ABSTRACTS

Abstracts of presentations from the Clinical and Scientific Meeting, the Royal College of Surgeons of Edinburgh, 4-6 October 2000

J.R.Coll.Surg.Edinb., 46 February 2001, 59-67

SYME PROFESSORSHIP 2000 - WINNING PRESENTATION

Colorectal neoplasia - serological risk stratification of symptomatic patients
N. G. HURST, M. J. WAKELAM and T. ISMAIL
Institute for Cancer Studies, University Hospital NHS Trust, Birmingham, U.K.

Background: Reliable identification of symptomatic patients who are at increased risk of colorectal neoplastic disease, compared with non-neoplastic pathology, remains challenging. Non-invasive assessment, by symptom-profiling and clinical examination may result in false reassurance being derived from negative results. The poor concordance of the classical colorectal triad of symptoms with the presence of malignancy is well documented. Most investigations available are invasive, with the exception of faecal occult blood testing, and have variable accuracy with a significant false negative rate. Serological analysis has hitherto not been of benefit in assessment of patients with colorectal symptoms. Matrix metalloproteinases (MMP's) are endopeptidases, which degrade the extracellular matrix, permitting tumour invasion and establishment of metastatic cell colonies. MMP2 and MMP9 degrade type IV collagen that is the principle component of the basement membrane. Methods and results: 2000 patients' data were retrospectively analysed following presentation to a colorectal clinic at which a nonneoplastic disease diagnosis was made, but who were referred for colorectal imaging. 11%, with a clinical impression of nonneoplastic disease, were shown to have colorectal neoplasia, rising to 13% when no clinical abnormality was identified. Similar proportions of frank cancers were identified in each subgroup (39% and 44%, respectively). The symptom profile was not shown to correlate with the eventual neoplastic or nonneoplastic diagnosis. No statistical differences were identifiable between diagnostic categories on uni- or multi-variant analysis. A sub-group of symptomatic patients were prospectively recruited over a 12-month period, and underwent standardised assessment as per clinic protocol. Patient serum samples were assayed by ELISA for MMP9, blinded to the diagnosis achieved by specialist investigations. Healthy volunteers provided control specimens. Serum quantification allowed prediction of over 95% of neoplastic lesions identified by standard imaging techniques, with a false negative rate of under 1.5%. No pre-malignant adenomatous polyps were overlooked. Risk stratification improved cancer-incidence from 1 in 7 in all investigated patients, to 1 in 3 in those with positive serology. Colorectal neoplasia rates rose from 1 in 4 overall, to 1 in 2 in those with positive blood tests. Conclusion: Serum MMP9 quantification can reliably identify symptomatic patients at increased risk of colorectal neoplasia. Further work is required to expand the present study, to include asymptomatic individuals. Similarly, encouraging results could lead to the first reliable non-invasive population screening for colorectal cancer.

REFERENCES

1. Mainprize KS, Mortensen NJMcC , Warren BF. Early colorectal cancer: recognition, classification and treatment. Brit J Surg 1998, 85: 469-76
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3. Atkin WS, Hart A, Edwards R, McIntyre P, Sutton S, Cuzick J, Northover JMA. Uptake, yield of neoplasia, and adverse effects of flexible sigmoidoscopy screening. Gut 1998, 42: 560-5
4. Parson SL, Watson SA, Brown PD, Collins HM and Steele RJC. Matrix metalloproteinases (review article). Brit J Surg 1997, 84, 160-6
5. Zucker, S, Lysik, RM, Zarrabi, MH and Moll, U. Mr 92,000 Type IV collagenase is increased in plasma of patients with colon cancer and breast cancer. Cancer Research 1993, 53; 140-6
6. Stetler-Stevenson WG, Hewitt R, Corcoran M. Matrix metalloproteinases and tumour invasion: from correlation and causality to the clinic. Seminars in Cancer Biology 1996, 7: 147-54

OTHER PRESENTATIONS

Are the anti-tumour effects of conjugated linoleic acid mediated by increased expression of key genes involved in the apoptopic pathway?

