Rapid Screening Using Pathomorphologic Interpretation to Detect BRAFV600E Mutation and Microsatellite Instability in Colorectal Cancer.

PURPOSE: Rapid decision-making is essential in precision medicine for initiating molecular targeted therapy for patients with cancer. This study aimed to extract pathomorphologic features that enable the accurate prediction of genetic abnormalities in cancer from hematoxylin and eosin images using deep learning (DL). EXPERIMENTAL DESIGN: A total of 1,657 images (one representative image per patient) of thin formalin-fixed, paraffin-embedded tissue sections from either primary or metastatic tumors with next-generation sequencing-confirmed genetic abnormalities-including BRAFV600E and KRAS mutations, and microsatellite instability high (MSI-H)-that are directly relevant to therapeutic strategies for advanced colorectal cancer were obtained from the nationwide SCRUM-Japan GI-SCREEN project. The images were divided into three groups of 986, 248, and 423 images to create one training and two validation cohorts, respectively. Pathomorphologic feature-prediction DL models were first developed on the basis of pathomorphologic features. Subsequently, gene-prediction DL models were constructed for all possible combinations of pathomorphologic features that enabled the prediction of gene abnormalities based on images filtered by the combination of pathomorphologic feature-prediction models. RESULTS: High accuracies were achieved, with AUCs > 0.90 and 0.80 for 12 and 27, respectively, of 33 analyzed pathomorphologic features, with high AUCs being yielded for both BRAFV600E (0.851 and 0.859) and MSI-H (0.923 and 0.862). CONCLUSIONS: These findings show that novel next-generation pathology methods can predict genetic abnormalities without the need for standard-of-care gene tests, and this novel next-generation pathology method can be applied for colorectal cancer treatment planning in the near future.

The survival impact of preoperative FOLFOX for resectable locally advanced rectal cancer: the R-NAC-01 study.

PURPOSE: In this follow-up of the R-NAC-01 study, we assessed the long-term oncological benefit of four courses of modified leucovorin, 5-fluorouracil (FU), and oxaliplatin (mFOLFOX6) chemotherapy before rectal surgery. METHODS: In this prospective, multicenter study (UMIN 000012559) involving 11 hospitals in Japan, patients with lower rectal cancer underwent four cycles of mFOLFOX6 chemotherapy and subsequent surgery within four to six weeks. The 3-year recurrence-free survival and local recurrence rates were then reported. RESULTS: Of 41 patients (36 males, 5 females; mean age: 60.8 years old) who received 4 courses of chemotherapy, 40 underwent total mesorectal excision, and 1 underwent total pelvic exenteration. R0 resection was achieved in 40 patients, but none showed a pathological complete response. Twenty-nine patients received adjuvant chemotherapy for an average of 4 months. The 3 year recurrence-free survival and local recurrence rates in patients undergoing curable resection were 72.8% and 8.5%, respectively. cStage III patients with adjuvant chemotherapy had a significantly higher 3 year recurrence-free survival than those without adjuvant chemotherapy (76.6 vs. 40.0%, log-rank p = 0.03). CONCLUSION: Four courses of mFOLFOX6 chemotherapy before surgery may be a promising treatment strategy for locally advanced rectal cancer. Adjuvant chemotherapy might be needed for cStage III patients, even after four courses of neoadjuvant mFOLFOX6.

Utility of double arterial cannulation for surgical repair of acute type A dissection.

