British Association of Surgical Oncology 55th Scientific Meeting

European J o u r n a l o f Surgical O n c o l o g y 1997; 2 3 : 5 8 0 - 5 9 9

ABSTRACTS British Association of Surgical Oncology 55th Scientific Meeting held jointly with BACR at The Royal College of Surgeons of England, 35-43 Lincoln's Inn Fields London, UK, on 27 and 28 November 1997 Abstracts of members' papers B A S O Symposium

The breast referral guidelines have cut down inappropriate referrals in the under 50s The Cardiff Breast Group: R. A. Cochrane', E. L. Davies ~, H. Singhal', H. M. SweetlandI, D. J. T. Webster3, I. J. Monypenny3, K. Lyonsa, R. E. ManseP

~The UniversiO, Department of Surgery I, Radiology2, UniversiO, Hospital of Wales and Llandough3 Hospital. Heath Park,'Cardiff CF4 4XN Background: When the National Breast Referral Guidelines were applied to our local GPs letters immediately prior to their release in January 1996, it was shown that on the basis of the GPs own conclusions 29% of symptomatic women could have been managed initially by their own GP without missing any carcinomas. The main benefit of the guidelines was intended to focus on the younger patients who had more inappropriate referrals and were at lower risk. Objective: To determine if the breast referral practices of local GPs have altered due to the breast referral guidelines. Setting: The Rapid Access Breast Clinic at the University Hospital of Wales. Subjects: 2332 referrals from the inception of the Rapid Access Clinic in May 1995 to the release of the guidelines, and 2421 referrals from May 1996 to the end of the year. Design: Audit of GP's referral letters, notes, clinic and pathological databases. Method: Random samples of 600 (1995) and 466 0966) were drawn and the referral letters were scored as within or outside the guidelines. The reason for referral (lump, pain, family history etc.) was also recorded. As no guidance is offered for patients with a family history of breast cancer in this edition of the guidelines, all patients with a family history mentioned in their referral letters were excluded. Results: Once again none oftbe GP referral letters for the 142 symptomatic carcinomas fell outside the guidelines. In the benign group after exclusion of the family history patients the level of inappropriate referrals fell from 24% in 1995 to 21% in 1966 (z2=not significant). Prior to the release of the guidelines GPs were significantly better at referring patients over 50 (14% over 50 vs 31% under 50 X2=<0.001) but after the guidelines there was no significant difference (16°/o over 50, 20°/0 under 50/.2=not significant). The 1I% fall in referrals outside the guidelines in the under 50s is significant/.2 = <0.01. Conclusions: The Breast Referral Guidelines seem to have been effective in reducing the higher level of inappropriate referrals in younger patients at less risk of carcinoma. They have again adequately covered referral for the diagnosis of malignant breast disease to our specialist clinic and have reduced the benign workload. However,'there is room for a greater application of the guidelines to the GP's own conclusions immediately prior to referral. (GP = General Practitioner) 0748-7983/97/060580+20 $12.0010

Selective replication of an E I B attenuated adenovirus, onyx-015 in p 5 3 ( - ) tumours - results of a phase 1 dose escalation trial of intratumoural injections in patients with recurrent p 5 3 ( - ) squamous cell cancer of the head and neck I. Ganly ~'2, D. Kiro4, D. Soutar z, R. Otto 3, A. G. Robertson I, O. Park ~, A. Balmain4, D. D. Von Hoff~, S. B. Kaye ~

~University Dept of Medical Oacology Beatson Oncology Centre. Glasgow: 2Dept of Plastic mid Reconstructive Surgery Caaniesburn Hospital, Glasgow Scotland; ~Cancer Therapy and Research Center. San Antonio, TX78229; ~ON)'X Pharmaceuticals, Richmond. CA 94806 Objective: ONYX-015 is an adenovirus which lacks the E IB gene coding for the 55 kDa protein. In vitro and in vivo studies show that it selectively replicates and lyses cells with non-functional or mutant p53. The objective of this study was to determine the safety and efficacy of direct intratumoural injection of ONYX-015. Methods: Patients with recurrent head and neck cancer were recruited. The p53 status of the tumours was assessed by immunohistochemistry and gene sequencing on a single core biopsy taken from the recurrent tumour. Only patients with abnormal p53 as assessed by IHC were treated. Patients received direct intratumoural injection of ONYX-015 over 8 cohorts with a minimum of 3 patients per cohort. Patients were assessed twice weekly for toxicity. Tumour response was assessed both clinically and radiologically 4 weeks postinjection. Patients with stable disease had repeat injections done every 4 weeks. Postinjection biopsies at days 8 and 22 were assayed for viral replication by in situ hybridization of viral DNA. Immune status of patients was assessed by measuring pretreatment CD4 counts. Immune response to virus ~'as monitored by measuring neutralizing antibody pre and post treatment. Results: 32 patients were treated - 23 in a single injection protocol and 9 in a multiple injection protocol. In the single dose injection - 5 patients were treated at 107pfu, 4 at 10~pfu, 4 at 109pfu, 3 at 3× 109pfu, 3 at 10~°pfu, and 4 at 10" pfu. In the multiple injection protocol, 4 patients were treated with 5 daily injections of l09 pfu (total dose 5 x l09 pfu) and 5 with 5 daily injections of 10I° pfu (total dose 5 x 10~°pfu). All patients had abnormal p53 as judged by IHC. 20125 tumours for which p53 sequencing results are available had mutations in exons 5-9. Most patients were immunosuppressed before treatment as assessed by low CD4 counts <500. No significant toxicities were observed. Thirteen patients received repeat treatment (up to 5 cycles). Ten patients had significant necrosis of injected tumours both clinically and radiologically. Efficacy was better with multiple injections compared to a single injection probably due to better viral distribution. Viral replication was detected in postinjection biopsies in 7/20 patients. 50% of patients had antibodies to adenovirus pretreatment and 100% had antibodies post treatment. Patients who showed necrosis of tumours continued to respond despite developing antibodies. Conclusions: lntratumoural injection of reeurrent head and neck squamous cell cancer with ONYX-015 is feasible and safe. In addition, clear biological © 1997 W.B. Saunders Company Limited

Abstracts effects of tumour necrosis are seen. Multiple injections have greater efficacy than single injections with no increase in toxicity. Phase I I studies are planned in order to better define the efficacy of ONYX-015.

T P R - M E T oncogenic rearrangement is involved early in gastric carcinogenesis

S. Ramesh, S. McMillan, C. Chadwick, P. McCulloch Department of Surger),. UniL,ersiO,of Liverpool, Liverpool c-met, which encodes the hepatocyte growth factor receptor, can occur in a truncated mutant form spliced to a translocated promoter region (TPRMET). This form of the receptor is constitutively activated, and is widely believed to be important in carcinogenesis, largely from evidence obtained #l vitro and transgenic expression in mice. The relative frequency of TPRMET expression in human gastric carcinomas and normal mucosa is unknown. We have studied TPR-MET m-RNA expression in gastric carcinomas, associated 'normal' mucosa, and mucosa from patients with histologically and bacteriologically normal stomachs. Amplifiable c-DNA was obtained by reverse transcription of t-RNA extracted from MNNG-HOS cells, 35 gastric cancers, 28 associated 'normal' ~tnd 18 histologically normal stomachs. 'Hot-start' nested PCR was performed, and the products electrophoresed, stained and examined under UV light. The PCR products were sequenced. Results are shown in the Table. TPR-MET+ Gastric cancer (n = 35) Associated mucosa (n = 28) Normal Mucosa (n = 18)

TPR-MET-

% + v e p value

26

9

74

19

9

67

0.13",0.26 cl

8

I0

44

0.04

*Fisher's exact test, t3Paired t-test. TPR-M ET rearrangement is found in a high proportion of gastric cancers and associated non-malignant mucosa. It is frequently present in normal mucosa without H. pylori infection. These findings implicate TPRMET in the very early stages of human gastric carcinogenesis. MNNG-HOS = N-methyl-N'-nit roso-guanidine HOS = human osteosareoma cell line.

