Transanal versus conventional total mesorectal excision for rectal cancer using the IDEAL framework for implementation.

BACKGROUND: Transanal total mesorectal excision (TaTME) is an innovative technique for distal rectal cancer dissection. It has been shown to have similar short-term outcomes to conventional open and laparoscopic total mesorectal excision (cTME), but recent studies have raised concern about increased morbidity and local recurrence rates. The aim of this study was to assess outcomes after TaTME versus cTME for rectal cancer. METHODS: TaTME was implemented in 2014 using IDEAL principles in a single institution. The institution maintains databases for all patients undergoing rectal cancer surgery. This retrospective review compared data collected from all patients who had TaTME with those from a propensity-matched cohort of patients who underwent cTME. The primary outcome was a composite pathological measure combining margin status and quality of total mesorectal excision (TME). Short-term clinical and survival outcomes were also measured. RESULTS: Propensity matching created 109 matched pairs for analysis. Nine patients (8.3 per cent) undergoing TaTME had positive margins and/or incomplete TME, compared with 11 (10.5 per cent) undergoing cTME (P = 0.65). There were no significant differences in morbidity between the TaTME and cTME groups, including number of anastomotic leaks (13.8 versus 18.3 per cent; P = 0.37). The estimated 3-year local recurrence-free survival rate was 96.3 per cent in both groups (P = 0.39). Estimated 3-year overall (93.6 per cent for TaTME versus 94.5 per cent for cTME; P = 0.09) and disease-free (88.1 versus 76.1 per cent; P = 0.90) survival rates were similar. CONCLUSION: TaTME provided similar outcomes to cTME for rectal cancer with the application of IDEAL principles.

Health and quality of life among a cohort of patients having lateral internal sphincterotomy for anal fissures.

AIM: The aim of this study is to report changes in health-related quality of life attributable to lateral internal sphincterotomy for treatment of anal fissure. There is very little evidence on whether the overall health-related quality of life of patients is detrimentally affected by the condition, or which aspects of self-perceived health status improve after lateral internal sphincterotomy. This study will articulate which aspects of health tend to improve and guide postoperative expectations appropriately. Knowledge gained from this study may also identify gaps in an individual patient's episode of care. METHOD: Patients were prospectively identified when they consented to surgical treatment of their anal fissure and were contacted by phone to participate. Participants completed a number of patient-reported outcomes preoperatively and 6 months postoperatively. Faecal incontinence-related quality of life, pain and depression were measured at both time points. The severity of faecal incontinence was measured at both times. RESULTS: Participants reported high levels of pain preoperatively. Postoperatively, improvement in pain exceeded the threshold of clinical relevance (P < 0.01). Thirty-five per cent of participants reported significant effects of faecal incontinence preoperatively, while 26% did so postoperatively. Participants with multiple comorbidities were more likely to report faecal incontinence postoperatively than preoperatively. CONCLUSION: This study reports that lateral internal sphincterotomy improved pain symptoms without adverse effects on continence. Not all domains of health-related quality of life were similarly positively affected by anal fissure repair.

Transanal endoscopic microsurgery as day surgery - a single-centre experience with 500 patients.

AIM: Transanal endoscopic microsurgery (TEM) is the current treatment of choice for rectal adenomas and early rectal cancer. Postoperative admission to hospital is common but possibly unnecessary. Our objective was to analyse predictors and outcomes of TEM patients having same day discharge (TEM-D) compared with those who were admitted to hospital (TEM-A). METHOD: At St Paul's Hospital (SPH), demographic, surgical, pathological and follow-up data have been collected prospectively since TEM was started in 2007. Trends in admission and readmission rates were analysed using the Cochran-Armitage trend test, and predictors of admission were analysed using univariate and multivariate logistic regressions. RESULTS: Between 2007 and 2016, 500 patients were treated by TEM at SPH. The overall admission rate was 29% (145/500), but this decreased to 19% in the last 3 years of the study (P < 0.001). The readmission rate was 5.2% (n = 26/500) and did not change significantly over the study period (P = 0.30). Reasons for admission included the following: surgeon discretion/monitoring (35%), urinary retention (26%), haemorrhage (10%), breach of peritoneal cavity (7%), infection (7%) and other (15%). The most common reasons for readmission were haemorrhage (54%, n = 14), pain (19%, n = 5) and infection (12%, n = 3). Factors associated with admission were as follows: tumour height (OR 1.09, 1.02-1.17), prolonged operative time (OR 1.25, 1.14-1.37), unsutured surgical defect (OR 1.99, 1.22-3.25) and surgeon experience (OR 4.62, 2.75-7.77). CONCLUSION: Outpatient TEM is safe and carries a low risk of readmission. In centres with an outpatient TEM strategy, predictors of hospital admission include proximal tumours, prolonged surgical time and open management of the surgical defect.

