Targeted Next Generation Sequencing as a Reliable Diagnostic Assay for the Detection of Somatic Mutations in Tumours Using Minimal DNA Amounts from Formalin Fixed Paraffin Embedded Material

Background

Targeted Next Generation Sequencing (NGS) offers a way to implement testing of multiple genetic aberrations in diagnostic pathology practice, which is necessary for personalized cancer treatment. However, no standards regarding input material have been defined. This study therefore aimed to determine the effect of the type of input material (e.g. formalin fixed paraffin embedded (FFPE) versus fresh frozen (FF) tissue) on NGS derived results. Moreover, this study aimed to explore a standardized analysis pipeline to support consistent clinical decision-making.

Method

We used the Ion Torrent PGM sequencing platform in combination with the Ion AmpliSeq Cancer Hotspot Panel v2 to sequence frequently mutated regions in 50 cancer related genes, and validated the NGS detected variants in 250 FFPE samples using standard diagnostic assays. Next, 386 tumour samples were sequenced to explore the effect of input material on variant detection variables. For variant calling, Ion Torrent analysis software was supplemented with additional variant annotation and filtering.

Results

Both FFPE and FF tissue could be sequenced reliably with a sensitivity of 99.1%. Validation showed a 98.5% concordance between NGS and conventional sequencing techniques, where NGS provided both the advantage of low input DNA concentration and the detection of low-frequency variants. The reliability of mutation analysis could be further improved with manual inspection of sequence data.

Conclusion

Targeted NGS can be reliably implemented in cancer diagnostics using both FFPE and FF tissue when using appropriate analysis settings, even with low input DNA.     

Biochemical and Immunological Characterization of Truncated Fragments of the Receptor-Binding Domains of C. difficile Toxin A

Clostridium difficile is an emerging pathogen responsible for opportunistic infections in hospitals worldwide and is the main cause of antibiotic-associated pseudo-membranous colitis and diarrhea in humans. Clostridial toxins A and B (TcdA and TcdB) specifically bind to unknown glycoprotein(s) on the surface of epithelial cells in the host intestine, disrupting the intestinal barrier and ultimately leading to acute inflammation and diarrhea. The C-terminal receptor-binding domain (RBD) of TcdA, which is responsible for the initial binding of the toxin to host glycoproteins, has been predicted to contain 7 potential oligosaccharide-binding sites. To study the specific roles and functions of these 7 putative lectin-like binding regions, a consensus sequence of TcdA RBD derived from different C. difficile strains deposited in the NCBI protein database and three truncated fragments corresponding to the N-terminal (residues 1–411), middle (residues 296–701), and C-terminal portions (residues 524–911) of the RBD (F1, F2 and F3, respectively) were designed and expressed in Escherichia coli. In this study, the recombinant RBD (rRBD) and its truncated fragments were purified, characterized biologically and found to have the following similar properties: (a) are capable of binding to the cell surface of both Vero and Caco-2 cells; (b) possess Toll-like receptor agonist-like adjuvant activities that can activate dendritic cell maturation and increase the secretion of pro-inflammatory cytokines; and (c) function as potent adjuvants in the intramuscular immunization route to enhance immune responses against weak immunogens. Although F1, F2 and F3 have similar repetitive amino acid sequences and putative oligosaccharide-binding domains, they do not possess the same biological and immunological properties: (i) TcdA rRBD and its fragments bind to the cell surface, but only TcdA rRBD and F3 internalize into Vero cells within 15 min; (ii) the fragments exhibit various levels of hemagglutinin (HA) activity, with the exception of the F1 fragment, which demonstrates no HA activity; and (iii) in the presence of alum, all fragments elicit various levels of anti-toxin A-neutralizing antibody responses, but those neutralizing antibodies elicited by F2 did not protect mice against a TcdA challenge. Because TcdA rRBD, F1 and F3 formulated with alum can elicit immune protective responses against the cytotoxicity of TcdA, they represent potential components of future candidate vaccines against C. difficile-associated diseases.     

Residues 240–250 in the C-Terminus of the Pirh2 Protein Complement the Function of the RING Domain in Self-Ubiquitination of the Pirh2 Protein

Pirh2 is a p53 inducible gene that encodes a RING-H2 domain and is proposed to be a main regulator of p53 protein, thus fine tuning the DNA damage response. Pirh2 interacts physically with p53 and promotes its MDM2-independent ubiquitination and subsequent degradation as well as participates in an auto-regulatory feedback loop that controls p53 function. Pirh2 also self-ubiquitinates. Interestingly, Pirh2 is overexpressed in a wide range of human tumors. In this study, we investigated the domains and residues essential for Pirh2 self-ubiquitination. Deletions were made in each of the three major domains of Pirh2: the N-terminal domain (NTD), Ring domain (RING), and C-terminal domain (CTD). The effects of these deletions on Pirh2 self-ubiquitination were then assessed using in vitro ubiquitination assays. Our results demonstrate that the RING domain is essential, but not sufficient, for Pirh2 self-ubiquitination and that residues 240–250 of the C-terminal domain are also essential. Our results demonstrate that Pirh2 mediated p53 polyubiquitination occurs mainly through the K48 residue of ubiquitin in vitro. Our data further our understanding of the mechanism of Pirh2 self-ubiquitination and may help identify valuable therapeutic targets that play roles in reducing the effects of the overexpression of Pirh2, thus maximizing p53''s response to DNA damage.     