581

Statistical Test: Mann-Whitney U. ICI 182780 a novel pure antioestrogen, n =number, E.,=oestradiol. Ki67= . antigen-proliferation marker.

A comparative analysis of C - M Y C oncoprotein expression in cutaneous and uveal melanoma J. S. Chana, I. A. Cree, A. J. E. Foss, J. L. Hungerford, G. D. Wilson Raft Institute of Plastic Surgery Mt Vernon Hospital and Department of Ophthalnmlogy, Moorfields Eye ltospital Introduction: We have shown that overexpression of c-myc provides an independent prognostic marker in both primary and metastatic cutaneous melanoma. However, although chromosomal abnormalities involving amplification of the 8q segment have been associated with uveal melanoma, the role of c-no,c oncogene expression has been little investigated in this turnout. Aim: The aim of this study was to undertake an analysis of the prognostic value~ of c-myc oneoprotein expression in posterior uveal melanoma and to compare these results with those previously obtained for cutaneous melanoma. Methods: c-myc expression was measured by dual parameter flow cytometry in 71 patients with posterior uveal melanoma. Results: c-myc oncoprotein was detected in uveal tumours with survival analysis revealing a significant association between high oncoprotein positivity arid improved survival (Log-Rank test, Z2=6.47, P=0.01). Multivariate analysis using Cox's proportional hazards revealed c-myc oncoprotein to be an independent prognostic marker more accurate than other clinicopathological parameters tested CLog-Rank test, Z-'=6.61, P= 0.01). Conclusion: c-myc promotes cellular proliferation by influencing transit through a cell cycle check point in the early G I phase of the cell cycle. Overexpression ofc-myc would therefore be expected to correlate with poor outcome as demonstrated in cutaneous melanoma. These results of high oncoprotein expression correlating with improved outcome in uveal melanoma are therefore surprising and in contrast to our previous studies using the same method in cutaneous melanoma. This study suggests that the pattern of oncogene expression is distinct from cutaneous melanoma and that the biology of these tumours is different.

Lectin histochemistry of Barrett's metaplasia

Effect of antioestrogens on BRCA-I mutated breast tissue compared with normal breast Maria Bramley~, R. B. Clarke3, A. Howellz, Elizabeth Anderson3, A. D. Baildam~ Departments of Surgerymand Medical Oncolog.v2, University Hospital of South Manchester mid Department of Clinical Research~. Christie Hospilal, Manchester Women found to have the BRCA-I genotype are randomized into tamoxifen versus placebo breast cancer prevention trials, but little is known of tamoxifen's effects on their breast tissue. We studied the effects of tamoxifen and a pure antioestrogen (ICI 182780) on breast tissue from women with known BRCA-I mutation (n=3), other 'high risk' women in whom genetic testing was not possible (n= 10) and normal women undergoing benign breast biopsies (n = 6). Tissue was implanted into the flanks of athymic nude mice (n=20) and 2 weeks later treated either with slow release oestradiol (2 mg E2 n= 12) or control pellets (n=8). One week later control mice were treated with vehicle in =4), tamoxifen ( I mg/day n = 2), or ICI 182780 (5 rag/ week n =2) injections fur 2 weeks and E2 mice with vehicle (n =4), tamoxifen (I rag/day n=4), or ICI 182780 (5 rag/week n=4). Implants were removed for histological examination and assessment of proliferation (Ki67) using immunocytochemistry at weeks 2, 3, 4, and 5. Of 1370 implants retrieved, 1008 contained normal breast lobules (73.6%). In all groups there was a significant rise in Ki67 expression after E,. In the normal risk group, tamoxifen could reduce Ki67 to near cpntrol levels (I.9 + 1.5 versus 1.9 + 1.9% cells positive). In the 'high risk' group, tamoxifen had no significant effect on Ki67 but after ICI 182780, Ki67 fell from 3.5+_3.4 in the E., alone group to 1.8+_2.6 (P=0.009). In the BRCA I mutated group Ki67 fell from 4.5_+6.3 in the E2 alone group to 2.2_+4 after tamoxifen and 2.1 _+2.7 after ICI 182780. In conclusion, the BRCA I mutated breast proliferates in response to oestrogen. This proliferation is abrogated by tamoxifen and ICI 182780 but the latter appears more potent. This work has ethical committee and Home Office approval.

J Wayman1, Julie Riehards2, S. A. RaimesI, M. K. Bennetlz, S. M. GriffinI ~The Oesophago-Gastric Cancer Unit at Nea,eastle General Hospital and 'Department of Histopalhologj: Freemml l'[ospital. Newcastle upon Tyne Barrett's metaplasia is associated with an increased risk of adenocareinoma of the lower oesophagus. Using Lectin histochemistry to characterize subtle changes in complex glycoconjugate expression within and on the surface of cells, we sought to elucidate changes associated with metaplastic differentiation of the lower oesophagus and adjacent epithelium. Forty three patients, 26 with Barret's metaplasia and the remainder normal controls underwent endoscopy and biopsy of the gastric cardia, ocsophageal mucosa and, where appropriate, area of macroscopic columnar epithelium. 4 tun serial sections were exposed to a panel of six biotinylated Lectins (UEAI. WGA, GSI, PNA, ERC) each with distinct carbohydrate binding properties selected from a much larger pilot panel. The product of the streptavidin/biotin reaction was interpreted by two observers using light microscopy. Statistical analysis was performed using Z2-test. Functional compartments within the squamous epithelium exhibited significantlydifferent Lectin binding characteristics, particularly with respect to I-IAA (P<0.0001) and PNA (P<0.0001) [basal vs. superficial compartment =2/43 vs. 32/43 and 1/43 vs. 21/43 respectively]. Corresponding functional compartments of squamous and gastric cardia and squamuus and Barrett's exhibited significantly different Lectin binding, particularly with respect to ECA [superficial squamous i) vs. cardia=3/43 vs. 36143; P<0.0001 & ii) vs. Barrett's=3/43 vs. 17126; P<0.0001]. No differences were observed between functional compartments of Barrett's compared with gastric cardia, nor between squamous or cardia epithelium from Bat'ret's patients compared with controls. Differences between functional compartments within squamous epithelium and between squamous and metaplastic epithelium are consistent with increasing complexity of glyeoconjugate expression in maturation and metaplastic differentiation. Similarity ol~ epithelium adjacent to Barrett's compared with controls is inconsistent with a 'field change' associated with Barrett's metaplasia.

