Regional Distribution of Nitric Oxide Synthase in Human Anorectal Tissue: A Pilot Study on the Potential Role for Nitric Oxide in Haemorrhoids.

OBJECTIVE: To study the distribution of nitric oxide synthase (NOS) isoforms and protein levels in human haemorrhoids and rectal tissue. METHODS: Protein expression of NOS1, NOS2 and NOS3 was compared between haemorrhoids (n=14) and normal rectal submucosa (n=6) using Western blot analysis. The localisation of all NOS isoforms to specific structures was determined by immunohistochemistry. RESULTS: Western blot analysis showed median (interquartile range) protein levels of all NOS isoforms were 1.5-2.4 times higher in haemorrhoids than rectal tissue; 121.4 (55.2-165.5) vs 50.0 (25.5-73.7) for NOS1 (p=0.020), 32.2 (23.8-140.6) vs 14.8 (9.6-34.0) for NOS2 (p=0.109), and 80.1 (62.0-139.5) vs 54.3 (48.7 -61.7) for NOS3 (p=0.015). Immunohistochemistry revealed a different distribution and location of all NOS isoforms in vascular and non-vascular structure of haemorrhoids and rectal tissues. The number of haemorrhoid specimens showing positive immunoreactivity of NOS in the vascular endothelium was significantly higher than that in rectal tissue for NOS1 (11/14 (79%) vs 1/6 (17%); p=0.018) and NOS3 (8/14 (57%) vs 0/6 (0%); p=0.042), but not for NOS2 (6/14 (43%) vs 4/6 (67%); p=0.63). CONCLUSION: Haemorrhoids have significantly higher protein levels of NOS1 and NOS3 than rectal tissue. The vascular endothelium of haemorrhoids also has significantly higher positive immunoreactivity of NOS1 and NOS3 than rectal tissue suggesting that blood vessels in haemorrhoids are exposed to higher NO concentrations than those of rectal tissue. Since haemorrhoids exhibit marked vascular dilatation and present with bleeding or swelling, a reduction in NOS - by applying NOS inhibitors - may potentially improve the symptoms of haemorrhoids.

Endothelin-1 and its receptors on haemorrhoidal tissue: a potential site for therapeutic intervention.

BACKGROUND AND PURPOSE: Haemorrhoids is a common anorectal condition affecting millions worldwide. We have studied the effect of endothelin-1 (ET-1) and the role of endothelin ETA and ETB receptors in haemorrhoid tissue. EXPERIMENTAL APPROACH: Protein expression of ET-1, ETA and ETB receptors were compared between haemorrhoids and normal rectal submucosa using Western blot analysis, with the localization of proteins determined by autoradiography and immunohistochemistry. Effects of ET-1 and sarafotoxin 6a on human colonic and rectal arteries and veins was assessed by wire myography and the involvement of receptor subtypes established by selective antagonists. KEY RESULTS: Dense binding of [125 I]-ET-1 to haemorrhoidal sections was reduced by selective receptor antagonists. A higher density of ETB than ETA receptors was found in haemorrhoidal, than in control rectal tissue and confirmed by Western blot analysis. ETA and ETB receptors were localized to smooth muscle of haemorrhoidal arteries and veins, with ETB receptors on the endothelium. Human colonic and rectal arteries and veins were similarly sensitive to ET-1 and affected by the ETA selective antagonist, but sarafotoxin S6a-induced contractions were more pronounced in veins and antagonized by a selective ETB receptor antagonist. CONCLUSIONS AND IMPLICATIONS: ETA and ETB receptors are present in human haemorrhoids with ETB receptors predominating. ETA receptors are activated by ET-1 to mediate a contraction in arteries and veins, but the latter are selectively activated by sarafotoxin S6a - a response that involves ETB receptors at low concentrations. Selective ETB agonists may have therapeutic potential to reduce congestion of the haemorrhoidal venous sinusoids.

Quality of Life and Medico-Legal Implications Following Iatrogenic Bile Duct Injuries.

