Gallbladder microbiota composition is associated with pancreaticobiliary and gallbladder cancer prognosis.

BACKGROUND: The microbial population of the intestinal tract and its relationship to specific diseases has been extensively studied during the past decade. However, reports characterizing the bile microbiota are rare. This study aims to investigate the microbiota composition in patients with pancreaticobiliary cancers and benign diseases by 16S rRNA gene amplicon sequencing and to evaluate its potential value as a biomarker for the cancer of the bile duct, pancreas, and gallbladder. RESULTS: We enrolled patients who were diagnosed with cancer, cystic lesions, and inflammation of the pancreaticobiliary tract. The study cohort comprised 244 patients. We extracted microbiome-derived DNA from the bile juice in surgically resected gallbladders. The microbiome composition was not significantly different according to lesion position and cancer type in terms of alpha and beta diversity. We found a significant difference in the relative abundance of Campylobacter, Citrobacter, Leptotrichia, Enterobacter, Hungatella, Mycolicibacterium, Phyllobacterium and Sphingomonas between patients without and with lymph node metastasis. CONCLUSIONS: There was a significant association between the relative abundance of certain microbes and overall survival prognosis. These microbes showed association with good prognosis in cholangiocarcinoma, but with poor prognosis in pancreatic adenocarcinoma, and vice versa. Our findings suggest that pancreaticobiliary tract cancer patients have an altered microbiome composition, which might be a biomarker for distinguishing malignancy.

Genome Editing Using Cas9 Ribonucleoprotein Is Effective for Introducing PDGFRA Variant in Cultured Human Glioblastoma Cell Lines.

Many variants of uncertain significance (VUS) have been detected in clinical cancer cases using next-generation sequencing-based cancer gene panel analysis. One strategy for the elucidation of VUS is the functional analysis of cultured cancer cell lines that harbor targeted gene variants using genome editing. Genome editing is a powerful tool for creating desired gene alterations in cultured cancer cell lines. However, the efficiency of genome editing varies substantially among cell lines of interest. We performed comparative studies to determine the optimal editing conditions for the introduction of platelet-derived growth factor receptor alpha (PDGFRA) variants in human glioblastoma multiforme (GBM) cell lines. After monitoring the copy numbers of PDGFRA and the expression level of the PDGFRα protein, four GBM cell lines (U-251 MG, KNS-42, SF126, and YKG-1 cells) were selected for the study. To compare the editing efficiency in these GBM cell lines, the modes of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) delivery (plasmid vs. ribonucleoprotein (RNP)), methods of transfection (lipofection vs. electroporation), and usefulness of cell sorting were then evaluated. Herein, we demonstrated that electroporation-mediated transfer of Cas9 with single-guide RNA (Cas9 RNP complex) could sufficiently edit a target nucleotide substitution, irrespective of cell sorting. As the Cas9 RNP complex method showed a higher editing efficiency than the Cas9 plasmid lipofection method, it was the optimal method for single-nucleotide editing in human GBM cell lines under our experimental conditions.

A tailored next-generation sequencing panel identified distinct subtypes of wildtype IDH and TERT promoter glioblastomas.

Central nervous system tumors are classified based on an integrated diagnosis combining histology and molecular characteristics, including IDH1/2 and H3-K27M mutations, as well as 1p/19q codeletion. Here, we aimed to develop and assess the feasibility of a glioma-tailored 48-gene next-generation sequencing (NGS) panel for integrated glioma diagnosis. We designed a glioma-tailored 48-gene NGS panel for detecting 1p/19q codeletion and mutations in IDH1/2, TP53, PTEN, PDGFRA, NF1, RB1, CDKN2A/B, CDK4, and the TERT promoter (TERTp). We analyzed 106 glioma patients (grade II: 19 cases, grade III: 23 cases, grade IV: 64 cases) using this system. The 1p/19q codeletion was detected precisely in oligodendroglial tumors using our NGS panel. In a cohort of 64 grade Ⅳ gliomas, we identified 56 IDH-wildtype glioblastomas. Within these IDH-wildtype glioblastomas, 33 samples (58.9%) showed a mutation in TERTp. Notably, PDGFRA mutations and their amplification were more commonly seen in TERTp-wildtype glioblastomas (43%) than in TERTp-mutant glioblastomas (6%) (P = .001). Hierarchical molecular classification of IDH-wildtype glioblastomas revealed 3 distinct groups of IDH-wildtype glioblastomas. One major cluster was characterized by mutations in PDGFRA, amplification of CDK4 and PDGFRA, homozygous deletion of CDKN2A/B, and absence of TERTp mutations. This cluster was significantly associated with older age (P = .021), higher Ki-67 score (P = .007), poor prognosis (P = .012), and a periventricular tumor location. We report the development of a glioma-tailored NGS panel for detecting 1p/19q codeletion and driver gene mutations on a single platform. Our panel identified distinct subtypes of IDH- and TERTp-wildtype glioblastomas with frequent PDGFRA alterations.

