Grade 3 Pancreatic Neuroendocrine Tumor Refractory to Chemotherapy Successfully Treated with Peptide Receptor Radionuclide Therapy Leading to Conversion Surgery.

A 59-year-old woman was diagnosed with a pancreatic neuroendocrine tumor (P-NET; Grade 3, Ki67: 25%) with multiple liver and lymph node metastases and started chemotherapy with streptozosin (500 mg/m2/day) in combination with lanreotide acetate (120 mg). After six courses of (daily) streptozosin, the patient had progressive disease, as assessed by computed tomography (CT), and peptide receptor radionuclide therapy (PRRT) was started as second-line treatment. As PRRT was remarkably successful and the tumor shrank, surgery was performed to resect the primary pancreatic tumor, liver metastases, and lymph node metastases. CT evaluation performed six months after the surgery showed a complete response.

Serum miR­29 is increased in mice with early liver fibrosis.

Non-alcoholic steatohepatitis (NASH) is a fatty liver disease that is not caused by alcohol consumption and is characterized by fatty degeneration, inflammation and hepatocellular damage. Therefore, predicting future fibrosis is critical in the early stages of NASH to prevent disease progression. The present study examined histological changes in the liver as well as microRNA (miR/miRNA) expression changes in the liver and serum of NASH mice model to identify potential biomarker candidates that could predict early fibrosis. This study used 6-week-old C57BL/6NJcl male mice and fed the control with a standard solid diet (CE-2) for breeding and propagation and NASH groups with a high-fat diet [choline-deficient high-fat and 0.1% (w/v) methionine supplemented diet], respectively. Agilent Technologies miRNA microarray was used to investigate microRNA expression in the liver and serum. Hematoxylin and eosin staining of the livers of the NASH group mice during the second week of feeding revealed fatty degeneration, balloon-like degeneration and inflammatory cell infiltration, confirming that the mice were in a state of NASH. The livers of the NASH group mice at 6 weeks of feeding showed fibrosis. Microarray analysis revealed that miRNAs were upregulated and 47 miRNAs were downregulated in the liver of the NASH group. Pathway analysis using OmicsNet predicted miR-29 to target collagen genes. Furthermore, miR-29 was downregulated in the livers of NASH-induced mice but upregulated in serum. These findings suggested that lower miR-29 expression in NASH-induced liver would increase collagen expression and fibrosis. Early liver fibrosis suggests that miR-29 leaks from the liver into the bloodstream, and elevated serum miR-29 levels may be a predictive biomarker for early liver fibrosis.

Examination and comparison of the RNA extraction methods using mouse serum.

Serum microRNAs (miRNAs) are considered useful as non-invasive biomarkers for different diseases. However, the optimal method for extracting RNAs from serum is currently unknown. In the present study, several RNA extraction kits were used to examine the optimal kit. RNAs were extracted from the serum of 8-week-old C57BL/6NJcl male mice following the protocol of each RNA extraction kit. The yield of the extracted RNA samples was calculated, and an Agilent Bioanalyzer was used to assess the electrophoretic patterns. An Agilent mouse miRNA microarray was utilized to confirm the expression patterns of the extracted RNA samples. The results revealed significant differences in RNA yields from the miRNeasy Serum/Plasma Advanced kit and mirVana™ PARIS™ RNA and Native Protein Purification Kit compared with almost all other samples. Further, two peaks were determined in the miRNeasy Serum/Plasma Advanced kit using a small RNAs kit of Agilent Bioanalyzer, including one at 20-40 nucleotides (nt) and another at ~40-100 nt, whereas the other reagents had a single peak. This revealed that the extracted RNAs may differ in composition based on the RNA extraction method. Some types of miRNAs were only detected with certain RNA extraction reagents. This suggested that different RNA extraction reagents may cause differences in the types of miRNAs detected. On the other hand, the miRNAs commonly expressed by the three RNA extraction reagents are highly correlated in expression levels.