In this review we aimed to evaluate quality of life after bile duct injury and the consequent medico-legal implications. A comprehensive English language literature search was performed on MEDLINE, Embase, Science Citation Index and Google™ Scholar databases for articles published between January 2000 and April 2016. The last date of search was 11 April 2016. Key search words included bile duct injury, iatrogenic, cholecystectomy, prevention, risks, outcomes, quality of life, litigation and were used in combination with the Boolean operators AND, OR and NOT. Long-term survival after bile duct injury is significantly impaired (all-cause long-term mortality approximately 21 %) along with the quality of life (especially psychological/mental state remains affected). Bile duct injury is associated with high rates of litigation. Monetary compensation varied from £2500 to £216,000 in the UK, €9826-€55,301 in the Netherlands and $628,138-$2,891,421 in the USA. Bile duct injuries have profound implications for patients, medical personnel and healthcare providers as they cause significant morbidity and mortality, high rates of litigation and raised healthcare expenditure.

Nuclear expression of phosphorylated focal adhesion kinase is associated with poor prognosis in human colorectal cancer.

AIM: To determine whether phosphorylated focal adhesion kinase (P-FAK) has prognostic value in colorectal cancer (CRC) and to test whether it has any association with Tensin 4 (TNS4) expression. MATERIALS AND METHODS: P-FAK expression was assessed using immunohistochemistry in 462 CRC cases arrayed on a tissue microarray. P-FAK and TNS4 expression were assessed by immunohistochemistry in 40 cases of paired primary colorectal cancer and corresponding hepatic metastases. RESULTS: Nuclear P-FAK expression was observed in 44% of studied cases. Positive nuclear P-FAK expression was associated with shorter disease-specific survival in univariate (p=0.005) and multivariate analysis (p=0.016). P-FAK expression was greater in metastases than the primary tumours (p<0.001) and showed significant association with nuclear TNS4 (p<0.001) in metastases. CONCLUSION: P-FAK expression is an independent prognostic marker in CRC. The present data suggest that the FAK signalling pathway may interact with TNS4, a known oncogene in CRC.

Results of laparoscopic colorectal surgery from a national training center.

BACKGROUND/PURPOSE: Trials have shown laparoscopic colorectal surgery to be safe. We aim to analyze the long-term results from a single national training center for laparoscopic surgery, especially in patients with high predicted mortality scores as well as in octogenarians. We also aim to explore the trend in the length of the learning curve among consultants and colorectal trainees, and determine whether or not laparoscopic colorectal surgery is amenable to surgical training. METHODS: All patients between July 2003 and July 2011 having laparoscopic colorectal surgery were included in a prospectively maintained database and analyzed retrospectively. We collected operative data (operation time, conversion), postoperative 30-day morbidity/mortality, cancer survival (including local/distant recurrences), postoperative incisional/port site hernia rates, and rates of reoperation. RESULTS: A total of 508 patients (258 males and 250 females) were enrolled in the study. The mean age of patients was 65.5 years and median body mass index (BMI) 27 kg/m(2); 70% of cases were malignant. Conversion rate was 15%, mean operation time was 175 minutes, and mean blood loss was 220 mL. The mean postoperative length of stay was 5.8 days, 30-day morbidity 23% (leak rate 1.38%), and 30-day mortality 1.57%. Operating time and conversion rates were significantly lower in right-sided resections compared to left-sided and rectal resections, and lymph node retrieval was significantly higher. Readmission and reoperation rates were 4.9% and 2.8%, respectively. The overall mean follow-up period was 1.8 years, rate of incisional/port site/parastomal hernia was 5.7% (n = 30), and readmission secondary to adhesions was <1% (n = 4). Readmission rates and 30-day surgical morbidity were significantly higher in patients with non-neoplastic disease compared to those with benign or malignant lesions. The mean follow-up period for cancer patients was 2.3 years. Local and distant recurrence rates were 4.2% and 13.2%, respectively. Overall death from cancer was 10.4%. Among the study participants, 74 were octogenarians and 23 had a predicted mortality of >5% (P-Possum tool). No statistically significant increases were observed in conversion, morbidity, or mortality rates in these groups (p > 0.05), but length of stay was statistically longer-7 days for octogenarians and 8 days for patients with >5% predicted mortality (p < 0.05). In 2003, two consultants operated on all cases; currently, twice as many procedures are performed by supervised trainees instead of consultants, with no change in outcome. Operating time was significantly higher in the consultant-led cases, but no other differences were noted in short- or long-term outcomes between consultant- and junior/senior trainee-led cases. CONCLUSION: We conclude that laparoscopic colorectal surgery should be the standard treatment option offered to all patients regardless of age and comorbidities and it is amenable to training.