Abstracts

582

Gastro-intestinal I

N M 2 3 gene product expression in colorectal cancers of young patients: predictor of tumour behaviour? I. C, Smith *, N. E. I. Langloisz, O. Eremin~, S. D. Heys I Surgical Nutrition mid Metabolism Unit. Department of Surger),~ and Pathology:. UniversiO, of Aberdeen. Foresterhill. Aberdeen Background: The Nm23 gene product is a putative metastasis suppressor gene which has structural homology to a nucleoside diphosphate kinase. Previous studies have demonstrated, in patients with breast cancer, an inverse correlation between Nm23 expression and prognosis. However, studies of colorectal cancer have revealed conflicting results. Aim: The aim of this study was to determine if expression of the Nm23 gene product was correlated with metastatic potential and surviwd in young patients (45 years and under) with colorectal cancer. Method: 81 patients with colorectal cancer were studied and staged using Dukes and Jass classifications, and overall survival assessed. Formalinfixed, wax-embedded material (tumour and regional nodes) was examined microscopically for the degree of tumour differentiation and evidence of extra-n~ural vascular invasion (shown previously to be prognostic factors). The presence of the Nm23 gene product (HI) was detected using standard immunohistochemical techniques. The percentage of cells expressing each receptor was evaluated. Results: Nm23 gent product expression did not correlate with tumour stage, lymph node involvement by tumour, presence of distant metastases, extramural vascular invasion or degree of tumour differentiation. Multivariate analysis (Cox stepwise regression model) identified the following as independent prognostic variables: Dukes stage (P = 0.0001 ) and extramural vascular invasion (P=0.007). Nm23 expression was not an independent prognostic indicator (P = 0.87). Conclusions: This study has identified that Nm23 expression does not correlate with existing indicators of tumour aggressiveness and behaviour nor is an independent predictor of survival in young palients with colorectal cancer.

Further results of a randomized, placebo-controlled double-blind survival study, using the anti-idiotypic monoclonal antibody 10SAD7 in patients with advanced colorectal cancer C. A. Maxwell Armstrong, Lindy G. Durrant, T. J. D. Buckley, R. A. Robins, J. H. Scholefield, J. Carmichael, J. D. Hardcnstle Deportment of Surgery Universit), Hospital, Noltin.fhonl Introduction: The colorectal cancer vaccine 105AD7 is an anti-idiotypic antibody thai mimics the tumour associated antigen gp72. A Phase 1 study using 105AD7 in 13 patients with advanced colorectal cencer has shown that the vaccine is non-toxic. In addition, nine of these patients had evidence of a T cell response with trial patients living three times as long as a contemporary group of unimmunized patients. Aim: A Phase II study comparing survival of patients with advanced colorectal cancer receiving either 105AD7 or placebo (alum). Materials and Methods: Patients with advanced colorectal cancer confi rmed radiologieally or histologically were randomized in I of 4 participating trial centres. No radiotherapy or chemotherapy was given in the preceding I and

Control Ave Chlor Ave T-test

the vaccine. Median survival from date on study in a univariate analysis wits 124 and 184 days in 105AD7 and placebo arms respectively (P=0.38). Survival from date of diagnosis of advanced disease was 456 and 486 dltys (P=0.82). Chemotherapy, radiotherapy and disease site all affected survival in a multivariate analysis (P<0.ff2). Discussion: Immunization with 105AD7 does not confer a survival advantage on patients with advanced colorectal cancer. Little toxicity was. however, noted and future studies will concentrate on its use as adjuvant therapy.

Differentiating effect o f chloroquine in colorectal c a n c e r cells in vitro

M. Collie, H. S. Mhairl, S. Toffa, M. C. Ilqnslett Department of Surger): Royal Free Itospital. London Colorectal cancer cells are known to be heterogeneous expressors of the tumour associated antigen Carcinoembryonic Antigen (CEA). which may be targetted by antibodies. The efficiency of this antibody guided detection and treatment is compromised by the heterogeneity of expression of the CEA. which is associated with the degree of difrerentiation of the cells. Altering pH has been shown to induce differentiation in lymphoblastoid cells *. A pH study was devised, using Chlorquine. Chlorquine is known to cross biological membranes and to enter lysosomes, where it becomes protonated, is no longer free to traverse tile membranes and so accumulates in the lysosomc, mopping up hydrogen ions '. The cytoplasmic pH being robbed of its hydrogen ions, thereby rises. Ht29 colorectal cancer cells were grown in standard monolayer culture in medium containing serial dilutions of Chloroquine, from 10 a M to 10 -9 M. After 5 days, the cells were counted, immunostained for membrane CEA and scanned using Fluorescein Activated Cell Sorting. Marked growth inhibition and increased membrane CEA expression was seen in all samples exposed to Chloroquine at concentrations > 10 ~ Molar. A further study was performed, with three human colorectal cancer cell lines Lovo. I-IT29 and Colo (high, low and non-expressors of CEA respectively) grown in standard monolayer cell culture medium cont~tining 10 s M Chloroquine. Total cell CEA was measured in addition to membrane CEA. by permeabolizing duplicates of each cell sample. Results: The proliferation rate of each cell line was significantly reduced (T-test, P<0.01). Membrane and total CEA expression measured by fluorescence on FACS, was increased in all three cells according to the nonparametric Wileoxon Rank Sum test. Using the T-test, total cell CEA wits increased in all three cell lines, and membrane CEA in the Colo cells. Conclusion: Chloroquine is effective as a CEA-inducer in colorectal cancer cells in vitro at a concentration of 10"5M, whether they are naturally high, low or non-expressors of CEA. Further studies on cell spheroids .'rod transfected colorectal cancer are needed, before the clinical potential of Chloroquine in tumour targetting can be realized.

References I. Bauer G, Hofler P, Zur Hausen H. 1982; Epstein-Barr Virus Induction by a Serum Factor. Virolog), 121: 184-94. 2. Devlin TM (Ed.). Textbook of Biochemistry with Clinical Correlations. John Wiley & Sons (1992). Chap 5.

Lovo Surface

Lovo Total

780.17 1382.96 0.17

477.7 1253.04 0.06

3 months respectively. Patients received 105ADT/placebo at 0, 6 and 12 weeks. Blood tests were assayed at each visit and concomitant tberapy, W H O performance status and any adverse events noted. CT and chest X-rays were performed at 0 and 12 weeks where possible. Further follow-up was by referring clinician and date of death recorded. Results: 162 patients were randomized between April 1994 and October 1996 with 85 receiving 105AD7 and 77 placebo. The mean ages and sexratios of the groups were comp~lrable as was the time from diagnosis of advanced disease to trial entry (172 vs. 179 days). One serious adverse event occurred in the 105AD7 arm and this was felt unlikely to be attributable to

Ht29 Surface 23.56 63.37 0.03

HT29 Total

Colo Surface

Colo Total

34.21 81.22 I x 10-5

15.86 60. I 0 3 x 10-9

22.62 73.13 2 x 10-7

Loss of perivascular nerves in the submucosa adjacent to colorectal cancer V. Chamary, T. Robson*, M. Loizidou, P. B. B0ulos, I. Taylor, G. Burnstock* Deportments of Surgery and Anotono,*. Universit), College London Medical School, London WI Various cancers lack perivascular innervation. However, blood vessel innervation has been observed in tissue adjacent to tumours, but no quantitative study exists.

