Molecular pathology quality control in Southeast Asia: Results of a multiregional quality assurance study from MASTER KEY Asia.

Tissue specimen quality assurance is a major issue of precision medicine for rare cancers. However, the laboratory standards and quality of pathological specimens prepared in Asian hospitals remain unknown. To understand the methods in Southeast Asian oncology hospitals and to clarify how pre-analytics affect the quality of formalin-fixed paraffin-embedded (FFPE) specimens, a questionnaire surveying pre-analytical procedures (Part I) was administered, quality assessment of immunohistochemistry (IHC) staining and DNA/RNA extracted from the representative FFPE specimens from each hospital (Part II) was conducted, and the quality of DNA/RNA extracted from FFPE of rare-cancer patients for genomic sequencing (Part III) was examined. Quality measurements for DNA/RNA included ΔΔCt, DV200, and cDNA yield. Six major cancer hospitals from Malaysia, Philippines, and Vietnam participated. One hospital showed unacceptable quality for the DNA/RNA assessment, but improved by revising laboratory procedures. Only 57% (n = 73) of the 128 rare-cancer patients' specimens met both DNA and RNA quality criteria for next-generation sequencing. Median DV200 was 80.7% and 64.3% for qualified and failed RNA, respectively. Median ΔΔCt was 1.25 for qualified and 4.89 for failed DNA. Longer storage period was significantly associated with poor DNA (fail to qualify ratio = 1579:321 days, p < 0.001) and RNA (fail to qualify ratio = 1070:280 days, p < 0.001). After improvement of pre-analytical factors, the qualification rate increased for hospitals A and E from 41.5% to 70.5% and 62.5% to 86%, respectively. This is the first report to elucidate the pre-analytical laboratory procedures of main Southeast Asian oncology hospitals. An external quality assessment program may improve factors associated with tumor FFPE specimen quality.

Combination of 1.1 mm flexible cryoprobe with conventional guide sheath and therapeutic bronchoscope in biopsy of apical upper lobe solitary pulmonary nodule.

BACKGROUND: Lung cancer is frequently situated peripherally in the upper lobes of the lung. Acquiring adequate tissue from this difficult-to-reach area remains a challenge. Transbronchial cryobiopsy (TBCB) has the ability to acquire larger specimens, but the rigidity of the standard 1.9 mm and 2.4 mm cryoprobes frequently poses challenges when used with a guide sheath (GS). The novel 1.1 mm cryoprobe, being both smaller and more flexible, may address this limitation. We describe the usage of this 1.1 mm flexible cryoprobe with GS in the biopsy of solitary pulmonary nodules (SPN) in the apical segment of the upper lobe in two cases. CASE REPORT: Both procedures were conducted with advanced airway under total intravenous anaesthesia. 2.6 mm GS was used in combination with a 2.2 mm rEBUS probe, using a therapeutic bronchoscope. Case 1 describes a SPN in the apical segment of the right upper lobe that was inconclusive by forceps biopsy due to GS displacement and inadequate biopsy depth. A steerable GS combined with the novel cryoprobe subsequently overcame this issue. Case 2 describes a SPN in the apical segment of the left upper lobe in which the standard cryoprobe failed to advance through the GS due to steep angulation. It also highlights with shorter activation time, the novel cryoprobe enable biopsied tissue to be retrieved through the GS while the bronchoscope-GS remains wedgend in the airway segment. There were no bleeding or pneumothorax complications in both cases, and histopathological examination confirmed adenocarcinoma of the lung. CONCLUSION: The 1.1 mm flexible cryoprobe in combination with GS and therapeutic bronchoscope offers an option to acquire adequate tissue in difficult-to-reach regions in the lung such as the apical segment of upper lobes. Further prospective series to evaluate its performance and safety in SPN biopsy is highly anticipated.

Peripheral transbronchial needle aspiration-guided pinpoint cryobiopsy of lung nodule without bronchus sign: A case report.

Peripheral transbronchial needle aspiration (pTBNA) allows the access of pulmonary nodules without bronchus sign but is limited to cytological examination. A 39-year-old man with left parotid carcinoma presented with an incidental lung nodule. Target localisation was performed with manual airway mapping, virtual bronchoscopic navigation, and pTBNA. Direct target validation using radial endobronchial ultrasound (rEBUS) was performed through the puncture defect. Targeted pinpoint biopsy with a 1.1 mm cryoprobe through the pTBNA puncture defect confirmed metastatic adenoid cystic carcinoma. Guided pTBNA with rEBUS validation followed by cryobiopsy of lung nodules without bronchus sign is potentially feasible for histological and molecular analyses.

