway, and thus promoted the TAM recruitment and polarization. Depleting cytosolic mtDNA using DNase I or blocking TLR9 pathway by TLR9 antagonist, siRNA for TLR9 or p65 in HCC cells with Drp1 overexpression significantly decreased the recruitment and polarization of TAMs. Blocking CCR2 by antagonist significantly reduced TAM infiltration and suppressed HCC progression in mouse model. In conclusion, our findings reveal a novel mechanism of TAM infiltration in HCC by mitochondrial fission-induced mtDNA stress.

BACKGROUND: Both circulating tumor cell (CTC) and total circulating cell-free DNA (ccfDNA) predict cancer patient prognosis. However, no study has explored the prognostic value of the combined use of CTC and ccfDNA. We aimed to investigate individual and joint effects of CTC and ccfDNA on clinical outcomes of metastatic breast cancer (MBC) patients. METHODS: We collected 227 blood samples from 117 MBC patients. CTCs were enumerated using the CellSearch System. ccfDNAs were quantified by quantitative real-time PCR and Qubit fluorometer. The individual and joint effects of CTC and ccfDNA levels on patient progression-free survival (PFS) and overall survival (OS) were analyzed using Cox proportional hazards models. RESULTS: Compared to patients with < 5 CTCs, patients with ≥ 5 CTCs had a 2.58-fold increased risk of progression and 3.63-fold increased risk of death. High level of ccfDNA was associated with a 2.05-fold increased risk of progression and 3.56-fold increased risk of death. These associations remained significant after adjusting for other important clinical covariates and CTC/ccfDNA levels. CTC and ccfDNA levels had a joint effect on patient outcomes. Compared to patients with low levels of both CTC and ccfDNA, those with high levels of both markers exhibited a > 17-fold increased death risk (P < 0.001). Moreover, longitudinal analysis of 132 samples from 22 patients suggested that the inconsistency between CTC level and outcome in some patients could possibly be explained by ccfDNA level. CONCLUSIONS: CTC and total ccfDNA levels were individually and jointly associated with PFS and OS in MBC patients.

Objectives: Hepatocellular carcinoma (HCC) accounts for 70%-85% of liver cancer, and about 85% of HCC are hepatitis B virus-related hepatocellular carcinoma (HBV-HCC) in china. Most patients are already in the middle or late stages of the disease at the time of diagnosis, trans-hepatic arterial chemoembolization (TACE) combine with traditional Chinese medicine (TCM) have been reported as an effective treatment, and effective prognostic molecular markers are helpful to predict therapeutic efficacy. In this study, we aim to explore whether circulating cell-free mitochondrial DNA (ccf-mtDNA) content is associated with the outcome for HCC patients receiving TACE combine with TCM treatment. Method: Retrospective analysis was conducted in a cohort with 141 HBV-HCC patients. Univariate and multivariate analysis was conducted with Cox proportional risk regression model to explore the correlation between ccf-mtDNA content and patient prognosis. Kaplan-Meier method was used to draw the survival curve of ccf-mtDNA content and the survival prognosis of patients. Results: (1) high content of serum ccf-mtDNA is an independent risk factor for the prognosis of HBV-HCC patients treated with TACE combined with TCM adjuvant therapy (HR=4.010, 95%IC=1.252-12.844, P = 0.019). (2) K-M survival analysis showed that patients with high ccf-mtDNA content had poor prognosis (Log Rank P=0.027). Conclusions: Our findings suggest that ccf-mtDNA is a potential novel non-invasive biomarker for prognosis of HCC patients receiving TACE combine with TCM treatment.

Background DNAs released from tumor cells into blood (circulating tumor DNAs, ctDNAs) carry tumor-specific genomic aberrations, providing a non-invasive means for cancer detection. In this study, we aimed to leverage somatic copy number aberration (SCNA) in ctDNA to develop assays to detect early-stage HCCs. Methods We conducted low-depth whole-genome sequencing (WGS) to profile SCNAs in 384 plasma samples of hepatitis B virus (HBV)-related HCC and cancer-free HBV patients, using one discovery and two validation cohorts. To fully capture the robust signals of WGS data from the complete genome, we developed a machine learning-based statistical model that is focused on detection accuracy in early-stage HCC. Findings We built the model using a discovery cohort of 209 patients, achieving an overall area under curve (AUC) of 0.893, with 0.874 for early-stage (Barcelona clinical liver cancer [BCLC] stage 0-A) and 0.933 for advanced-stage (BCLC stage B-D). The performance of the model was then assessed in two validation cohorts (76 and 99 patients) that only consisted of patients with stage 0-A HCC. Our model exhibited a robust predictive performance, with an AUC of 0.920 and 0.812 for the two validation cohorts. Further analyses showed the impact of tumor sample heterogeneity in model training on detecting early-stage tumors, and a refined model addressing the heterogeneity in the discovery cohort significantly increased model performance in validation. Interpretation We developed an SCNA-based, machine learning-driven model in the non-invasive detection of early-stage HCC in HBV patients and demonstrated its performance through strict independent validations.

Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease, resulting in important economic losses in pig farming. Prompt detection of PRRS virus (PRRSV) in the field samples is important for effective PRRS control. Droplet digital PCR (ddPCR) is a novel PCR technology, which offers good precision and direct quantification without using calibration curves. In this study, we established a ddPCR system for the sensitive and accurate quantification of PRRSV. Specificity of the assay was determined by the failure of amplification of other relevant viruses. Quantitative linearity, sensitivity and accuracy of ddPCR were compared to those of real time PCR for PRRSV testing. Both methods showed a high degree of linearity (R2=∼1) and quantitative correlation, although ddPCR showed somewhat higher sensitivity than real time PCR. Collectively, our findings indicate that ddPCR might offer improved analytical sensitivity and specificity for PRRSV measurements.

Adjuvant chemotherapy treatment for different endometrial cancer stages is still debated. We aimed to evaluate the outcome of early (FIGO I–II) vs late stage (FIGO III–IV) endometrial cancer in an institutional experience using chemotherapy only after surgery. Charts of patients with endometrial carcinoma who underwent surgery with postoperative chemotherapy between February 2012 and December 2017 were retrospectively identified, and the recurrence as well as prognosis were assessed. Of the 272 eligible endometrioid adenocarcinoma (EA) patients, 127 had received chemotherapy, 145 did not receive chemotherapy; 37 were in late stage (FIGO III–IV) and 235 were in early stage (FIGO I–II). In the late stage group, patients with no chemotherapy had worse overall survival (OS) and recurrence-free survival (RFS) as compared to the patients taking chemotherapy (OS, 28.6% vs 76.4%, P = 0.059; RFS, 17.1% vs 66.4%, P = 0.053). However, in the early stage group, there was no significant difference between the OS and RFS between the patients that were receiving and not receiving chemotherapy (OS, 84.1% vs 93.3%, P = 0.789; RFS, 76.7% vs 72.4%, P = 0.924). Independent predictive factors of recurrence were age over 53 years, histological grade G3, as well as late stages (FIGO III–IV), while independent predictive factors of OS were age over 53 years, deeper depth of myometrial invasion, and late stages (FIGO III–IV). In late stages, patients with chemotherapy had lower recurrence rate and favorable OS as compared to patients not taking chemotherapy, which was the benefit of postoperative adjuvant chemotherapy, and chemotherapy might be strongly considered in late stage EA.

The lack of accurate biomarkers makes it difficult to determine whether organs are suitable for transplantation. Mitochondrial DNA (mtDNA) correlates with tissue damage and kidney disease, making it a potential biomarker in organ evaluation.Donors who had experienced cardiac death and successfully donated their kidneys between January 2015 and May 2017 were included this study. We detected the level of mtDNA in the plasma of the donor using quantitative real-time polymerase chain reaction and then statistically analyzed the relationship between the level of mtDNA and the delayed graft function (DGF) of the recipient.The incidence of DGF or slowed graft function (SGF) increased by 4 times (68% versus 16%, P0.001) when the donor mtDNA (dmtDNA) level was0.114. When dmtDNA levels were0.243, DGF and primary nonfunction were approximately 100% and 44%, respectively. Moreover, dmtDNA was an independent risk factor for slowed graft function and DGF. A prediction model for DGF based on dmtDNA achieved an area under the receiver operating characteristic curve for a prediction score as high as 0.930 (95% confidence interval 0.856-1.000), and the validation cohort results showed that the sensitivity and specificity of the model were 100% and 78%, respectively. dmtDNA levels were correlated with 6-month allograft function (R=0.332, P0.001) and 1-year graft survival (79% versus 99%, P0.001).We conclusively demonstrated that plasma dmtDNA was an independent risk factor for DGF, which is valuable in organ evaluation. dmtDNA is a possible first predictive marker for primary nonfunction and worth further evaluation.

Exosomes are now accepted as potential biomarkers in cardiovascular disease development, especially in atherosclerosis. Atherosclerosis is a leading cause of cardiovascular disease-related death and morbidity, accounting for one-fifth of all deaths globally. Therefore, the biomarkers for the management of atherosclerosis is urgently needed. Exosomes are reported to play key roles cell-to-cell communication in atherosclerosis with lipid bilayer membranous vesicles containing nucleic acids, proteins, and lipid contents, which are released from all most of multiple kinds of living cells. This review aims to discuss the potential roles of exosome-derived miRNA, protein, and DNA as biomarkers in atherosclerosis pathogenesis, diagnosis, and therapy.