Extracellular miRNAs in the serum and feces of mice exposed to high­dose radiation.

Exposure to high-dose radiation causes life-threatening intestinal damage. Histopathology is the most accurate method of judging the extent of intestinal damage following death. However, it is difficult to predict the extent of intestinal damage. The present study investigated extracellular microRNAs (miRNAs or miRs) in serum and feces using a radiation-induced intestinal injury mouse model. A peak of 25-200 nucleotide small RNAs was detected in mouse serum and feces by bioanalyzer, indicating the presence of miRNAs. Microarray analysis detected four miRNAs expressed in the small intestine and increased by >2-fold in serum and 19 in feces following 10 Gy radiation exposure. Increased miR-375-3p in both serum and feces suggests leakage due to radiation-induced intestinal injury and may be a candidate for high-dose radiation biomarkers.

Changes in cognitive ability and serum microRNA levels during aging in mice.

Mild cognitive impairment (MCI) is an early stage that can result in dementia. MCI can be reversed, and diagnosis at an early stage is crucial to control the progression to dementia. Dementia is currently diagnosed based on interviews and screening tests; however, novel biomarkers must be identified to allow early MCI detection. Therefore, the present study aimed to identify novel biomarkers in the form of blood microRNAs (miRNAs/miRs) for the diagnosis of MCI or early dementia. Blood samples were collected from C57BL/6NJcl male mice at four time points, including 4-week-old (4W), 8-week-old (8W), 36-week-old (36W) and 58-week-old (58W), and serum was isolated. Body weight and blood total cholesterol levels were increased, and blood alkaline phosphatase was decreased with aging. The 8W mice exhibited the highest cognitive ability in the Morris water maze test, whereas the 58W mice demonstrated decreased cognitive ability. The serum RNA concentrations of the 4W, 8W, 36W and 58W mice demonstrated no significant differences. Furthermore, small RNA levels were detected in the serum of all mice. miRNA microarray analysis revealed a >1.5-fold increase in the serum expression of two miRNAs (miR-21a-5p and miR-92a-3p) and a >1.5-fold decrease in the serum expression of two other miRNAs (miR-6769b-5p and miR-709) in 58W mice compared with those in 8W mice. In the future, we aim to further analyze aged mice to discover novel MCI biomarkers.

Analysis of gene expression changes during lipid droplet formation in HepG2 human liver cancer cells.

Fatty liver is a condition of excessive triglyceride accumulation in hepatocytes. Additionally, hepatocytes exhibit a high degree of fat droplet accumulation during excessive alcohol consumption and metabolic syndrome. However, the molecular mechanisms involved in fat droplet formation remain unknown. The present study used an in vitro fatty liver formation model of the human liver cancer cell line, HepG2, to comprehensively search for fat droplet formation-related genes, and which exhibit changes in expression during fat droplet formation. Microarray analysis with extracted total RNA determined the genes that are involved in fat droplet formation and their expression was confirmed using quantitative polymerase chain reaction following the culture of the HepG2 cells in culture medium containing 0, 50, 200 and 500 µM of oleic acid for 24 h. The results revealed 142 genes demonstrating increased expression levels by >2.0-fold with oleic acid treatment and 426 genes demonstrating decreased expression levels. Perilipin 2 (PLIN2) was estimated as the gene most closely associated with fatty liver. Lipid droplet formation in the HepG2 cells induced by oleic acid led to the upregulation of PLIN2 in a concentration-dependent manner. On the whole, the findings of the present study indicate the involvement of genes in oleic acid-induced lipid droplet formation in HepG2 cells; PLIN2 in particular may play a crucial role in this process.

Peroral cholangioscopy for the evaluation of bile duct stricture in hepatocellular carcinoma on a preoperative examination.