Study protocol evaluating the use of bowel stimulation before loop ileostomy closure to reduce postoperative ileus: a multicenter randomized controlled trial.

AIM: Postoperative ileus is the most commonly observed morbidity following ileostomy closure. Studies have demonstrated that the defunctionalized bowel of a loop ileostomy undergoes a series of functional and structural changes, such as atrophy of the intestinal villi and muscular layers, which may contribute to ileus. A single-centre study in Spain demonstrated that preoperative bowel stimulation via the distal limb of the loop ileostomy decreased postoperative ileus, length of stay and time to gastrointestinal function. METHOD: A multicentre randomized controlled trial involving patients from Canadian institutions was designed to evaluate the effect of preoperative bowel stimulation before ileostomy closure on postoperative ileus. Stimulation will include canalizing the distal limb of the ileostomy loop with an 18Fr Foley catheter and infusing it with a solution of 500 ml of normal saline mixed with 30 g of a thickening agent (Nestle© Thicken-Up© ). This will be performed 10 times over the 3 weeks before ileostomy closure in an outpatient clinic setting by a trained Enterostomal Therapy nurse. Surgeons and the treating surgical team will be blinded to their patient's group allocation. Data regarding patient demographics, and operative and postoperative variables, will be collected prospectively. Primary outcome will be postoperative ileus, defined as an intolerance to oral food in the absence of clinical or radiological signs of obstruction, that either requires nasogastric tube insertion or is associated with two of the following on or after post-operative day 3: nausea/vomiting; abdominal distension; and the absence of flatus. Secondary outcomes will include length of stay, time to tolerating a regular diet, time to first passage of flatus or stool and overall morbidity. A cost analysis will be performed to compare the costs of conventional care with conventional care plus preoperative stimulation. DISCUSSION: This manuscript discusses the potential benefits of preoperative bowel stimulation in improving postoperative outcomes and outlines our protocol for the first multicenter study to evaluate preoperative bowel stimulation before ileostomy closure. The results of this study could have considerable implications for the care of patients undergoing ileostomy closure.

Transcriptome analysis of Inbred Long Sleep and Inbred Short Sleep mice.

Many studies have utilized the Inbred Long Sleep and Inbred Short Sleep mouse strains to model the genetic influence on initial sensitivity to ethanol. The mechanisms underlying this divergent phenotype are still not completely understood. In this study, we attempt to identify genes that are differentially expressed between these two strains and to identify baseline networks of co-expressed genes, which may provide insight regarding their phenotypic differences. We examined the whole brain and striatal transcriptomes of both strains, using next generation RNA sequencing techniques. Many genes were differentially expressed between strains, including several in chromosomal regions previously shown to influence initial sensitivity to ethanol. These results are in concordance with a similar sample of striatal transcriptomes measured using microarrays. In addition to the higher dynamic range, RNA-Seq is not hindered by high background noise or polymorphisms in probesets as with microarray technology, and we are able to analyze exome sequence of abundant genes. Furthermore, utilizing Weighted Gene Co-expression Network Analysis, we identified several modules of co-expressed genes corresponding to strain differences. Several candidate genes were identified, including protein phosphatase 1 regulatory unit 1b (Ppp1r1b), prodynorphin (Pdyn), proenkephalin (Penk), ras association (RalGDS/AF-6) domain family member 2 (Rassf2), myosin 1d (Myo1d) and transthyretin (Ttr). In addition, we propose a role for potassium channel activity as well as map kinase signaling in the observed phenotypic differences between the two strains.