Dentatin Induces Apoptosis in Prostate Cancer Cells via Bcl-2, Bcl-xL, Survivin Downregulation, Caspase-9, -3/7 Activation, and NF-κB Inhibition.

This study was set to investigate antiproliferative potential of dentatin (a natural coumarin isolated from Clausena excavata Burm. F) against prostate cancer and to delineate the underlying mechanism of action. Treatment with dentatin dose-dependently inhibited cell growth of PC-3 and LNCaP prostate cancer cell lines, whereas it showed less cytotoxic effects on normal prostate epithelial cell line (RWPE-1). The inhibitory effect of dentatin on prostate cancer cell growth was due to induction of apoptosis as evidenced by Annexin V staining and cell shrinkage. We found that dentatin-mediated accumulation of reactive oxygen species (ROS) and downregulated expression levels of antiapoptotic molecules (Bcl-2, Bcl-xL, and Survivin), leading to disruption of mitochondrial membrane potential (MMP), cell membrane permeability, and release of cytochrome c from the mitochondria into the cytosol. These effects were associated with induction of caspase-9, -3/7 activities, and subsequent DNA fragmentation. In addition, we found that dentatin inhibited TNF-α-induced nuclear translocation of p65, suggesting dentatin as a potential NF-κB inhibitor. Thus, we suggest that dentatin may have therapeutic value in prostate cancer treatment worthy of further development.

Development of a novel database to review and assess the clinical effects of EPA and DHA omega-3 fatty acids.

Due to their multiple mechanisms of biological action, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been the focus of ongoing active research for decades. In spite of the resulting body of knowledge, there remain significant gaps in our understanding of EPA/DHA health effects. Further, the volume of existing research makes it challenging to conduct systematic investigations to identify or resolve those gaps. The purpose of this article is to introduce the GOED Clinical Study Database (CSD), a comprehensive, manually-curated relational database that catalogs this research.

Risk-adjusted hospital clinical management issue rates using data from the Victorian Audit of Surgical Mortality.

BACKGROUND: In recent years, there has been a concerted drive for an increase in public reporting of hospital-level outcomes as a means of identifying strategies to improve patient safety. Surgical care, as a high-risk area of medical practice, has come under sharp scrutiny. This study uses data from the Victorian Audit of Surgical Mortality (VASM) in conjunction with data from the Victorian Admitted Episode Dataset to compare hospital rates of clinically identified serious clinical management issues that were definitely or probably preventable and caused or contributed to the death of the patient who would otherwise be expected to survive. METHODS: Cases where the date of death was between 1 July 2015 and 30 June 2017 that completed the full VASM audit process were extracted from the VASM database and combined with data extracted from the Victorian Admitted Episode Dataset, where a surgical admission occurred in the same time period. A logistic regression model was used as a method of indirect standardization to derive the probability of preventable clinical management issues, which was then used to calculate the standardized incident rate for all Victorian surgical hospitals. Hospitals were compared by plotting the standardized incident rates on three funnel plots. RESULTS: There were five hospitals (8.3%) of the 60 that deviated significantly from the state-wide rate of 0.00012. CONCLUSION: The risk adjustment model identified several hospitals that may have a systematic issue which warrant further clinical quality assurance investigation.

Chemical Compositional Changes in Over-Oxidized Fish Oils.

A recent study has reported that the administration during gestation of a highly rancid hoki liver oil, obtained by oxidation through sustained exposure to oxygen gas and incident light for 30 days, causes newborn mortality in rats. This effect was attributed to lipid hydroperoxides formed in the omega-3 long-chain polyunsaturated fatty acid-rich oil, while other chemical changes in the damaged oil were overlooked. In the present study, the oxidation condition employed to damage the hoki liver oil was replicated, and the extreme rancidity was confirmed. A detailed analysis of temporal chemical changes resulting from the sustained oxidative challenge involved measures of eicosapentaenoic acid/docosahexaenoic acid (EPA/DHA) omega-3 oil oxidative quality (peroxide value, para-anisidine value, total oxidation number, acid value, oligomers, antioxidant content, and induction time) as well as changes in fatty acid content, volatiles, isoprostanoids, and oxysterols. The chemical description was extended to refined anchovy oil, which is a more representative ingredient oil used in omega-3 finished products. The present study also analyzed the effects of a different oxidation method involving thermal exposure in the dark in contact with air, which is an oxidation condition that is more relevant to retail products. The two oils had different susceptibility to the oxidation conditions, resulting in distinct chemical oxidation signatures that were determined primarily by antioxidant protection as well as specific methodological aspects of the applied oxidative conditions. Unique isoprostanoids and oxysterols were formed in the over-oxidized fish oils, which are discussed in light of their potential biological activities.