OBJECTIVES: Outcomes of planned and unplanned (rescue) double arterial cannulation (DAC) in surgery for acute type A aortic dissection were investigated retrospectively. METHODS: The study involved 805 patients who were divided into 4 groups according to the cannulation strategy: single cannulation of the femoral artery (n = 338), axillary artery (n = 256), left ventricular apex (n = 52) or ascending aorta (n = 5) (total, n = 57), and DAC (n = 154). Patients who underwent DAC were divided between planned (n = 132) and rescue (n = 22) usage. Characteristics and outcomes were compared between groups. Both unmatched and propensity score-matched analyses were performed. RESULTS: Shock (39%, 19%, 33% and 14%, in the femoral artery, axillary artery, left ventricular apex/ascending aorta and DAC, respectively) and leg malperfusion (5%, 16%, 16% and 26%, respectively) differed significantly (P < 0.001), but in-hospital mortality did not (9%, 8%, 18% and 7%, respectively; P = 0.096). The 5-year survival rates were 79.4%, 79.7%, 78.6% and 82.2%, respectively. Propensity score-matched analysis showed no statistically significant differences in in-hospital mortality rates (10%, 12%, 14% and 9%, respectively; P = 0.78) and 5-year survival rates (78.4%, 72.3%, 82.3% and 78.0%, respectively). The leading vessel combination and indications for planned and rescue DAC were the femoral and axillary arteries (98%) and true lumen narrowing and/or leg malperfusion (34%), and the axillary followed by femoral (77%) artery and low cardiopulmonary bypass flow (36%). In-hospital mortality in the planned and rescue DAC groups was 7% and 9%, respectively. CONCLUSIONS: DAC seems effective for both prevention and management of intraoperative malperfusion.

Correction to: Short-Term Symptomatic Relief in Gastroesophageal Reflux Disease: A Comparative Study of Esomeprazole and Vonoprazan.

The original version of the article unfortunately contained percentage errors in second and third paragraphs of GerdQ Score section. Below is the corrected version.

Preoperative FOLFOX in resectable locally advanced rectal cancer can be a safe and promising strategy: the R-NAC-01 study.

PURPOSE: The aim of this study was to assess the safety of rectal surgery after 5-fluorouracil-leucovorin-oxaliplatin chemotherapy (FOLFOX6). METHODS: This was a prospective, multicenter study in 11 Japanese hospitals. We included patients with rectal cancer who received 4 courses of modified FOLFOX6 (mFOLFOX6) before rectal surgery and examined the postoperative complication rate, the clinicopathological response, and the rate of chemotherapy-related adverse events (UMIN 000012559). RESULTS: The study population included 36 men and 5 women. The average age of the patients was 60.8 years and the average body mass index was 23.1 kg/m2. After 4 courses of chemotherapy, grade 2 peripheral nerve disorder and other grade 3 adverse events were seen in 3 patients each (7.3%). Twenty-eight (73.7%) and 8 (21.1%) patients underwent low anterior resection and abdominoperineal resection, respectively. The pelvic nerves were preserved in 35 patients. Surgical morbidity (grade ≥ 3) occurred in 4 patients (10.5%). Anastomotic leakage occurred after surgery in 2 patients (7.1%). No patients achieved pathologically complete remission. However, downstaging of the clinical stage and N stage was seen in 17 (41.5%) and 22 (53.7%) patients, respectively. CONCLUSIONS: Surgery after four courses of mFOLFOX6 chemotherapy can be a safe and promising strategy for patients with locally advanced rectal cancer.

Short-Term Symptomatic Relief in Gastroesophageal Reflux Disease: A Comparative Study of Esomeprazole and Vonoprazan.

BACKGROUND AND AIM: Proton pump inhibitors and vonoprazan (a potassium-competitive acid blocker) are recommended as first-line treatments for gastroesophageal reflux disease (GERD). However, few reports have investigated the onset of action of these agents for GERD symptom relief. The present study compared the symptom relief of esomeprazole with that of vonoprazan via monitoring self-reported GERD symptoms after treatment initiation. METHODS: This was a prospective, multicenter, randomized, open-label, parallel group, comparative clinical study between esomeprazole (20 mg/day) and vonoprazan (20 mg/day) administered for 4 weeks to patients with GERD symptoms. Patients who had scores ≥ 8 on the Gastroesophageal Reflux Disease Questionnaire (GerdQ) were defined as having GERD and enrolled in this study. Sixty patients were randomly assigned to either the esomeprazole group (n = 30) or the vonoprazan group (n = 30). Treatment response rates in each drug group were evaluated according to the GerdQ. The Frequency Scale for the Symptoms of GERD (FSSG) scores from the 1st day after treatment initiation and the Global Overall Symptom (GOS) scale scores during treatment were also evaluated. RESULTS: At 4 weeks, the treatment response rates for symptom relief according to the GerdQ were 88.0% in the esomeprazole group and 81.8% in the vonoprazan group. The GOS scales, which reflect the impact of GERD symptoms, were similar for both groups. The FSSG scores decreased from the 1st to the 14th day in both groups. CONCLUSIONS: There were no substantial differences in the symptom relief between the two groups at any time point in this short-term study.

