Translating colorectal cancer genetics into clinically useful biomarkers.

Colorectal cancer (CRC) is a major health problem worldwide accounting for over a million deaths annually. While many patients with Stage II and III CRC can be cured with combinations of surgery, radiotherapy and chemotherapy, this is morbid costly treatment and a significant proportion will suffer recurrence and eventually die of CRC. Increased understanding of the molecular pathogenesis of CRC has the potential to identify high risk patients and target therapy more appropriately. Despite increased understanding of the molecular events underlying CRC development, established molecular techniques have only produced a limited number of biomarkers suitable for use in routine clinical practice to predict risk, prognosis and response to treatment. Recent rapid technological developments, however, have made genomic sequencing of CRC more economical and efficient, creating potential for the discovery of genetic biomarkers that have greater diagnostic, prognostic and therapeutic capabilities for the management of CRC. This paper reviews the current understanding of the molecular pathogenesis of CRC, and summarizes molecular biomarkers that surgeons will encounter in current clinical use as well as those under development in clinical and preclinical trials. New molecular technologies are reviewed together with their potential impact on the understanding of the molecular pathogenesis of CRC and their potential clinical utility in classification, diagnosis, prognosis and targeting of therapy.

Association of angiopoietin-2, C-reactive protein and markers of obesity and insulin resistance with survival outcome in colorectal cancer.

BACKGROUND: This study investigated the relationship of obesity, insulin resistance, inflammation and angiogenesis with cancer progression and survival in a colorectal cancer cohort. METHODS: Clinical and pathological data, along with anthropometric and follow-up data, were collected from 344 consecutive colorectal cancer patients. Serum samples at diagnosis were analysed by immunoassay for adiponectin, C-reactive protein (CRP), vascular endothelial growth factor-A (VEGF-A), angiopoietin-2 (Ang-2), insulin-like growth factor-1 (IGF-1), insulin and C-peptide. RESULTS: Serum Ang-2 and VEGF-A levels increased with tumour T stage (P=0.007 and P=0.025, respectively) and N stage (P=0.02 and P=0.03, respectively), and correlated with CRP levels (r=0.43, P<0.001 and r=0.23, P<0.001, respectively). Angiopoietin-2 correlated with C-peptide (r=0.14, P=0.007) and VEGF-A with IGF-1 in males (r=0.25, P=0.001). Kaplan-Meier analysis showed that patients with high serum levels of CRP and Ang-2 had significantly reduced survival (both P≤0.001). After adjusting for tumour stage and age, Ang-2 remained a significant predictor of survival. The CRP levels were inversely associated with survival in American Joint Committee on Cancer stage II patients (P=0.038), suggesting that CRP could be used to support treatment decisions in this subgroup. Serum markers and anthropometric measures of obesity correlated with each other, but not with survival. CONCLUSION: Our study supports the concept that obesity-related inflammation, rather than obesity itself, is associated with colorectal cancer progression and survival. The study confirms serum Ang-2 as a predictive marker for outcome of colorectal cancer.

Evaluation of physician assistants in National Health Service Scotland.

Physician assistants (PAs) have medical training and work supervised by a doctor. In 2006-2008 the Scottish Government piloted use of USA-trained PAs. The aim of the paper is to evaluate the impact and contribution made by PAs to delivering effective health care in National Health Service (NHS) Scotland. Mixed methods, longitudinally, including interviews, feedback forms and activity data collection. Data analysis used nVivo, SPSS and Excel. Participants were 15 USA-trained PAs, medical supervisors and team members, 20 patients, four NHS senior managers and three trade union representatives. Settings were four Scottish NHS Boards where PAs worked in primary care, out of hours clinics, emergency medicine, intermediate care and orthopaedics. Two minor patient safety issues arose. Patients were satisfied with PAs. Scope of practice did not replicate US working. Inability to prescribe was a hindrance. PAs tended to have longer consultations, but provided continuity and an educational resource. They were assessed to be mid-level practitioners approximating to nurse practitioner or generalist doctor. Valued features were generalism, medical background, confidence differential diagnosis and communication. Interviewees suggested PAs could fulfil roles currently filled by medical staff, potentially saving resources. In conclusion, there is potential for PAs to fulfil distinctive mid-level roles in the Scottish NHS adding value in continuity, communication and medical approach.

