HBV integrations reshaping genomic structures promote hepatocellular carcinoma.

OBJECTIVE: Hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC), mostly characterised by HBV integrations, is prevalent worldwide. Previous HBV studies mainly focused on a few hotspot integrations. However, the oncogenic role of the other HBV integrations remains unclear. This study aimed to elucidate HBV integration-induced tumourigenesis further. DESIGN: Here, we illuminated the genomic structures encompassing HBV integrations in 124 HCCs across ages using whole genome sequencing and Nanopore long reads. We classified a repertoire of integration patterns featured by complex genomic rearrangement. We also conducted a clustered regularly interspaced short palindromic repeat (CRISPR)-based gain-of-function genetic screen in mouse hepatocytes. We individually activated each candidate gene in the mouse model to uncover HBV integration-mediated oncogenic aberration that elicits tumourigenesis in mice. RESULTS: These HBV-mediated rearrangements are significantly enriched in a bridge-fusion-bridge pattern and interchromosomal translocations, and frequently led to a wide range of aberrations including driver copy number variations in chr 4q, 5p (TERT), 6q, 8p, 16q, 9p (CDKN2A/B), 17p (TP53) and 13q (RB1), and particularly, ultra-early amplifications in chr8q. Integrated HBV frequently contains complex structures correlated with the translocation distance. Paired breakpoints within each integration event usually exhibit different microhomology, likely mediated by different DNA repair mechanisms. HBV-mediated rearrangements significantly correlated with young age, higher HBV DNA level and TP53 mutations but were less prevalent in the patients subjected to prior antiviral therapies. Finally, we recapitulated the TONSL and TMEM65 amplification in chr8q led by HBV integration using CRISPR/Cas9 editing and demonstrated their tumourigenic potentials. CONCLUSION: HBV integrations extensively reshape genomic structures and promote hepatocarcinogenesis (graphical abstract), which may occur early in a patient's life.

Downregulation of CAMK2N1 due to DNA Hypermethylation Mediated by DNMT1 that Promotes the Progression of Prostate Cancer.

Calcium/calmodulin-dependentprotein kinase II inhibitor I (CAMK2N1) as one of the tumor suppressor genes is significantly downregulated in prostate cancer (PCa). Reduced expression of CAMK2N1 is positively correlated with PCa progression. However, the mechanisms of CAMK2N1 downregulation in PCa are still unclear. The promoter region of CAMK2N1 contains a large number of CG loci, providing the possibility for DNA methylation. Consequently, we hypothesized that DNA methylation can result in the reduced expression of CAMK2N1 in PCa. In the presented study, the DNA methylation level of CAMK2N1 in prostate cells and clinical specimens was determined by bisulfite sequencing (BS), pyrosequencing, and in silico analysis. Results showed that CAMK2N1 was highly methylated in PCa cells and tissues compared to normal prostate epithelial cells and nonmalignant prostate tissues, which was associated with the clinicopathological characteristics in PCa patients. Afterwards, we explored the expression of CAMK2N1 and its DNA methylation level by qRT-PCR, western blot, BS, and methylation-specific PCR in PCa cells after 5-Aza-CdR treatment or DNMT1 genetic modification, which demonstrated that the reduced expression of CAMK2N1 can be restored by 5-Aza-CdR treatment via demethylation. Moreover, DNMT1 formed a positive feedback loop with CAMK2N1 in PCa cells. The expression of CAMK2N1 was downregulated by DNMT1-mediated DNA methylation, which reversely induced DNMT1 expression through activating AKT or ERK signaling pathway. Finally, functional assays including wound healing, invasion, and migration assay, as well as the xenograft model in nude mice indicated that CAMK2N1 inhibited the invasion, migration, and proliferation of PCa cells and these effects were reversed by DNMT1 overexpression. In conclusion, DNMT1-mediated hypermethylation of CAMK2N1 not only downregulates the gene expression but also promotes the progression of PCa.

Epidemiology of adenosarcoma and the inverse probability of treatment weighting (IPTW) adjusted survival analysis of lymph node dissection in uterine adenosarcoma.