Purification, characterization, and gene analysis of cellulase (Cel8A) from Lysobacter sp. IB-9374

An enzyme that has both beta-1,4-glucanase and chitosanase activities was found in the culture medium of the soil bacterium Lysobacter sp. IB-9374, a high lysyl endopeptidase-producing strain. The enzyme was purified to homogeneity from the culture filtrate using five purification steps and designated Cel8A. The purified Cel8A had a molecular mass of 41 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A pH optimum of 5.0 was found for the beta-1,4-glucanase activity, and pH optima of 5.0 and 7.0 were found for the chitosanase activity. Nucleotide sequencing of the Cel8A gene yielded a deduced amino acid sequence that comprises a 33-amino acid, N-terminal signal peptide and a mature enzyme consisting of a 381-residue polypeptide with a predicted molecular mass of 41,241 Da. The amino acid sequence of the Cel8A, which contains the catalytic module of glycosyl hydrolase family 8, is homologous to beta-1,3-1,4-D-glucanase from Bacillus circulans WL-12 and endoglucanase N-257 from B. circulans KSM-N257.     

Ketoprofen resolution by enzymatic estirification and hydrolysis of the ester product

ImmobilizedCandida antarctica lipase was used to catalyze the separation of ketoprofen into its components by means of esterification followed by the enzymatic hydrolysis of the ester product. In this study, ketoprofen underwent esterification to ethanol in the presence of isooctane. When the reaction was complete, 58.3% of the ketoprofen had been transformed into an ester. The ketoprofen remaining in solution after the reation was complete consisted primarily of itsS-enantiomer (83.0%), while the 59.4% of the ketoprofen component of the ester consisted of itsR-enantiomer. We then subjected the ester product to enzymatic hydrolysis in the presence of the same enzyme and produced a ketoprofen product rich in theR-enantiomer; 77% of this product consisted of theR-enantiomer when 50% of the ester had been hydrolyzed, and 90% of it consisted of theR-enantiomer when 30% of the ester had been hydrolyzed. By contrast, theR-enantiomer levels only reached approximately 42 and 65%, respectively, when 50 and 30% of the racemic ester was hydrolyzed under the same conditions.     

Characterization of a hypertriglyceridemic transgenic miniature pig model expressing human apolipoprotein CIII

Hypertriglyceridemia has recently been considered to be an independent risk factor for coronary heart disease, in which apolipoprotein (Apo)CIII is one of the major contributory factors, as it is strongly correlated with plasma triglyceride levels. Although ApoCIII transgenic mice have been generated as an animal model for the study of hypertriglyceridemia, the features of lipoprotein metabolism in mice differ greatly from those in humans. Because of the great similarity between pigs and humans with respect to lipid metabolism and cardiovascular physiology, we generated transgenic miniature pigs expressing human ApoCIII by the transfection of somatic cells combined with nuclear transfer. The expression of human ApoCIII was detected in the liver and intestine of the transgenic pigs. As compared with nontransgenic controls, transgenic pigs showed significantly increased plasma triglyceride levels (83 ± 36 versus 38 ± 4 mg·dL(-1), P < 0.01) when fed a chow diet. Plasma lipoprotein profiling by FPLC in transgenic animals showed a higher peak in large-particle fractions corresponding to very low-density lipoprotein/chylomicrons when triglyceride content in the fractions was assayed. There was not much difference in cholesterol content in FPLC fractions, although a large low-density lipoprotein peak was identified in both nontransgenic and transgenic animals, resembling that found in humans. Further analysis revealed markedly delayed clearance of plasma triglyceride, accompanied by significantly reduced lipoprotein lipase activity in post-heparin plasma, in transgenic pigs as compared with nontransgenic controls. In summary, we have successfully generated a novel hypertriglyceridemic ApoCIII transgenic miniature pig model that could be of great value for studies on hyperlipidemia in relation to atherosclerotic disorders.     

Up‐regulation of ZFAS1 indicates dismal prognosis for cholangiocarcinoma and promotes proliferation and metastasis by modulating USF1 via miR‐296‐5p

LncRNAs has been demonstrated to modulate neoplastic development by modulating downstream miRNAs and functional genes. In this study, we aimed to detect the interaction among lncRNA ZFAS1 miR‐296‐5p and USF1. We explored the proliferation, migration and invasion of cholangiocarcinoma. The differentially expressed ZFAS1 was discovered in both tissues and cell lines by qRT‐PCR. The targeting relationship between miR‐296‐5p and ZFAS1 or USF1 was validated by dual‐luciferase assay. The impact of ZFAS1 on CCA cell proliferation was observed by CCK‐8 assay. The protein expression of USF1 was determined by Western blot. The effects of ZFAS1, miR‐296‐5p and USF1 on tumour growth were further confirmed using xenograft model. LncRNA ZFAS1 expression was relatively up‐regulated in tumour tissues and cells while miR‐296‐5p was significantly down‐regulated. Knockdown of ZFAS1 significantly suppressed tumour proliferation, migration, invasion and USF1 expression. Overexpressed miR‐296‐5p suppressed cell proliferation and metastasis. Knockdown of USF1 inhibited cell proliferation and metastasis and xenograft tumour growth. In conclusion, ZFAS1 might promote cholangiocarcinoma proliferation and metastasis by modulating USF1 via miR‐296‐5p.