Bayesian estimation of weak material dispersion: theory and experiment.

This work considers the estimation of dispersion in materials via an interferometric technique. At its core, the problem involves extracting the quadratic variation in phase over a range of wavelengths based on measured optical intensity. The estimation problem becomes extremely difficult for weakly dispersive materials where the quadratic nonlinearity is very small relative to the uncertainty inherent in experiment. This work provides a means of estimating dispersion in the face of such uncertainty. Specifically, we use a Markov Chain Monte Carlo implementation of Bayesian analysis to provide both the dispersion estimate and the associated confidence interval. The interplay between various system parameters and the size of the resulting confidence interval is discussed. The approach is then applied to several different experimental samples.

Breast cancer and cytomegalovirus.

PURPOSE: To determine whether cytomegalovirus is causally associated with breast cancer and whether cytomegalovirus should be categorised as an oncogenic virus. METHODS: We undertook a review of published epidemiological and laboratory studies, using established causal criteria: Bradford Hill criteria to determine whether cytomegalovirus is associated with breast cancer; and Evans/Mueller criteria to determine whether cytomegalovirus should be categorised as an oncogenic virus. RESULTS: Although there are inconsistencies in the findings of published epidemiological and laboratory studies, these may be explained by factors such as: differences in timing of blood samples, differences in selection of cases and controls, or high cytomegalovirus seroprevalence among participants in the epidemiological studies; and, in the laboratory studies, differences in sample preparations, age of sample, whether or not paired breast cancer and normal breast tissue samples were used, differences in the tests, primers and/or antibodies used, differences in histological types of breast cancer studied, and/or features of the virus. CONCLUSIONS: Overall, the results of published studies of cytomegalovirus and breast cancer suggest cytomegalovirus is a causal factor for at least some types of breast cancer. If the evidence for a link between cytomegalovirus and breast cancer continues to strengthen, further research could lead to: targeted screening; therapy using antiviral drugs; and, perhaps, primary prevention of a significant proportion of breast cancer. Vaccination against viruses has already been shown to be effective in preventing cervix and liver cancer; cytomegalovirus vaccines are already under development.

Atriopeptin-immunoreactive neurons in the brain: presence in cardiovascular regulatory areas.

Antisera to atriopeptin III and to a cyanogen bromide fragment of the precursor molecule atriopeptigen were prepared and used to examine the distribution of atriopeptin-like immunoreactive material in the heart and brain of the rat. Granules of this material were seen in myocytes throughout the right and left atria and were densest in the perinuclear region. The distribution of atriopeptin-like immunoreactive material in the heart is consistent with previous reports of atrial secretory granules. In the brain neurons containing the material were observed in the hypothalamus and the pontine tegmentum. Atriopeptin in the brain may serve as a neurotransmitter in neural systems controlling blood volume and composition, the same physiological functions regulated by blood-borne atriopeptin.

Bioactive cardiac substances: potent vasorelaxant activity in mammalian atria.

Mammalian atrial extracts possess natriuretic and diuretic activity. In experiments reported here it was found that atrial, but not ventricular, extract also causes relaxation of isolated vascular and nonvascular smooth muscle preparations. The smooth muscle relaxant activity of atrial extract was heat-stable and concentration-dependent and could be destroyed with protease. Rabbit aortic and chick rectum strips were used for the detection of atrial biological activity. The atrial activity was separated by column chromatography into two peaks having apparent molecular weights of 20,000 to 30,000 and less than 10,000. The atrial substance that copurified with the smooth muscle relaxant activity in both peaks caused natriuresis when injected into conscious rats. It appears that atria possess at least two peptides that elicit smooth muscle relaxation and natriuresis, suggesting an endogenous system of fluid volume regulation.

