External validation of the Surgical Outcome Risk Tool (SORT) in 3305 abdominal surgery patients in the independent sector in the UK.

BACKGROUND: Assessing the risk of post-surgical mortality is a key component of pre-surgical planning. The Surgical Outcome Risk Tool (SORT) uses pre-operative variables to predict 30-day mortality. The aim of this study was to externally validate SORT in patients undergoing major abdominal surgery. METHODS: Data were collected from patients treated in five independent hospitals in the UK. Individualised SORT scores were calculated, and area under the receiver operating characteristic (AUROC) and precision-recall curves (PRC) plus 95% confidence intervals (CI) were drawn to test the ability of SORT to identify in-hospital death. Outcomes of patients with a SORT predicted risk of mortality of ≥ 5% (high risk) were compared to those with a predicted risk of < 5% (standard risk). RESULTS: The study population comprised 3305 patients, mean age 51 years, 2783 (84.2%) underwent elective surgery most frequently involving the colon (24.6%), or liver, pancreas or gallbladder (18.2%). Overall, 1551 (46.9%) patients were admitted to ICU and 29 (0.88%) died. The AUROC of SORT for discriminating patients at risk of death in hospital was 0.899 (95% CI 0.849 to 0.949) and the PRC 0.247. In total, 72 (2.18%) patients were stratified as high risk. There were more unplanned ICU admissions and deaths in this group compared to the standard risk group (25.0% and 3.3%, versus 3.1% and 0.5%, respectively). CONCLUSION: We externally validated SORT in a large population of abdominal surgery patients. SORT performed well in patients with lower risk profiles, but underpredicted adverse outcomes in the higher risk group.

Multimedia educational tools for cognitive surgical skill acquisition in open and laparoscopic colorectal surgery: a randomized controlled trial.

AIM: Conventional teaching in surgical training programmes is constrained by time and cost, and has room for improvement. This study aimed to determine the effectiveness of a multimedia educational tool developed for an index colorectal surgical procedure (anterior resection) in teaching and assessment of cognitive skills and to evaluate its acceptability amongst general surgical trainees. METHOD: Multimedia educational tools in open and laparoscopic anterior resection were developed by filming multiple operations which were edited into procedural steps and substeps and then integrated onto interactive navigational platforms using Adobe® Flash® Professional CS5 10.1. A randomized controlled trial was conducted on general surgical trainees to evaluate the effectiveness of online multimedia in comparison with conventional 'study day' teaching for the acquisition of cognitive skills. All trainees were assessed before and after the study period. Trainees in the multimedia group evaluated the tools by completing a survey. RESULTS: Fifty-nine trainees were randomized but 27% dropped out, leaving 43 trainees randomized to the multimedia group (n = 25) and study day group (n = 18) who were available for analysis. Posttest scores improved significantly in both groups (P < 0.01). The change in scores (mean ± SD) in the multimedia group was not significantly different from the study day group (6.02 ± 5.12 and 5.31 ± 3.42, respectively; P = 0.61). Twenty-five trainees completed the evaluation survey and experienced an improvement in their decision making (67%) and in factual and anatomical knowledge (88%); 96% agreed that the multimedia tool was a useful additional educational resource. CONCLUSION: Multimedia tools are effective for the acquisition of cognitive skills in colorectal surgery and are well accepted as an educational resource.

Interrelationship of depression, stress and inflammation in cancer patients: a preliminary study.