B.MAJUMDER*, S.MOIR*, K.W.J.WAHLE# and S.D.HEYS*
*Department of Surgery, University of Aberdeen, #Rowett Research Institute, Aberdeen, U.K.

Background: Dietary fatty acids are believed to play an important role in carcinogenesis. Oleic acid (in olive oil), eicosapentaenoic and docosahexaenoic acids (in fish oil) may have anti-tumour effects as suggested by epidemiological and laboratory studies. Attention has focused on the fatty acid conjugated linoleic acid (CLA), known to have the most potent anti-tumour effects of all fatty acids (maximum effectiveness at 1% in animal diet). The effects of CLA may be mediated through enhanced apoptosis. However, its effects on genes involved in apoptosis are unknown. This study has examined the effects of CLA on p53, p21WAF1and bcl-2 expression, which are believed to play a key role in apoptosis. Method: Breast cancer cells (MCF7, MDA-MBA-231) were grown in RPMI media; the media was supplemented with CLA in different concentrations (0mM to 200mM) for 24 hours. At the end of this period, the effects on cell growth were assessed using a standard MTT assay. The absorbance at 540nm was used as a measure of density of live cells. Following treatment with CLA,RNA was extracted. Northern blotting was also performed to determine the effects on the expression of p53, p21WAF1 and bcl-2. The oligonucleotide probes were labelled using P32. Autoradiography was performed and densitomertric analysis was carried out; all samples are quantified with 18S. All experiments were carried out in triplicate. Results: Exposure to CLA resulted in a dose dependent reduction in growth of MCF7 cells - 20% reduction at 6.25mM, 50% at 100mM and 65% at 200mM (p<0.001). Similar results were obtained for the MDA-MBA-231 cells (up to a 39% reduction in growth at 200mM of CLA) (p<0.002). Northern blot analysis showed that after treatment with CLA there was a dose dependent effect on wild type p53 expression (MCF7 cells) with an increase by 284% at 12.5mM, 347% at 100mM, and 523% at 200mM of CLA (p<0.01). In contrast, there was a non-significant reduction in bcl-2 expression. In addition, expression of p21WAF1, a key downstream regulator of p53, was raised up to 203%(p<0.01). CLA did not change the expression of mutant p53 (present in MDA-MBA-231 cells) nor did it change the expression of p21WAF1, which require a wild type p53 for activation. But it did, however, increase the expression of bcl-2 by 103% at 12.5mM, 201% at 50mM, and 207% at 100mM of CLA (P<0.01). Conclusion: This study is the first demonstration of the effect of CLA on gene expression in breast cancer cells. These results indicate a possible mechanism for the anti-tumour activity of a dietary nutrient, CLA, working by increasing the expression of the wild type p53 gene. However, in those cells with mutant p53, CLA inhibits cell growth, but this is through a p53- independent pathway.

Predicting the outcome of anterior sphincter repair using artificial neural networks 
A. GARDINER and G.S. DUTHIE 
Academic Surgical Unit, Castle Hill Hospital, Cottingham, U.K.

Background: Prediction of success after anterior sphincter repair (ASR) for incontinence is difficult. Standard multivariate analysis techniques have only 75-80% accuracy. Artificial intelligence, including Artificial Neural networks (ANN), have been used in the analysis of the complex clinical data and have also proved to be successful in predicting the probability of success following ASR. Method: Prospective ano-rectal physiology data of patients undergoing anterior sphincter repair was collected. Complete data sets of 75% of the series were used to train an ANN; the remaining 25% were used for the data validation. The output was continence grading, ranging from 0-4 (worse to continent). Results: The outcome at 3,6 and 12 months post-operatively was obtained and assessed. The best correlation between actual data value and ANN value was found at 12 months, (r=0.31; P =.0001). Clear correlation's were also found at 3 months (r=0.898; P=0.0001) and 6 months (r=0.742; P=0.0002). Conclusions: Artificial Neural Networks are more accurate (93%) than statistics. (75%) when applied to the prediction of outcome following anterior sphincter repair. This assessment confirms the usefulness of pudendal latency in the prediction of ASR outcome. The results obtained highlight the obvious usefulness of ANN's which can now be used in a prospective evaluation of the application of the technique.