Purification and sequence analysis of bioactive atrial peptides (atriopeptins).

Mammalian cardiac atria have several biologically active peptides that exert profound effects on sodium excretion, urine volume, and smooth muscle tone. In the present study two such peptides of low molecular weight were purified and separated from each other on the basis of differences in charge, hydrophobicity, and biological profile. The first peptide, designated atriopeptin I, exhibits natriuretic and diuretic activity and selectivity relaxes intestinal smooth muscle but not vascular smooth muscle strips. The second peptide, atriopeptin II, is a potent natriuretic and diuretic that relaxes both intestinal and vascular strips. Sequence analysis of atriopeptin I indicates that it is composed of 21 amino acids, of which serine and glycine residues predominate. The amino terminal sequence of atriopeptin II up to residue 21 is the same as that of atriopeptin I, with the addition of the Phe-Arg extension at the carboxyl terminus. Both peptides appear to be derived from a common high molecular weight precursor (designated atriopeptigen); their biological selectivity and potency may be determined by the site of carboxyl terminal cleavage.

A nonpeptidyl mimic of superoxide dismutase with therapeutic activity in rats.

Many human diseases are associated with the overproduction of oxygen free radicals that inflict cell damage. A manganese(II) complex with a bis(cyclohexylpyridine)-substituted macrocyclic ligand (M40403) was designed to be a functional mimic of the superoxide dismutase (SOD) enzymes that normally remove these radicals. M40403 had high catalytic SOD activity and was chemically and biologically stable in vivo. Injection of M40403 into rat models of inflammation and ischemia-reperfusion injury protected the animals against tissue damage. Such mimics may result in better clinical therapies for diseases mediated by superoxide radicals.

Successful Heart-Lung Transplant for a Patient on Continuous-Flow Left Ventricular Assist Device Support Complicated With Amiodarone-Induced Pulmonary Fibrosis.

In this case report, we present a successful case of en bloc heart-lung transplant in a patient with advanced cardiopulmonary respiratory failure from amiodarone-associated pulmonary fibrosis that occurred post-left ventricular assist device implantation.

Prevalence of Mycoplasma genitalium in health clinic attendees complaining of vaginal discharge in Bangladesh.

The objective of the study was to determine the prevalence of Mycoplasma genitalium in a sample of health clinic attendees complaining of vaginal discharge. A subsample of 399 vaginal and cervical swabs was randomly selected from 2579 samples collected during a study to determine the causes of vaginal discharge in women attending primary health-care clinics in Dhaka, Bangladesh. Cervical samples were tested for M. genitalium by polymerase chain reaction. In addition, the samples were tested for Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, bacterial vaginosis and candida. M. genitalium was detected in three samples (0.8%; 95% confidence interval: 0.00-1.6). The prevalence of C. trachomatis, N. gonorrhoeae T. vaginalis, bacterial vaginosis and candida was 1.3, 3.8, 8, 23.25 and 32.5%, respectively. Two women with M. genitalium were co-infected with T. vaginalis or candida. This is the first study to document the existence of M. genitalium in Bangladesh. Although the prevalence of this infection is low in the population tested, further research into this pathogen in other Bangladeshi populations is justified.

Successful Outcome Following Orthotopic Heart Transplantation With a Donor Half Way Across The Country.

Orthotopic heart transplantation is the criterion standard treatment for end-stage heart failure and the number of recipient candidates has been increasing. Despite this increasing demand, there is limited donor organ supply. In order to surmount this challenge, we propose harvesting donor hearts from more distant locations and accepting longer cold ischemic times. The usual accepted total ischemic time limit for the transplanted human heart is up to 4 hours. Here, we report the successful use of a donor heart from 1268 miles away with a total allograft ischemic time greater than 6 hours.

Comparison of multi-institutional pre-treatment verification for VMAT of nasopharynx with delivery errors.