OBJECTIVE: Depression is common in cancer patients and detrimentally affects patients' quality of life. Both depression and stress are associated with raised inflammatory marker levels. This prospective study of cancer patients focuses on childhood trauma, recent life events and inflammatory marker levels as risk factors for high post-surgery depressive symptoms. METHODS: Ninety cancer patients (56 head and neck, 34 colorectal) completed the Hospital Anxiety and Depression Scale, pre-surgery and six, 12 and 24 weeks post-surgery. Recent life events and childhood trauma were assessed at six and 12 weeks respectively. Blood samples were taken pre- and one and six weeks post-surgery to measure C-reactive protein (CRP) and pro-inflammatory cytokine levels. RESULTS: Childhood trauma and recent life events were risk factors for higher depressive symptom levels. In colorectal cancer patients, baseline CRP levels were associated with depressive symptom levels at six (p=0.008) and 12 weeks (p=0.038). Baseline and six week Tumour Necrosis Factor-alpha (TNFα) levels were significantly associated with higher depressive symptoms at later time points after adjusting for cancer-related variables. Childhood trauma was positively associated with TNFα and CRP levels in colorectal cancer patients. The associations between inflammatory markers and depressive symptoms were not significant after adjusting for childhood trauma. LIMITATIONS: Small sample size. CONCLUSIONS: Raised inflammatory mediator levels may be risk factors for depressive symptoms in colorectal cancer patients and thus worth considering as a potential therapeutic target. These pilot data support recent findings demonstrating long-term effects of childhood adversity on adult health.

Microsatellite and chromosomal stable colorectal cancers demonstrate poor immunogenicity and early disease recurrence.

OBJECTIVE: Colorectal cancers may demonstrate chromosomal instability (CSI) or microsatellite instability (MSI-H). A third group of microsatellite and chromosome stable (MACS) colorectal cancer has been described more recently. Patients with MSI-H colorectal cancers demonstrate improved outcome and a pronounced inflammatory infiltrate. Enhanced host immune response and increased immunogenicity might explain these observations. This study aims to further characterize colorectal cancer immunogenicity. METHOD: Microsatellite stability status was determined in resected tumour samples. Microsatellite stable (MSS) tumour samples were stratified by DNA ploidy status, as determined by flow cytometry into aneuploid MSS (CSI) and diploid MSS (MACS) cancers. Lymphocyte proliferation, quantified by bromodeoxyuridine incorporation assays assessed tumour protein immunogenicity and ELISA assays quantified inflammatory cytokine release. Kaplan-Meier survival curves and multivariate analyses were used to determine prognostic value. RESULTS: Patients with MSI-H colorectal cancer had improved outcome but those with MACS cancers undergoing curative surgery had significantly poorer disease-free survival (P = 0.002). The MACS phenotype was an independent predictor of poor outcome (HR = 2.44, 1.33-4.47, P = 0.004). Lymphocyte proliferation assays confirmed enhanced immunogenicity of MSI-H proteins and reduced immunogenicity of MACS proteins (P < 0.0001). In vitro levels of IFN-gamma (P = 0.004) and IL-18 (P < 0.0001) mirrored these differences in lymphocyte activity. CONCLUSIONS: Stratification of colorectal cancer by MSI and ploidy status may have prognostic value in patients undergoing curative surgery. MSI-H cancers display enhanced immunogenic properties but the immune response to MACS cancers appears to be absent and this may contribute to their poor prognosis.

Microarray profiling of colorectal cancer in Bangladeshi patients.

OBJECTIVE: We have carried out a retrospective analysis of all cases of colorectal cancer at the Royal London Hospital between April 1998 and March 2002 and determined the differences in presentation and outcome between Bangladeshi and Non-Bangladeshi patients. DNA microarrays were used to explain any potential genetic differences between these two groups that may explain the different phenotypes. MATERIALS AND METHODS: We examined the colorectal database at our institution. Microarray profiles, using Affymetrix HU133A Genechips (Santa Clara, CA USA) were obtained from 10 Bangladeshi patients and an age-, sex- and stage-matched group of 10 Non-Bangladeshi patients. RESULTS: Three hundred and sixty-three patients have been treated for colorectal cancer at the Royal London Hospital. Eighteen (5%) patients were of Bangladeshi origin. The prevalence was 27/100,000 compared to 342/100,000 of the Non-Bangladeshi population. Eleven (61%) of 18 Bangladeshi patients were under the age of 40 and 4 (22%) patients presented with locally advanced or metastatic disease. In comparison 39/345 (11%) of non-Bangladeshi patients presented with advanced disease. None of the Bangladeshi patients gave a positive family history. Microarray profiling between these two groups demonstrated 1203 differentially expressed genes (P < 0.05). CONCLUSION: Colorectal cancer is uncommon in the Bangladeshi patients compared to the non-Bangladeshi population. This cancer presents in younger patients and at a more advanced stage. There is no positive family history within this ethnic community and therefore the cancers are sporadic. However, microarray profiling is able to delineate different gene expression between these two groups. Therefore, there should be a low threshold for investigating young Bangladeshi patients with symptoms of colorectal neoplasia and any future national screening programme should allow for ethnic variation.