Aberrant expression of genes in de novo lipogenesis (DNL) pathway were associated with various cancers, including hepatocellular carcinoma (HCC). Single nucleotide polymorphisms (SNPs) of DNL genes have been reported to be associated with prognosis of some malignancies. However, the effects of SNPs in DNL genes on overall survival of HCC patients receiving transarterial chemoembolization (TACE) treatment are still unknown. In present study, nine SNPs in three genes (ACLY, ACACA and FASN) in DNL pathway were genotyped using the Sequenom iPLEX genotyping system in a hospital-based cohort with 419 HCC patients treated with TACE, and their associations with HCC overall survival were evaluated by Cox proportional hazard regression analysis under three genetic models (additive, dominant and recessive). Although we did not find any significant results in total analysis (all p>0.05), our stratified data showed that SNP rs9912300 in ACLY gene was significantly associated with overall survival of HCC patients with lower AFP level and SNP rs11871275 in ACACA gene was significantly associated with overall survival of HCC patients with higher AFP level. We further identified the significant interactions between AFP level and SNP rs9912300 or rs11871275 in the joint analysis. Conclusively, our data suggest that genetic variations in genes of DNL pathway may be a potential biomarker for predicting clinical outcome of HCC patients treated with TACE.

Accumulating evidence has indicated that variations of mitochondrial DNA (mtDNA) content may affect the susceptibility to hepatocellular carcinoma (HCC). However, no study has been conducted to evaluate the association of circulating mtDNA content and the risk of liver cirrhosis, a leading cause of HCC. We conducted a nested case–control study including 136 cirrhotic hepatitis B virus (HBV) cases and 136 frequency-matched non-cirrhotic HBV controls. We determined mtDNA content in serum DNA using quantitative real-time PCR and analyzed its association with cirrhosis risk. We found that cirrhotic HBV patients had significantly lower levels of mtDNA content than non-cirrhotic HBV controls (P = 0.0184). Compared to patients with high mtDNA content, those with low mtDNA content had a 2.25-fold increased risk of cirrhosis [odds ratio (OR) 2.25, 95 % confidence interval (CI) 1.26–4.02]. This association exhibited a significant dose relationship as evidenced in both tertile and quartile analyses (P for trend = 0.0018 and 0.0008, respectively). Stratified analyses showed that the association was prominent in younger patients (P = 0.0122), males (P = 0.0069), never smokers (P = 0.0063), never drinkers (P = 0.0078), patients with a family history of HBV infection (P = 0.0062), and patients with low values of aspartate aminotransferase to platelet ratio index (APRI), a commonly used noninvasive marker for cirrhosis (P = 0.0109). Moreover, a joint effect was observed between low mtDNA content and high APRI values on cirrhosis risk (OR 24.07, 95 % CI 6.72–86.24). Low circulating mtDNA content may confer an increased cirrhosis risk in HBV patients. Further prospective studies are warranted to validate these findings and explore the clinical significance.

Background and Aim APRI (aspartate aminotransferase [AST] to platelet ratio index) is widely used to assess fibrosis and cirrhosis risk, especially in hepatitis C virus (HCV)-infected patients. Few studies have evaluated APRI and hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) risk. Prospective evidence is needed to assess whether APRI predicts HCC risk in HBV patients. Method In a prospectively enrolled clinical cohort of 855 HBV patients with a 1-year exclusion window (followed for > 1 year and did not develop HCC within 1 year), the predictive value of APRI in HCC risk was evaluated by Cox proportional hazards model using univariate and multivariate analyses and longitudinal analysis. Results Higher APRI prospectively conferred a significantly increased risk of HCC in univariate analysis (quartile analysis, P trend = 2.9 × 10−7). This effect remained highly significant after adjusting for common host characteristics but not cirrhosis (P trend = 7.1 × 10−5), and attenuated when cirrhosis is adjusted (P trend = 0.021). The effect remained prominent when the analysis was restricted to patients with a more stringent 2-year exclusion window (P trend = 0.008 in quartile analysis adjusting all characteristics including cirrhosis), indicating that the association was unlikely due to including undetected HCC patients in the cohort, thus minimizing the reverse-causation limitation in most retrospective studies. Longitudinal comparison demonstrated a persistently higher APRI value in HBV patients who developed HCC during follow-up than those remaining cancer free. Conclusion APRI might be a marker of HCC risk in HBV patients in cirrhosis-dependent and -independent manners. Further studies are warranted to validate this finding and test its clinical applicability in HCC prevention.

The functional abnormality of circadian regulation genes is involved in the development and progression of hepatocellular carcinoma (HCC). However, the association between functional single nucleotide polymorphisms (SNPs) in circadian gene NPAS2 and the overall survival of HCC patients treated with transcatheter arterial chemoembolization (TACE) has never been investigated. Six functional SNPs in the NPAS2 gene were genotyped using the Sequenom iPLEX genotyping system in a cohort of 448 unresectable Chinese patients with HCC treated with TACE. Multivariate Cox proportional hazards model and Kaplan–Meier curves were used for the prognosis analysis. We found that two SNPs, rs1053096 and rs2305160, in the NPAS2 gene showed significant associations with overall death risk in HCC patients in the recessive model (hazard ratio [HR] = 1.48; 95% confidence interval [CI], 1.13–1.94; P = 0.004) and in the dominant model (HR = 1.63; 95% CI, 1.29–2.07; P < 0.001), respectively. Moreover, we observed a cumulative effect of these two SNPs on HCC overall survival, indicating a significant trend of increasing death risk with increasing number of unfavorable genotypes (P for trend < 0.001). Compared with the patients without any unfavorable genotypes, the HRs for patients with one and two unfavorable genotypes were 1.41 (95% CI, 1.10–1.82; P = 0.007) and 2.09 (95% CI, 1.46–2.97, P < 0.001), respectively. The haplotype and diplotype analyses further characterized the association between NPAS2 genotype and survival of HCC patients. Our results for the first time suggest that NPAS2 gene polymorphisms may serve as an independent prognostic marker for HCC patients treated with TACE.