Molecular profiling reveals prognostically significant subtypes of canine lymphoma.

We performed genomewide gene expression analysis of 35 samples representing 6 common histologic subtypes of canine lymphoma and bioinformatics analyses to define their molecular characteristics. Three major groups were defined on the basis of gene expression profiles: (1) low-grade T-cell lymphoma, composed entirely by T-zone lymphoma; (2) high-grade T-cell lymphoma, consisting of lymphoblastic T-cell lymphoma and peripheral T-cell lymphoma not otherwise specified; and (3) B-cell lymphoma, consisting of marginal B-cell lymphoma, diffuse large B-cell lymphoma, and Burkitt lymphoma. Interspecies comparative analyses of gene expression profiles also showed that marginal B-cell lymphoma and diffuse large B-cell lymphoma in dogs and humans might represent a continuum of disease with similar drivers. The classification of these diverse tumors into 3 subgroups was prognostically significant, as the groups were directly correlated with event-free survival. Finally, we developed a benchtop diagnostic test based on expression of 4 genes that can robustly classify canine lymphomas into one of these 3 subgroups, enabling a direct clinical application for our results.

Trajectory clustering: a non-parametric method for grouping gene expression time courses, with applications to mammary development.

Trajectory clustering is a novel and statistically well-founded method for clustering time series data from gene expression arrays. Trajectory clustering uses non-parametric statistics and is hence not sensitive to the particular distributions underlying gene expression data. Each cluster is clearly defined in terms of direction of change of expression for successive time points (its 'trajectory'), and therefore has easily appreciated biological meaning. Applying the method to a dataset from mouse mammary gland development, we demonstrate that it produces different clusters than Hierarchical, K-means, and Jackknife clustering methods, even when those methods are applied to differences between successive time points. Compared to all of the other methods, trajectory clustering was better able to match a manual clustering by a domain expert, and was better able to cluster groups of genes with known related functions.

Hormone interactions to Leu-rich repeats in the gonadotropin receptors. III. Photoaffinity labeling of human chorionic gonadotropin with receptor Leu-rich repeat 4 peptide.

Human chorionic gonadotropin (hCG) binds to the extracellular N-terminal domain, exodomain, of its receptor, and the resulting hCG-exodomain complex is thought to modulate the membrane associated domain, endodomain, of the receptor to generate hormone signal. The bulk of the exodomain is speculated to assume a crescent structure consisting of eight to nine Leu-rich repeats (LRRs), which may provide the hormone contact sites. Unfortunately, little experimental evidence is available for the precise hormone contact points in the exodomain and the endodomain. The two preceding articles (Song, Y., Ji, I., Beauchamp, J., Isaacs, N., and Ji, T. (2001) J. Biol. Chem. 276, 3426-3435; Song, Y., Ji, I., Beauchamp, J., Isaacs, N., and Ji, T. (2001) J. Biol. Chem. 276, 3436-3442) show that putative LRR2 and LRR4 are crucial for hormone binding. In particular, the N-terminal region of LRR4 assumes the hydrophobic core of the LRR4 loop, whereas the C-terminal region is crucial for signal generation. However, it is unclear whether LRR4 interacts hCG and the endodomain and how it might be involved in signal generation. In this article, our affinity labeling results present the first evidence that the N-terminal region of LRR4 interacts with hCG, preferentially the hCGalpha subunit and that the hCG/LRR4 complex interacts with exoloop 2 of the endodomain. This interaction offers a mechanism to generate hormone signal.

The role of the hinge region of the luteinizing hormone receptor in hormone interaction and signal generation.