Tumour-infiltrating lymphocytes in colorectal cancer with microsatellite instability are activated and cytotoxic.

BACKGROUND: Patients with colorectal cancer that display high-level microsatellite instability (MSI-H) appear to have a better prognosis. This may be explained by the pronounced T cell infiltrate seen in MSI-H tumours that is related to a specific antigen-driven immune response. The nature of tumour-infiltrating lymphocytes in colorectal cancers was investigated using quantitative real-time polymerase chain reaction (PCR) and immunohistochemistry. METHODS: Quantitative fluorescent hydrolysis probe-based reverse transcriptase-PCR assays were used to detect levels of mRNA specifying T cell markers in fresh frozen colorectal tissue from MSI-H tumours and those with little or no microsatellite instability (microsatellite stable (MSS) tumours). In addition, immunohistochemistry was performed on paraffin-embedded sections to compare expression of the same T cell markers and the activation markers granzyme B and interleukin 2 receptor alpha-subunit (IL-2Ralpha) in MSI-H and MSS tumours. RESULTS: MSI-H tumours contained higher ratios of CD8/CD3 mRNA copy numbers than MSS tumours (P = 0.016), confirming the cytotoxic nature of lymphocyte infiltrates in this subset of colorectal cancers. Furthermore, immunohistochemistry confirmed that MSI-H tumours contained more infiltrating lymphocytes than MSS tumours, as shown by increased expression of CD3 (P = 0.003) and CD8 (P = 0.008). Consistent with other studies, the lymphocytes in MSI-H tumours were activated as indicated by significantly higher granzyme B counts (P = 0.020) and a significantly higher level of expression of IL-2Ralpha (P = 0.017). CONCLUSION: The results support the hypothesis that MSI-H colorectal cancers may be more immunogenic than MSS tumours.

Expression of 25-hydroxyvitamin D-1-alpha-hydroxylase mRNA in individuals with colorectal cancer.

Vitamin D prevents proliferation, promotes differentiation, and induces apoptosis of colon cells, and reduced intake or insufficiency of the vitamin in the body are associated with increased risk of colorectal cancer. Results of previous studies have suggested that mRNA that codes for 25-hydroxyvitamin D-1-alpha-hydroxylase (1 alpha OHase), which converts 25-hydroxyvitamin D to its active metabolite, might be up regulated in human colon carcinomas. We used real-time reverse transcription PCR assays to measure absolute 1 alpha OHase mRNA concentrations in the colonic mucosa of 44 individuals without cancer, and in paired healthy colon and cancerous colon samples taken from 27 individuals with the disease, to ascertain whether or not such up regulation takes place. Our results suggest that concentrations of 1 alpha OHase mRNA in tumour samples and in healthy colon samples from individuals without cancer are similar, but that concentrations are significantly lower in the paired, phenotypically healthy mucosa of individuals with cancer.

Expression of HLA class II in colorectal cancer: evidence for enhanced immunogenicity of microsatellite-instability-positive tumours.