Abstracts

Serum Albumin Albumin age Dukes Stage

583

Range

Regression Coefficient

Proportional Hazard

95% CI for Proportional Hazard

p value

22-51 g/I 35-80 A-C

-0.046 0.0367 0.790

0.955 1.037 2.203

0.914-0.998 1.013-1.062 1.601-3.031

0.038 0.003 <0.001

The perivascular innervation of arterioles in colorectal cancer (o = 15) and adjacent submucosa was studied using a panel of immunohistocbemical markers of transmitters: Neuropeptide Y (NPY), Tyrosinc Hydroxylase (TH), Vasoactive Intestinal Peptide (VIP), Substance P (SP) and Calcitonin G e n t Related Peptide (CGRP). Immunorcactivity was compared to that of normal colonic tissue (controls). Furthemlore. distance from the tumour edge over which immunoreactivity was reduced was measured. The absence of perivascular nerves within coloreetal cancers was confirmed, and in addition loss of periv,'tseular nerves in areas of submucosa adjacent to cancers w~,s noted. The loss was progressively greater with advancing tumour stage (Dukes A to C) for TH. NPY and VIE The pattern of loss varied for different transmitters: in the submucosa of Dukes A cancers, 5% (mean) of the total number of arterioles showed TH immunoreaetivity (controls, mean=75%). 69% showed NPY immunoreactivity (controls= 94.85%) and 2Y¼,showed VI P immunoreactivity (controls = 57%). For Dukes B submucosal arterioles, immunoreactivity dropped to 7%. 12%, 20% and for Dukes C, to 0%. 9%, 17%, (TH, NPY. VIE respectively). There was no decrease in SP in Dukes A submucosa (40%) compared to controls, but immunoreactivity fell in Dukes B (19%) and C (0%). C G R P immunoreactivity was never above 4%. Reduced NPY immunoreactivity was observed over 4.3 mm (mean, Dukes A / B = 1.3 mm Dukes C/D=~.7 mm) and 4.45 mm for VIP (Dukes A/B = 3.9 mm, Dukes C / D = 6 . 0 ram). TH and SP measurements did not return to normal within the available tissue (8.28 mm). These results suggest the tumour itself may influence neural integrity, perhaps via the secretion of humoral factors. T H = T y r o s i n e Hydroxylase, VIP=Vasoactive Intestinal Peptide, S P = Substance P, NPY = Neuropeptide Y, C G R P = C a l c i t o n i n Gene Related Peptide.

Serum albumin is an independent prognostic indicator in patients with colorectal cancer C. Sutton~, S. S. Ubhi~, J. Imeson~, Carol Barrt, P. S. Veilch z Deparouent of Surgery, Leicester General Hospital~, Leicester Royal Infirmaryz and ULCCSG, Department of Epldemiology & Public Health, University of Leicestera The aim of this study was to determine the prognostic indicators for survival in patients undergoing surgery for colorectal cancer. 260 patients undergoing potentially curative surgery for colorectal cancer in the Leicester Hospitals between 1986 and 1991 were prospectively studied and have been followed up for a minimum of five years. Using the Cox proportional hazard regression model, the prognostic value for survival of age, tumour stage (Dukes), tumour differentiation, pre-operative serum albumin and haemoglobin levels were determined. The results are summarized in the table:

Pre-operative serum albumin, age and tumour stage were tbund to be independent prognostic factors for survival in patients with colorectal cancer. Conclusion: For each I gram dt,x:rcase in serum albumin there is a 5% increase in the risk of dying. Patients with low pre-operative serum albumin levels should be considered for adjuvant therapy irrespective of their age or tumours Dukes stage.

O m e p r a z o l e induced h y p e r g a s t r i n a e m i a promotes the progression of adenomas and decreases survival in the Min mouse model of F A P

A. M. Smith, J. D. I-lardcastle,S. A. Watson The Academic Unit of Cancer Studies, Dept of Surgery, Nottingham Universit.I, Introduction: Gastrin is a potent mitogen for colorectal epithelium and tumours. Over-expression of the gastrin receptor has been shown in hyperproliferative colonic epithelium and all grades of adenomas. Thus a hyper-gastrinaemie state may promote progression through the adenomacarcinoma sequence. Aim: To determine the effect of omeprazole-induced hypergastrinaemia on the progression of intestinal neoplasia in the Mitt (multiple intestinal neoplasia) mouse model of polyposis coil Methods: Mice were entered into the study when PCR analysis confirmed the Min-genotype. Min mice were randomised to 4 groups to receive: omeprazole (75 mg/kg, daily oral treatment); omeprazole + Gastrimmune (a gastrin immunogen inducing the in sire formation of anti-gastrin antibodies 100 tie/mouse, day 0 and every 3 weeks). Control mice received oral vehicle control + / - a control immunogen. Serum gastrin levels were measured by radioimmunoassay. Prior to termination, bromodeoxyuridine was administered to generate a proliferative index. Results: Treatment with omeprazole resulted in serum gastrin levels of 236 pg/ml compared to 67 pg/ml in the untreated group. Hypergastrinaemia significantly decreased survival compared to the appropriate control (p= 0.0001, Log rank test) with mice in the control group having a 50% survival of 9.5 weeks compared to 6 weeks in the omeprazole treated group. Gastrimmune induced the formation of serum antibodies with an antigen binding capacity of >20ng/ml resulting in effective neutralization of the bypergastrinaemic state. Gastrin neutralization resulted in a reversal of the survival disadvantage induced by omeprazole (,o=0.0017). The hypergastrinaemie mice had enhanced proliferation of normal colonic mucosa (9.46% proliferating cells increased to 20.1"A,,p<0.05, Mann Whitney) and colonic neoplasia (22.3% increased to 35.0%, p<0.01). Conclusions: The level of hypergastrinaemia was in the range attained in humans on a maintenance dose of omeprazole and resulted in enhanced progression through the adenoma--careinoma sequence. Gastrin was confirmed as the mediator inducing a state of hyper-proliferation within both normal and neoplastic colonic epithelium. This study highlights the need to re-assess the safety of hypergastrinaemia.

Population Studies The impact of the N.H.S. breast screening program on disease stage in a population invited for screening D. B. Kingsmore*, D. Hole**, C. R. Gillis**, W. D. George* *Department of Surgery Western h~rmary, Glasgou: ** West of Scotkmd Cancer Surveillance Unit, Glasgow Whether the N.H.S. breast screening program will lower the mortality from breast cancer remains unknown. The aim of this study was to examine the impact of the screening program on the distribution of stage of disease and thus estimate the future effect on mortality.

All 7566 women under 75 diagnosed with invasive breast cancer from 1980-1996 in it defned area (population 1.5 million) were identified. I n formation on t umour size and nodal stat us was obtained from the pathology reports. Women were grouped according to age and the screening round active in the area in which they lived. Incidence rates were calculated by areas and screening round. These were amalgamated to determine the incidence of turnouts by size and nodal status by screening round. There was a marked increase in the incidence of node negative and small tumours in the two screening rounds. The incidence of large turnouts decreased in the last round but there was no corresponding decrease in the incidence of the 4 + node positive tumours.

Abstracts

584

Incidence (per I00,000) of Breast Cancer By Screening Round In Women Aged 50-64 Screening Round

PreScrecning Ist Round 2nd Round

Tumour Size (mm)

Nodal Status

<20

20-39

40+

Neg.

1-3+

4+

36.8 87.9 98.2

55.0 70.2 71.5

22.7 23.7 14.0

51.1 59.2 101.1

35.8 46.9 42.1

19.0 20.2 23.3

We conclude that screening on a population basis has effected the presenting tumour size but while the number of node negative women has doubled, the incidence of node positive women remains unaltered. The impact on mortality is therefore unlikely to be seen for some years yet.

Should soft tissue sarcomas be treated in a district hospital?