Luteinizing hormone receptor, a G protein-coupled receptor, consists of two halves, the N-terminal extracellular hormone binding domain (exodomain) and the C-terminal membrane-associated, signal-generating domain (endodomain). The exodomain has seven to nine Leu-rich repeats, which are generally thought to form a 1/3 donut-like structure and interact with human choriogonadotropin (hCG). The resulting hCG-exodomain complex adjusts the structure and its association with the endodomain, which results in signal generation in the endodomain. It is unclear whether the rigid 1/3 donut structure could provide the agility and versatility of this dynamic action. In addition, there is no clue as to where the endodomain contact point (the signal modulator) in the exodomain is. To address these issues, the exodomain was examined by Ala scan and multiple substitutions, while receptor peptides were used for photoaffinity labeling and affinity cross-linking. Our results show that the C-flanking sequence (hinge region), Thr(250)-Gln(268), of the Leu-rich repeats (LRRs) specifically interacts with hCG, preferentially hCGalpha. This interaction is inhibited by exoloop 2 of the endodomain but not by exoloops 1 and 3, suggesting an intimate relationship between Thr(250)-Gln(268), exoloop 2, and hCG. Taken together, our observations in this article suggest a new paradigm that the LRRs contact the front of hCG, while both flanking regions of the LRRs interact with the sides of hCG. This would trap hCG in the 1/3 donut structure of the LRRs and enhance the binding affinity. In addition, mutations of conserved Ser(255) in the sequence can constitutively activate the receptor. This provides a clue for the signal modulator in the exodomain. In contrast, a phenyl or phenolic group is necessary at conserved Tyr(253) for targeting the receptor to the surface.

Risk assessment in upper gastrointestinal haemorrhage: implications for resource utilisation.

AIMS: Acute upper gastrointestinal (GI) bleeding is a common and serious problem with an associated mortality of some 10%. It is desirable, both for optimising outcomes and for the efficient use of resources, that some form of risk assessment be made early and management be stratified accordingly. A risk scoring system was recently proposed and validated by Rockall and colleagues from the UK. We wished to assess its validity in a New Zealand setting. METHODS: 565 consecutive patients treated for acute upper gastrointestinal bleeding at Wellington Hospital between 1988 and 1991 were the subject of a major prospective study. All patients were retrospectively assigned a score of 0-7 based on presentation criteria as suggested by Rockall and colleagues. The score is a composite one having regard to age, haemodynamic variables on presentation and associated serious co-morbidity. Correlation was sought between the score and the in hospital mortality risk. RESULTS: The overall 30-day mortality was 11%. Of the 60.5% of patients with a total score of less than 4 ('low risk'), the group mortality was 3.2%. Of the 39.5% of patients with a total score of 4-7 ('high risk'), the group mortality was 22.4%. CONCLUSIONS: The scoring system appears as valid in a New Zealand patient population as in the UK. We suggest that this scoring system be adopted in hospitals for patients with acute upper GI bleeding to efficiently direct the use of 'intensive care' type facilities to those most at risk, and thereby optimise management.

The amino-terminal region of the luteinizing hormone/choriogonadotropin receptor contacts both subunits of human choriogonadotropin. I. Mutational analysis.

The luteinizing hormone/choriogonadotropin receptor is a seven-transmembrane receptor. Unlike most seven-transmembrane receptors, it is composed of two halves of equal size, the N-terminal extracellular exodomain and the C-terminal membrane-associated endodomain. The exodomain is exclusively responsible for high affinity hormone binding, whereas receptor activation occurs only in the endodomain. This mutually exclusive physical separation of the two functional domains sets the lutropin receptor and its subfamily of receptors apart from all other seven-transmembrane receptors. The mechanisms of hormone binding and receptor activation also appear to be different from those of other receptors in that binding occurs in at least two steps. However, the precise hormone contact sites in the exodomain are unknown. To determine the hormone/receptor contact sites, we have examined the receptor using progressive truncation from the C terminus, Ala scanning, immunofluorescence microscopy, and antibody binding. Progressive truncation from the C terminus of the receptor indicates several discrete regions that impact hormone binding. These regions are around the boundaries of exons 1-2, 4-5, 6-7, and 9-10. Ala scanning of the Asp17-Arg26 region near the exon 1-2 junction uncovered three alternating residues (Leu20, Cys22, and Gly24) crucial for hormone binding. Ala substitution for any one of these residues abolished hormone binding, although the resulting mutant receptors were successfully expressed on the cell surface. In contrast, Ala substitution for their flanking and intervening residues did not impair hormone binding. These results and the data in the accompanying article (Phang, T., Kundu, G., Hong, S., Ji, I., and Ji, T. (1998) J. Biol. Chem. 273, 13841-13847) indicate that this region directly contacts the hormone and suggest a novel mode of embracing the hormone.