Methylation-Specific Electrophoresis.

Methylation of CpG sites in the promoter region of genomic DNA is an important epigenetic modification that plays a critical role in gene regulation, particularly in gene silencing. Epigenetic abnormalities, along with genetic alterations, are implicated in carcinogenesis and cancer progression. Numerous studies have investigated the role of epigenetics in cancer using various tools to assess DNA methylation. However, conventional analysis methods for DNA methylation require a large amount of DNA but lack higher sensitivity, making them unsuitable for analysis of samples with high heterogeneity, such as tumor tissues. In this study, we introduce a novel electrophoresis method named "methylation-specific electrophoresis (MSE)," which utilizes a denaturing gradient acrylamide gel. We demonstrate the applicability of the MSE method for DNA methylation analysis of the mucin gene as an example.

Distribution and favorable prognostic implication of genomic EGFR alterations in IDH-wildtype glioblastoma.

BACKGROUND: We aimed to evaluate the mutation profile, transcriptional variants, and prognostic impact of the epidermal growth factor receptor (EGFR) gene in isocitrate dehydrogenase (IDH)-wildtype glioblastomas (GBMs). METHODS: We sequenced EGFR, evaluated the EGFR splicing profile using a next-generation sequencing oncopanel, and analyzed the outcomes in 138 grade IV IDH-wildtype GBM cases. RESULTS: EGFR mutations were observed in 10% of GBMs. A total of 23.9% of the GBMs showed EGFR amplification. Moreover, 25% of the EGFR mutations occurred in the kinase domain. Notably, EGFR alterations were a predictor of good prognosis (p = 0.035). GBM with EGFR alterations was associated with higher Karnofsky Performance Scale scores (p = 0.014) and lower Ki-67 scores (p = 0.005) than GBM without EGFR alterations. EGFRvIII positivity was detected in 21% of EGFR-amplified GBMs. We identified two other EGFR variants in GBM cases with deletions of exons 6-7 (Δe 6-7) and exons 2-14 (Δe 2-14). In one case, the initial EGFRvIII mutation transformed into an EGFR Δe 2-14 mutation during recurrence. CONCLUSIONS: We found that the EGFR gene profiles of GBM differ among cohorts and that EGFR alterations are good prognostic markers of overall survival in patients with IDH-wildtype GBM. Additionally, we identified rare EGFR variants with longitudinal and temporal transformations of EGFRvIII.

An oncogenic splice variant of PDGFRα in adult glioblastoma as a therapeutic target for selective CDK4/6 inhibitors.