The objective for the study was to analysis the epidemiology of adenosarcoma, and independent prognostic factors and impact of lymph node dissection (LND) of uterine adenosarcoma. Cases of patients with primary adenosarcoma were obtained from the Surveillance, Epidemiology, and End Results (SEER) database from 2000 to 2016. Overall survival was analyzed by the Kaplan-Meier method and log-rank test. The differences in baseline covariates between the 2 groups were adjusted by inverse probability of treatment weighting method. The prognostic factors were identified by univariate and multivariate Cox regression analysis and hazard ratio and 95% confidence interval (CI) of covariates were also estimated. 1129 patients with pathological primary adenosarcoma between 2000 and 2016 were identified from the surveillance, epidemiology, and end results database. The only 4 patients were male. 1027 patients with primary uterine adenosarcoma, and 53.1% underwent LND and only 3.5% patients were with positive lymph node. Age, marital status, largest tumor size, tumor grade, T stage and chemotherapy were significantly correlated with survival. Race, tumor number, LND, and radiotherapy did not affect overall survival in patients. Inverse probability of treatment weighting-adjusted K-M curve showed that LND did not improve survival and lymph node metastasis (LNM) did not affect survival. The majority of primary adenosarcoma patients are female with high incidence of uterus and rare incidence of distant metastasis. Age, marital status, tumor size, T stage, grade, and chemotherapy are independent prognostic factors of uterine adenosarcoma. LNM was not a significant prognostic risk factor, and LND did not benefit survival.

Neoadjuvant radiotherapy for resectable hepatocellular carcinoma with portal vein tumor thrombus: a systematic review.

Background: The prognosis of hepatocellular carcinoma (HCC) with portal vein tumor thrombus (PVTT) is extremely poor. The clinical outcome of preoperative radiotherapy (RT) is still controversial. This study aimed to compare the clinical outcomes of combined neoadjuvant RT and hepatectomy with hepatectomy alone for HCC with PVTT. Methods: Comprehensive database searches were performed in PubMed, the Cochrane Library, EMBASE, and the Web of Science to retrieve studies published from the database creation to July 1, 2020. Only comparative studies that measured survival between neoadjuvant RT followed by hepatectomy and hepatectomy alone were included. The characteristics of the included studies and patients were extracted, and the included data are presented as relative ratio (RR) estimates with 95% confidence intervals (CIs) for all outcomes. The RRs of each study were pooled using a fixed or random effects model with Review Manager (the Cochrane Collaboration, Oxford, UK) version 5.3. The response rate to RT and the overall survival (OS) rate in neoadjuvant RT followed by hepatectomy and hepatectomy alone were measured. Results: One randomized and two non-randomized controlled trials with 302 patients were included. Most patients were classified as Child-Pugh A, and Type II and III PVTT were the most common types. After RT, 29 (22.8%) patients were evaluated as partial response (PR) and had a positive RT response, but nine (7.1%) had progressive disease (PD). Neoadjuvant RT followed by hepatectomy was received by 127 (42.1%) patients after excluding 15 (5.0%) patients with severe complications or PD after RT, and 160 (53.0%) patients received hepatectomy alone. In the randomized controlled trial (RCT), the 1-year OS rate in the neoadjuvant RT group and the surgery alone group was 75.2% and 43.1%, respectively (P<0.001). In the two non-randomized studies, a meta-analysis with a fixed effects model showed a longer OS in patients undergoing neoadjuvant RT followed by hepatectomy compared with hepatectomy alone at 1-year follow-up (RR =2.02; 95% CI: 1.45-2.80; P<0.0001). Conclusions: This systematic review showed that neoadjuvant RT followed by hepatectomy in patients with resectable HCC and PVTT was associated with a longer OS than patients who received hepatectomy alone.

Serum alpha-fetoprotein as a predictive biomarker for tissue alpha-fetoprotein status and prognosis in patients with hepatocellular carcinoma.

Background: Alpha-fetoprotein (AFP) expression is closely related to hepatocarcinogenesis, and it is an important prognostic factor for hepatocellular carcinoma (HCC). We aimed to investigate the relationship between serum AFP concentration and tissue AFP status and identify the prognostic value of serum and tissue AFP for HCC. Methods: This is a retrospective review of 248 patients with HCC from January 2012 to December 2018. Receiver operating characteristic (ROC) curves were plotted to investigate the predictive value of serum AFP for tissue AFP status. Overall survival (OS) was analyzed using the Kaplan-Meier method and log-rank tests were used for comparison between two groups. Multivariate Cox proportional hazards regression analysis was performed for various risk factors. Results: The serum AFP level in patients with tissue AFP-positive HCC was higher than those with tissue AFP-negative HCC (506.7 vs. 7.7 ng/mL, P<0.0001). Youden's index yielded an optimal cut-off value of serum AFP for tissue AFP status of 92.33 ng/mL with a sensitivity and specificity of 0.84 (95% CI: 0.74-0.90) and 0.88 (95% CI: 0.82-0.92), respectively. Moreover, high serum AFP concentrations (≥92.33 ng/mL) were significantly correlated with positive hepatitis B virus (HBV, P=0.012), tumor size (P=0.025) and histological grade (P=0.001); tissue AFP-positive status was associated with positive HBV (P=0.006), tumor number (P=0.033) and histological grade (P<0.001). Further, serum AFP level ≥92.33 ng/mL and tissue AFP-positive status were associated with poorer OS, and positive HBV (Positive: HR 3.496; 95% CI: 1.349-9.064; P=0.010) and larger tumor size (≥5; HR 2.617; 95% CI: 1.372-4.992; P=0.003) were independent factors of OS. Conclusions: This study showed that serum AFP level could be a highly predictive biomarker for tissue AFP status in patients with HCC. Furthermore, serum AFP levels ≥92.33 ng/mL and tissue AFP-positive status were associated with poorer OS but were not independent factors of OS.