Role of epidermal growth factor receptor tyrosine kinase inhibition in the chemoprevention of breast cancer 
K.C. CHAN*†, W.F. KNOX#, J.M. GEE‡, R.I. NICOLSON‡, C.S. POTTEN† and N.J. BUNDRED*
*Department of Surgery, #Department of Pathology, University Hospital of South Manchester, †Department of Epithelial Biology, Paterson Institute for Cancer Research, Manchester,‡Department of Biology, Tenovus Cancer Centre, Cardiff, U.K.

Background and results: The factors controlling epithelial proliferation in the human breast and in situ breast cancer are unclear. Anti-estrogens inhibit the growth of around 50% of breast cancers, but do not inhibit estrogen receptor negative breast carcinogenseis or proliferation in ductal carcinoma in situ (DCIS). The epidermal growth factor receptor (EGFR) is expressed in normal and pre-malignant breast epithelium. In an in vivo model epithelial proliferation decreased by 80% in normal breast xenografts and by 61% in DCIS xenografts after treatment with the novel EGFR tyrosine kinase inhibitor (EGFR TKI), ZD1839. Apoptosis increased by 104% after 7 days of treatment in DCIS. Efficacy was achieved by a wide range of doses with minimal signs of toxicity, and was associated with a reduction in expression of activated mitogen-activated protein kinase in both normal and DCIS xenografts. Additionally, estrogen-stimulated increase in progesterone receptor expression was inhibited by EGFR-TKI therapy. ZD1839 interfered with growth factor and steroid signalling pathways in the breast. Conclusion: Based on these observations, we conclude that EGFR-TK inhibition offers an approach to chemo-prevention of breast cancer.

Post transplantation malignancy: 32-year experience in 1666 allografts 
G. MORRIS-STIFF, H. BEER#, J. STEWARD#, P. GRIFFIN, J. SALAMAN, C. DARBY, R. LORD and W.A. JUREWICZ 
Welsh Regional Transplant Unit, University Hospital of Wales* and Welsh Cancer Surveillance/Intelligence Unit, Cardiff#

Aims: The development of a malignancy is the most feared sequel of transplantation. The aim of this study, therefore, was to report the incidence of this complication in our transplant population over a 32 year-period. Methods: Data was collated from, in-house transplant database, pathology archives, regional Cancer Surveillance and Intelligence Unit, Cincinnati Transplant Tumour Registry, European Dialysis and Transplantation Association, in-patient records, GP questionnaire and patient interview. Results: During the period June 1967-October 1999, 1666 transplants were performed in 1436 patients using azathioprine/prednisolone (AP, n=461) or calcineurin inhibitor based immunosuppression (CI, n=1205). 397 tumours were identified (AP n=154, CI n= 243) consisting of 390 de-novo tumours, 4 transmitted tumours [2 melanomas (1 donor), 1 renal, 1 colonic] and 3 recurrences of pre-transplant neoplasia [3 renal]. 83% of de-novo tumours were of dermal origin: SCC (n=124), BCC )n=117), Bowen's disease (n=79) and melanoma (n=2). Others included lymphoma (n=21), gastrointestinal (n=16), breast (n=6), uncertain primary (n=5), bronchus (n=4), thyroid/renal/cervix(n=3),bladder/melanoma/leukaemia/ myeloma (n=2) and epiglottis (n=1). The relative risks compared with the general population were: Bowen's x174, SCC x173, lymphoma x101, thyroid x84, BCC x28 and renal x21. The time from transplant to tumour occurrence was significantly less for the CI group [5.58 vs 18; p<0.05]. 44 of 73 patients with non-skin tumours but only 1 of 324 patients with dermal neoplasia died of carcinomatosis. Conclusions: The incidence of neoplasia in our transplant cohort was 28%. Data would suggest that with prompt management, dermal tumours which account for 81% of malignancies may not be as aggressive as previously believed.