Detection and analysis of Lactobacillus paracasei penicillin-binding proteins revealed the presence of cholate-sensitive penicillin-binding protein 3 and an elongated cell shape in a cholate-sensitive strain.

Penicillin-binding proteins (PBPs) are responsible for peptidoglycan synthesis. By using biotinylated ampicillin, we detected PBPs of Lactobacillus paracasei strains. Ten PBPs were identified, 7 of which had apparent molecular sizes similar to those of Escherichia coli. In the presence of cholate, strain NRIC 0625 showed an elongated shape, and its putative PBP3 showed cholate-sensitive penicillin-binding activity. Furthermore, this strain was highly sensitive to cefalexin, which is known to inhibit cell division by inactivating PBP3. These results suggest that the septum synthetase PBP3 of lactic acid bacteria can be one of the targets of intestinal bile acid.

A novel transmembrane protein defines the endoplasmic reticulum stress-induced cell death pathway.

Mitochondrial membrane potential (ΔΨm) maintenance is physiologically critical in cells; its loss causes apoptotic signalling and cell death. Accumulating DNA mutations and unfolded proteins in stressed cells activate signalling pathways for cell death induction. Cancer cells often fail to die even in the presence of some death signalling proteins. Here, we report a short hairpin RNA (shRNA) with an artificial sequence, denoted Psi1 shRNA, which leads to ΔΨm loss in HCT116 cells. The Psi1 shRNA target gene was shown to encode transmembrane protein 117 (TMEM117). TMEM117 knockdown led to ΔΨm loss, increased reactive oxygen species levels, up-regulation of an endoplasmic reticulum (ER) stress sensor C/EBP homologous protein and active caspase-3 expression, and cell growth impairment, altering homeostasis towards cell death. TMEM117 levels were down-regulated in response to the ER stressor thapsigargin and decreased when cells showed ΔΨm loss. These results suggested that TMEM117 RNAi allowed apoptotic cell death. Therefore, TMEM117 probably mediates the signalling of ΔΨm loss in ER stress-mediated mitochondria-mediated cell death.

Comparative study: Vonoprazan and proton pump inhibitors in Helicobacter pylori eradication therapy.

AIM: To compare the effectiveness and safety of vonoprazan-based therapy with proton pump inhibitor (PPI)-based therapies to treat Helicobacter pylori (H. pylori). METHODS: We retrospectively analysed data from first-line (vonoprazan or PPI with 200 mg clarithromycin and 750 mg amoxicillin twice daily for 7 d) (n = 1353) and second-line (vonoprazan or PPI with 250 mg metronidazole and 750 mg amoxicillin twice daily for 7 d) (n = 261) eradication treatments for H. pylori -positive patients with associated gastrointestinal diseases from April 2014 to December 2015 at Hattori Clinic, Japan. The primary endpoint was the eradication rate, which was assessed with a full analysis set. The secondary endpoints were adverse events and related factors. RESULTS: After the first-line treatments, the eradication rates for vonoprazan, esomeprazol, rabeprazole, and lansoprazole were 87.9% (95%CI: 84.9%-90.5%), 71.6% (95%CI: 67.5%-75.5%), 62.9% (95%CI: 52.0%-72.9%), and 57.3% (95%CI: 50.4%-64.1%), respectively. The vonoprazan eradication rate was significantly higher than that of the PPIs (P < 0.01). Interestingly, smoking did not affect the H. pylori eradication rate in the vonoprazan group (P = 0.34), whereas it decreased the rates in the PPI groups (P = 0.013). The incidence of adverse events in the vonoprazan group was not different from the PPI group (P = 0.054), although the vonoprazan group exhibited a wider range of adverse events. Vonoprazan-based triple therapy was highly effective as a second-line treatment, with an eradication rate similar to that of PPI-based therapy. CONCLUSION: Vonoprazan might be superior to PPIs in first-line H. pylori therapy, particularly for smokers. However, caution is required due to possible adverse events.

shRNA target prediction informed by comprehensive enquiry (SPICE): a supporting system for high-throughput screening of shRNA library.

