Molecular mechanism of the miR-7/BCL2L1/P53 signaling axis regulating the progression of hepatocellular carcinoma.

Background: To investigate the roles of miR-7 and its potential mechanisms in hepatocellular carcinoma (HCC). Methods: The functions of miR-7 were identified and measured by MTT [3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide], colony formation, transwell, and flow cytometry assays. A luciferase assay was applied to verify the direct binding of miR-7 on BCL2L1 3'untranslated region (3'UTR). An in vitro experiment was then used to investigate the biological effects of miR-7 and BCL2L1. A co-immunoprecipitation (COIP) assay was used to detect the protein interaction between BCL2L1 and P53. Results: We found that miR-7 overexpression suppressed cell proliferation, migration, and invasion in HCC. BCL2L1 was also demonstrated as a direct target gene of miR-7. This study showed that BCL2L1 could partially rescue the inhibitory effect of miR-7 on the proliferation, migration, and invasion of HCC cells. Our research showed that miR-7 could inhibit the epithelial-mesenchymal transition (EMT) pathway by regulating BCL2L1. We also further confirmed that miR-7 inhibits the proliferation, migration, and invasion of Hep3B and Huh7 cells by targeting BCL2L1. Furthermore, we observed that the BCL2L1 protein interacts with the P53 protein and BCL2L1 affects the development of liver cancer through P53. We also found that BCL2L1 could promote the invasion and migration of liver cancer cells through P53 inhibition. BCL2L1 also inhibited the expression of Caspase 3/7 in hepatoma cells by inhibiting the expression of P53. Conclusions: Our study demonstrated that miR-7/BCL2L1/P53 may serve as a regulatory molecular axis for HCC treatment. Our results suggest that miR-7/BCL2L1/P53 may have predictive value and represent a new treatment strategy for liver cancer.

A pilot study to evaluate tissue- and plasma-based DNA driver mutations in a cohort of patients with pancreatic intraductal papillary mucinous neoplasms.

Intraductal papillary mucinous neoplasms (IPMNs) are precursor lesions to pancreatic ductal adenocarcinoma that are challenging to manage due to limited imaging, cytologic, and molecular markers that accurately classify lesions, grade of dysplasia, or focus of invasion preoperatively. The objective of this pilot study was to determine the frequency and type of DNA mutations in a cohort of surgically resected, pathologically confirmed IPMN, and to determine if concordant mutations are detectable in paired pretreatment plasma samples. Formalin-fixed paraffin-embedded (FFPE) tissue from 46 surgically resected IPMNs (31 low-grade, 15 high-grade) and paired plasma from a subset of 15 IPMN cases (10 low-grade, 5 high-grade) were subjected to targeted mutation analysis using a QIAseq Targeted DNA Custom Panel. Common driver mutations were detected in FFPE from 44 of 46 (95.6%) IPMN cases spanning all grades; the most common DNA mutations included: KRAS (80%), RNF43 (24%), and GNAS (43%). Of note, we observed a significant increase in the frequency of RNF43 mutations from low-grade to high-grade IPMNs associated or concomitant with invasive carcinoma (trend test, P = 0.01). Among the subset of cases with paired plasma, driver mutations identified in the IPMNs were not detected in circulation. Overall, our results indicate that mutational burden for IPMNs is a common occurrence, even in low-grade IPMNs. Furthermore, although blood-based biopsies are an attractive, noninvasive method for detecting somatic DNA mutations, the QIAseq panel was not sensitive enough to detect driver mutations that existed in IPMN tissue using paired plasma in the volume we were able to retrieve for this retrospective study.

Identification of potential prognostic biomarkers among gene models for coiled-coil domain-containing family members in hepatocellular carcinoma elucidates their influence on the hypoxia pathway and immune microenvironment.