A clinical study of a single pre-operative injection of Onyx-015 attenuated adenovirus in squamous carcinoma of the head and neck 
S.E. MORLEY*, D.S. SOUTAR*, R. BROWN#, D KIRN† and S. KAYE#
*Department of Plastic Surgery, Canniesburn Hospital, #Department of Medical Oncology, University of Glasgow, U.K., †Onyx Pharmaceuticals, Richmond, California, USA

Objectives: Onyx-015 is a gene-deleted adenovirus designed to kill cancer cells deficient in the tumour suppresser p53 protein, which is mutated in >50% of human solid malignancies including intra-oral squamous carcinoma (SCC). Little is known about spread and replication of Onyx-015 within a tumour mass or harmful effects on normal tissue. We report a study investigating a pre-operative and intra-tumoural and normal tissue injection of Onyx-015 in patients with operable intra-oral SCC looking at viral replication, cell death, immune response (systemic and in-situ) and expression of p53 and the p21 tumour suppresser protein. Methods: Fifteen patients with intra-oral SCC were assigned to receive an injection of Onyx-015 into each hemi-tumour 1, 3 or 14 days prior to resection, with the other half acting as a control, and an injection of Onyx-015 into normal tissue. P53 status of tumours was determined along with viral presence and evidence of immune response within each hemi-tumour and the normal tissue following resection. Expression of p21 protein and Tunel stain for apoptosis was also determined. Results: Fourteen patients have been treated to date with no damage to the injected normal tissue. Evidence of viral replication has been found in 6 0f 12 tumours evaluated with evidence of p53 mutation on gene sequencing in two of these (further results of p53 analysis awaited). Virus was detected in one normal tissue biopsy that had abnormal p53 on immuno-histochemistry. Evidence of a systemic and in-situ CD8 mediated immune response was noted along with evidence of apoptotic cell death. Conclusion: Onyx-015 attenuated adenovirus represents a successful gene therapy model currently undergoing phase III trials in head and neck cancer. We present novel data indicating that the virus can be detected preferentially in p53 negative tumour tissues and does not harm normal tissue supporting its role as a selective treatment for p53 defective tumours.

Prevention of ischaemia-reperfusion injury in aortic aneurysm surgery with fluid optimization using hydroxyethyl starch 
R. VOHRA 
Department of Vascular Surgery, Selly Oak Hospital, Birmingham, U.K.