S. S. Mudan, P..C. Weaver Department of Surgery Queen dlexandra Hospital. Hampshire Introduction: Soft tissue sarcomas are rare turnouts and are generally the domain .of the tertiary level centre. We have examined the outcome from these tumours in a district hospital under the care of an interested surgeon. Methods: Prospective data base spanning 1985-1996. 100% follow up was secured through the hospital notes and by contact with the primary physician. Demographic and turnout related factors were analysed for disease-frec interval, and disease-specific survival using the Kaplan-Meier method and assessed for signifcance by the log-rank test. Results: There were 128 cases seen in our hospital's clinics, the male/female ratio was 70:58 and the medium age 59 yrs (range 14-96). All patients were white. 56"/,,were without disease. 30% dead of disease, 10% alive with disease and 2% dead of other causes for a median institutional follow up time of 25 months (range < I--440). 4 I% were lower extremity and the remainder about evenly divided between upper extremity, gastrointestinal, retroperitoneal and gynaecologic. Only two limb amputations were performed. The median size was 10 cm. Median disease-free survival time was 40 months (range < 1~40) and the median tumour-specific mortality time was 69 months. Surviwd for dxtremity lesions was significantly better than other sites. Conclusion: Our results show that multidiscii~linary management of these turnouts in a district hospital are equivalent to specialised centres and supports the requirement for specialist training in surgical oncology.

Colorectal cancer in a district general hospital: what are the effects of referral pathways and treatment delay? How does current management compare with national guidelines? Naomi MacKenzie, K. Akhtar, Babita Jyoti, S. Ravi, S. J. Walker Department of Surgery Blackpool Iqctoria Hospital. Whinne.t, l'lt:vs Road, Blackpool, Lancashire FY3 8NR. UK A prospective study of all patients undergoing elective surgery for colorectal cancer in a district general hospital was undertaken over a 6 month period with particular reference to the effects of referral patterns, management and

short term outcome. Out of 60 patients, 31 were initially referred to a surgeon (Group I, 14 male, 17 female; median age 71 (37-89]) and 29 to a physician (Group II, 18 male, I1 female; median age 71 [54--92]). Following surgical "referral the median time to a definitive operation was 36 days (3-167) and following medical referral 105 days (13-322) (P<0.002). There were no significant differences in pre-operative anxiety levels, operative morbidity. mortality or Duke's stage between Groups 1 and II or between patients whose treatment was or was not delayed by more than eight weeks. Prior to surgery 37 (62%) patients had the whole of their colon visualized and 17 (28%) underwent abdominal/pelvic ultrasound or CT. Five of 34 (15%) with rectal turnouts underwent preoperative radiotherapy and 7 of 16 (44%) patients with a Dukes C carcinoma were referred for adjuvant chemotberapy. Conclusion: In this study the time to definitive surgery for patients with coloreclal cancer was dependent on the pattern of referral. Delay did not appear to affect the short-term outcome. Adherence to national guidelines was variable.

Macrophage function pneumoperitoneum

is

suppressed

by

a

carbon

dioxide

M. I. Puttick, C. C. Nduka, Lynette Yong, A. Darzi Academic Surgical Unit. hnperial Callege School of Medicine at St. Mary's. London. UK The safety of laparascopic surgery for malignant disease has been questioned because of the incidence of port-site metastases and the possible risk of recurrence. Several experimental studies have demonstrated enhanced cancer growth following CO: laparoscopy. Possible mechanisms include a direct effect of CO,, on tumour biology or an indirect effect on anti-turnout defences. Due to its anatomical location, the peritoneal macrophage (PM.~ plays a pivotal role in post-surgical immune responses. This study examined the effect of the CO2 pneumoperitoneum on PMO activation and anti-tumour cytotoxicity. Rodent PMIZis (2 x 10~/ml in RPMI), were incubated in 95% air/5%,CO,, (control), 100% helium or I0(Y'A,CO,, for 3 hours at 37°C. Following exposure, cells were stimulated with LPS (1 ug/ml) and TNF-a production measured by ELISA at 0 and 6hrs. To assess cancer cell lysis, BrdU-labelled CC531s colon cancer cells were co-cuhured for 18 hrs with gas-exposed PM ~ s . DNA fragments in the supernatant were measured using anti-BrdU antibody and cell lysis expressed as a ratio to control. Results: mean (+s.d.)

Control

Helium

CO.,

0hrs PMO TNF pg/ml 6 hrs P M O TNF pg/ml Relative cancer cell lysis PM.~ viability

116.5 05.53) 108.5 (98.7) 1.0 >90%

61.7 (27.1) 905.8 (49.2)* 0.89 >90%

41.9 (23.4)* 628.6 (46.4)** 0.75** >9ffV.

"ONE WAY ANOVA 100% helium vs. 100% CO: P<0.001. **ONE WAY ANOVA control vs. CO2 P<0.01 *ONE WAY ANOVA control vs. 100% CO,, and 100'7o helium P<0.001. Macrophage exposure to both helium and CO= caused a significant impairment in responsiveness to stimulation by LPS without affecting cell viability. Furthermore, CO: exposure caused significant attenuation of cancer cell lysis compared with controls. These data may have important implications for laparoscopy in the presence of intra-peritoneal cancer cells.

Genes to Therapy

Role of RET proto--oncogene mutation analysis in the diagnosis and prognosis of multiple endocrine neoplasia type 2A gene carriers Roberta Rossi, Barbara PasiniZ, Maria R. Ambrosio, Paola Franeeschetti, Maria C. Zatelli, G. C. PansinP, A. LibonP, E. C. degli Uberli

Sezione di Endocrinologia and Ilstitu;o di Chirurgia Generale, Universitd degli Studi di Ferrara, 44100 Ferrara, =Direzione Sciendfica. Istituto Nazionale Tunwri, 20133 Milano. Italy Distinct germ-line mutations in the RET proto-oncogenc were found to be the causative genetic event of the dominantly inherited multiple endocrine neoplusia (MEN) 2A and 2B, familial medullary thyroid carcinoma (FMTC) and Hirschsprung's disease (HD). in the present study we describe a family (51 members) with MEN 2A and HD phenotype associated with a point mutation in one of the cysteine

codon at the extracellular domain of the RET proto-oncogene. A 41-yearold man was referred for screening of MEN 2A after surgery for MTC with metasasis to the cervical lymph nodes and associated parathyroid hyperplasia in absence of pbeochromocytoma. Clinical screening of family members revealed 7 subjects with abnormal pentagastrin-stimulated caleitonin (CT) test; a 73-year-old man had an unilateral pheochromocytoma and a 8-yearold child with normal CT test was affected with liD and nodular goiter. • Mutation analysis of the RET proto-oncogene was performed by restriction enzyme digestion of the PCR products generated from genomic DNA isolated from peripheral blood lymphocytes. DNA sequence analysis showed a heterozygote Cys618Arg mutation in exon l0 of the RET proto-oncogene in the proband as well as in I I family members at risk. MTC associated with C-cell hyperplasia was found in 3 adult gene 'mutation carriers with pathologic CT values who underwent surgery. Seven gene carriers were

Abstracts identified in unaffected children at risk. C-cell hyperplasia was found in 4 children (13, 10, 9, and 8-year-old) with elevated levels of stimulated plasma Ct who underwent surgery. In the g-year-old child mutation carrier with HD and normal CT values, histological examination did not demonstrate MTC or C-cell hyperplasia. Although the association of MEN 2A with HD is infrequent, the occurrence in this family of both phenotypes with a single mutation of the RET protooncogene, suggests that HD should be considered as part of the MEN 2A disease spectrum, and screening for MTC and pheochromocytoma is indicated in patients with HD associated with mutations in exon 10 of the RET protooneogene. Our data indicate that genetic screening of MEN 2A pedigrees allows the early identification or germ-line mutation in the RET proto-oncogene and suggest a prophylactic thyroidectomy in gene carriers, as early as possible, before the development of biochemical or clinical evidence of MTC.