Background: The family of coiled-coil domain-containing (CCDC) proteins participates in a wide range of physiological functions and plays a pivotal role in governing the invasion and metastasis of malignant tumor cells. Nonetheless, the precise mechanism governing the interaction among the immune microenvironment, hypoxia pathway, and proliferation in hepatocellular carcinoma (HCC) remains elusive. In this study, our objective was to identify the prognostic significance of CCDC family genes in HCC. Methods: We conducted an analysis of RNA-seq data from HCC patients sourced from The Cancer Genome Atlas (TCGA) database. Our analysis involved comparing the expression profiles of 168 CCDC family genes between tumor and normal tissues to identify differentially expressed genes (DEGs). The prognostic value of these genes was verified using overall survival (OS) data from TCGA-LIHC patients, employing Univariate and multivariate Cox proportional hazards regression models and Kaplan-Meier plots. Subsequently, we constructed a prognostic signature known as the CCDC score and validated it using additional datasets (ICGC-LIRI-JP and GSE14520). Additionally, we performed functional enrichment analysis and conducted an assessment of the tumor immune microenvironment (TIME). Results: We identified 34 DEGs of the CCDC family. Among them, six DEGs (CCDC6/22/51/59/132/134) were upregulated and associated with poor prognosis. Higher CCDC score was an independent predictor of poor OS in TCGA-HCC patients (P<0.001, HR =2.37), which was validated in the ICGC-LIRI-JP (P=0.021, HR =2.15) and GSE14520 (P=0.002, HR =2.23) datasets. Functional enrichment analysis showed that hypoxia pathway genes were enriched in the high CCDC score group. Furthermore, immune microenvironment analysis demonstrated that high CCDC score was associated with a suppressed TIME caused by the extrinsic immune escape. Conclusions: The CCDC score, derived from six CCDC genes, exhibits remarkable expression levels in liver cancer and holds promise as an independent prognostic indicator. Our bioinformatics analysis revealed a high CCDC score is strongly associated with activation of the hypoxia pathway and an immunosuppressive tumor microenvironment in HCC. This profound finding may serve as a cornerstone for innovative targeted drug therapies and pave the way for further investigations into the underlying mechanisms of CCDC-related carcinogenesis in liver cancer.

Glycolysis-related gene dihydrolipoamide acetyltransferase promotes poor prognosis in hepatocellular carcinoma through the Wnt/ß-catenin and PI3K/Akt signaling pathways.

Background: Recent research suggests that dihydrolipoamide acetyltransferase (DLAT), which is a copper-induced cell death-related gene, is involved in multiple biological events in tumors. This study sought to investigate the relationship between DLAT and hepatocellular carcinoma (HCC). Methods: In the Cancer Genome Atlas (TCGA) database, we first identified the differentially expressed gene (i.e., DLAT), then confirmed DLAT expression, and found a link between it and the prognosis of HCC patients. An internal validation nomogram was built based on a multivariate Cox regression analysis. Data from the Tumor Immune Estimation Resource (TIMER) database was used to examine the association between DLT and immunological cells. A gene set enrichment analysis (GSEA) was conducted to investigate the probable mechanism of action. Finally, in vitro cytological research was conducted to further examin the involvement of DLAT in HCC-related unfavorable biological events. Results: The database screenings showed that DLAT was a differentially expressed molecule; that is, DLAT was more highly expressed in the cancer tissues than normal tissues. TCGA results and Kaplan-Meier-plotter data sets showed that HCC patients with reduced DLAT expression had greater disease-specific survival (DSS), overall survival (OS), and progression-free interval (PFI). The prediction model had a concordance index of 0.659 (0.614-0.704), which indicates high accuracy. According to the TIMER database, tumor cells in the HCC microenvironment may be able to bypass the immune system due to the expression of DLAT. The in vitro cytological tests showed that DLAT knockdown significantly decreased the proliferation and invasion of the HCC cells. It also inhibited the activity of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase B (Akt) and Wnt/ß-catenin signaling pathways. Conclusions: Decreased DLAT expression significantly prolongs the OS, PFI, and DSS of HCC patients. DLAT may be employed as a new predictive biomarker for HCC, and may be linked to the immune system in HCC patients. The tumor microenvironment (TME) may have a significant effect on the ability of tumor cells to evade the immune system. The PI3K/Akt and Wnt/ß-catenin signaling pathways may affect the prognosis of HCC by interfering with DLAT. Given these findings, HCC may be an ideal target for the development of anti-cancer therapies.

Multilevel stenting of malignant colonic obstructions from multilevel breast cancer colonic metastasis.

Video 1A video depicting the enclosed case, procedure, and discussion.

Intracystic Glucose and Carcinoembryonic Antigen in Differentiating Histologically Confirmed Pancreatic Mucinous Neoplastic Cysts.