Pharmacological characteristics of endothelin receptors on sheep rectal blood vessels.

Haemorrhoids is associated with high blood flow of the anorectal region. The question of whether pharmacological manipulation of vascular supply can relieve the symptoms of haemorrhoids has been raised. In order to undertake this type of clinical investigation, it is first essential to gain a better understanding of the properties of vascular receptors that may regulate blood flow into anal cushions and haemorrhoids. Due to the limited availability of human anorectal specimens and the good reliability of sheep tissue as an experimental model of human anorectal diseases, we studied the properties of endothelin receptors in sheep rectal artery (SRA) and vein (SRV), the vessels contributing to the blood flow of haemorrhoidal plexus, using isometric tension recordings. We found that endothelin-1 and sarafotoxin 6a were very potent constrictor agents in both SRA and SRV. The selective ET(A) receptor antagonist PD156707 (100 nM) produced a parallel rightward displacement of ET-1-induced contractions in both vessels and abolished sarafotoxin 6a-induced contractions in the SRA. PD156707 (3 µM) practically abolished contractions to ET-1 in the SRA, suggesting that the response is entirely mediated by ET(A) receptors. While, the selective ET(B) receptor antagonist BQ788 (100 nM) caused no significant change in ET-1-induced contractions in both vessels, a minor role for ET(B) receptor subtype to responses to sarafotoxin 6a in the artery was suggested.

Intratumoral T cell infiltration, MHC class I and STAT1 as biomarkers of good prognosis in colorectal cancer.

OBJECTIVE: To evaluate immunosurveillance/editing in colorectal cancer. DESIGN: Transformation stimulates the production of interferon gamma (IFNgamma) which signals via the IFNgamma receptor (IFNGR1) on tumours. This results in stimulation of nuclear STAT1 (nSTAT1), inhibition of tumour growth and upregulation of major histocompatibility complex (MHC) while promoting T cell extravasation. In contrast, downregulation of MHC class I by allele loss results in loss of T cell recognition. A tissue microarray of 462 colorectal tumours with mean follow-up of 42 months (range 1-116) was stained by immunohistochemistry for markers which predict immunosurveillance/editing. RESULTS: The presence of a high level of intratumoral T cells (ITTC) correlated with improved survival compared with a low level of ITTC, with a mean difference in survival of 16.3 months (p=0.006). There was a direct correlation between nSTAT1 expression and ITTC (p<0.001). Patients whose tumours had a high level of ITTC and nSTAT1 survived 20 months longer than those whose tumours had a low level of ITTC and no nSTAT1. A strong correlation was seen between ITTC and MHC class I expression (p=0.0002). A mean survival advantage of 26.1 months was seen in patients whose tumours had strong MHC I expression and high levels of ITTC over those who had weak MHC I and low levels of ITTC (log-rank test=12.023, p=0.034). Both MHC I and ITTC are independent predictors of good survival. CONCLUSIONS: ITTC, nSTAT1 and strong MHC class I expression on tumours identify patients with improved survival and an intact tumour immune system that may benefit from immunotherapy. Conversely, loss of these markers identifies patients whose tumours have escaped immunosurveillance and are unlikely to benefit from immunotherapy.

Guidelines for colorectal cancer screening and surveillance in moderate and high risk groups (update from 2002).

The British Society of Gastroenterology (BSG) and the Association of Coloproctology for Great Britain and Ireland (ACPGBI) commissioned this update of the 2002 guidance. The aim, as before, is to provide guidance on the appropriateness, method and frequency of screening for people at moderate and high risk from colorectal cancer. This guidance provides some new recommendations for those with inflammatory bowel disease and for those at moderate risk resulting from a family history of colorectal cancer. In other areas guidance is relatively unchanged, but the recent literature was reviewed and is included where appropriate.

Overexpression of FLIPL is an independent marker of poor prognosis in colorectal cancer patients.