Understanding human genome alterations is necessary to optimize genome-based cancer therapeutics. However, some newly discovered mutations remain as variants of unknown significance (VUS). Here, the mutation c.1403A > G in exon 10 of the platelet-derived growth factor receptor-alpha (PDGFRA) gene, a VUS found in adult glioblastoma multiforme (GBM), was introduced in human embryonal kidney 293 T (HEK293T) cells using genome editing to investigate its potential oncogenic functions. Genome editing was performed using CRISPR/Cas9; the proliferation, drug sensitivity, and carcinogenic potential of genome-edited cells were investigated. We also investigated the mechanism underlying the observed phenotypes. Three GBM patients carrying the c.1403A > G mutation were studied to validate the in vitro results. The c.1403A > G mutation led to a splice variant (p.K455_N468delinsN) because of the generation of a 3'-acceptor splice site in exon 10. PDGFRA-mutated HEK293T cells exhibited a higher proliferative activity via PDGFRα and the cyclin-dependent kinase (CDK)4/CDK6-cyclin D1 signaling pathway in a ligand-independent manner. They showed higher sensitivity to multi-kinase, receptor tyrosine kinase, and CDK4/CDK6 inhibitors. Of the three GBM patients studied, two harbored the p.K455_N468delinsN splice variant. The splicing mutation c.1403A > G in PDGFRA is oncogenic in nature. Kinase inhibitors targeting PDGFRα and CDK4/CDK6 signaling should be evaluated for treating GBM patients harboring this mutation.

Prognostic impact of PDGFRA gain/amplification and MGMT promoter methylation status in patients with IDH wild-type glioblastoma.

Background: Platelet-derived growth factor receptor alpha (PDGFRA) is the second most frequently mutated tyrosine kinase receptor in glioblastoma (GBM). However, the prognostic impact of PDGFRA amplification on GBM patients remains unclear. Herein, we evaluated this impact by retrospectively analyzing outcomes of patients with IDH wild-type GBM. Methods: Using a custom-made oncopanel, we evaluated PDGFRA gain/amplification in 107 GBM samples harboring wild-type IDH, along with MGMT promoter (MGMTp) methylation status. Results: We detected PDGFRA gain/amplification in 31 samples (29.0%). PDGFRA gain/amplification predicted poor prognosis (P = .003). Compared to unamplified PDGFRA, PDGFRA gain/amplification in GBM was associated with higher patient age (P = .031), higher Ki-67 score (P = .019), and lower extent of surgical resection (P = .033). Unmethylated MGMTp also predicted poor prognosis (P = .005). As PDGFRA gain/amplification and unmethylated MGMTp were independent factors for poor prognosis in multivariate analyses, we grouped GBM cases based on PDGFRA and MGMTp status: poor (PDGFRA gain/amplification and unmethylated MGMTp), intermediate (PDGFRA gain/amplification or unmethylated MGMTp), and good (PDGFRA intact and methylated MGMTp) prognosis. The Kaplan-Meier survival analysis indicated that these groups significantly correlated with the OS of GBM patients (P < .001). Conclusions: Here we report that PDGFRA gain/amplification is a predictor of poor prognosis in IDH wild-type GBM. Combining PDGFRA gain/amplification with MGMTp methylation status improves individual prognosis prediction in patients with IDH wild-type GBM.

High Pathological Reproducibility of Diet-induced Atherosclerosis in Microminipigs via Cloning Technology.

BACKGROUND/AIM: The reproducibility of athero - sclerotic lesions was evaluated after the production of cloned-microminipigs and their offspring. MATERIALS AND METHODS: Cloned-microminipig-parents were produced by microminipigsomatic cell nuclei. These parents were crossbred and delivered males (F1-offspring) were divided into two groups: normal chow diet (NcD)-fed and high-fat/high-cholesterol diet (HcD)-fed groups. One of the F1-offsprings was subjected to cloning, and delivered males (F1-clones) were fed with HcD. After 8 weeks, all animals were necropsied for patho - physiological studies compared to non-cloned-microminipigs. RESULTS: HcD-fed F1-offspring and F1-clones, but not NcD-fed F1-offspring, exhibited increased serum lipid levels and systemic atherosclerosis, which were comparable to those of HcD-fed non-cloned-microminipigs. Homogeneity of variance analysis demonstrated that standard deviation values of serum lipoprotein and aortic atherosclerosis area from HcD-fed animals decreased in F1-offspring and F1-clones. CONCLUSION: HcD-induced atherogenesis was highly reproducible in F1-offsprings and F1-clones, indicating that the atherosclerosis-prone genomic background was preserved in the cloned-microminipigs, which can be used for studies on human atherosclerosis and related diseases.

Predicted Prognosis of Patients with Pancreatic Cancer by Machine Learning.