RNA interference (RNAi) screening is extensively used in the field of reverse genetics. RNAi libraries constructed using random oligonucleotides have made this technology affordable. However, the new methodology requires exploration of the RNAi target gene information after screening because the RNAi library includes non-natural sequences that are not found in genes. Here, we developed a web-based tool to support RNAi screening. The system performs short hairpin RNA (shRNA) target prediction that is informed by comprehensive enquiry (SPICE). SPICE automates several tasks that are laborious but indispensable to evaluate the shRNAs obtained by RNAi screening. SPICE has four main functions: (i) sequence identification of shRNA in the input sequence (the sequence might be obtained by sequencing clones in the RNAi library), (ii) searching the target genes in the database, (iii) demonstrating biological information obtained from the database, and (iv) preparation of search result files that can be utilized in a local personal computer (PC). Using this system, we demonstrated that genes targeted by random oligonucleotide-derived shRNAs were not different from those targeted by organism-specific shRNA. The system facilitates RNAi screening, which requires sequence analysis after screening. The SPICE web application is available at http://www.spice.sugysun.org/.

Draft Genome Sequence of Lactobacillus sucicola JCM 15457T, a Motile Lactic Acid Bacterium Isolated from Oak Sap.

Here, we report the draft genome sequence of a motile lactic acid bacterium, Lactobacillus sucicola JCM 15457(T), isolated from oak sap. Motility-related genes and their organization in the annotated genome were broadly similar to those in the sequenced genomes of related lactobacilli.

Study of prolonged administration of lanthanum carbonate in dialysis patients.

Data of 36 months were accumulated regarding the effects of lanthanum carbonate (LA) on serum phosphate concentrations in dialysis patients. Fifty-three patients (average age and dialysis history 58.4 years and 9.1 years) were included in this study who have been receiving outpatient treatment since March 2009, and who have been unable to maintain serum phosphate concentrations of ≤6.0 mg/dL via traditional therapeutic agents used for hyperphosphatemia. Patients were given dosage of LA in addition to, or instead of, co-hyperphosphatemia treatments already being received. Mean dosages of calcium carbonate (CC) and sevelamer hydrochloride (SH) before starting LA administration were 1301.9 mg and 2462.3 mg, respectively. Dosage of LA for all cases was 750 mg at initial dose; 1528.3 mg at 5 months; and 1416.7 mg at 30 months. Dosage of other phosphate binders were 905.7 mg of CC and 820.8 mg of SH at 5 months; and 687.5 mg of CC and 1031.3 mg of SH at 30 months. Serum phosphorus levels (P levels) were significantly decreased at 1 month of LA administration, and continued until 30 months of La treatment. These results suggest that LA successfully controlled serum P and Ca concentrations simultaneously within target ranges without affecting serum intact parathyroid hormone concentration, although further long-term prospective cohort study on LA would be required.

Proportion of flat- and depressed-type and laterally spreading tumor among advanced colorectal neoplasia.

BACKGROUND & AIMS: Flat- and depressed-type neoplasias along with laterally spreading tumors (LSTs) have been reported in colorectal neoplasias. We estimated the prevalence of flat and depressed types and LSTs along with their proportion among advanced neoplasias in a large average-risk population undergoing screening colonoscopy. METHODS: This was a cross-sectional study performed at a single, general community institution, with subjects who were 40 to 79 years old, asymptomatic, and who had undergone their first colonoscopy for screening between 2003 and 2009 (n = 4910). Among the neoplasias detected, advanced neoplasias were morphologically classified as the polypoid type, flat and depressed type, or LST. We determined the prevalence and proportion for each type among the advanced neoplasias, with morphologies defined according to the Japanese endoscopic classification. RESULTS: Advanced neoplasias were detected in 7.9% of men, 4.7% of women, and 6.1% of overall subjects. The polypoid type, the flat and depressed types, and the LSTs accounted for 75.3%, 7.5%, and 17.2% of advanced neoplasia, respectively. There was a high proportion of T1 cancers among the depressed types (40%). Approximately 80% of LSTs were located on the right side of the colon and more than 30% of LSTs showed high-grade dysplasia or T1 cancer. CONCLUSIONS: Most advanced neoplasias detected were of the polypoid type. LSTs accounted for a considerable proportion among advanced neoplasia and tended to be located on the right side of the colon. The influences of any LSTs need to be taken into consideration for preventing colorectal cancer.