Microsatellite-instability-positive (MSI+) colorectal cancers are characterised by an accumulation of deletion and insertion mutations at simple repeated sequences caused by inactivation of mismatch repair proteins. They share several clinicopathological features, including a better prognosis and a pronounced stromal inflammatory reaction. We have used suppression subtraction hybridisation between normal colonic epithelium and paired carcinomas to generate differential gene expression profiles in MSI+ and MSI- tumours. Following reverse northern blotting analysis, 11 genes were found to be up-regulated in the MSI+ tumour, and one of these specified the HLA-DM gene. This was in contrast to the MSI(-) tumour screen, where none of the clones analysed expressed this class of gene. Our results confirm that MSI(+) tumours may have a greater potential for the efficient presentation of antigens to the helper arm of the immune system and lend further support to the theory that the better prognosis of patients with MSI+ tumours may be linked to an enhanced immunogenicity of these tumours.

Expression of the Ca2+-activated chloride channel genes CLCA1 and CLCA2 is downregulated in human colorectal cancer.

The role of ion channels in carcinogenesis and tumor progression remains unclear. We have used suppression subtractive hybridization of mRNA from paired normal colon epithelium and tumor, followed by quantitative kinetic RT-PCR, to demonstrate that the transcription of two members of a novel Ca(2+)-dependent chloride channel family, CLCA1 and CLCA2, was significantly downregulated in approximately 80% of colorectal carcinomas. This figure rose to >90% when expression was adjusted for tumor cell proliferation. In normal colon epithelium, CLCA1 mRNA levels were significantly associated with c-myc transcription but became decoupled in the tumor samples. There was no association between CLCA2 and either CLCA1 or c-myc mRNA levels. Transcription of both genes in three colorectal cancer cell lines, T84, HT29, and Caco2, was barely detectable. Illegitimate transcription of CLCA1 was detected in 12 of 15 blood samples taken from healthy volunteers, making its use as a marker for the detection of tumor spread unreliable. Our results suggest that CLCA1 could specify a new tumor suppressor and that, as in breast cancer, CLCA2 may function as a tumor suppressor in colorectal cancer.

Molecular quantification and mapping of lymph-node micrometastases in cervical cancer.

BACKGROUND: A proportion of patients with cancer and lymph nodes negative on histology will develop recurrence. Reverse-transcriptase PCR (RT-PCR) is a highly sensitive method for detection of lymph-node micrometastases, but accurate quantitative assessment has been difficult. METHODS: We studied primary tumours and 156 lymph nodes from 32 patients with cervical cancer (stage IA2, IB1, and IB2) and 32 lymph nodes from nine patients with benign disease. A fully quantitative, real-time RT-PCR assay was used to document absolute copy numbers of the epithelial marker cytokeratin 19. Primers and probe were designed not to amplify either of the two cytokeratin 19 pseudogenes. FINDINGS: All primary tumours and histologically involved lymph nodes (six) had more than 106 copies of cytokeratin 19 mRNA per microg total RNA. Expression of cytokeratin 19 (up to 1.1 x 10(5) copies per microg RNA) was detected in 66 (44%) of 150 histologically uninvolved lymph nodes, and in nodes from 16 of 32 patients with cervical cancer. 15 of these 16 patients with evidence of micrometastases had the highest cytokeratin 19 transcription level in a first lymph-node drainage station (three obturator, six internal, and six external iliac node). Transcription of cytokeratin 19 was found at a low level in just one of 32 lymph nodes obtained from nine patients with benign disease. Median copy number of cytokeratin 19 transcription was significantly higher (>10(3) copies) in association with adverse prognostic features. INTERPRETATION: The results suggest that about 50% of early-stage cervical cancers shed tumour cells to the pelvic lymph nodes. The amount of cytokeratin 19 expression was related to clinicopathological features. Further studies are required to document the clinical implications of molecular micrometastases.

Elevated levels of RanBP7 mRNA in colorectal carcinoma are associated with increased proliferation and are similar to the transcription pattern of the proto-oncogene c-myc.