Anti-ulcerogenic activity of dentatin from clausena excavata Burm.f. against ethanol-induced gastric ulcer in rats: Possible role of mucus and anti-oxidant effect.

BACKGROUND: Clausena excavata Burm.f. (Rutaceae) has been used for the treatment of stomach disorders including peptic ulcer. PURPOSE: In this study, we aimed to investigate dentatin isolated from C. excavata Burm.f., for anti-ulcer activity against ethanol ulcer model in rats. METHODS: Gastric acid output, ulcer index, serum profile, histological evaluation using Hematoxylin and eosin (HE), periodic acid Schiff base stainings and immunohistochemical localization for heat shock proteins 70 (HSP70) were all investigated. Possible involvement of reduced glutathione (GSH), lipid peroxidation, prostaglandin E2 (PGE2), superoxide dismutase (SOD) enzymes, radical scavenging, and anti-Helicobacter pylori activity were investigated. RESULTS: Dentatin showed anti-secretory activity against the pylorus ligature model and protected the gastric mucosa from ethanol ulceration, as revealed by the improved macroscopic and histological appearance. Dentatin significantly increased the gastric homogenate content of PGE2 GSH and SOD. Dentatin inhibited the lipid peroxidation as revealed by the reduced gastric content of malondialdehyde (MDA). Moreover, dentatin up-regulated HSP70 expression. However, dentatin showed insignificant anti-H. pylori activity. CONCLUSION: Dentatin possesses gastro-protective activity, which could be attributed to the anti-secretory, mucus production, anti-oxidant, and HSP70 activities.

Examination of marine and vegetable oil oxidation data from a multi-year, third-party database.

Fish oil (FO) products constitute good sources of omega-3 fats. Oxidation data from a large third-party database of 1900 + globally-sourced FO samples were assessed. In FO products, for peroxide value (PV), 13.9% exceeded 5 mEq O2/kg (2.2% >10); for acid value (AcV) 2.1% exceeded 3 mg KOH/g, while for p-anisidine value (pAV) in unflavoured oils, 6.1% exceeded 20, (3.8% >30), and 8.8% exceeded TOTOX limits (26). Additionally, we compared FO with other dietary oils. The FO median PV was similar to those of algal and sunflower oils, 4.8-fold greater than krill oil, and 5.2-fold less than extra-virgin olive oil. The median pAV differed non-significantly among oils. The FO median AcV was similar to those of algal and extra-virgin olive oils, 3.4-fold greater than sunflower oil, and 11.9-fold less than krill oil. This study has provided new insight that retail FO products predominantly meet regulatory guidelines and are comparable in oxidative status to other dietary oils.

Omega-3 Long-Chain Polyunsaturated Fatty Acid Content and Oxidation State of Fish Oil Supplements in New Zealand.

Forty-seven fish oil products available on the New Zealand market were analyzed for eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) content, as well as for oxidative status in a collaborative effort by several analytical laboratories. Of the tested products, 72%, 86% and 77% complied with voluntary industry-set maximum limits on Peroxide Value (PV), para-Anisidine Value (p-AV), and TOTOX, respectively. 91% of the products complied with EPA/DHA content claims. All fish oils complied with a p-AV limit of 30, 98% with a PV limit of 10 meq O2/kg, and 96% with a calculated TOTOX value of 50, which are less stringent limits used by the European and British Pharmacopeia and the Australian authorities. The results are in stark contrast to the very low percentage of fish oil products reported to be in compliance with primary oxidation limits and EPA/DHA content by a recently published assessment of fish oil supplements in New Zealand. Possible reasons for this discrepancy are evaluated and discussed.

Dentatin from Clausena excavata Induces Apoptosis in HepG2 Cells via Mitochondrial Mediated Signaling.