PURPOSE: Colorectal cancer is one of the most common cancers. The tumor necrosis factor-related apoptosis inducing ligand (TRAIL) pathway transmits apoptotic signals and anticancer agents that activate this system, which are in clinical development. We sought to determine the prognostic value of the clinically most relevant members of this pathway in colorectal cancer patients. EXPERIMENTAL DESIGN: We used an arrayed panel of colorectal cancer tissue to assess the protein expression of the functional TRAIL receptors (TRAIL-R1 and TRAIL-R2) and both the long and short forms of FLICE inhibitory protein (FLIP(L) and FLIP(S)). Disease-free survival was examined by Kaplan-Meier estimates and the log-rank test. Prognostic factors were determined by Cox multivariate analysis. RESULTS: The TRAIL receptors and FLIP(S) were not associated with survival. On univariate analysis, strong FLIP(L) expression was associated with a significantly higher survival (P = 0.0082). On multivariate analysis using the Cox proportional hazards model, FLIP(L) phenotype was significantly associated with a poor prognosis in this series (hazard ratio, 2.04; 95% confidence interval, 1.18-3.56; P = 0.011). CONCLUSIONS: Overexpression of FLIP(L), but not TRAIL-R1 or TRAIL-R2, provides stage-independent prognostic information in colorectal cancer patients. This may indicate a clinically more aggressive phenotype and a subset of patients for whom more extensive adjuvant treatment would be appropriate.

The role of MUC1 and MUC3 in the biology and prognosis of colorectal cancer.

BACKGROUND: MUC1 and MUC3 are from a large family of glycoproteins with an aberrant expression profile in various malignancies. Much interest has been focused on the role of these proteins in the development and progression of colorectal cancer; however, no previous studies have included the highly confounding variable of vascular invasion in their survival analysis. Using high throughput tissue microarray technology we assessed the prognostic value of MUC1 and MUC3 expression in the largest cohort of colorectal cancer patients to date. We propose that tumours lacking expression of MUC1 and MUC3 will be more likely to metastasise, due to previously observed loss of cell-cell adhesion, and this will therefore lead to more aggressive cancers with poorer prognosis. METHODS: A tissue micro-array was prepared from tumour samples of 462 consecutive patients undergoing resection of a primary colorectal cancer. A comprehensive prospectively recorded data base with mean follow up of 75 months was collected and included common clinicopathological variables and disease specific survival. Immunohistochemical analysis of MUC1 and MUC3 expression was performed using antibodies NCL-MUC1 and 1143/B7 respectively, results were correlated with the variables within the database. RESULTS: Positive expression of MUC1 and MUC3 was seen in 32% and 74% of tumours respectively. On univariate analysis no correlation was seen with either MUC1 or MUC3 and any of the clinicopathological variables including tumour grade and stage, vascular invasion and tumour type. Kaplan-Meier analysis demonstrated a significant reduction in disease specific survival with MUC1 positive tumours (p = 0.038), this was not seen with MUC3 (p = 0.552). On multivariate analysis, using Cox proportional hazards model, MUC1 expression was shown to be an independent marker of prognosis (HR 1.339, 95%CI 1.002-1.790, p = 0.048). CONCLUSION: MUC1 expression in colorectal cancer is an independent marker of poor prognosis, even when vascular invasion is included in the analysis. These results support previous studies suggesting a role for MUC1 in colorectal cancer development possibly through its effects on cell adhesion.

A neoadjuvant/adjuvant randomized trial of colorectal cancer patients vaccinated with an anti-idiotypic antibody, 105AD7, mimicking CD55.

PURPOSE: To assess the tolerability and effectiveness of 105AD7 vaccination in colorectal cancer patients. 105AD7 is a human anti-idiotypic antibody mimicking CD55, a glycoprotein, which is more than expressed on colorectal cancer cells and protects them from attack by complement. EXPERIMENTAL DESIGN: Colorectal cancer patients (n = 67) eligible for primary surgery were randomized to receive the anti-idiotypic antibody 105AD7+/-Bacillus Calmette-Guerin/alum or to no treatment (control group). The immunizations were given i.d./i.m. before surgery and continued for a period of 2 years. The patients were monitored in enzyme-linked immunospot (ELISPOT; gamma-IFN), proliferation assay, and Luminex cytokine assays. RESULTS: No serious adverse events were recorded. Of the 32 investigated immunized patients, 14 (44%) were considered to be responders in the ELISPOT assay. Induced proliferative responses were noted in 17 of 40 (43%) monitored patients. There was no correlation between the ELISPOT and proliferation assays. Luminex analyses revealed tumor necrosis factor-alpha and granulocyte macrophage colony-stimulating factor responses not only to the vaccine but also toward the native antigen CD55 in 9 of 13 (69%) patients. CONCLUSIONS: Immune responses to vaccination were induced in a majority of monitored patients measured by ELISPOT and proliferation assay. The lack of correlation between the ELISPOT and proliferation assays may reflect the fact that the two methods measure different T-cell responses and highlights the importance of multiple readouts in evaluating a potential cancer vaccine. Responses to both the anti-idiotype and the CD55 antigen were measurable, adding support to the use of CD55 as a target in cancer treatment.