PURPOSE: Measurement-based pre-treatment verification with phantoms frequently uses gamma analysis to assess acceptable delivery accuracy. This study evaluates the sensitivity of a commercial system to simulated machine errors for three different institutions' Volumetric Modulated Arc Therapy (VMAT) planning approaches. METHODS: VMAT plans were generated for ten patients at three institutions using each institution's own protocol (manually-planned at institution 1; auto-planned at institutions 2 and 3). Errors in Multi-Leaf Collimator (MLC) field size (FS), MLC shift (S), and collimator angle (C) of -5, -2, -1, 1, 2 and 5 mm or degrees were introduced. Dose metric constraints discriminated which error magnitudes were considered unacceptable. The smallest magnitude error treatment plans deemed clinically unacceptable (typically for a 5% dose change) were delivered to the ArcCHECK for all institutions, and with a high-dose point ion chamber measurement in 2 institutions. Error detection for different gamma analysis criteria was compared. RESULTS: Not all deliberately introduced VMAT plan errors were detected using a typical 3D 3%/3 mm global gamma pass rate of 95%. Considering all institutions, gamma analysis was least sensitive to negative FS errors. The most sensitive was a 2%/2 mm global analysis for institution 1, whilst for institution 2 it was 3%/3 mm global analysis. The majority of errors (58/59 for institution 1, 54/60 for institution 3) were detected using ArcCHECK and ion chamber measurements combined. CONCLUSIONS: Not all clinically unacceptable errors are detected. Combining ion chamber measurements with gamma analysis improved sensitivity and is recommended. Optimum gamma settings varied across institutions.

Nitric oxide-mediated cyclooxygenase activation. A key event in the antiplatelet effects of nitrovasodilators.

We have evaluated the contributions of nitric oxide (NO) and prostacyclin (PGI2) in the in vivo antiplatelet effects of clinically useful nitrovasodilators. In rats, intravenous infusion of three NO donors, glyceryl trinitrate, sodium nitroprusside, or 3'-morpholinosydnonimine, the stable metabolite of molsidomine, released 6-keto PGF1alpha (the stable metabolite of PGI2) and inhibited ex vivo human platelet aggregation to adenosine diphosphate by at least 80%. In in vitro studies, glyceryl trinitrate, sodium nitroprusside, and 3'-morpholinosydnonimine, at clinically attainable concentrations, increased cyclooxygenase activity in endothelial cells (EC), which resulted in a four- to sixfold release of 6-keto PGF1alpha. Pretreatment of the EC with hemoglobin which binds to and inactivates the biological actions of NO, but not by methylene blue (MelB), attenuated the NO-mediated PGI2 from the EC by at least 70%. Release of 6-keto PGF1alpha by the NO donors increased the ability of these compounds to inhibit thrombin-induced human platelet aggregation by at least 10 times; this potentiation was inhibited by hemoglobin but not by MeB. MeB blocked the direct anti-platelet effect of the NO donors in the absence of EC. In summary, we have demonstrated that NO, directly as well as together with an NO-driven cyclooxygenase activation (and hence PGI2), release contributes to the marked anti-platelet effects observed after the in vivo administration of clinically used nitrovasodilators.

Activation of human complement by immunoglobulin G antigranulocyte antibody.

The ability of antigranulocyte antibody to fix the third component of complement (C3) to the granulocyte surface was investigated by an assay that quantitates the binding of monoclonal anti-C3 antibody to paraformaldehyde-fixed cells preincubated with Felty's syndrome serum in the presence of human complement. The sera from 7 of 13 patients with Felty's syndrome bound two to three times as much C3 to granulocytes as sera from patients with uncomplicated rheumatoid arthritis. The complement-activating ability of Felty's syndrome serum seemed to reside in the monomeric IgG-containing serum fraction. For those sera capable of activating complement, the amount of C3 fixed to granulocytes was proportional to the amount of granulocyte-binding IgG present in the serum. Thus, complement fixation appeared to be a consequence of the binding of antigranulocyte antibody to the cell surface. These studies suggest a role for complement-mediated injury in the pathophysiology of immune granulocytopenia, as has been demonstrated for immune hemolytic anemia and immune thrombocytopenia.