INTRODUCTION: Differentiating mucinous neoplastic pancreatic cysts (MNPC) from cysts without malignant potential can be challenging. Guidelines recommend using fluid carcinoembryonic antigen (CEA) to differentiate MNPC; however, its sensitivity and specificity vary widely. Intracystic glucose concentration has shown promise in differentiating MNPC, but data are limited to frozen specimens and cohorts of patients without histologic diagnoses. This study aimed to compare glucose and CEA concentrations in differentiating MNPC using fresh fluid obtained from cysts with confirmatory histologic diagnoses. METHODS: This multicenter cohort study consisted of patients undergoing endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) for pancreatic cysts during January 2013-May 2020. Patients were included if the cyst exhibited a histologic diagnosis and if both CEA and glucose were analyzed from fresh fluid. Receiver operating curve (ROC) characteristics were analyzed, and various diagnostic parameters were compared. RESULTS: Ninety-three patients, of whom 59 presented with MNPC, met the eligibility criteria. The area under the receiver operating curve (AUROC) was 0.96 for glucose and 0.81 for CEA (difference 0.145, P = 0.003). A CEA concentration of ≥192 ng/mL had sensitivity of 62.7% and specificity of 88.2% in differentiating MNPC, whereas glucose concentration of ≤25 mg/dL had sensitivity and specificity of 88.1% and 91.2%, respectively. DISCUSSION: Intracystic glucose is superior to CEA concentration for differentiating MNPC when analyzed from freshly obtained fluid of cysts with histologic diagnoses. The advantage of glucose is augmented by its low cost and ease of implementation, and therefore, its widespread adoption should come without barriers. Glucose has supplanted CEA as the best fluid biomarker in differentiating MNPC.

The Role of EUS in Liver Biopsy.

PURPOSE OF REVIEW: EUS-guided liver biopsy (EUS-LB) is being used with increased frequency to perform parenchymal liver biopsy. Evolution of the technique can now achieve excellent liver tissue cores. This review covers important developments in this procedure. RECENT FINDINGS: Clinical studies have recently demonstrated that the 19G EUS core biopsy needle is superior to non-core needles for liver tissue acquisition. In addition, wet suction provides more robust tissue samples than dry suction. Heparin priming of the needle (instead of saline) can prevent blood clogging within the needle lumen. A 1-hour recovery time after the EUS-LB is sufficient in almost all cases. The EUS-LB can deliver bilobar biopsies, which can decrease sampling error. Patients who need a liver biopsy in addition to an endoscopy or EUS are best served by the EUS-LB, as the combination procedure saves time and cost. The EUS-LB is a safe and effective means for procuring good liver core biopsies. Incremental improvements in technique have increased quality of the resulting specimen. Future directions of this technique are discussed.

Endoscopic ultrasound-guided biopsy in chronic liver disease: a randomized comparison of 19-G FNA and 22-G FNB needles.

Background and study aims Endoscopic ultrasound-guided liver biopsy uses a 19-gauge (G) needle for parenchymal liver biopsies. We evaluated tissue yields with a 22G fine-needle biopsy (FNB) versus 19G FNA fine-needle aspirate (FNA) device. Patients and methods Biopsies were obtained from 20 patients using the 19G FNA and 22G FNB randomizing each in a cross-over fashion with a blinded outcome assessor. Tissue adequacy for histologic evaluation was the primary outcome, or the proportion of specimens obtaining pathologic diagnosis (portal structures ≥ 5 or length of the longest piece ≥ 15 mm). Additional secondary outcomes included portal and centrilobular inflammation/fibrosis, length of the longest piece, aggregate specimen length, and small (< 5 mm), medium (5 - 8 mm) and large (> 8 mm) fragments. Results were compared in a per needle basis. Patients with cirrhosis were excluded. Results Eighty biopsies (40 each 19G FNA and 22G FNB) were obtained. Tissue adequacy was greater for the 19G FNA (88 %) versus 22G FNB (68 %), ( P  = 0.03). There was no difference in total portal structures for the 19G FNA (7.4) and 22G FNB (6.1), ( P  = 0.28). There was no difference in pre-processing outcomes. After processing, length of the longest piece was higher for the 19G FNA (9.1 mm) versus 22G FNB (6.6 mm), ( P  = 0.02). More total post-processing small fragments 29.9 versus 20.7, ( P  = 0.01) and fewer large fragments 1.0 versus 0.4 for the 22G FNB ( P  = 0.01) were detected. Conclusions Tissue adequacy was higher for the 19G FNA versus 22G FNB needle. The 22G FNB needle produced samples more prone to fragmentation during specimen processing.

A prospective pilot comparison of wet and dry heparinized suction for EUS-guided liver biopsy (with videos).