Cytoplasmic expression of p27(kip1) is associated with a favourable prognosis in colorectal cancer patients.

AIM: To evaluate the prognostic significance of p27(kip1) in colorectal cancer patients. METHODS: Cytoplasmic and nuclear p27(kip1) expression was evaluated in 418 colorectal cancers using tissue microarrays. Data were associated with known patient and tumor variables and long-term patient outcomes, providing further insight into the mechanisms by which p27(kip1) may influence tumor development. RESULTS: Nuclear and cytoplasmic p27(kip1) expressions were detected in 59% and 19% of tumors respectively. Cytoplasmic p27(kip1) was almost invariably associated with positive nuclear p27(kip1) expression. Neither case correlated with known clinical or pathological variables, including tumor stage, grade or extramural vascular invasion. Furthermore, nuclear p27(kip1) expression had no impact on survival. However, we identified a significant correlation between expression of cytoplasmic p27(kip1) and longer disease-specific survival times. On multivariate analysis, TNM stage and extramural vascular invasion were highly significant independent prognostic factors, with positive cytoplasmic p27 expression showing a trend towards improved patient survival (P = 0.059). CONCLUSION: These findings support the recent evidence that cytoplasmic p27(kip1) has a distinct and important biological role that can influence tumor outcome.

Immunosurveillance is active in colorectal cancer as downregulation but not complete loss of MHC class I expression correlates with a poor prognosis.

Many colorectal tumors lose or downregulate cell surface expression of MHC class I molecules conferring resistance to T-cell-mediated attack. It has been suggested that this phenomenon is due to in vivo immune-tumor interactions. However, evidence of the impact of MHC class I loss on outcomes from colorectal cancer is scarce. In our study of more than 450 colorectal cancers in tissue microarray format, we have shown that both high levels of MHC class I expression and absent MHC class I expression are associated with similar disease-specific survival times, possibly due to natural killer cell-mediated clearance of MHC class I-negative tumor cells. However, tumors with low level expression of MHC class I were found to confer a significantly poorer prognosis, retaining independent significance on multivariate analysis. The existence of these poor prognosis tumors, which may avoid both NK- and T-cell-mediated immune surveillance, has important implications for the design of immunotherapeutic strategies in colorectal cancer.

Expression of the stress-related MHC class I chain-related protein MICA is an indicator of good prognosis in colorectal cancer patients.

The realization of targeted cancer therapy has driven the need to improve selection of patients with colorectal cancer for adjuvant therapy, leading to a search for potential new prognostic markers. There is accumulating evidence that immunosurveillance acts as an extrinsic tumor suppressor. As genetic instability is an early event in colorectal cancer, this can lead to altered expression of molecules conferring resistance to immune attack. Hence, molecules up or downregulated in this process may impact on patient survival. In our study, 449 colorectal tumors were screened for expression of the stress-related protein MICA, which functions as a ligand for the NKG2D receptor and whose expression confers susceptibility to both T- and NK-cell attack. Intensity of MICA expression was quantified using automated image analysis and MICA expression showed no correlation with conventional clinicopathological variables. In contrast, survival analysis showed a significant correlation between higher levels of MICA expression and improved disease-specific survival, with independent prognostic significance in multivariate analysis. Thus, patients with low levels of MICA and a poor prognosis may be good candidates for aggressive chemotherapy. In contrast, patients with high expression of MICA may be candidates for the antibody therapies, as they should be susceptible to NK killing by antibody dependent cellular cytotoxicity.

Expression of the membrane complement regulatory protein CD59 (protectin) is associated with reduced survival in colorectal cancer patients.