Guanylin stimulation of Cl- secretion in human intestinal T84 cells via cyclic guanosine monophosphate.

Intestinal salt and fluid secretion is stimulated by Escherichia coli heat-stable enterotoxins (ST) through activation of a membrane guanylate cyclase found in the intestine. Guanylin is an endogenous intestinal peptide that has structural similarity to the bacterial peptides. Synthetic preparations of guanylin or E. coli ST 5-17 stimulated Cl- secretion in T84 cells cultured on semipermeable membranes as measured by increases in short circuit current (Isc). The guanylin/ST receptors appeared to be on the apical surface of T84 cells, since addition of guanylin to the apical, but not basolateral, reservoir stimulated Isc. Bumetanide added to the basolateral side effectively inhibited the Isc responses of T84 cells to either guanylin or ST 5-17. Guanylin appeared to be about one-tenth as potent as ST in stimulating transepithelial Cl- secretion. Guanylin and E. coli ST 5-17 both caused massive (> 1,000-fold) increases in cGMP levels in T84 cells, but guanylin was less potent than ST. Both peptides fully inhibited the binding of 125I-ST to receptor sites on intact T84 cells. The radioligand binding data obtained with guanylin or ST 5-17 best fit a model predicting two receptors with different affinity for these ligands. The Ki values for guanylin were 19 +/- 5 nM and 1.3 +/- 0.5 microM, whereas the Ki values for ST 5-17 were 78 +/- 38 pM and 4.9 +/- 1.4 nM. We conclude that guanylin stimulated Cl- secretion via the second messenger, cGMP, in T84 human colon cells. At least two guanylin receptors with different affinities for these ligands may exist in the cultured T84 cells. It may be postulated that guanylin is an endogenous hormone that controls intestinal Cl- secretion by a paracrine mechanism via cGMP and that E. coli ST stimulates Cl- secretion by virtue of an opportunistic mechanism through activation of guanylin receptors.

Endogenous nitric oxide enhances prostaglandin production in a model of renal inflammation.

The interaction between nitric oxide (NO) and cyclooxygenase (COX) was studied in a rabbit model of renal inflammation, the ureteral obstructed hydronephrotic kidney (HNK). Ex vivo perfusion of the HNK but not the control kidney (e.g., unobstructed contralateral kidney, CLK), led to a time-dependent release of nitrite (NO2-), a breakdown product of NO. Stimulation of the HNK with bradykinin (BK) evoked a time-dependent increase in prostaglandin E2 (PGE2) production. NG-monomethyl-L-arginine (L-NMMA), which blocks the activity of both constitutive and inducible nitric oxide synthase (cNOS and iNOS), aminoguanidine, a recently described selective iNOS inhibitor, dexamethasone, or cycloheximide abolished the release of NO2- and attenuated the exaggerated BK-induced PGE2 production. This supports the existence of iNOS and COX-2 in the HNK. In the CLK, BK elicited release of both NO2- and PGE2 but this did not augment with time. L-NMMA but not aminoguanidine, dexamethasone, or cycloheximide attenuated NO2- and PGE2 release indicative of the presence of constitutive but not inducible NOS or COX. The current study suggests that the endogenous release of NO from cNOS in the CLK activates a constitutive COX resulting in optimal PGE2 release by BK. In addition, in the HNK, NO release from iNOS activates the induced COX resulting in markedly increased release of proinflammatory prostaglandin. The broader implication of this study is that the cyclooxygenase isozymes are potential receptor targets for nitric oxide.

Dual inhibition of nitric oxide and prostaglandin production contributes to the antiinflammatory properties of nitric oxide synthase inhibitors.