Hepatocellular carcinoma (HCC) is a primary liver cancer with high global incidence and mortality rates. Current candidate drugs to treat HCC remain lacking and those in use possess undesirable side effects. In this investigation, the antiproliferative effects of dentatin (DTN), a natural coumarin, were evaluated on HepG2 cells and DTN's probable preliminary molecular mechanisms in apoptosis induction were further investigated. DTN significantly (p<0.05) suppressed proliferation of HepG2 cells with an IC50 value of 12.0 µg/mL, without affecting human normal liver cells, WRL-68 (IC50>50 µg/mL) causing G0/G1 cell cycle arrest via apoptosis induction. Caspase colorimetric assays showed markedly increased levels of caspase-3 and caspase-9 activities throughout the treatment period. Western blotting of treated HepG2 cells revealed inhibition of NF-κB that triggers the mitochondrial-mediated apoptotic signaling pathway by up-regulating cytoplasmic cytochrome c and Bax, and down-regulating Bcl-2 and Bcl-xL. The current findings suggest DTN has the potential to be developed further as an anticancer compound targeting human HCC.

Considerations for incorporating eicosapentaenoic and docosahexaenoic omega-3 fatty acids into the military food supply chain.

The U.S. military may consider exploring the inclusion of the long-chain omega-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), in the diets of active duty military personnel. To be successful, certain challenges must be overcome including determining appropriate dosage, ensuring cost efficiency, and optimizing stability. To increase EPA and DHA intake, the military should consider using one of three strategies, including mandates or recommendations on omega-3 supplement usage, contracts to purchase commercially available foods for distribution in the food supply chain, or direct addition of EPA and DHA into currently consumed foods. This review presents the challenges and strategies and provides potential suggestions to the military to increase the likelihood of success.

α-Mangostin from Cratoxylum arborescens demonstrates apoptogenesis in MCF-7 with regulation of NF-κB and Hsp70 protein modulation in vitro, and tumor reduction in vivo.

Cratoxylum arborescens is an equatorial plant belonging to the family Guttiferae. In the current study, α-Mangostin (AM) was isolated and its cell death mechanism was studied. HCS was undertaken to detect the nuclear condensation, mitochondrial membrane potential, cell permeability, and the release of cytochrome c. An investigation for reactive oxygen species formation was conducted using fluorescent analysis. To determine the mechanism of cell death, human apoptosis proteome profiler assay was conducted. In addition, using immunofluorescence and immunoblotting, the levels of Bcl-2-associated X protein (Bax) and B-cell lymphoma (Bcl)-2 proteins were also tested. Caspaces such as 3/7, 8, and 9 were assessed during treatment. Using HCS and Western blot, the contribution of nuclear factor kappa-B (NF-κB) was investigated. AM had showed a selective cytotoxicity toward the cancer cells with no toxicity toward the normal cells even at 30 µg/mL, thereby indicating that AM has the attributes to induce cell death in tumor cells. The treatment of MCF-7 cells with AM prompted apoptosis with cell death-transducing signals. This regulated the mitochondrial membrane potential by down-regulation of Bcl-2 and up-regulation of Bax, thereby causing the release of cytochrome c from the mitochondria into the cytosol. The liberation of cytochrome c activated caspace-9, which, in turn, activated the downstream executioner caspace-3/7 with the cleaved poly (ADP-ribose) polymerase protein, thereby leading to apoptotic alterations. Increase of caspace 8 had showed the involvement of an extrinsic pathway. This type of apoptosis was suggested to occur through both extrinsic and intrinsic pathways and prevention of translocation of NF-κB from the cytoplasm to the nucleus. Our results revealed AM prompt apoptosis of MCF-7 cells through NF-κB, Bax/Bcl-2 and heat shock protein 70 modulation with the contribution of caspaces. Moreover, ingestion of AM at (30 and 60 mg/kg) significantly reduced tumor size in an animal model of breast cancer. Our results suggest that AM is a potentially useful agent for the treatment of breast cancer.

Dentatin isolated from Clausena excavata induces apoptosis in MCF-7 cells through the intrinsic pathway with involvement of NF-κB signalling and G0/G1 cell cycle arrest: a bioassay-guided approach.