PURPOSE: Pancreatic cancer remains a disease of high mortality despite advanced diagnostic techniques. Mucins (MUC) play crucial roles in carcinogenesis and tumor invasion in pancreatic cancers. MUC1 and MUC4 expression are related to the aggressive behavior of human neoplasms and a poor patient outcome. In contrast, MUC2 is a tumor suppressor, and we have previously reported that MUC2 is a favorable prognostic factor in pancreatic neoplasia. This study investigates whether the methylation status of three mucin genes from postoperative tissue specimens from patients with pancreatic neoplasms could serve as a predictive biomarker for outcome after surgery. EXPERIMENTAL DESIGN: We evaluated the methylation status of MUC1, MUC2, and MUC4 promoter regions in pancreatic tissue samples from 191 patients with various pancreatic lesions using methylation-specific electrophoresis. Then, integrating these results and clinicopathologic features, we used support vector machine-, neural network-, and multinomial-based methods to develop a prognostic classifier. RESULTS: Significant differences were identified between the positive- and negative-prediction classifiers of patients in 5-year overall survival (OS) in the cross-validation test. Multivariate analysis revealed that these prognostic classifiers were independent prognostic factors analyzed by not only neoplastic tissues but also nonneoplastic tissues. These classifiers had higher predictive accuracy for OS than tumor size, lymph node metastasis, distant metastasis, and age and can complement the prognostic value of the TNM staging system. CONCLUSIONS: Analysis of epigenetic changes in mucin genes may be of diagnostic utility and one of the prognostic predictors for patients with pancreatic ductal adenocarcinoma.

The Association of Peroxiredoxin 4 with the Initiation and Progression of Hepatocellular Carcinoma.

AIMS: Peroxiredoxin 4 (PRDX4) is a member of the peroxiredoxin family of antioxidant enzymes. Previously, we reported that PRDX4 can restrain the initiation and progression of nonalcoholic steatohepatitis by reducing local and systemic reactive oxygen species (ROS) levels. Oxidative stress is recognized as a key factor in hepatocarcinogenesis, and a high ROS level has also been found in hepatocellular carcinoma (HCC). Here, our aim is to investigate roles of PRDX4 in the initiation and progression of HCC. RESULTS: In this study, for hepatocarcinogenesis, wild-type (WT), PRDX4 knockout (PRDX4-/y), and human PRDX4 transgenic (hPRDX4+/+) mice were given a weekly intraperitoneal injection of diethylnitrosamine for 25 weeks. The HCC incidence was higher in PRDX4-/y mice than in WT or hPRDX4+/+ mice. Intrahepatic and circulating oxidative stress and inflammatory cell infiltration in the liver were obviously decreased in hPRDX4+/+ mice, compared with WT mice. Furthermore, in our cohort study, human HCC specimens with low expression of PRDX4 had higher ROS levels and a highly malignant phenotype, which was associated with a reduced overall survival, compared with those with high PRDX4 expression. However, in human HCC cell lines, PRDX4 knockdown led to a rapidly increased intracellular ROS level and suppressed cell proliferation, inducing cell death. Innovation and Conclusion: Our results clearly indicate that PRDX4 has an inhibitory effect in the initiation of HCC, but a dual (inhibitory or promoting) role in the progression of HCC, suggesting the potential utility of PRDX4 activators or inhibitors as therapy for different stages and phenotypes of HCC.

Cholic Acid Enhances Visceral Adiposity, Atherosclerosis and Nonalcoholic Fatty Liver Disease in Microminipigs.