We have used suppression subtractive hybridisation, "in silico" cloning and reverse Northern dot blot analysis to identify significant up-regulation of RanBP7 transcription in a human colorectal carcinoma. Quantitative RT-PCR analyses using the Taqman system demonstrated that RanBP7 mRNA levels were elevated in 47/75 colorectal tumours. There was no significant difference in 17 matched normal and tumour pairs and reduced levels in 11. Since RanBP7 specifies a key member of nuclear transport receptors responsible for the nuclear import of histone H1 and ribosomal proteins, we investigated whether this up-regulation might be proliferation-associated. RanBP7 mRNA copy numbers were significantly correlated with those of proliferating cell nuclear antigen in both normal and cancer tissue. Interestingly, the transcription pattern of the proto-oncogene c-myc showed a similar correlation with PCNA mRNA. Our results highlight the need for the careful interpretation of quantitative data that compare mRNA levels in normal and cancer tissue.

Transcription of the inositol polyphosphate 1-phosphatase gene (INPP1) is upregulated in human colorectal cancer.

We have used suppression subtractive hybridization to demonstrate significant overexpression of the inositol polyphosphate 1-phosphatase gene (INPP1) in colorectal cancer compared with matched normal colon epithelium. Its gene product catalyses the hydrolysis of inositol 1,3,4-trisphosphate and inositol 1, 4-bisphosphate, a key molecule in the phosphoinositide metabolic and signaling pathways. Following confirmation of the differential expression by reverse Northern dot blot analysis, fully quantitative Taqman reverse transcriptase-polymerase chain reaction assays showed that its transcription was upregulated in 42/49 colorectal tumors. There was no significant difference in four tumors and reduced transcription was observed in three. This is the first study to report the upregulation of the INPP1 gene in a human cancer and should facilitate further studies looking at the role of phosphatidylinositol signaling reactions in human colorectal cancer.

Cytokeratin 20 is not a tissue-specific marker for the detection of malignant epithelial cells in the blood of colorectal cancer patients.

BACKGROUND: The clinical benefit of using reverse transcription polymerase chain reaction (RT-PCR)-based assays to detect circulating tumour cells during post-operative surveillance of cancer patients remains unclear. Cytokeratin 20 has been proposed as a tissue-specific marker for the detection of micrometastases in the blood of colorectal cancer patients. However, recent reports have challenged its specificity, and hence the validity of its use. AIMS: The aim of this study was to evaluate the tissue-specificity of ck20 mRNA transcription and its use for detecting circulating colon epithelial cells. PATIENTS AND METHODS: RNA was isolated from the peripheral blood of 51 colorectal cancer patients, four patients with benign gastrointestinal disease and 42 healthy controls. In addition, it was prepared from 32 colorectal cancers, from a pituitary cancer, from normal kidney, liver, fibroblasts, keratinocytes and from 24 lymph nodes obtained from eight patients with benign gastrointestinal disease. Real-time RT-PCR assays were used to quantitative and compare ck20 transcription. RESULTS: Significant levels of ck20 mRNA were detected in all 42 blood samples from healthy volunteers and in all pre- and post-operative blood samples from colorectal cancer patients regardless of the presence of metastatic disease. It was also detected in all other mRNA samples analysed. CONCLUSION: The lack of colon tissue-specificity renders ck20 useless as a marker for the post-operative surveillance of colorectal cancer patients.

Detection of cytokeratins 19/20 and guanylyl cyclase C in peripheral blood of colorectal cancer patients.

The clinical significance of detecting supposed tumour cell-derived mRNA transcripts in blood using the polymerase chain reaction (PCR) remains unclear. We have used a fully quantitative 5'-nuclease RT-PCR assay to screen for the expression of cytokeratins (ck) 19 and 20 and guanylyl cyclase C (GCC) in the peripheral blood of 21 healthy controls and 27 colorectal cancer patients. Expression of cytokeratin 19 and 20 mRNA was detected in 30% and 100% of samples, respectively, taken from healthy volunteers. There was no apparent difference in ck19 and ck20 mRNA transcription levels between controls and patients, or between patients with different Dukes' stages. While GCC mRNA was detected in only 1/21 control samples, it was expressed in approximately 80% of patients, although again there was no correlation between GCC levels and disease stage. Transcription levels of all three markers varied considerably between samples, even between samples taken from the same person at different times. We conclude that neither ck19 nor ck20 are reliable markers for the detection of colon epithelial cells in peripheral blood and that an evaluation of the usefulness of GCC awaits further longitudinal studies.