Background Numerous studies indicate that the leukocyte telomere length is associated with the risk of cancers, including colorectal cancer (CRC). However, the prognostic value of leukocyte telomere length in CRC patients has not been investigated. Patients and methods Relative telomere length (RTL) of peripheral blood leukocytes (PBLs) from 571 CRC patients receiving surgical resection was measured using a polymerase chain reaction-based method. The Cox proportional hazards ratio model and the Kaplan–Meier curve were used to estimate the association between RTL and the clinical outcome of CRC patients in the training set (90 patients) and the testing set (86 patients). Finally, an independent cohort of 395 patients was used as an external validation set. The immunophenotype of PBLs and the plasma concentration of several immune-related cytokines were determined by flow cytometry and enzyme-linked immunosorbent assay, respectively. Results Patients with shorter RTL had significantly poorer overall survival and relapse-free survival than those with longer RTL in the training, testing and validation sets. Furthermore, leukocyte RTL and Tumor-Node-Metastasis (TNM) stage exhibited a significant joint effect in the prognosis prediction of combined CRC patients, indicating that patients with both short RTL and advanced stages had the worst prognosis, when compared with other subgroups. In addition, patients with short RTL showed the higher percentage of CD4+ T cell and the lower percentage of B cell in peripheral blood mononuclear cells, as well as the lower concentration of plasma transforming growth factor-β1, suggesting a possibility that the immune functions changed with RTL alteration. Conclusions Our study for the first time demonstrates that leukocyte RTL is an independent prognostic marker complementing TNM stage and associated with the immune functions in CRC patients.

BACKGROUND Compelling epidemiological evidence indicates that alterations of telomere length are associated with risks of many malignancies in a tumor-specific manner, such as lung cancer, breast cancer, and non-Hodgkin's lymphoma. However, the association between leukocyte telomere length and glioma risk has not been investigated. METHODS Relative telomere length (RTL) of peripheral blood leukocytes from 467 glioma patients and 467 healthy controls, matched by age and sex, was measured using the real-time PCR-based method in a case-control study. An unconditional multivariate logistic regression model was applied to estimate the association between RTL and glioma risk. RESULTS Glioma patients showed notably longer RTL than controls (median, 0.555 vs 0.444; P > .04). RTL was negatively correlated with age in both cases (ρ = -0.430; P < .001) and controls (ρ = -0.388; P < .001). After adjusting for age, sex, smoking status and family history of cancer, multivariate logistic regression analysis showed that there was a U-shaped association between RTL and glioma risk (P for nonlinearity

The epithelial cell adhesion molecule (EpCAM) is overexpressed in a wide variety of human cancers and is associated with patient prognosis, including those with lung cancer. However, the association of single nucleotide polymorphisms (SNPs) in the EpCAM gene with the prognosis for non-small-cell lung cancer (NSCLC) patients has never been investigated. We evaluated the association between two SNPs, rs1126497 and rs1421, in the EpCAM gene and clinical outcomes in a Chinese cohort of 506 NSCLC patients. The SNPs were genotyped using the Sequenom iPLEX genotyping system. Multivariate Cox proportional hazards model and Kaplan–Meier curves were used to assess the association of EpCAM gene genotypes with the prognosis of NSCLC. We found that the non-synonymous SNP rs1126497 was significantly associated with survival. Compared with the CC genotype, the CT+TT genotype was a risk factor for both death (hazard ratio, 1.40; 95% confidence interval [CI], 1.02–1.94; P = 0.040) and recurrence (hazard ratio, 1.34; 95% CI, 1.02–1.77; P = 0.039). However, the SNP rs1421 did not show any significant effect on patient prognosis. Instead, the AG+GG genotype in rs1421 was significantly associated with early T stages (T1/T2) when compared with the AA genotype (odds ratio for late stage = 0.65; 95% CI, 0.44–0.96, P = 0.029). Further stratified analysis showed notable modulating effects of clinical characteristics on the associations between variant genotypes of rs1126497 and NSCLC outcomes. In conclusion, our study indicated that the non-synonymous SNP rs1126497 may be a potential prognostic marker for NSCLC patients.