The amino-terminal region of the luteinizing hormone/choriogonadotropin receptor contacts both subunits of human choriogonadotropin. II. Photoaffinity labeling.

The luteinizing hormone/choriogonadotropin receptor, a seven-transmembrane receptor, is composed of two equal halves, the N-terminal extracellular exodomain and the C-terminal membrane-associated endodomain. Unlike most seven-transmembrane receptors, the exodomain alone is responsible for high affinity hormone binding, whereas signal is generated in the endodomain. These physical separations of hormone-binding and receptor activation sites are attributed to unique mechanisms for hormone binding and receptor activation of this receptor and its subfamily members. However, the precise hormone contact sites in the exodomain are unclear. In the preceding article (Hong, S., Phang, T., Ji, I., and Ji, T. H. (1998) J. Biol. Chem. 273, 13835-13840), a region immediately downstream of the N terminus of the exodomain was shown to be crucial for hormone binding. To test if the region interacts with the hormone, human choriogonadotropin (hCG) was photoaffinity-labeled with a peptide mimic corresponding to Gly18-Tyr36 of the receptor. This peptide mimic specifically photoaffinity-labeled both the alpha- and beta-subunits of hCG. Interestingly, hCGalpha was preferentially labeled. On the other hand, denatured hCG was not labeled, and a mutant analog of the peptide failed to label hCG. Furthermore, the affinity labeling was UV-dependent and saturable, indicating the specificity of the photoaffinity labeling. Our results indicate that the region of the exodomain interacts with hCG and that the contact points are near both subunits of hCG. Particularly, the alternate residues (Leu20, Cys22, and Gly24) are crucial for hCG binding. In addition, the results underscore the fact that there is a crucial hormone contact site outside of the popularly believed primary hormone-binding site that is composed of Leu-rich repeats and is located in the middle of the exodomain. Our observations are crucial for understanding the molecular mechanism through which the initial high affinity hormone binding leads to receptor activation in the endodomain.

Continuous infusion of methylene blue for septic shock.

Nitric oxide has been determined to be a potential mediator of the haemodynamic changes associated with sepsis. The haemodynamic eects of nitric oxide can be partially antagonised by methylene blue, through inhibition of the enzyme, guanylate cyclase. The case report presented here demonstrates a beneficial haemodynamic eect of continuous infusion of methylene blue during sepsis. These findings could be extrapolated to other patients or prompt additional research.

Codon 249 mutation of the p53 gene is a rare event in hepatocellular carcinomas from ethnic Chinese in Singapore.

The present study characterised p53 mutations in 44 hepatocellular carcinomas (HCCs) from Chinese patients residing in a high-incidence area. Twelve point mutations (27%) were detected in tumour tissues using single-strand conformation polymorphism analysis followed by direct DNA sequencing. Remarkably, no mutations were observed at codon 249. This is in contrast to HCCs from other high HCC incidence areas with endemic aflatoxin exposures, in which codon 249 is a mutational hot spot. It is therefore suggested that risk factors other than dietary exposure to aflatoxin may contribute to the high HCC incidence in Singapore.

Mutations of the tumour suppressor gene p53 in colorectal and hepatocellular carcinomas.