We have recently put forward the hypothesis that the dual inhibition of proinflammatory nitric oxide (NO) and prostaglandins (PG) may contribute to the antiinflammatory properties of nitric oxide synthase (NOS) inhibitors. This hypothesis was tested in the present study. A rapid inflammatory response characterized by edema, high levels of nitrites (NO2-, a breakdown product of NO), PG, and cellular infiltration into a fluid exudate was induced by the administration of carrageenan into the subcutaneous rat air pouch. The time course of the induction of inducible nitric oxide synthase (iNOS) protein in the pouch tissue was found to coincide with the production of NO2-. Dexamethasone inhibited both iNOS protein expression and NO2- synthesis in the fluid exudate (IC50 = 0.16 mg/kg). Oral administration of N-iminoethyl-L-lysine (L-NIL) or NG-nitro-L-arginine methyl ester (NO2Arg) not only blocked nitrite accumulation in the pouch fluid in a dose-dependent fashion but also attenuated the elevated release of PG. Finally, carrageenan administration produced a time-dependent increase in cellular infiltration into the pouch exudate that was inhibited by dexamethasone and NOS inhibitors. At early times, i.e., 6 h, the cellular infiltrate is composed primarily of neutrophils (98%). Pretreatment with colchicine reduced both neutrophil infiltration and leukotriene B4 accumulation in the air pouch by 98% but did not affect either NO2- or PG levels. In conclusion, the major findings of this paper are that (a) selective inhibitors of iNOS are clearly antiinflammatory agents by inhibiting not only NO but also PG and cellular infiltration and (b) that neutrophils are not responsible for high levels of NO and PG produced.

Verification of nose irradiation using orthovoltage x-ray beams.

Determination of dose distributions from superficial and orthovoltage irradiations of basal cell carcinoma of the nose has been performed using a nose shaped phantom constructed from paraffin wax. EBT type radiochromic film was used for dose measurements. A 2 cm diameter 50 kVp anterior field was used to irradiate the nose phantom, with sheets of film placed at 7 mm, 14 mm and 23 mm physical depth. The percentage depth doses at these points were measured to be 84% +/- 1.6%, 66% +/- 2.7% and 50% +/- 1.2% respectively, compared to the expected percentage depth doses of 72%, 52% and 34%, measured in full scatter conditions. This discrepancy is believed to be due to the steep drop off at the sides of the nose phantom, resulting in reduced attenuation at the edges of the beam, which in turn results in an increase in the scatter contribution to the dose at depth on the central axis. Measured dose profiles from this technique showed a reasonably uniform distribution. A second technique using a 250 kVp tangent-like field to irradiate the tip of the nose was also tested. Radiochromic film was placed against the edges of the phantom for dose measurement. The dose at the surface was measured to be 27% +/- 1.5% less than the expected dose. It is believed that this discrepancy is due to a combination of the lack of backscatter from the phantom, and a small offset between the phantom and the treatment cone. Dose measurements and profiles showed that this technique results in a variation in dose across the treated volume of 7%. However, the difficulty in predicting the delivered dose prohibited it from clinical use.

Optical polarization of excitons and trions under continuous and pulsed excitation in single layers of WSe2.

The potential for valleytronic operation has stimulated much interest in studying polarized emission from transition metal dichalcogenides. In most studies, however, little regard is given to the character of laser excitation. We measure the circularly polarized photoluminescence of WSe2 monolayers as a function of excitation energy for both continuous-wave (cw) and pulsed laser excitation sources. Using cw excitation, the temperature dependence of the depolarization of the trion follows the same trend as that of the neutral exciton and involves collisional broadening. However, the polarization of the trion is nearly twice the polarization of the neutral exciton at low temperatures. When a pulsed laser with the same average fluence is used as the excitation source, the degrees of polarization become very similar, in stark contrast to the cw results. The difference in polarization behaviors is linked to the different amounts of energy deposited in the system during these measurements for similar average fluences. At a moderate fluence, pulsed excitation also has the potential to fundamentally alter the emission characteristics of WSe2.