Prospective and longitudinal epidemiological evidence is needed to assess the association between telomere length and risk of hepatocellular carcinoma (HCC). In 323 cancer-free Korean-American HBV patients with 1-year exclusion window (followed for >1 year and did not develop HCC within 1 year), we measured the relative telomere length (RTL) in baseline serum DNAs and conducted extensive prospective and longitudinal analyses to assess RTL-HCC relationship. We found that long baseline RTL conferred an increased HCC risk compared to short RTL [hazard ratio (HR) = 4.93, P = 0.0005). The association remained prominent when the analysis was restricted to patients with a more stringent 5-year exclusion window (HR = 7.51, P = 0.012), indicating that the association was unlikely due to including undetected HCC patients in the cohort, thus minimizing the reverse-causation limitation in most retrospective studies. Adding baseline RTL to demographic variables increased the discrimination accuracy of the time-dependent receiver operating characteristic analysis from 0.769 to 0.868 (P = 1.0 × 10-5). In a nested longitudinal subcohort of 16 matched cases-control pairs, using a mixed effects model, we observed a trend of increased RTL in cases and decreased RTL in controls along 5 years of follow-up, with a significant interaction of case/control status with time (P for interaction=0.002) and confirmed the association between long RTL and HCC risk [odds ratio [OR] = 3.63, P = 0.016]. In summary, serum DNA RTL may be a novel non-invasive prospective marker of HBV-related HCC. Independent studies are necessary to validate and generalize this finding in diverse populations and assess the clinical applicability of RTL in HCC prediction.

Increasing evidence suggests that alterations in mitochondrial DNA (mtDNA) copy number (mtDNAcn) and relative telomere length (RTL) may be implicated in the tumorigenesis of several malignancies. Alterations of both RTL and mtDNAcn are generally accepted as independent biomarkers for predicting risk and prognosis in various cancers. The aim of this study was to evaluate the prognostic value of combining leukocyte RTL with mtDNAcn (RTL-mtDNAcn) in hepatocellular carcinoma (HCC).RTL and mtDNAcn in peripheral blood leukocytes (PBLs) were measured using a real-time PCR-based method in a total of 250 HCC patients treated with transcatheter arterial chemoembolization (TACE). We evaluated the associations between RTL and/or mtDNAcn and HCC overall survival using Kaplan-Meier curve analysis and Cox proportional hazards regression model.We found that patients with longer leukocyte RTL or lower mtDNAcn had shorter overall survival time. The univariate analysis (HR 1.63, 95 % CI 1.23-2.17, P = 7.7 × 10(-4)) and multivariate analysis (HR 1.78, 95 % CI 1.31-2.42, P = 2.4 × 10(-4)) indicated that longer leukocyte RTL was significantly associated with poorer OS in HCC patients. Kaplan-Meier curve analysis showed that patients with longer RTL had shorter overall survival time than those with shorter RTL (log-rank P = 0.001). Patients with lower mtDNA copy number was significantly associated with poorer OS by Cox proportional hazards model using both univariate (HR 1.60, 95 % CI 1.21-2.13, P = 0.001) and multivariate analyses (HR 1.77, 95 % CI 1.30-2.41, P = 2.8 × 10(-4)). Kaplan-Meier curve analysis showed that patients with lower mtDNA content had significantly shorter overall survival time than those with higher mtDNA content (log-rank P = 0.001). Furthermore, combination of leukocyte RTL and mtDNAcn significantly improved the efficacy of predicting HCC prognosis. Patients with longer RTL and lower mtDNAcn exhibited a significantly poorer overall survival in both the univariate analysis (HR 2.21, 95 % CI 1.52-3.22, P = 3.5 × 10(-5)) and multivariate analysis (HR 2.60, 95 % CI 1.73-3.90, P = 4.3 × 10(-6)). The effect on patient prognosis was more evident in patients with longer RTL and lower mtDNAcn than in those with shorter RTL and lower mtDNA (HR 2.11, 95 % CI 1.34-3.32, P = 0.001) or in those with longer RTL and higher mtDNA (HR 2.10, 95 % CI 1.34-3.27, P = 0.001).Our data suggest that combination of leukocyte RTL-mtDNAcn may be a potential efficient prognostic marker for HCC patients receiving the TACE treatment.