BACKGROUND AND AIMS: As EUS-guided liver biopsy sampling (EUS-LB) becomes more widely used, further studies have investigated ways to improve tissue yields. Use of a heparin-primed needle may lead to less clotting of blood within the needle, improve tissue recovery, and decrease fragmentation. The purpose of this study was to prospectively evaluate wet suction using a heparin-primed needle for EUS-LB. METHODS: This was a prospective crossover study evaluating wet suction for EUS-LB in parenchymal liver disease. The primary outcome was specimen adequacy, defined by an aggregate specimen length ≥15 mm and ≥5 complete portal tracts (CPTs). Secondary outcomes included number of CPTs, length of the longest piece, aggregate specimen length, and number of small (≤4 mm), medium (5-8 mm), and large (≥9 mm) fragments. Adverse events were tracked at 7 and 30 days. RESULTS: One hundred twenty biopsy specimens were collected from 40 participants (3 specimens per patient). Specimen adequacy occurred in 39 wet heparin (98%), 37 dry heparin (93%), and 30 dry needle biopsy samples (80%; 95% confidence interval [CI], .14-.18; P = .01). There was no difference between dry needle techniques. Length of the longest piece was 8.9 mm for wet heparin and 5.8 mm for dry techniques (95% CI, .33-1.53; P = .003). Aggregate specimen length was 49.2 mm for wet heparin and 23.9 mm for dry heparin (95% CI, -46.34 to 44.94; P = .003). Mean CPT count was 7.0 for wet heparin versus 4.0 for dry (95% CI, .74-6.26; P = .01). There were more medium (2.0 vs 1.0; 95% CI, .06-1.24; P = .03) and large (1.0 versus 0.0; 95% CI, .33-1.53; P = .003) fragments with wet suction with no difference in small fragments between groups. CONCLUSIONS: The use of wet suction EUS-LB demonstrated improved tissue adequacy compared with dry needle techniques. (Clinical trial registration number: NCT03103997.).

Heparin priming of EUS-FNA needles does not adversely affect tissue cytology or immunohistochemical staining.

BACKGROUND AND STUDY AIMS: Endoscopic ultrasound (EUS)-guided fine-needle aspiration (FNA) or biopsy (FNB) is an indispensable diagnostic tool. Improvements in needling technique have led to increasing tissue yields. Blood clogging of the needle can cause difficulties with specimen handling and stylet passage, which improves when the needle is primed with heparin before use. However, the effect of heparin on cytology, histology or immunochemistry (IHC) of FNA and FNB specimens is unknown. The goal of the study was to evaluate heparin priming on cytologic/histologic appearance, IHC staining, ease of stylet passage, and specimen bloodiness. PATIENTS AND METHODS: This was a retrospective study of patients undergoing EUS-FNA/FNB. Needle sizes were 25 gauge (g), 22 g, and 19 g. Heparin priming of the needle was done and the stylet replaced ("dry heparin") or suction attached without replacing the stylet ("wet heparin"). Smears and cellblocks were examined by pathologists, and IHC staining were done as needed. Specimen bloodiness was compared with matched controls. RESULTS: Adequate tissue yields were obtained in all samples (37 heparin, 36 no heparin). Heparin priming did not exhibit negative effects on cytologic or histologic interpretation of the specimens, nor IHC. There was no difference in cellblock bloodiness between the heparin primed needle specimens and the non-heparin control group. . CONCLUSIONS: Heparin priming of EUS-FNA or FNB needles does not negatively affect cytologic or histologic interpretation, nor interfere with IHC. In addition, heparin priming does not increase specimen bloodiness. When the "wet suction" technique is used for EUS-FNA, heparin priming can be used instead of saline priming of the EUS needle.

Therapeutic Endoscopy Can Be Performed Safely in an Ambulatory Surgical Center: A Multicenter, Prospective Study.

Background. Even amongst experienced endoscopists, endoscopic retrograde cholangiopancreatography (ERCP) and endoscopic ultrasound with fine needle aspiration (EUS-FNA) carry a potential risk for complications. These procedures are typically performed in a hospital-based endoscopy unit with general anesthesia. Aims. The goal of our study was to evaluate the feasibility of ERCP and EUS-FNA in an ambulatory surgical center (ASC). Methods. From June to November of 2014, we prospectively enrolled consecutive subjects undergoing ERCP and/or EUS-FNA in an ASC. An anesthesiologist, who was not involved in our study group, screened all subjects prior to their scheduled procedure. In order to monitor for adverse events (AE), all subjects received a telephone call at day 1 and 30 days after procedure. Results. 375 subjects (98 inpatients and 277 from an ASC) were enrolled. In the total population, a high proportion of subjects underwent procedures for neoplasms (21 (23.3%) inpatients versus 44 (17.1%) from an ASC) and for sphincter of Oddi dysfunction (SOD) (27 (27.5%) versus 48 (17.3%)) and had the American Society for Anesthesiologists (ASA) class ≥III (75 (76.5%) versus 140 (50.5%)) and high-risk features (17 (17.3%) versus 75 (27.1%)). Overall ERCP-related AE (10 (13.2%) versus 12 (7.5%), p = 0.2), pancreatitis (7 (9.2%) versus 11 (6.9%), p = 0.6), and hemorrhage (3.9% versus 0.6%, p = 0.25) were not different between inpatients and ASC subjects. There was also no difference between inpatients and ASC subjects' EUS-related AE (1 (4.5%) versus 4 (3.4%), p = 0.6), pancreatitis (1 (4.5%) versus 3 (2.6%), p = 0.2), and hemorrhage (0% versus 1 (0.9%), p = 0.9). Conclusions. ERCP and EUS can be performed in a higher risk population under the supervision of anesthesia in ASCs. Overall, the AE are equivalent between inpatients and ASC subjects.