AIM: We have recently established a novel swine model for studies of atherosclerosis using MicrominipigsTM (µMPs) fed a high-fat/high-cholesterol diet (HcD). Using this swine model, we re-evaluated the effects of dietary cholic acid (CA) on serum lipid profile, atherosclerosis and hepatic injuries. METHODS: The µMPs were fed HcD supplemented with 0.7% CA (HcD＋CA) for eight weeks, and the effect of CA on serum lipoprotein levels, expression of oxidative stress markers, adiposity and lesion formation in the aorta, liver, and other organs was investigated. RESULTS: The HcD＋CA-fed group exhibited more visceral adiposity, progression of atherosclerosis and higher serum levels of oxidative stress markers than the HcD-fed group, even though they showed similar serum lipid levels. The liver demonstrated increased lipid accumulation, higher expression of oxidative stress markers, accelerated activation of foamy Kupffer cells and stellate cells, and increased hepatocyte apoptosis, indicating non-alcoholic fatty liver disease (NAFLD). Intriguingly, foamy macrophage mobilization was observed in various organs, including the reticuloendothelial system, pulmonary capillary vessels and skin very often in HcD＋CA-fed µMPs. CONCLUSION: To our knowledge, this is the first large animal model, in which visceral obesity, NAFLD and atherosclerosis are concomitantly induced by dietary manipulation. These data suggest the detrimental effects of CA, potentially through local and systemic activation of oxidative stress-induced signaling to macrophage mobilization, on the acceleration of visceral adiposity, atherosclerosis and NAFLD.

TET1-mediated DNA hypomethylation regulates the expression of MUC4 in lung cancer.

Lung cancer remains a disease of high mortality, despite advanced diagnostic techniques. Mucins (MUC) play crucial roles in carcinogenesis and tumor invasion in lung neoplasms. Our immunohistochemistry (IHC) studies have shown that high MUC4 expression correlates with a poor outcome. We have also shown that the expression of several mucin genes in cancer cell lines is regulated by DNA methylation. We evaluated the expression level of MUC4, mRNA and several DNA hypomethylation factors in lung tissue samples from 33 patients with various lung lesions. The results indicated that the DNA methylation status of MUC4 matched the expression level of mRNA. In addition, the TET1 (Ten-Eleven Translocation) mRNA showed a significant correlation with the status of DNA methylation of MUC4. Furthermore, the treatment of a lung cancer cell line with TET1 siRNA caused a reduction in MUC4 mRNA expression. Thus, we suggest that TET1 mediated DNA hypomethylation plays a key role in the expression of MUC4. This is the first report that TET1 mediated DNA hypomethylation regulates the expression of MUC4 in lung cancer. The analysis of these epigenetic changes may be useful for diagnosing carcinogenic risk.

Embryonic expression of a decapentaplegic gene in the oligochaete annelid Tubifex tubifex.

We have cloned and characterized the expression of a decapentaplegic homologue (designated Ttu-dpp) from the oligochaete annelid Tubifex tubifex. RT-PCR analysis and in situ hybridization revealed that Ttu-dpp begins to be expressed around the time of the onset of ectodermal germ band (GB) elongation (i.e., the onset of gastrulation). At this time, Ttu-dpp expression is detected in the anteriormost part of the GBs. As development proceeds and the GBs elongate, the domain of Ttu-dpp-expressing cells extends posteriorly. Then Ttu-dpp-expressing cells within the GB are divided into two groups: one group occurs along the ventral midline and coincides with the domain of ventral ganglia; the other is located more dorsally. The latter group of Ttu-dpp-expressing cells subsequently undergoes dorsalward expansion, which results in the formation of a lateral stripe of cells in every segment except the first (i.e., segment I). In embryos that undergo body elongation (that is one of the last morphogenetic movements occurring prior to hatchout), Ttu-dpp expression in the lateral region is confined to setal sacs, which are arranged in the same transverse plane around the periphery of each segment (except segment I).

Percutaneous sclerotherapy for intractable external biliary fistula after hepatectomy.

A 59-year-old man was diagnosed with cholangitis and a liver abscess caused by intrahepatic stones and underwent a hepatectomy of the left lobe and a side-to-side hepaticojejunostomy. After the operation, the patient developed an intractable external biliary fistula in the left remnant medial region. For the purpose of closing the fistula as a conservative treatment, an injection of 95% dehydrated ethanol was started after confirming the absence of any communication with the central bile duct; 1.5-5 ml was used for each injection, and the tube was clamped for 2 hours after injection. The excretion of bile juice decreased from the second injection, the excretion became serous, and the fistula completely closed after about 2 months without any particular complications. Percutaneous sclerotherapy by the injection of ethanol was found to be useful for closing a noncommunicating external biliary fistula.