The present study describes mutations of the tumour suppressor gene p53 in a local collection of colorectal and hepatocellular carcinomas (HCCs). Tumour DNA was extracted from both fresh and paraffin-embedded tissues and exons 5-8 of the p53 gene were amplified by polymerase chain reaction (PCR). Mutations were detected by single-strand conformation polymorphism (SSCP) analysis followed by direct DNA sequencing. Of the 38 colorectal carcinomas and 42 HCCs examined, 15 (39%) and 13 (31%), respectively, showed p53 mutations. Two-thirds (10/15) of the mutations in colorectal carcinomas were base transitions with a predominance at CpG dinucleotide sites--a pattern characteristic to an endogenous process in cancer development. Three mutational hotspots at codons 175, 248 and 282 were also identified. Mutations did not correlate with histological grade, Dukes stage, or metastasis. However, tumours at the distal site of the colorectum showed a higher proportion of mutations than the proximal site. In the case of HCCs, majority (9/13) of the mutations were base transitions and no mutations were observed at codon 249. This is in contrast to results from other high-incidence areas such as Africa and China, where aflatoxin is believed to be a major aetiologic factor for liver cancers. The results therefore suggest that other risk factors, rather than dietary exposure to aflatoxin, may contribute to the high HCC incidence in Singapore.

Receptor binding dependent structural changes in human choriogonadotropin: photochemical inter-subunit crosslinking.

Activation of surface receptors is thought to occur in multiple transient steps with conformational adjustments of hormones and receptors beginning from the initial hormone-receptor contact. In this study, we have established a sensitive photochemical crosslinking method to detect structural change of hCG upon receptor binding. hCG consists of an α subunit and a ß subunit. Free α subunit was derivatized with photosensitive reagents and reassociated with unmodified ß subunit. Reassociated hCG αß dimer was capable of high affinity receptor binding and activation. The reagents attached to the α subunit were capable of crosslinking the α subunit to the ß subunit. However, the extent of inter-subunit cross-linking in solution was two-three fold greater than inter-subunit crosslinking after hCG bound to the receptor. This difference indicates a novel structural change at the subunit interface in response to hCG binding to the receptor. Although highly unlikely, other microenvironmental factors might have interfered with the crosslinking efficiency without impacting the structure of hCG. This study lays the ground work to precisely define the location and nature of the change. Such information will be crucial for the understanding of the molecular mechanism of the hormone-receptor interaction and receptor activation.

K-ras mutation in colorectal carcinomas from singapore.

54 sporadic colorectal cancers were analyzed for aberrations in the K-ras oncogene. DNA was extracted from frozen tissues obtained from surgical resection and analyzed for mutations in codons 12, 13 and 61 of the K-ras oncogene using single strand conformation polymorphism analysis (SSCP) and direct sequencing. Point mutations in the K-ras oncogene were found in 26/54 (48%) cases, all of which resulted in amino acid substitutions. No other types of mutations (e.g. insertions or deletions) were found. 4 of the mutations were at codon 12, 22 in codon 13 and only 1 was a codon 61 mutant. G-->A transitions were found to be predominant. A remarkable finding was the high preponderance of (13)Gly-(13)Ser mutations (54%). No correlation was observed between K-ras mutations and tumor location, Dukes' stage, differentiation levels, age or sex of the patient.

Characterization of p53 gene mutations in colorectal carcinomas from an Asian population.

A series of colorectal carcinomas from an Asian population in Singapore were analyzed for mutations in the tumor suppressor gene p53. Based on single-strand conformation polymorphism (PCR-SSCP) analysis and direct DNA sequencing, 15 of 38 tumors (39%) were found to contain mutations in exons 5-8 of the p53 gene. The point mutations were predominantly base transitions. Among the 10 transitions, 5 were at CpG dinucleotide sites. One-third (5/15) of the mutations were found at previously identified hotspot codons 175, 248 and 282. In one case, an insertion of a 6-base pair sequence was found. p53 mutations did not correlate with tumor histological grade, Dukes' stage, or metastases. However, tumors at the distal site showed a higher proportion of mutations than the proximal site. Further, no mutation was found in the normal mucosa adjacent to tumor site, suggesting that no germ-line mutations were present. The results were compared with those from other studies and are discussed in connection to possible etiological factors that are specific to the local population.