Loss of P I 6 protein expression correlates with tumour progression in cutaneous melanoma

.I.S. Chana, R. Grovel-, G. D. Wilson, R. Sanders Raft Institute of Plastic Surgery and Gray I..aboratory Cancer Research Trust, Mt Vernon Hospital Northwood Introduction: Inactivation or the p16 tumour suppressor gene has been reported frequently in melanoma cell lines and mutations have been detected in familial melanoma kindreds. The aim of this study was to assess the role of p16 inactivation in melanocytic progression by measuring the level of p16 protein in a range of sporadic benign and malignant melanocytic lesions. Methods: Using dual parameter flow cytometry p16 protein expression was measured in 30 benign melanocytic naevi (BMN), 38 primary and 51 metastatic melanomas. Results: A high level of p16 expression was demonstrated in BMN (96% medmn nuclear pos,tlvlty) with a slgmficant reduction when compared to primary melanomas (69%, P
Drug

resistance

evaluation

by

functional

assay

and

immunofluorescence in bladder cancer Irene Y-S Chong, Victoria Bradley, Claire Davies, M. Hayes*, Marilena Loizidou, A. J. Cooper*, I. Taylor

Department of Surgery University College London Medical School and Department of Uralogy*, Southampton General Ho.~pital Superficial bladder cancer frequently recurs and can evoke repeated chemotherapy. However, multidrug resistance (MDR) limits the effectiveness of chemotherapy.

585

We assessed MDR in recurrent bladder cancer ( n = l l ) by (i) immunofluorescent staining of three proteins associated with MDR, Pglycoprotein (P170), MDR related protein (MRP) and lung resistance protein (LRP) in frozen sections and (ii) by confocal microscopy of epirnbicin accumulation (20 mg/ml, 2 hours) in primary explants in cultures. Specifically, intracellular drug distribution was determined including changes under the influence of the MDR reversing agents, verapamil and PSC833. Five cell lines (including resistant variants) were used for method development and afforded the 'gold standard' results for both assays. 7/11 specimens exhibited positive immunoflaorescenee, 5 for P170 with or without MDR proteins, I for MRP and l for LRE with the percentage of stained cells usually <15%. Of the 7/11 immunohistochemically resistant cancers, only 5 showed a resistant pattern of epirubicin uptake in culture: ie, lesser amounts of total drug uptake compared to sensitive cultures (3x-10x), nuclear exclusion of drug, reversal of the latter pattern using verapamil and/or PSC833. Of the 4Ill specimens with negative immunofluorescence, 2 grew resistant explants hi vitra. Overall, only 7Ill specimens gave matching results using both assays (sensitive/sensitive or resistant/resistant). These early results show that both immunofluorescence and functional assay give information on MDR status. The majority of turnouts showed at least small populations of resistant cells. The clinical implication is that reversal strategy should be developed for early or prophylactic implementation. MDR=Multidrug resistance, PI70=P-glycoprotein. MRP=Muhidrug resistance related protein, LRP = Lung resistance protein.

Suicide gene therapy for colorectal liver metastases: evidence of a systemic anti tumor immune response I. M. Pope H, F. Campbell2, J. YatesN, G. Lepts~, G. Mellor2, R. B. Jones~, S. E. Christmasz, A. R. Kinsella~, G. J. Peston I

University Departments of ISurgery 2Patholagy and ~lnmamology, Royal Liverpool University Hospital. Liverpool UK Expression of the HSVI-TK gene in localized turnouts results in tumour regression following the administration of ganciclovir. A localized anti tumour immune response contributes to this regression. The aim or this study was to determine if the treatment of localized HSVI-TK expressing coloreetal liver metastases resulted in a systemic anti turnout immune response and the regression of distant metastases. The effect of HSV I-TK expression in the absence of drug selection was also investigated. Liver metastases were generated by the subcapsular injection of 5 x l0 e ceils or the K I 2TK cell line (Groups I and 2) and KI2 cell line (Group 3) in BDIX rats. All animals received a second injection or the K.12 cell line in an adjacent lobe or the liver. After 7 days, Group 2 were treated with intraperitonea] GCV 755 rag/ kg b.d. for 5 days. Following sacrifice turnout volumes were determined and T cell cytotoxicity assays performed. HSVI-TK expressing tumours showed marked regression following GCV (Group 2), mean tumour volume 0.6 mm ~ (P<0.0001, Mann-Whitney) compared to 98.4mm 3 for controls (Group 3). However, HSVI-TK protein expression alone also resulted in tamour regression (Group I), mean turnout volume 22.4mm j (P<0.05). Adjacent tamours in Group 2 had a mean tumoar volume of 45.2 mm 3 (NS), whilst Group I showed significant regression, mean tumour volume 20.2ram ~ (p<0.05). A cytotoxic T cell population recognizing the umodified KI2 cell line was identified in Group 2. Conclusions: HSVI-TK mediated therapy ofexperimental liver metastases results in a systemic anti turnout immune response. The TK protein appears to have a role in the generation of anti tumour immunity. HSVI-TK: Herpes Simplex Virus Type I Thymidine Kinase. GCV: Ganciclovir. Experiments were performed under the Animals (Scientific Procedures) Act, 1986.

Breast Cancer New stereotactic excision of abnormalities: the ABB! system

non-palpable

ma~mographic

P. J. Drew, M. J. imrie, J. N. Fox, P. J. Catleton, J. R. T. Monson, M. J. Kerin

The University of Hall, Academic Surgical Unit, Castle Hill Hospital, Hulll HUI6 5JQ Screen detected suspicious or malignant mammographic abnormalities have traditionally been managed by wire Iocalisation and excision. This has

associated morbidity due to the need for general anaesthesia, excision of significant breast tissue and the requirement for multiple procedure in some patients. We present our initial experience with the ABBI (Advanced Breast Biopsy Instrumentation) system (Auto Suture International, USSC, Norwalk, USA), used for core biopsy, diagnostic and therapeutic procedures in-patients with screen detected mammographic abnormalities. All procedures were done under local anaesthesia as day cases. Fifty-seven patients with screen detected abnormalities were evaluated with this system. Fourteen 04) patients underwent ABBI excision biopsies:

586

Abstracts

invasive ductal carcinoma (5), ductal carcinoma-in-situ (3) and complex sclerosing lesion (6) (including 2 radial scars). Median procedure times were 50 minutes (40-60). The maximum specimen weight in our current series was 14 g (7-14 g). The size of the invasive cancers ranged from 8-13 ram. All patients with carcinoma have had wide excision following their ABI excision, although no further carcinoma was found in 2 cases. All have been node negative. Core biopsies were performed on 43 patients. Histology revealed 28 benign lesions, 9 DCIS, I invasive Iobular carcinoma and 5 invasive ductal carcinoma. The patient with DCIS and invasive carcinoma subsequently underwent further definitive surgery. The mean duration of these procedures was 15 minutes (13-17). ABB1 is a new stereotactic biopsy instrumentation device that works in conjunction with the breast screening programme. It is well-toleruted, minimally invasive procedure and as such offers significant potential advantages oyer conventional techniques.