// Chun Wang 1, 2, * , Hie-Won Hann 3, * , Zhong Ye 1, * , Richard S. Hann 3 , Shaogui Wan 1, 4 , Xishan Ye 1 , Peter D. Block 1 , Bingshan Li 5 , Ronald E. Myers 1 , Xiaowei Wang 6 , Hee-Soon Juon 1 , Jesse Civan 3 , Mimi Chang 7 , Ho S. Bae 7 , Jinliang Xing 8 , Hushan Yang 1 1 Division of Population Science, Department of Medical Oncology, Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, USA 2 Department of Environmental Health, School of Public Health, Nantong University, Nantong, Jiangsu 226000, China 3 Liver Disease Prevention Center, Division of Gastroenterology and Hepatology, Department of Medicine, Thomas Jefferson University, Philadelphia, PA 19107, USA 4 Institute of Pharmacy, Pharmaceutical College, Henan University, Kaifeng, Henan 475004, China 5 Center for Human Genetics Research, Department of Molecular Physiology & Biophysics, Vanderbilt University, Nashville, TN 37232, USA 6 Department of Radiation Oncology, Washington University School of Medicine, Saint Louis, MO 63108, USA 7 Asian Pacific Liver Center, Saint Vincent Medical Center, Los Angeles, CA 90057, USA 8 Experimental Teaching Center, School of Basic Medicine, Fourth Military Medical University, Xi’an 710032, China * These authors have contributed equally to this work Correspondence to: Hushan Yang, e-mail: hushan.yang@jefferson.edu Keywords: hepatocellular carcinoma, risk, microRNA, serum Received: December 15, 2015 Accepted: May 4, 2016 Published: May 24, 2016 ABSTRACT The predictive value of circulating microRNAs (miRNAs) in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) has been demonstrated in retrospective studies, but it has rarely been tested in prospective studies. In a cohort of 373 cancer-free HBV patients with a median follow-up of 4.5 years, we measured the expression of 24 retrospectively identified HCC-related miRNAs in baseline serum samples. When we analyzed the prospective associations of miRNA expression with HCC risk using the Cox proportional hazards model, we found that 15 of the 24 miRNAs exhibited a significant association with HCC risk. In particular, 7 miRNAs (miR-122, miR-99a, miR-331, miR-125b, miR-23b, miR-92a, and miR-26a) were associated with an increased risk, and 8 miRNAs (miR-652, miR-23a, miR-27a, miR-34a, miR-145, miR-10a, miR-150, and let-7f) were associated with a decreased risk. Compared to HBV patients with a low miRNA-based risk score, those with a high miRNA-based risk score exhibited a significantly elevated HCC risk in both univariate (hazard ratio [HR] 6.56, 95% confidence interval [CI] 2.74-15.70) and multivariate (HR 3.57, 95% CI 1.34-9.48) analyses. The risk score significantly increased the HCC prediction performance of alpha-fetoprotein (concordance index increased from 0.68 to 0.82, P < 0.0001). In silico analyses indicated that the genes targeted by the 15 miRNAs are mainly enriched in the transforming growth factor-beta signaling pathway. Collectively, these results provide prospective evidence that circulating miRNAs serve as non-invasive markers for risk prediction of HCC in HBV patients.

Recent studies have demonstrated a potential link between circulating cell-free mitochondrial DNA (mtDNA) content and cancers. However, there is no study evaluating the association between circulating mtDNA as a non-invasive marker of hepatocellular carcinoma (HCC) risk. We conducted a nested case-control study to determine circulating mtDNA content in serum samples from 116 HBV-related HCC cases and 232 frequency-matched cancer-free HBV controls, and evaluate the retrospective association between mtDNA content and HCC risk using logistic regression and their temporal relationship using a mixed effects model. HCC cases had significantly lower circulating mtDNA content than controls (1.06 versus 2.47, P = 1.7 × 10−5). Compared to HBV patients with higher mtDNA content, those with lower mtDNA content had a significantly increased risk of HCC with an odds ratio (OR) of 2.19 (95% confidence interval [CI] 1.28–3.72, P = 0.004). Quartile analyses revealed a significant dose-dependent effect (Ptrend = 0.001) for this association. In a pilot longitudinal sub-cohort of 14 matched cases-control pairs, we observed a trend of dramatically decreased mtDNA content in cases and slightly decreased mtDNA content in controls, with a significant interaction of case-control status with time (Pinteraction = 0.049). Our findings suggest that circulating mtDNA is a potential novel non-invasive biomarker of HCC risk in HBV patients.

Background and aims Telomere length has emerged as a promising risk predictor of various cancers including hepatocellular carcinoma (HCC). However, the majority of studies in this area measured telomere length in hepatocytes and one in lymphocytes with conflicting results. Moreover, no studies have been reported on using circulating DNA telomere length as a non-invasive HCC biomarker. Methods We conducted a nested case-control study to determine the relative telomere length (RTL) in serum DNA from 140 hepatitis B virus (HBV)-related HCC cases and 280 frequency-matched cancer-free HBV controls. Results Cases had a significantly longer RTL (median, 0.31; range, 0.02–2.31) than controls (median, 0.20; range, 0.01–1.60) (P = 0.003). Consistently, longer RTLs conferred a significantly increased HCC risk compared to short RTLs in a univariate logistic regression analysis (odds ratio [OR] = 1.55, 95% confidence interval [CI] = 1.02–2.33, P = 0.038). This association attenuated after multivariate adjustment (OR = 1.40, 95% CI = 0.90–2.19, P = 0.132). In a quartile analysis, a significant dose-response relationship was noted in univariate analysis (Ptrend = 0.017) which was again attenuated in multivariate analysis (Ptrend = 0.079). Further analyses revealed that the significant association between serum RTL and HCC risk was evident in non-cirrhotic (OR = 3.54, 95% CI 1.58–7.93 P = 0.002), but not cirrhotic (OR = 0.95, 95% CI 0.55–1.64, P = 0.860) HBV patients. Moreover, the significantly increased HCC risk conferred by cirrhosis was modulated by RTL with a significant interaction effect (Pinteraction = 0.013). Conclusions RTL in circulating cell-free serum DNA could potentially be used as a novel non-invasive biomarker for non-cirrhotic HCC. Prospective cohort studies are warranted to validate this finding and assess its clinical significance in HCC prevention.