Differential expression of caudal and dorsal genes in the teloblast lineages of the oligochaete annelid Tubifex tubifex.

We have cloned homologues (designated Ttu-cdx and Ttu-dl) of caudal and dorsal genes from the oligochaete annelid Tubifex tubifex. In situ hybridization revealed that Ttu-cdx begins to be expressed around the time of completion of ectodermal teloblastogenesis (i.e., the onset of gastrulation). At this time, Ttu-cdx expression is detected in a few cells that are located in the anteriormost part of the mesodermal germ bands (GBs). As development proceeds and the GBs elongate, the domain of Ttu-cdx-expressing cells in the GBs extends posteriorly. During this process, M teloblasts and primary blast cells remained negative for Ttu-cdx expression. This Ttu-cdx expression pattern is in sharp contrast to the posterior caudal expression in other organisms. Expression of Ttu-dl is detected from the one-cell stage through to the gastrula stage. Although Ttu-dl is expressed in most blastomeres, there are regional differences in Ttu-dl expression levels. During early cleavage, a large amount of Ttu-dl mRNA, which is supplied maternally, is inherited by D-cell line micromeres 2d and 4d; the remaining micromeres and macromeres inherit relatively small amounts of Ttu-dl transcripts. Another regional difference in Ttu-dl expression levels is seen in the ectodermal GB that is comprised of four bast-cell bandlets. The ventralmost bandlet (N lineage) exhibits the highest level of Ttu-dl expression with the lowest level in the two middle bandlets (O and P lineages) and an intermediate level in the dorsalmost bandlet (Q lineage).

Genetic alterations in gallbladder carcinoma.

Gallbladder carcinoma is an aggressive cancer associated with a poor prognosis. Unfortunately, the precise molecular mechanisms of development and progression of this highly malignant tumor remain unknown. It is still unclear whether loss of heterozygosity (LOH) plays a significant role in gallbladder carcinogenesis, but recent studies have found a high incidence of LOH at several chromosomes in gallbladder carcinoma. In particular, LOH on chromosomes 1p, 3p, 5p, 8p, 9p, 9q, 13q, 16q, and 17p has been highlighted and LOH on 3p, 9p, 13q, 16q, and 17p has been detected in preneoplastic lesions and in the early phase of gallbladder carcinoma during multistep carcinogenesis. The proto-oncogene, K-ras, is the best known genetic alteration in several human neoplasms, including gallbladder carcinoma. The accumulation of these genetic changes leads to a disruption in cell-cycle regulation and also continuous cell proliferation. We present an overview of K-ras alteration and LOH at several chromosome loci in gallbladder carcinoma. Further studies of the molecular mechanism in gallbladder carcinoma and the delineation of the genetic influence involved should promote our understanding of gallbladder carcinogenesis.

Loss of heterozygosity of chromosome 16q in gallbladder carcinoma.

BACKGROUND: The present study was planned to investigate cumulative genetic changes during development and progression of gallbladder carcinoma (GBC) in clinical patients. MATERIALS AND METHODS: We examined GBC DNA from resected tissue isolated from 56 cases of GBC for loss of heterozygosity (LOH) at six loci on five chromosomal arms (1p36, 9p21, 13q14, 16q24, 17p13), using highly polymorphic microsatellite markers. RESULTS: High incidences of LOH at 1p36 (19/36: 53%), 9p21 (12/32: 38%), 13q14 (20/36: 56%), 16q24 (31/54: 61%), and 17p13 (15/36: 42%) were detected. When comparing genetic features with clinicopathological stages of these tumors, it appeared that only LOH at 16q24 had a high incidence (5/6: 83%) at an early stage (T1a: tumor invades lamina propria) of the disease, although large numbers of LOH were found on all chromosomal arms in tumors of more advanced stages (T1b, T2, T3, and T4). CONCLUSION: These results suggested that the putative tumor suppressor gene on 16q24 may be strongly related to an early step of carcinogenesis in GBC and that GBC acquires a high malignant potential when the tumor invades the muscle layer.