A genetic database can be utilized to identify potential biomarkers for biphenotypic hepatocellular carcinoma-cholangiocarcinoma.

BACKGROUND: Biphenotypic hepatocellular carcinoma-cholangiocarcinoma (HCC-CC) is an uncommon primary liver neoplasm. Due to limitations in radiologic imaging for the diagnosis of this condition, biopsy is a common method for diagnosis, which is invasive and holds potential complications. To identify alternative means for obtaining the diagnosis and assessing the prognosis of this condition, we evaluated biomarkers for biphenotypic HCC-CC using a genetic database. METHODS: To evaluate the genetic associations with each variable we utilized GeneCards(®), The Human Gene Compendium (http://www.genecards.org). The results of our search were entered into the Pathway Interaction Database from the National Cancer Institute (PID-NCI) (http://pid.nci.nih.gov), to generate a biomolecule interaction map. RESULTS: The results of our query yielded 690 genes for HCC, 98 genes for CC and 50 genes for HCC-CC. Genes depicted in this analysis demonstrate the role of hormonal regulation, embryonic development, cell surface adhesion, cytokeratin stability, mucin production, metalloproteinase regulation, Ras signaling, metabolism and apoptosis. Examples of previously described markers included hepatocyte growth factor (HGF), mesenchymal epithelial transition (MET) and Kirsten rat sarcoma viral oncogene homolog (KRAS). Novel markers included phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA), GPC3, choline kinase alpha (CHKA), prostaglandin-endoperoxide synthase 2 (PTGS2), telomerase reverse transcriptase (TERT), myeloid cell leukemia 1 (MCL1) and N-acetyltransferase 2 (NAT2). CONCLUSIONS: GeneCards is a useful research tool in the genetic analysis of low frequency malignancies. Utilizing this tool we identified several biomarkers are methods for diagnosing HCC-CC. Finally, utilizing these methods, HCC-CC was found to be predominantly a subtype of CC.

Does the urgency of endoscopic retrograde cholangiopancreatography (ercp)/percutaneous biliary drainage (pbd) impact mortality and disease related complications in ascending cholangitis? (deim-i study).

BACKGROUND: The Tokyo Guidelines have greatly impacted the management of ascending cholangitis. Though ERCP is the favored modality for biliary decompression, no evidence exists for the timing of ERCP. The DEIM-I study set out to determine if the time from patient presentation to biliary decompression impacted in hospital all cause mortality in ascending cholangitis. METHOD: DEIM-I cohort study was a single-blinded and consisted of 250 subjects with moderate to severe ascending cholangitis who underwent ERCP/PBD. Subjects were randomized into quartiles based upon time from presentation until ERCP/PBD. The primary outcome utilized logistic regression to estimate relative risk (RR) of all cause, in hospital mortality with time to procedure as the predictive covariate. Secondary outcomes were analyzed using multivariate logistic regression and included; multiple organ failure (MOF), sepsis, systemic inflammatory response syndrome (SIRS), surgical incidence, hospital readmission and length of stay (LOS). RESULTS: The risk for hospital mortality was significantly less when biliary drainage was performed within 11 h, compared to >42 h (RR 0.34, 95%CI 0.12 to 0.99, p=0.049). Hospital readmission was lower in subjects who underwent biliary decompression less than 11 h, when compared to those greater than 22 h. Subjects who underwent biliary decompression within 21 h had significant higher risk for surgery compared to those 22-42 h. CONCLUSION: The relative risk of all cause in hospital mortality was lower in subjects who underwent biliary decompression in under 11 h compared to greater than 42 h.