Background & Aims Previous studies have indicated that telomere length is associated with altered risk of various tumours including hepatitis B virus (HBV)-related hepatocellular carcinoma. However, the association between telomere length and the risk of cirrhosis has not been reported. Methods In this nested case–control study, we used real-time quantitative PCR to determine the relative telomere length (RTL) in serum DNA samples from 100 HBV-related cirrhosis cases and 100 frequency-matched HBV controls, and evaluated the associations between RTL and cirrhosis risk by logistic regression analyses. Results We found that cirrhotic cases had a significantly longer RTL (median, 0.36; range, 0.08–1.87) than non-cirrhotic controls (median, 0.20; range, 0.05–1.11) (P

Background: Cumulative data have shown that microRNAs (miRNA) are involved in the etiology and prognosis of colorectal cancer (CRC). Genetic polymorphisms in pre-miRNA genes may influence the biogenesis and functions of their host miRNAs. However, whether these polymorphisms are associated with CRC prognosis remains unknown. Methods: We analyzed the effects of seven single-nucleotide polymorphisms (SNP) in pre-miRNA genes on the prognosis of a Chinese population with 408 CRC patients with surgically-resected adenocarcinoma. Results: Two SNPs were identified to be significantly associated with recurrence-free survival and overall survival of the patients. The most significant SNP was rs6505162 in pre-miR-423. Compared with the homozygous wild-type genotype, the variant-containing genotypes of this SNP were significantly associated with both the overall survival (HR = 2.12, 95% CI = 1.34–3.34, P = 0.001) and the recurrence-free survival (HR = 1.59, 95% CI = 1.08–2.36, P = 0.019). Another SNP, rs4919510 in pre-miR-608, was also associated with altered recurrence-free survival (HR = 0.61, 95% CI = 0.41–0.92, P = 0.017). These effects were evident only in patients receiving chemotherapy but not in those without chemotherapy. In addition, the combined analysis of the two SNPs conferred a 2.84-fold (95% CI = 1.50–5.37, P = 0.001) increased risk of recurrence and/or death. Similarly, this effect was only prominent in those receiving chemotherapy (P < 0.001) but not in those without chemotherapy (P = 0.999). Conclusions: Our data suggest that genetic polymorphisms in pre-miRNA genes may impact CRC prognosis especially in patients receiving chemotherapy, a finding that warrants further independent validation. Impact: This is one of the first studies showing a prognostic role of pre-miRNA gene SNPs in CRC. Cancer Epidemiol Biomarkers Prev; 21(1); 217–27. ©2011 AACR.

Zinc (Zn)-deficiency (ZD) is implicated in the pathogenesis of human oral-esophageal cancers. Previously, we showed that in ZD mice genetic deletion of cyclooxygenase-2 (Cox-2) enhances N-nitrosomethylbenzylamine-induced forestomach carcinogenesis. By contrast, Cox-2 deletion offers protection in Zn-sufficient (ZS) mice. We hypothesize that ZD activates pathways insensitive to COX-2 inhibition, thereby promoting carcinogenesis. This hypothesis is tested in a Cox-2(-/-) mouse tongue cancer model that mimics pharmacologic blockade of COX-2 by firstly examining transcriptome profiles of forestomach mucosa from Cox-2(-/-) and wild-type mice on a ZD vs. ZS diet, and secondly investigating the roles of identified markers in mouse forestomach/tongue preneoplasia and carcinomas. In Cox-2(-/-) mice exposed to the tongue carcinogen 4-nitroquinoline 1-oxide, dietary ZD elicited tongue/esophagus/forestomach carcinomas that were prevented by ZS. The precancerous ZD:Cox-2(-/-) vs. ZS:Cox-2(-/-) forestomach had an inflammatory signature with upregulation of the proinflammation genes S100a8 and S100a9. Bioinformatics analysis revealed overrepresentation of inflammation processes comprising S100a8/a9 and an nuclear factor (NF)-κB network with connectivity to S100A8. Immunohistochemistry revealed co-overexpression of S100A8, its heterodimeric partner S100A9, the receptor for advanced glycation end-products (RAGE), NF-κB p65, and cyclin D1, in ZD:Cox-2(-/-) forestomach/tongue preneoplasia and carcinomas, evidence for the activation of a RAGE-S100A8/A9 inflammatory pathway. Accumulation of p53 in these carcinomas indicated activation of additional inflammatory pathways. Zn-replenishment in ZD:Cox-2(-/-) mice reversed the inflammation and inhibited carcinogenesis. Thus, ZD activates alternative inflammation-associated cancer pathways that fuel tumor progression and bypass the antitumor effect of Cox-2 ablation. These findings have important clinical implications, as combination cancer therapy that includes Zn may improve efficacy.