ETHNOPHARMACOLOGICAL RELEVANCE: Clausena excavata Burm. f. has been used in folk medicines in eastern Thailand for the treatment of cancer. MATERIALS AND METHODS: To investigate the apoptosis mechanism, we isolated dentatin (DTN) from this plant using a bioassay-guided approach. DTN-induced cytotoxicity was observed with the MTT assay. Acridine orange/propidium iodide staining was used to detect cells in early apoptosis and high content screening (HCS) to observe nuclear condensation, cell permeability, mitochondrial membrane potential (MMP) and cytochrome c release. Apoptosis was confirmed with a clonogenic assay, DNA laddering and caspase 3/7 and 9 assays. Reactive oxygen species (ROS) formation, Bcl-2 and Bax expression, and cell cycle arrest were also investigated. The involvement of nuclear factor-kappa B (NF-κB) was analysed with the HCS assay. RESULTS: A significant increase in chromatin condensation in the cell nucleus was observed by fluorescence analysis. Apoptosis was confirmed by the reduced number of colonies in the clonogenic assay and the increased number of cellular DNA breaks in treated cells observed as a DNA ladder. Treatment of MCF-7 cells with DTN encouraged apoptosis with cell death-transducing signals that reduced MMP by down-regulation of Bcl-2 and up-regulation of Bax, triggering cytochrome c release from the mitochondria to the cytosol. The released cytochrome c triggered the activation of caspase 9 followed by the executioner caspase 3/7. DTN treatment significantly arrested MCF-7 cells at the G0/G1 phase (p<0.05) and ROS was significantly elevated. Moreover, DTN significantly blocked the induced translocation of NF-κB from cytoplasm to nucleus. CONCLUSION: Together, the results demonstrated that the DTN isolated from Clausena excavata inhibited the proliferation of MCF-7 cells, leading to cell cycle arrest and programmed cell death, which was confirmed to occur through the mitochondrial pathway with involvement of the NF-κB signalling pathway.

Inclusion Complex of Zerumbone with Hydroxypropyl- ß -Cyclodextrin Induces Apoptosis in Liver Hepatocellular HepG2 Cells via Caspase 8/BID Cleavage Switch and Modulating Bcl2/Bax Ratio.

Zerumbone (ZER) isolated from Zingiber zerumbet was previously encapsulated with hydroxypropyl- ß -cyclodextrin (HP ß CD) to enhance ZER's solubility in water, thus making it highly tolerable in the human body. The anticancer effects of this new ZER-HP ß CD inclusion complex via apoptosis cell death were assessed in this study for the first time in liver hepatocellular cells, HepG2. Apoptosis was ascertained by morphological study, nuclear stain, and sub-G1 cell population accumulation with G2/M arrest. Further investigations showed the release of cytochrome c and loss of mitochondrial membrane potential, proving mitochondrial dysfunction upon the ZER-HP ß CD treatment as well as modulating proapoptotic and anti-apototic Bcl-2 family members. A significant increase in caspase 3/7, caspase 9, and caspase 8 was detected with the depletion of BID cleaved by caspase 8. Collectively, these results prove that a highly soluble inclusion complex of ZER-HP ß CD could be a promising anticancer agent for the treatment of hepatocellular carcinoma in humans.

Induction of apoptosis in cancer cells by NiZn ferrite nanoparticles through mitochondrial cytochrome C release.

The long-term objective of the present study was to determine the ability of NiZn ferrite nanoparticles to kill cancer cells. NiZn ferrite nanoparticle suspensions were found to have an average hydrodynamic diameter, polydispersity index, and zeta potential of 254.2 ± 29.8 nm, 0.524 ± 0.013, and -60 ± 14 mV, respectively. We showed that NiZn ferrite nanoparticles had selective toxicity towards MCF-7, HepG2, and HT29 cells, with a lesser effect on normal MCF 10A cells. The quantity of Bcl-2, Bax, p53, and cytochrome C in the cell lines mentioned above was determined by colorimetric methods in order to clarify the mechanism of action of NiZn ferrite nanoparticles in the killing of cancer cells. Our results indicate that NiZn ferrite nanoparticles promote apoptosis in cancer cells via caspase-3 and caspase-9, downregulation of Bcl-2, and upregulation of Bax and p53, with cytochrome C translocation. There was a concomitant collapse of the mitochondrial membrane potential in these cancer cells when treated with NiZn ferrite nanoparticles. This study shows that NiZn ferrite nanoparticles induce glutathione depletion in cancer cells, which results in increased production of reactive oxygen species and eventually, death of cancer cells.