AHR mediates the aflatoxin B1 toxicity associated with hepatocellular carcinoma.

Aflatoxin exposure is a crucial factor in promoting the development of primary hepatocellular carcinoma (HCC) in individuals infected with the hepatitis virus. However, the molecular pathways leading to its bioactivation and subsequent toxicity in hepatocytes have not been well-defined. Here, we carried out a genome-wide CRISPR-Cas9 genetic screen to identify aflatoxin B1 (AFB1) targets. Among the most significant hits was the aryl hydrocarbon receptor (AHR), a ligand-binding transcription factor regulating cell metabolism, differentiation, and immunity. AHR-deficient cells tolerated high concentrations of AFB1, in which AFB1 adduct formation was significantly decreased. AFB1 triggered AHR nuclear translocation by directly binding to its N-terminus. Furthermore, AHR mediated the expression of P450 induced by AFB1. AHR expression was also elevated in primary tumor sections obtained from AFB1-HCC patients, which paralleled the upregulation of PD-L1, a clinically relevant immune regulator. Finally, anti-PD-L1 therapy exhibited greater efficacy in HCC xenografts derived from cells with ectopic expression of AHR. These results demonstrated that AHR was required for the AFB1 toxicity associated with HCC, and implicate the immunosuppressive regimen of anti-PD-L1 as a therapeutic option for the treatment of AFB1-associated HCCs.

The prognostic factors of primary colorectal sarcoma and the clinical outcomes of negative lymph node dissection.

BACKGROUND: Primary colorectal sarcoma is an extremely rare malignancy that is associated with poor patient outcomes. The aim of this study was to identify the prognostic factors of primary colorectal sarcoma and evaluate the clinical outcomes associated with these prognostic factors. METHODS: Between January 1, 2000 and December 31, 2016, the clinical data of 315 patients with primary colorectal sarcoma were obtained from the Surveillance, Epidemiology, and End Results (SEER) database. Cancer-specific survival (CSS) was analyzed by the Kaplan-Meier method and by log-rank test. The prognostic factors were identified by univariate and multivariate Cox regression analysis and hazard ratio (HR) and 95% confidence interval (CI) of covariates were also estimated. The optimal cutoff value for NLN count at dissection was identified using X-tile software and validated by univariate Cox regression analysis. RESULTS: Of the 315 patients with primary colorectal sarcoma identified, 88.6% received surgery. The median follow-up time was 34 months with an interquartile range (IQR) of 9-79 months. The 5-year rate of CSS was 76.73% and 27.8% for the surgery group and the non-surgery group, respectively (P<0.0001). Univariate and multivariate Cox regression analysis performed on the data of nonmetastatic patients demonstrated that sex, race, radiotherapy, and chemotherapy had no effect on patient CSS, with age, tumor site, tumor grade, and NLN dissection being independent prognostic factors. A significant correlation was found between advanced age (>80 years old) and poor CSS (HR 1.964; 95% CI: 1.005-3.839; P=0.048). There were also significant correlations between colonic tumors and poor CSS (HR 2.903; 95% CI: 1.348-6.250; P=0.006) and grade IV tumors and poor CSS (HR 3.431; 95% CI: 1.725-6.823; P<0.001), while NLN dissection was associated with improved CSS (HR 0.946; 95% CI: 0.911-0.983; P=0.004). X-tile software analysis was used to determine that the optimal cutoff value for NLN count was 13. Patients who received NLN dissection with a cutoff value of 13 or more displayed better CSS than those who did not (P=0.016). CONCLUSIONS: Primary colorectal sarcoma patients can benefit significantly from primary tumor surgery. Age, tumor site, grade and NLN dissection are independent prognostic factors for CSS in nonmetastatic patients. Importantly, nonmetastatic patients treated with NLN dissection with an NLN count of 13 or more have significantly better CSS.