SIMCOMP/SUBCOMP: chemical structure search servers for network analyses.

One of the greatest challenges in bioinformatics is to shed light on the relationship between genomic and chemical significances of metabolic pathways. Here, we demonstrate two types of chemical structure search servers: SIMCOMP (http://www.genome.jp/tools/simcomp/) for the chemical similarity search and SUBCOMP (http://www.genome.jp/tools/subcomp/) for the chemical substructure search, where both servers provide links to the KEGG PATHWAY and BRITE databases. The SIMCOMP is a graph-based method for searching the maximal common subgraph isomorphism by finding the maximal cliques in the association graph. In contrast, the SUBCOMP is an extended method for solving the subgraph isomorphism problem. The obtained links to PATHWAY or BRITE databases can be used to interpret as the biological meanings of chemical structures from the viewpoint of the various biological functions including metabolic networks.

PathPred: an enzyme-catalyzed metabolic pathway prediction server.

The KEGG RPAIR database is a collection of biochemical structure transformation patterns, called RDM patterns, and chemical structure alignments of substrate-product pairs (reactant pairs) in all known enzyme-catalyzed reactions taken from the Enzyme Nomenclature and the KEGG PATHWAY database. Here, we present PathPred (http://www.genome.jp/tools/pathpred/), a web-based server to predict plausible pathways of muti-step reactions starting from a query compound, based on the local RDM pattern match and the global chemical structure alignment against the reactant pair library. In this server, we focus on predicting pathways for microbial biodegradation of environmental compounds and biosynthesis of plant secondary metabolites, which correspond to characteristic RDM patterns in 947 and 1397 reactant pairs, respectively. The server provides transformed compounds and reference transformation patterns in each predicted reaction, and displays all predicted multi-step reaction pathways in a tree-shaped graph.

A therapeutic DNA vaccination strategy for autoimmunity and transplantation.

De novo autoimmunity induced by an allograft may play a significant role in chronic organ rejection, which remains a major barrier to successful transplantation. Accordingly, immunization with non-polymorphic antigens found in both donor allograft and recipient would be an attractive means to prevent long-term graft rejection, because it would rely on recipient mechanisms of immune homeostasis and could minimize the need to identify appropriate donor polymorphic antigens for induction of graft tolerance. Here we show that intradermal injection of plasmid DNA encoding glutamic acid decarboxylase (GAD) polypeptide, which is synthesized in both pancreatic islet and skin tissue, ameliorated new-onset type 1 diabetes in NOD mice and increased skin allograft survival in a BALB/c-C57BL/6 model system in a donor-specific manner. Successful therapy of autoimmune diabetes required CpG-methylation of plasmid DNA and co-delivery of a cDNA coding for the pro-apoptotic BAX protein, which was shown previously to induce Foxp3(+) regulatory T cells in NOD mice. In contrast, significantly increased skin allograft survival after immunization of recipient only required CpG-methylation of plasmid DNA coding for GAD alone. Injection of unmethylated plasmid DNA coding for BAX alone near the allograft also promoted graft survival, but induced a pro-inflammatory response to self-antigens. Our results reveal a promising potential for autoimmunity-targeting DNA vaccination to be applied to transplantation.

Reaction graph kernels predict EC numbers of unknown enzymatic reactions in plant secondary metabolism.

BACKGROUND: Understanding of secondary metabolic pathway in plant is essential for finding druggable candidate enzymes. However, there are many enzymes whose functions are not yet discovered in organism-specific metabolic pathways. Towards identifying the functions of those enzymes, assignment of EC numbers to the enzymatic reactions they catalyze plays a key role, since EC numbers represent the categorization of enzymes on one hand, and the categorization of enzymatic reactions on the other hand. RESULTS: We propose reaction graph kernels for automatically assigning EC numbers to unknown enzymatic reactions in a metabolic network. Reaction graph kernels compute similarity between two chemical reactions considering the similarity of chemical compounds in reaction and their relationships. In computational experiments based on the KEGG/REACTION database, our method successfully predicted the first three digits of the EC number with 83% accuracy. We also exhaustively predicted missing EC numbers in plant's secondary metabolism pathway. The prediction results of reaction graph kernels on 36 unknown enzymatic reactions are compared with an expert's knowledge. Using the same data for evaluation, we compared our method with E-zyme, and showed its ability to assign more number of accurate EC numbers. CONCLUSION: Reaction graph kernels are a new metric for comparing enzymatic reactions.