Objective: Bile duct tumor thrombosis in hepatocellular carcinoma (HCC) is a relatively rare event with a poor prognosis. Furthermore, bile duct tumor thrombus in HCC may be misdiagnosed when only imaging modalities are used. The efficiency of peroral cholangioscopy (POCS) in evaluating bile duct lesions has been reported. Patients: We present three cases of HCC with bile duct strictures in which POCS was performed as a preoperative evaluation. Results: In these three cases, diagnosing whether the lesion was a bile duct tumor thrombus on CT and endoscopic retrograde cholangiopancreatography was difficult. We performed POCS in three cases and were able to diagnose the presence of bile duct tumor thrombus of HCC, including differentiation from extrinsic compression of the bile duct. Conclusion: POCS for HCC with bile duct features is useful for the preoperative diagnosis of bile duct tumor thrombus, especially in cases where the surgical procedure depends on the presence of bile duct tumor thrombus.

A Preoperative Diagnosis of Advanced Cystic Duct Carcinoma Using SpyGlass DS Cholangioscopy: A Report of Two Cases.

Primary cystic duct carcinoma is a rare tumor. The curative treatment of cystic duct carcinoma is complete surgical resection, for which the evaluation of local extension is important. We herein report two cases of cystic duct carcinoma in which a preoperative examination was performed using per-oral cholangioscopy (POCS). Both patients underwent POCS due to suspicion of cystic duct carcinoma based on imaging findings. A visual analysis and biopsy were performed to evaluate local extension, which led to surgery. These cases suggest that POCS is useful for the preoperative assessment of local extension in advanced cystic duct carcinoma.

Multiomics and artificial intelligence enabled peripheral blood-based prediction of amnestic mild cognitive impairment.

BACKGROUND: Since dementia is preventable with early interventions, biomarkers that assist in diagnosing early stages of dementia, such as mild cognitive impairment (MCI), are urgently needed. METHODS: Multiomics analysis of amnestic MCI (aMCI) peripheral blood (n = 25) was performed covering the transcriptome, microRNA, proteome, and metabolome. Validation analysis for microRNAs was conducted in an independent cohort (n = 12). Artificial intelligence was used to identify the most important features for predicting aMCI. FINDINGS: We found that hsa-miR-4455 is the best biomarker in all omics analyses. The diagnostic index taking a ratio of hsa-miR-4455 to hsa-let-7b-3p predicted aMCI patients against healthy subjects with 97% overall accuracy. An integrated review of multiomics data suggested that a subset of T cells and the GCN (general control nonderepressible) pathway are associated with aMCI. INTERPRETATION: The multiomics approach has enabled aMCI biomarkers with high specificity and illuminated the accompanying changes in peripheral blood. Future large-scale studies are necessary to validate candidate biomarkers for clinical use.

Hepatic gene expression and functional changes associated with nonalcoholic steatohepatitis.

Non­alcoholic steatohepatitis (NASH) is a pathological condition of the liver in which hepatocyte steatosis, invasion of inflammatory cells and hepatic injury occur without alcohol abuse. Despite the known risk of liver cancer and liver fibrosis that may progress to liver cirrhosis that exists with NASH, an understanding of related gene expression and associated functional changes remains insufficient. The present study used a mouse model of NASH induced by a high­fat diet to examine gene expression in the liver and to search for transcripts that could predict early liver fibrosis in the future. Mice fed a high­fat diet for 2 weeks showed typical NASH liver histology by hematoxylin and eosin staining, and increased fibrosis was confirmed by Sirius red staining after 6 weeks. Functional changes associated with liver damage, liver inflammation, liver steatosis and liver fibrosis were predicted by toxicological ontology analysis using Ingenuity Pathways Analysis. Downregulated microRNA (miR)­21 and upregulated collagen type III α1 mRNA in the liver and upregulated exosomal miR­21 in serum of mice fed a high­fat diet for 1 and/or 2 weeks were confirmed by reverse transcription­quantitative PCR, suggesting that these changes occur prior to histological confirmation of fibrosis. Therefore, it may be possible to predict future liver fibrosis by analyzing fibrosis­related genes that shift prior to pathological findings.