Rapid adenoviral transduction of freshly resected tumour explants with therapeutically useful genes provides a rationale for genetic immunotherapy for colorectal cancer.

To develop protocols for the molecular immunotherapy of colorectal cancer, we compared the efficacy of three separate classes of therapeutic genes to induce antitumour responses in a murine colorectal cell model. Thus, the effects of two cytokines (IL-2 and GM-CSF) were compared with those of a costimulatory gene (B7.1) and a suicide gene (HSVtk). The rank order of efficacy against primary tumour growth was HSVtk[GCV], B7.1 > puro, IL-2 > GM-CSF, neo whereas the order of efficacy in inducing antitumour immunity was GM-CSF, IL-2, > B7.1, HSVtk[GCV] > puro, neo in a prophylactic vaccination model. To exploit these data in a clinically relevant and realistic way, we also demonstrated that colorectal tumours can reproducibly be explanted and established in short-term culture. Finally, a rapid transduction protocol has been developed by which, using adenoviral vectors, as many as 90% of the cells in these fresh tumour explants can be engineered to express high levels of the clinically relevant genes (GM-CSF or IL-2) within 1-2 weeks of surgery. Adenovirus-mediated gene delivery was reproducibly and significantly more efficient than retroviral transduction using the MFG-beta-Gal retroviral vector over the time-frame of importance for vaccination. Hence, combination of the animal model data with the ex vivo modification protocol suggests that vaccination of colorectal patients of the appropriate stage will be possible and effective.

Molecular assessment of tumour stage and disease recurrence using PCR-based assays.

Solid cancers arise as a consequence of the accumulation of genetic and epigenetic alterations within a single cell or group of cells. Their ongoing characterization is providing a range of acid-based molecular markers for neoplasia. This, together with continuous refinements to the polymerase chain reaction (PCR), had led to the emergence of PCR-based assays as potential aids in the clinical management of cancer patients. Although the sensitivity of molecular diagnosis has the potential to aid clinicians in therapeutic decision making, problems with its specificity mean that the predictive value of molecular staging is still unproved. Its role in the identification of minimal residual disease after curative surgical resection requires clinical validation in further prospective studies.

Genetic detection of lymph node micrometastases in patients with colorectal cancer.

BACKGROUND: Undetected micrometastases represent the single most important cause of treatment failure in patients undergoing putatively curative resection for colorectal cancer because current staging techniques are unable to identify patients with minimal residual disease. METHODS: A highly sensitive reverse transcription-polymerase chain reaction technique has been used to amplify tissue-specific messenger RNA from lymph nodes classified as tumour-free using both conventional histopathology and immunohistochemistry. RESULTS: Four of 15 patients were restaged after genetic diagnosis of lymph node micrometastases, while in a further two additional positive nodes were detected. CONCLUSION: Sensitive genetic techniques that detect minimal residual disease merit further study, particularly as there is evidence that patients may benefit from adjuvant chemotherapy.

Level of expression of E-cadherin mRNA in colorectal cancer correlates with clinical outcome.

A series of colorectal carcinomas (n = 49) resected from patients with known clinical outcomes were analysed for E-cadherin expression using in situ hybridisation to measure mRNA. Patients surviving 5 years or longer (n = 31) exhibited significantly higher levels of E-cadherin mRNA than those surviving less than 5 years (n = 18, P = 0.003). These preliminary results from this small sample suggest that E-cadherin expression may be a useful prognostic marker in colorectal cancer patients.