Background & Aims Zinc deficiency is implicated in the pathogenesis of human esophageal cancer. In the rat esophagus, it induces cell proliferation, modulates genetic expression, and enhances carcinogenesis. Zinc-replenishment reverses proliferation and inhibits carcinogenesis. The zinc-deficient rat model allows the identification of biological differences affected by zinc during early esophageal carcinogenesis. Methods We evaluated gene expression profiles of esophageal epithelia from zinc-deficient and replenished rats vs zinc-sufficient rats using microarray analysis. We characterized the role of the top–up-regulated gene S100A8 in esophageal hyperplasia/reversal and in chemically induced esophageal carcinogenesis in zinc-modulated animals by immunohistochemistry and real-time quantitative polymerase chain reaction. Results The hyperplastic-deficient esophagus has a distinct expression signature with the proinflammation genes S100 calcium binding protein A8 (S100A8) and A9 (S100A9) up-regulated 57-fold and 5-fold, respectively. Zinc replenishment rapidly restored to control levels the expression of S100A8/A9 and 27 other genes and reversed the hyperplastic phenotype. With its receptor for advanced glycation end products (RAGE), colocalization and overexpression of S100A8 protein occurred in the deficient esophagus that overexpressed nuclear factor κΒ p65 and cyclooxygenase-2 (COX-2) protein. Zinc replenishment, but not a COX-2 inhibitor, reduced the overexpression of these 4 proteins. Additionally, esophageal S100A8/A9 messenger RNA levels were associated directly with the diverse tumorigenic outcome in zinc-deficient and zinc-replenished rats. Conclusions In vivo zinc regulates S100A8 expression and modulates the link between S100A8–RAGE interaction and downstream nuclear factor κΒ/COX-2 signaling. The finding that zinc regulates an inflammatory pathway in esophageal carcinogenesis may lead to prevention and therapy for this cancer.

Background Genetic alterations in tricarboxylic acid (TCA) cycle metabolic enzymes were recently linked to various cancers. However, the associations of single nucleotide polymorphisms (SNPs) in genes of these enzymes have not been well studied. Methods We genotyped 16 SNPs from 7 genes encoding TCA cycle metabolic enzymes in 697 colorectal carcinoma (CRC) patients receiving surgical resection and analyzed their associations with clinical outcomes by multivariate Cox proportional hazard model. Then, the significant results were validated in another cohort of 256 CRC patients. Results We identified 4 SNPs in 2 genes had significant associations with CRC death risk and 5 SNPs in 3 genes had significant associations with CRC recurrence risk. Similar significant results were confirmed for rs4131826 in SDHC gene, rs544184 in SDHD gene and rs12071124 in FH gene in a validation cohort. Further analysis indicated that unfavorable genotypes exhibited a significant cumulative effect on overall and recurrence-free survival in a dose-dependent manner. Moreover, survival tree analysis indicated that SNP rs4131826 in SDHC gene and SNP rs12071124 in FH gene were the primary factors contributing to the different overall survival time and recurrence-free survival time of CRC patients, respectively. Immunohistochemical analysis further validated the effect of rs4131826 and rs544184 on expression of SDHC and SDHD in tissue samples. Conclusions Our study suggests that SNPs in TCA cycle metabolic enzymes might be significantly associated with clinical outcomes in Chinese population diagnosed with CRC. Further functional and validated studies are warranted to expend our results to clinical utility. Electronic supplementary material The online version of this article (doi:10.1186/s12943-015-0442-x) contains supplementary material, which is available to authorized users.

Genetic polymorphisms of thymidylate synthase (TYMS) gene have been reported to be associated with development or prognosis of several cancers. However, the association between polymorphisms of TYMS gene and clinical outcomes of non-small cell lung cancer (NSCLC) patients are still unknown. In the present study, we investigated the associations between single nucleotide polymorphisms (SNPs) of TYMS gene and response to chemotherapy as well as clinical outcomes in NSCLC patients. Five SNPs in TYMS gene were genotyped using the Sequenom iPLEX genotyping system in a hospital-based cohort with 500 NSCLC patients, and their associations with NSCLC outcomes were evaluated by Cox proportional hazard regression analysis under three genetic models (additive, dominant, and recessive models). Our data showed that there was no significant association between individual SNP and overall survival of NSCLC patients. However, SNP rs2847153 was significantly associated with NSCLC recurrence under recessive model. We further identified a significant interaction between rs2847153 and chemotherapy in modifying clinical outcome of patients. Our data showed that individuals carrying GG/GA genotypes of rs2847153 had a significantly better response to chemotherapy when comparing to those carrying AA genotype. Conclusively, our data suggest that SNPs rs2847153 in TYMS gene may be a potential biomarker for predicting clinical outcome and personalized treatment in NSCLC patients.