Dose-dependent expression of extracellular microRNAs in HCT116 colorectal cancer cells exposed to high-dose-rate ionising radiation.

Biomarkers of tumour response to radiotherapy may help optimise cancer treatment. The aim of the present study was to identify changes in extracellular microRNAs (miRNAs) as a biomarker of radiation-induced damage to human colorectal cancer cells. HCT116 cells were exposed to increasing doses of X-rays, and extracellular miRNAs were analysed by microarray. The results were correlated with the frequency of micronuclei. A total of 59 miRNAs with a positive correlation and 4 with a negative correlation between dose (up to 6 Gy) and extracellular miRNA expression were identified. In addition, for doses between 0 and 10 Gy, 12 miRNAs among those 59 miRNAs with a positive correlation were identified; for these extracellular miRNAs, a significantly positive correlation was observed between their expression and the frequency of micronuclei for doses up to 10 Gy. These results suggest that specific miRNAs may be considered as cell damage markers and may serve as secreted radiotherapy response biomarkers for colorectal cancer; however, the results must be further validated in serum samples collected from patients undergoing radiotherapy.

Extremely Late Recurrence of Ovarian Carcinoma Diagnosed by an Endoscopic Ultrasound-guided Fine-needle Biopsy.

We herein report a case of recurrence of epithelial ovarian carcinoma 41 years after the primary surgery that was diagnosed by an endoscopic ultrasound-guided fine-needle biopsy (EUS-FNB). The differential diagnosis based on the imaging findings was difficult. We performed an EUS-FNB and compared the EUS-FNB specimen to the surgical specimen that had been resected in the primary surgery for ovarian carcinoma 41 years earlier, including immunohistochemical staining. Finally, we made a definitive diagnosis of extremely late recurrence of ovarian carcinoma of the retroperitoneum. An EUS-FNB enables an accurate histological diagnosis by obtaining a sample that is large enough to perform immunohistochemical staining.

HER2-positive breast cancer that resists therapeutic drugs and ionizing radiation releases sphingomyelin-based molecules to circulating blood serum.

Breast cancer is the second most common cancer in the world based on incidence, reaching more than 2 million new cases in 2018, while continuing to increase. Invasive ductal carcinoma is the most common type of this cancer, making up approximately 70-80% of all breast cancer diagnoses. In particular, the type of breast cancer overexpressing human epidermal growth factor receptor 2 (HER2) has potential of strong proliferation, migration and invasion and early treatment is necessary. The authors identified and studied a single patient displaying complete therapeutic resistance to monoclonal anti-HER2 antibody therapy, chemotherapy and radiotherapy. A patient who exhibited resistance to postoperative adjuvant therapy after mastectomy was selected from HER2-positive breast cancer, and this patient had the grade of T4bN2aM0, Stage IIIB. The patient samples, blood serum and cancer tissue, were analyzed by metabolome and immunostaining technique, respectively. The characteristics of peripheral blood serum and solid tumor were investigated, aiming to find new serum biomarker(s) using the metabolomics technique. A correlation between the appearance of HER2-positive cancer tissue and serum concentration of the sphingomyelin family was found. In addition, HER2-positive tumor tissue in both the primary and recurrent cancer express the sphingomyelinase. These results suggest that sphingomyelins from this cancer tissue leads to therapy resistance, induction of invasion and strong proliferation.

Sarcoidosis with Splenic Involvement Diagnosed with Endoscopic Ultrasound-guided Fine-needle Aspiration.