Characterizing common substructures of ligands for GPCR protein subfamilies.

The G-protein coupled receptor (GPCR) superfamily is the largest class of proteins with therapeutic value. More than 40% of present prescription drugs are GPCR ligands. The high therapeutic value of GPCR proteins and recent advancements in virtual screening methods gave rise to many virtual screening studies for GPCR ligands. However, in spite of vast amounts of research studying their functions and characteristics, 3D structures of most GPCRs are still unknown. This makes target-based virtual screenings of GPCR ligands extremely difficult, and successful virtual screening techniques rely heavily on ligand information. These virtual screening methods focus on specific features of ligands on GPCR protein level, and common features of ligands on higher levels of GPCR classification are yet to be studied. Here we extracted common substructures of GPCR ligands of GPCR protein subfamilies. We used the SIMCOMP, a graph-based chemical structure comparison program, and hierarchical clustering to reveal common substructures. We applied our method to 850 GPCR ligands and we found 53 common substructures covering 439 ligands. These substructures contribute to deeper understanding of structural features of GPCR ligands which can be used in new drug discovery methods.

Comprehensive genomic analysis of sulfur-relay pathway genes.

Many cofactors and nucleotides containing sulfur atoms are known to have important functions in a variety of organisms. Recently, the biosynthetic pathways of these sulfur containing compounds have been revealed, where many enzymes relay sulfur atoms. Increasing evidence also suggests that the prokaryotic sulfur-relay enzymes might be the evolutionary origin of ubiquitination and the related systems that control a wide range of physiological processes in eukaryotic cells. However, these sulfur-relay enzymes have been studied in only a small number of organisms. Here we carried out comparative genomic analysis and examined the presence and absence of sulfurtransferases utilized in the biosynthetic pathways of molybdenum cofactor (Moco), 2-thiouridine (S(2)U), and 4-thiouridine (S(4)U), and IscS, a cysteine desulfurase. We found that all eukaryotes and many other organisms lack the intermediate enzymes in S(2)U biosynthesis. It is also found that most genes lack rhodanese homology domain (RHD), a catalytic domain of sulfurtransferase. Some organisms have a conserved sequence composed of about 100 residues in the C terminus of TusA, different from RHD. Host-associated organisms have a tendency to lose Moco biosynthetic enzymes, and some organisms have MoaD-MoaE fusion protein. Our findings suggest that sulfur-relay pathways have been so diversified that some putative sulfurtransferases possibly function in other unknown pathways.

E-zyme: predicting potential EC numbers from the chemical transformation pattern of substrate-product pairs.

MOTIVATION: The IUBMB's Enzyme Nomenclature system, commonly known as the Enzyme Commission (EC) numbers, plays key roles in classifying enzymatic reactions and in linking the enzyme genes or proteins to reactions in metabolic pathways. There are numerous reactions known to be present in various pathways but without any official EC numbers, most of which have no hope to be given ones because of the lack of the published articles on enzyme assays. RESULTS: In this article we propose a new method to predict the potential EC numbers to given reactant pairs (substrates and products) or uncharacterized reactions, and a web-server named E-zyme as an application. This technology is based on our original biochemical transformation pattern which we call an 'RDM pattern', and consists of three steps: (i) graph alignment of a query reactant pair (substrates and products) for computing the query RDM pattern, (ii) multi-layered partial template matching by comparing the query RDM pattern with template patterns related with known EC numbers and (iii) weighted major voting scheme for selecting appropriate EC numbers. As the result, cross-validation experiments show that the proposed method achieves both high coverage and high prediction accuracy at a practical level, and consistently outperforms the previous method. AVAILABILITY: The E-zyme system is available at http://www.genome.jp/tools/e-zyme/.