Hormone replacement therapy does not influence pathological stage of breast cancer: a study of 1113 tumours S. Stallard*~, J. Litherland*, C. Cordiner*, E. Mallon'[', H. Dobson*, W. D. George,, D. J. Holes

West of Scotland Breast Screening Centre*. Depts of Surgery~ aad Pathologyt. Wesleru Infirmary West of Scotland Cancer Sorveillance Unit~. Glasgow Women using Hormone Replacement Therapy (H RT) at the time of diagnosis of breast cancer have been reported as having a lower mortality from breast cancer compared with non users. Similarly, women using HRT when they are diagnosed have also been reported as having smaller tumours of better grade with less nodes involved compared to non-users. Favourable pathologicad prognostic features have also been reported in HRT users compared with non users with screen detected breast cancer. In a recent study however we have shown that significantly more women using HRT when they were screened developed interval cancers between screens when compared with non users. This implies that HRT users are more likely to have a cancer missed at screening compared to non users. It is therefore important to include interval c a n e r s in any study of the possible HRT effect on tumour type, size, grade and nodal status, I 113 women aged between 50 and 65 years were screened by the National B]-east Screening Programme (NBSP) between [988 and 1993 and either had a screen detected cancer (SDC) (816 women) or developed an interval cancer (297 women) between 1988 and the end of 1996. Current HRT usage was recorded by radiographers at the time of screening and also at the presentation with breast cancer (at assessment or hospital clinic). Of the 816 women with SDC 100 (12.3%) wcrc using HRT when they were screened. Of the 297 women who developed interval cancers 66 (22.2%) were currently taking HRT at diagnosis. Of the 1113 women studied therefore 166 women (15%) were HRT users wben they developed breast cancer. Tumour type was classified as DCIS, invasive ductal carcinoma (Grade I, 2, or 3), invasive Iobular, tubular, mucoid or other. Size was categorised as <10, 10-19.9, 20-29.9, 30-39.9, 40+ ram. Nodal status was classified as 0, I-3, 4 or more nodes involved. In the 1113 women studied there wcre no significant differences in the type, size or grade of turnouts or nodal status comparing HRT users with non users. When the 816 screen detected cancers alone were studied, HRT users were found to have significantly more better grade tumours, but there was no difference in size or nodal status comparing HRT users with non users.

at 6.6 (5-10) days. Complications* delaying discharge included skin envelope necrosis (4), infection (2) and haematoma (I). Complications were not increased in those receiving adjuvant chemotherapy or radiotherapy (11.1% chemotherapy alone, 20% radiotherapy, 15.3% no adjuvant therapy). Tissue expansion was completed in 4.7 (2-10) months, requiring 3.9 (2-7) visits. Further procedures were performed in 23/40 patients, including nipple reconstruction (38.1%), contralateral surgery ((11.9%), expander exchange (26.2%), capsulotomy (2.4%) and expander removal (2.4%). Relapses were recorded in skin flaps (2.5%), axilla (2.5%) and distant sites (5.0%). Ultraconservative SSM and IBR is a novel technique associated with acceptable rates of complication, recovery, recurrence and re-operation which compare favourably with more traditional skin-sacrificing procedures. *excluding seromas Values = mean (range)

The use of cytological grading in the selection of patients for neoadjuvant chemotherapy in breast cancer

M. J. A. Perry, Valerie Nufiez, K. Hollowood, Frances Davies, W. H. Allum

Departments of Surgery and Patholok:v Epsmn General Hospital. Epson The aims of this study were to compare fine needle aspiration cytology grade with histological grade of breast cancers and to determine whether cytology alone can be sufficiently reliable to select patients for neoadjuvant chemotherapy. Method: Patients attending the breast clinic underwent fine needle aspiration cytology as part of their primary assessment. Cytological grading of those with malignancy was undertaken according to the criteria of Robinson and McKec (l). In those treated surgically, cancers were graded using standard histological criteria. The results of preoperative cytological grading were compared with final histological grade. Results: We studied l l4 patients with C5 pathology who subsequently went on for histological grading. Histological Grade I 2 Cytological Grade

I 2 3

24 8 0 32

18 28 3 49

3 4 17 12 33

46 53 15 114

Discussion: The table shows the results of the comparison between the two methods of assessment. The positive predictive value of cytology, i.e. correct predictor of histology, was Grade I 52%, Grade 2 53%, and Grade 3 80%. Although cytology grade was a poor predictor for histology in low and moderate grade turnouts, the prediction in Grade 3 disease was 80%. These results are similar to those previously reported except our cytological Grade 3 corresponded to a histological Grade 3 in a higher percentage of cases. However, if cytological grading alone were to be used to determine the selection of patients for neoadjuvant chemotherapy for Grade 3 cancer, 1 in 5 patients would receive treatment unnecessarily. Reference: Robinson IA, McKee G, Nicholson A, D'Arcy J, Jackson PA, Cook MG, Kissin MW. Prognostic value of cytological grading of fine needle aspirates from breast Carcinomas. Laacet 1994; 343: 947-49.

Thesc resultsdo not support the commonly held beliefthat I'[RT users develop tumours with favourable prognostic Features.

Serum vascular endothelial growth factor in breast cancer Early experience of ultraconservative skin saving mastectomy and immediate breast and areolar reconstruction P. M. Peyser, R. M. Rainsbury

Dept of Surger); Royal Hampshire Cmmo' Hospital. Winchester Greater appreciation of the technical and oncological principles of skin saving mastectomy (SSM) and immediate breast reconstruction (IBR) is generating interest in new procedures. A technique of ultraconservative periareolar S S M and simultaneous reconstruction of the breast and areolus which allows 'seamless' breast reconstruction has been evaluated. Between 1994-1997, 40 patients (45 (31-61) yr) underwent 42 SSM, axillary dissection, IBR and areolar reconstruction using a latissimus dorsi (LD) pedicle flap and tissue expander (follow up 17.7 0-35) months). Breast resection and reconstruction was performed eitller through a single 5-6cm periareolar incision (39 cases) or via separate periareolar and axillary incisions (3 cases). Procedures lasted 4.0 (3.5-5.5) hr and were associated with 799 (390-1900) ml blood loss. Drains were removed at 6.3 (4-9) days shortly before discharge

K. Heer, H. Kumar, P. J. Drew, J. N. Fox, P. J. Carleton, J. R. T. Monson, M. J. Kerin

The University of Hull, Academic Sargical Unit, Castle Hill Ifospilal. Cottingham, East YorkMure HUI6 5JQ, UK introduction: VEGF is one of the most potent angiogenic cytokines. Its intratumouml levels correlate with MVD, nodal metastasis and disease free survival. This study evaluates the relation between serum VEGF and various pathological indices of breast cancer. Methods: Serum VEGFI65 was assayed preoperatively in 135 patients with breast malignancies and compared to serum VEGF levels in 72 healthy female controls. All tumours were staged by a single pathologist according to the TNM classification. Statistical analysis was done using the MannWhitney U Test. Results: Serum VEGF was elevated in all stages of breast cancer. Levels in nodal, recurrent and metastatic disease were significantly higher than controls. Patients with DCIS had elevated levels compared to controls. 67.5% patients had elevated VEGF levels compared to I 1.2% patients with raised

Abstracts

Controls (n=72) DCIS (n = 85 Ductal Carcinoma (n = 95) Lobular Carcinoma (n = 16) ER + VE (n = 62) E R - V E (n=20)

Median (IQR) VEGF pg/ml

P-Value vs. controls

173.8 (92.5-252.5) 450.4 (301.8-795.25 307.7 ( 155.5-483.05 207.1 ( I 18.7-379.8) 305.9 (179.7-485.2) 267.8 (94.2-523.7)

P = 0.002 P<0.0005 P=0.32 P<0.0005

P=O.I

CAI5-3 ;rod 13% with abnormal CEA levels. VEGF levels were not significantly elevated in patients with Iobular cancer or those with ER-ve tumours. Conclusion: Serum VEGF shows a correlation with not only tumour stage and cancer cell type, but also with ER status. This may have implications in the study of hormones in the aetiology, pathogenesis and prognosis of breast cancer. As serum VEGF is more sensitive than currently employed tumour markers, it may have a role in monitoring disease prognosis in breast cancer patients. VEGF-Vaseular Endothelial Growth Factor; M V D - M i c r o Vessel Density; ER-oestrogen receptor: IQR-Interquartile range: DCIS-Ductal carcinoma in situ.