Splenic sarcoidosis is often diagnosed by splenectomy or an ultrasound-guided splenic biopsy. However, splenectomy is invasive and costly, and a percutaneous biopsy is sometimes difficult. We herein report a case of splenic sarcoidosis diagnosed with endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA). A 71-year-old man was referred to our hospital for abnormal shadows on a chest roentgenogram. Computed tomography showed multiple lesions in the spleen and pulmonary consolidations. Bronchoscopy revealed no definitive diagnosis. We therefore performed EUS-FNA for a splenic lesion that led to the diagnosis. This case suggests that EUS-FNA is useful in confirming the diagnosis of sarcoidosis with suspected splenic lesions.

Changes in miRNA expressions in the injured small intestine of mice following high­dose radiation exposure.

The small intestine is one of the most highly regenerative and radiosensitive tissues in mammals, including humans. Exposure to high doses of ionizing radiation causes serious intestinal damage. Recently, several investigations have been conducted using radioprotective agents to determine ways for reducing intestinal damage caused by radiation exposure. However, a thorough understanding of functional changes occurring in the small intestine of mice exposed to high­dose radiation is necessary for developing novel and more potent radioprotective agents. In this study, we examined changes in microRNA (miRNA/miR) expressions in the small intestine of mice at 72 h after X­ray exposure (10 Gy). We identified seven upregulated miRNAs and six downregulated miRNAs in the small intestine of mice following radiation exposure using miRNA microarray analysis. Particularly, miR­34a­5p was highly expressed, which was confirmed by reverse transcription-quantitative PCR. Forkhead box P1 (Foxp1) was predicted to be a target of the mRNA of miR­34a­5p using OmicsNet. Decreased Foxp1 expression in the small intestine following radiation exposure was confirmed, suggesting that Foxp1 expression recovery may induce the suppression of radiation­induced enteritis. Therefore, miR­34a­5p is a potential target molecule for developing novel radioprotective agents.

4-methylumbelliferone inhibits clonogenic potency by suppressing high molecular weight-hyaluronan in fibrosarcoma cells.

The inflammatory response is closely associated with cancer cell survival. It has been reported that inflammatory signaling cascades promote tumor survival and exert detrimental effects in normal tissue. Hyaluronans have different cellular functions depending on their molecular weights and high molecular weight-hyaluronan (HMW-HA) exhibits anti-inflammatory effects. A previous study determined that the co-administration of 4-methylumbelliferone (4-MU) and X-ray irradiation enhanced anti-tumor and anti-inflammatory effects in HT1080 human fibrosarcoma cells. However, many mechanisms underlie the effect of hyaluronan molecular weight on cells and the induction of anti-inflammatory effects via 4-MU. The present study aimed to determine the relationship between hyaluronan synthesis inhibition by 4-MU and its anti-inflammatory and radio-sensitizing effect in the context of hyaluronan molecular weight. The hyaluronan concentration following 2 Gy X-ray irradiation and/or 4-MU administration was analyzed via ELISA. Additionally, the mRNA expressions of hyaluronan synthase (HAS) by 4-MU and various inflammatory cytokines and interleukins (IL) following exogenous HMW-HA administration were evaluated via Reverse transcription-quantitative PCR. Invasive potential was assessed by matrigel transwell assays and cell survival following exposure to 4-MU with HMW-HA was determined using a clonogenic potency assay. The results of the present study demonstrated that 4-MU suppressed HMW-HA production by inhibiting HAS2 and HAS3 expression. In addition, the surviving fraction of fibrosarcoma cells were rescued from the cell-killing effect of 4-MU via the exogenous administration of HMW-HA. The mRNA levels of certain inflammatory cytokines, including IL-1α, IL-36γ and IL-37 were elevated following HMW-HA administration. The surviving fraction of cells irradiated with 2 Gy alone did not increase following exogenous HMW-HA administration. The results of the present study indicated that the radio-sensitizing effect of 4-MU and the inhibitory effect on hyaluronan synthesis were not closely associated. It was also revealed that IL-1α, IL-36γ and IL-37 were associated with the cell-killing effect of 4-MU in HT1080 cells.