It has been known for some time that the immune system can recognise growing tumours, and that tumours may respond by modulation of molecules, which make them resistant to further attack. Expression, over-expression, or loss of these molecules may function as markers of tumour progression and prognosis. Among such molecules are the membrane-bound complement regulatory proteins (mCRP), which protect cells from bystander attack by autologous complement. These include CD59 (protectin), which prevents formation of the MAC complex in the terminal stages of complement activation. In the present study, we evaluated immunohistochemical expression of CD59 in a series of over 460 well-characterised colorectal cancers using tissue microarrays (TMA), and related this information to known tumour and patient variables and to survival. The CD59 expression was observed in 69 (15%) of cases overall, and was significantly associated with tumour grade. In contrast, no associations were noted with tumour site, stage or histological type. On survival analysis, a further correlation was observed between expression of CD59 by the colorectal tumours and a reduction in disease-specific patient survival. This observation was strongest for patients with early stage disease. However, a negative impact on survival was also seen in those patients with late stage disease. These results indicate that TMA linked to good clinicopathological databases with good long term follow up are useful tools for determining new prognostic indicators that can be used in future patient management. Immune surveillance may result in immune-editing that induces variable expression of a range of target antigens, and these may be useful prognostic markers. This study has identified CD59 expression as a marker of poor prognosis in colorectal cancer patients.

Pharmacological advancements in the treatment of chronic anal fissure.

Chronic anal fissure is a tear in the lining of the anal canal that, if not treated appropriately at an early stage, causes considerable anal pain during defaecation. Surgery is no longer considered the first-line treatment of this common condition, as recent advancements in medical treatment has produced promising results in the healing of fissures, thus avoiding the unwanted complications that frequently occur following operative treatment. This review looks at those pharmacological agents used commonly in the treatment of chronic anal fissures and explores alternative therapies that may be of benefit in the future.

Evidence that the p53 negative / Bcl-2 positive phenotype is an independent indicator of good prognosis in colorectal cancer: a tissue microarray study of 460 patients.

BACKGROUND: Advances in our understanding of the molecular biology of colorectal cancer have fuelled the search for novel molecular prognostic markers to complement existing staging systems. Markers assessed in combination may perform better than those considered individually. Using high-throughput tissue microarray technology, we describe the prognostic value of combined p53 / Bcl-2 status in colorectal cancer. PATIENTS AND METHODS: Tumour samples from 462 patients who underwent elective surgery to resect a primary colorectal cancer between 1994 and 2000 (mean follow-up of 75 months) were assembled in tissue microarray format. Clinico-pathological data including tumour grade, stage, vascular invasion status along with disease specific survival data has been collected prospectively. Immunohistochemical analysis of p53 and Bcl-2 expression was performed using antibodies DO-7 (p53) and 124 (Bcl-2), and results correlated with known clinico-pathological variables and outcomes. RESULTS: Abnormal nuclear p53 accumulation and Bcl-2 overexpression were detected in 221/445 (49.6%) and199/437 (45.5%) tumours respectively, with a significant inverse correlation between the two markers (p = 0.023). On univariate analysis no correlations were found between either marker and standard clinico-pathological variables, however nuclear p53 expression was associated with a significantly reduced survival (p = 0.024). Combined analysis of the two markers indicated that 112/432 (24.2%) cases displayed a p53(-)/Bcl-2(+) phenotype, this occurring more frequently in earlier stage tumours. Kaplan-Meier analysis revealed a significant survival advantage in these p53(-)/Bcl-2(+) tumours compared with the remaining cases (p = 0.0032). On multivariate analysis using the Cox proportional hazards model, neither p53 expression nor Bcl-2 expression alone were of independent prognostic significance, however the combined p53(-)/Bcl-2(+) phenotype was significantly associated with a good prognosis in this series (HR 0.659, 95%CI 0.452-0.959, p = 0.029). CONCLUSION: Patient stratification by combined p53 / Bcl-2 phenotype provides stage-independent prognostic information in colorectal cancer. Specifically, that up to a quarter of patients display a good prognosis p53(-)/Bcl-2(+) phenotype. This may indicate a more clinically indolent phenotype and a subset of patients for whom less aggressive adjuvant treatment appropriate.