Interleukin-6 increases adrenal androgen release by regulating the expression of steroidogenic proteins in NCI-H295R cells.

The purpose of this study was to investigate the potential mechanism of interleukin-6 (IL-6) on the stimulation of excessive androgen secretion in human NCI-H295R adrenocortical cells. We performed transcriptome sequencing of cancer and paracancerous tissues obtained from functional adrenal cortical adenomas. The secretion of dehydroepiandrosterone sulfate (DHEAS) in NCI-H295R cells was detected by a chemiluminescence assay. The expression of messenger RNA (mRNA) was detected by real-time polymerase chain reaction and that of protein was detected by western blotting. The expression of secretogranin II (SCG2) and IL-6 were significantly increased in cancer tissues. Upregulation of mRNA and protein levels of AKR1C3, CYP11A, CYP17A1, 3ßHSD, and SULT2A1 was observed after stimulation with IL-6. IL-6 could also increase the expression of StAR mRNA and proteins. Our results suggest that IL-6 can promote androgen secretion by regulating the expression of genes related to androgen pathways.

The appropriate number of negative lymph nodes dissection for nonmetastatic penile cancer.

Negative lymph nodes status has been attached more attention as a prognostic indicator for nonmetastatic penile cancer. We aimed to identify the appropriate number of negative lymph nodes dissection for nonmetastatic penile cancer using the Surveillance, Epidemiology and End Results database. A total of 1,470 nonmetastatic patients with penile squamous cell carcinoma were identified during 2004 and 2013. All patients were categorised according to different risk levels and lymphadenectomy. Univariate and multivariate Cox regression analyses were performed to evaluate the relationship between prognostic risk factors and cancer-specific survival. The optimal cut-off value of negative lymph nodes dissection was determined using the X-Tile program. A total of 1,470 patients were categorised into low- (pT1G1), intermediate- (pT1G2) or high-risk (pT1G3 and all higher stages) groups. In multivariate Cox analysis, lymphadenectomy improved the cancer-specific survival for patients in high-risk group (p = 0.014). Further, the optimal cut-off value of negative lymph nodes dissection for high-risk patients was 5 and patients with >5 negative lymph nodes had a higher cancer-specific survival (χ2  = 9.3676, p < 0.05). Therefore, lymphadenectomy improved survival for high-risk penile cancer and the removal of more than five negative lymph nodes was correlated with higher cancer-specific survival for high-risk patients who underwent lymphadenectomy.

Comparison of intracytoplasmic sperm injection outcome with fresh versus frozen-thawed testicular sperm in men with nonobstructive azoospermia: a systematic review and meta-analysis.

PURPOSE: The purpose of the study is to explore testicular sperm cryopreservation in patients with nonobstructive azoospermia (NOA) whether affect the outcome of subsequent intracytoplasmic sperm injection (ICSI). METHODS: A systematic review and meta-analysis was conducted by searching the MEDLINE and EMBASE databases for relevant published studies in English language (1997-2017). Studies were eligible if they included the comparison of using fresh and frozen-thawed testicular sperm followed by ICSI. Two reviewers independently performed data extraction, quality assessment and assessed the risk of bias. The overall summary risk estimated the number of events. A meta-analysis was conducted using a random effects or fixed effects model analysis according to the test of heterogeneity. RESULTS: A total of 17 studies with 1,261 ICSI cycles were identified. Analysis of the present data showed no difference in the fertilization outcome when comparing fresh versus frozen-thawed spermatozoa (RR = 1.02, 95% CI 0.86-1.09). Similarly, no difference in CR (RR = 1.01, 95% CI 0.96-1.05), good embryo rate (RR = 1.01, 95% CI 0.95-1.09), and IR (RR = 0.93, 95% CI 0.66-1.30) was observed if the spermatozoa was fresh or frozen-thawed. Finally, no difference in CPR or LBR was noted when using fresh or frozen-thawed cycles were analyzed separately (RR = 1.03, 95% CI 0.86-1.24; RR 1.11, 95% CI 0.88-1.41, respectively). CONCLUSIONS: In men with NOA, the ICSI outcome is not affected by whether the retrieved testicular sperm is fresh or frozen. Sperm cryopreservation ought to be considered in every surgical sperm retrieval case, which remain feasible even in patients with few testicular sperm retrieved.