Effects of voluntary and forced exercises on motor function recovery in intracerebral hemorrhage rats.

Motor paralysis is a severe consequence of intracerebral hemorrhage (ICH) that reduces patient quality of life. Rehabilitation is beneficial for stroke patients. However, functional recovery depends on the exercise type, and which factors are effective during rehabilitation are unknown. We aimed to clarify the effect of voluntary and forced exercises for functional recovery in ICH rats. Male Sprague-Dawley rats were divided into three groups: forced treadmill running (F-Ex.), voluntary wheel cage running (V-Ex.) and no exercise (Non-Ex.). The effects of the two exercises on motor recovery were analyzed by determining the motor deficit score and using the beam walking test. Stress and motivation status after rehabilitation were determined by corticosterone concentrations (ELISA) and immunoreactivity of ΔFosB (immunohistochemistry) in the nucleus accumbens, respectively. Significantly enhanced motor functional recovery was observed in the two trained groups compared with that in the Non-Ex. group. Of note, recovery in the V-Ex. group was greater than that in the F-Ex. group. To investigate the motivation and stress related to the exercises, the expression of ΔFosB in the nucleus accumbens and corticosterone concentration were compared after rehabilitation. In the V-Ex. group, there was a significant increase of ΔFosB, and in the F-Ex. Group, there was a high concentration of corticosterone. These data suggest that the effect of training for motor recovery was enhanced by motivation and reduced by stress.

Injured pancreatic ß cells enhance the release of miR-375-3p into the extracellular space.

miR-375-3p is a highly expressed microRNA in pancreatic ß cells. We have previously reported that when mice were exposed to 7 Gy X-ray irradiation, miR-375-3p was increased in the serum and there was cytotoxicity in pancreatic ß cells. However, it was unknown whether miR-375-3p is then released from injured pancreatic ß cells to the extracellular space. The present study investigated the effect of ionizing radiation and streptozotocin (STZ) treatment on the expression of extracellular miR-375-3p into culture supernatants using the rat pancreatic ß cell line RIN-5F. Cell growth was reduced, and cell death was increased at 24 h following exposure to 7 Gy irradiation as well as 24 h following treatment with 30 mM STZ compared with the control. Expression levels of miR-375-3p were significantly increased 24 h after 30 mM STZ treatment, yet this was only observed at 48 h following exposure to 7 Gy compared with the control. This suggests that the mechanism of cell death in RIN-5F is different between 7 Gy irradiation and 30 mM STZ treatment. The results of the present study suggest that injured pancreatic ß cells enhance the release of miR-375-3p from cells into extracellular space.

Regulation of radiosensitivity by 4-methylumbelliferone via the suppression of interleukin-1 in fibrosarcoma cells.

Tumor recurrence and distant metastasis following radiotherapy, which can lead to poor prognosis, are caused by residual cancer cells that acquire radioresistance. Chemotherapy or a combination of targeted inhibitors can potentially enhance radiation sensitivity and prevent metastasis. It was previously reported that co-administration of the hyaluronan synthesis inhibitor 4-methylumbelliferone (4-MU) enhanced the lethality of X-ray irradiation in HT1080 human fibrosarcoma cells and decreased their invasiveness to a greater extent than either treatment alone. To clarify the molecular basis of these effects, the present study conducted mRNA expression profiling by cDNA microarray to identify the signaling pathways that are altered under this combination treatment. The activation state of the signaling pathways was classified by z-scores in the Ingenuity Pathway Analysis. The results revealed that the pro-inflammatory cytokines interleukin (IL)-6 and IL-8 were activated by 2 Gy X-ray irradiation, an effect that was abolished by co-administration of 4-MU. Similar trends were observed for the upstream signaling component IL-1. These results indicate that the radiosensitivity of fibrosarcoma cells is improved by suppressing inflammation through the administration of 4-MU.