Aims: Reperfusion of ischaemic tissues is associated with microvascular dysfunction that manifests as an increase in endothelial permeability with enhanced protein and fluid flux across the endothelial layer. The inflammatory mediators released as a consequence of reperfusion can also activate endothelial beds in remote organs, resulting in leukocyte dependent endothelial injury characteristic of multiple organ failure. Recently it has been suggested that hydroxyethyl starch solution used as a plasma expander can reduce the effects of ischaemia-reperfusion.¹ This study tested this hypothesis for the first time in humans using abdominal aortic aneurysm repair as a reproducible model of ischaemia-reperfusion injury. Materials and Methods: Forty consecutive patients undergoing elective aneurysm repair were randomized to receive either hydroxyethyl starch (HES) or gelatine as plasma expanders. All operations were carried out under a standardized general anaesthetic. Microvascular permeability was assessed by microalbuminuria. The lung injury was assessed by the pO2/FiO2 ratio and the static lung compliance. Renal dysfunction was assessed by the differential excretion of urinary proteins: IgG for glomerular permeability and alpha-1microglobulin for tubular function. Gastric tonometry (pHi) was performed to assess the adequacy of splanchnic oxygenation. The systemic inflammation was assessed by the serum levels of neutrophil elastase, pro-inflammatory cytokines (IL-6, IL-8) and C-reactive protein (CRP). Results: The gas exchange was significantly better (50.6 kPa/% vs. 36.0 kPa/%, p< 0.05) in the HES treated patients as was the static lung compliance (77.2 ml/cm H2O vs. 71 ml/cm H2O, p < 0.05). Microalbuminuria was significantly elevated in the gelatine-treated patients, compared with the HES treated patients (16.3 mg/mmol vs. 6.95 mg/mmol, p < 0.05); differences persisted for 4 hours after reperfusion. The tubular function was also better preserved in the HES treated patients (8 mg/mmol vs. 14 mg/mmol, p < 0.05). The change in gastric pHi was less severe after reperfusion in the HES treated patients (7.33 vs. 7.29 p = 0.003). There was a significant correlation between the lowest pHi and the peakIL-6 on the first postoperative day (rs = -0.47, p = 0.03). There was no significant difference in the neutrophil elastase early after reperfusion, but differences became apparent after 48 hours (116.1 ng/ml in the HES group vs. 146.3 ng/ml in the gelatine group, p = 0.03). The serum level of IL-8 was not significantly different in the two groups during the study period. The peak CRP level was significantly lower in the HES treated group (142 mg/l vs. 246 mg/l, p < 0.05). Conclusion: HES protects against the remote effects of ischaemia-reperfusion injury. HES appears to exert its beneficial effects through modulation of the inflammatory response. Further studies are required to unravel these mechanisms.

REFERENCE

1. Zikria BA, Subbarao C, Oz MC, Popilkis SJ, Sachdev R, Chauhan P, et al. Hydroxyethyl starch macromolecules reduce myocardial reperfusion injury. Arch Surg 1990; 125: 930-4

A prospective randomized trial of early laparoscopic cholecystectomy versus early minilaparotomy cholecystectomy for acute cholecystitis 
PAUL B. S. LAI*, JAMES Y. W. LAU*, W. T. SIU#, S. W. LEE†, K. H. KWONG†, S. P. Y. KWOK†, MICHAEL K. W. LI#, C. K. LEOW*, SYDNEY S. C. CHUNG* and JOSEPH W. Y. LAU*
*Department of Surgery, Prince of Wales Hospital, #Chinese University of Hong Kong; Pamela Youde Nethersole Eastern Hospital & United Christian Hospital†, Hong Kong SAR, China

Objective: Our center had previously shown that early instead of delayed laparoscopic cholecystectomy would be the more preferable management of choice in the treatment of acute cholecystitis. The aim of this prospective randomized study was to compare the two techniques, laparoscopic cholecystectomy and minilaparotomy cholecystectomy, as an early intervention for patients with acute cholecystitis. Patients and methods: From February 1997 to October 1999, 159 patients from 3 regional hospitals with the clinical diagnosis of acute cholecystitis confirmed by ultrasonography or E-HIDA scan were randomized after informed consent to have laparoscopic cholecystectomy or minilaparotomy cholecystectomy within 24 hours of diagnosis. Patients who had previous upper abdominal surgeries or significant medical diseases that rendered them unfit for laparoscopic surgery were excluded from the study. There were 5 post-randomization exclusions. Clinical endpoints were monitored and recorded prospectively. Conclusion: Patients who underwent laparoscopic cholecystectomy had significantly less pain after surgery. Other endpoints were very similar between the two techniques. Apart from a shorter operating time, treating patients with mini-laparotomy cholecystectomy does not offer additional benefit.

©2001 The Royal College of Surgeons of Edinburgh, J.R.Coll.Surg.Edinb.