587

with palpable benign tumours (by ultrasound and/or Fine Needle Aspiration criteria) underwent excision biopsy under general anaesthesia in a 0.5T• Interventional MR machine (IM R). Lesions were localized with pre-and post contrast (intravenous Gadolinium DTPA, 0.2mmol/kg5 Fast Multiplanar Spoiled Gradient static sequences (FSPGR), Pre-operative 'real-time' Fast Gradient sequences were obtained using the Flashpoint Tracking device. Aprototype three-point fat-suppression Dixon subtraction technique was also used in two lesions for pre-operative localization. Excision was performed within the IMR using titanium instruments and an ultrasonic scalpel, lntraoperative real-time scanning was performed to demonstrate the resection margin. Static FSPGR and real-time images were obtained'for all lesions at the end of the procedure to confirm complete excision. The three point Dixon method was used to assess complete resection in two patients. Resells: All tumours were visualized with both static and real-time imaging and all enhanced with contrast. Four (25%) were visualized only after contrast enhancement. All but two lesions showed late enhancement (>4 mins). Intraoperative imaging demonstrated a resection margin in all cases. Maximum lesion dimensions were from 8-50mm. All post-procodurc scans clearly demonstrated complete excision. The Dixon sequence demonstrated the lesions before surgery and confirmed excision. There were I I fibroadenomas, 2 loci of sclerosing adenosis, 1 Schwannoma, 1 lymph node and I unexpected Grade 1 ductal carcinoma with high-grade DCIS in this series. This lesion was one of the two with marked early enhancement. Conclusion: This study represents the first reported series of breast surgery within an I M R unit. I ntra-operative M R scanning reliably identifies palpable breast turnouts, demonstrates the resection margin and confirms complete excision of the tumour. Further work can now be performed to investigate the use of intra-operative MR guidance in the excision of impalpable or malignant lesions.

T h e influence o f a g e on a x i l l a r y s u r g e r y a n d survival from breast cancer D. B. Kingsmore*, A. Ssemwogererc**, D. Hole**, C. R. Gillls**, W. D. George*

C a n r e l a x a t i o n a n d guided i m a g e r y alter the Type C personality?

* Departnwnt of Snrger): Western Infirmar),. Glasgow. ** H/est of Scotland Cancer Surveilkmce Unit. Glasgow

L. G. Walker I, M. B. Walker t, K. Ogston I, S. D. Heys2, A. K. Ah-See =, A. W. Hutcheon~, T. K. Sarkar 4, O. Eremln 2

The necessity of axillary surgery is increasingly under question as the indications for chemotherapy broaden and the number of small node negative tumours increases due to the screening program. The aim of this study was to assess the adequacy of axillary surgery in breast cancer by age and to investigate whether this influences survival outcome. All 7144 cases of histologically confirmed invasive breast cancer diagnosed from 1980-1994 in a defined geographical area were identified. From the original pathology reports tumour size, number of nodes removed and number positive was obtained. Mean follow up was 10 years. Inadequate axillary surgery was defined as removal of less than four lymph nodes. Inadequate axiflary surgery was performed significantly more frequently in older women. The risk of death in those women who had inadequate surgery was significantly higher in any age group that included younger women. In contrast, thc trend in older women was of a decreasing risk with increasing age category. This became not significant at age 55. Age

No. nodes

5year srvl

10 year srvl

R.H.R.

Sig.

<55

4+

73%

59%

1.00

1-3

66%

5 I%

1.46

4+ I-3

72% 72"/,

57% 57%

1.00 1.13

baseline P<0.0 I baseline n.s.

55-74

We conclude that older women have received different treatment of the axilla. In younger women inadequate axillary surgery is associated with a poorer survival from breast cancer seen but this is not seen in older women.

Behavioural Oncology U n i t ~ and Departments of Snrgery:, Medical OncologjP mid Radiotherapy4. University of Aberdeen and Aberdeen Royal Infirmary NHS Trust Aim: The aim of the study was to assess the effects of relaxation and guided imagery on the Type C (cancer prone) personality. Methods: 96 women with large (>4cm) or locally advanced (T~, "1"4, T,N.,) breast cancer were randomized to standard support with, or without, relaxation and guided imagery. The L scale of the Eysenck Personality Questionnaire (EPQ-L) is a measure of social conformity and the 3 subscales and total scale of the Courtauld Emotiomd Control Scale (CECS) measure emotional suppression. The EPQ-L and CECS were administered before the first cycle of primary chemotherapy and after 5 cycles. In addition, the EPQ was administered 12 weeks after the completion of radiotherapy. Data were analysed on an intention to treat basis. Results: A repeated measures MANOVA for the four CECS scales and the EPQ-L scale at baseline and after 5 cycles of chemotherapy was significant (Wilk's lambda F=3.91, P=0.006). Specifically, ANCOVAs revealed that the intervention reduced emotional suppression as assessed by the CECS total score (F=6.96. P=0.01), the CECS anxiety subseale (F=4.96, P = 0.03) and the CECS unhappiness subscale (F=9.57, P = fi.fi03). The largest effects were found in those practicing at least daily. When all 3 time points for the EPQ-L scale were included the difference between the 2 groups was significant (F =4.12, P<0.05). Conclusions: Relaxation and guided imagery reduce emotional suppression and conformity (Type C characteristics). Several studies have found that psychosocial interventions enhance survival: the present findings may help to explain this phenomenon.

Is invasive Iobular carcinoma associated with enhanced risk of margin Excision of breast turnouts in an interventional magnetic resonance unit S. Gould, G. Lamb, W. Gedroyc, D. Lomax, A. Mansfield, A. Darzi

Academic Surgical Unit, Imperiol College School of Medicine at St. Mary's, London Background: Intervcntional Magnetic Resonance (MR) machines produce unique opportunities for image guided tumour surgery. Fast pulse sequences producing a new image every 1.5 s allow virtually 'real-time' intra-operative scanning to monitor the progress of resection. Potential advantages include confirming complete tumour excision and identification of vital structures to avoid injury during the procedure. This study was undertaken to assess the potential use of the machine for image-guidod breast surgery. Materials and Methods: Sixteen female patients (median age 28.4 years)

positivity a n d local recurrence a f t e r b r e a s t conservation? H. Y. Chan I, A. L. Harris3, A. Jonesa, M. J. GreenalP

Deparmwnt of Surgery i, John Radcliffe Hospital. Oxford mid Department of Radiotherapy~and ICRF Clinical Oncology Unit~. Churchill Hospital. Oxford We reported (BASO - Aberdeen 19975 in 684 cases of invasive doctal carcinoma, that positive margins were associated with increased risk of local relapse (LR). If the surgical cavity biopsies were negative, the 5 year LR rate was 5.2°/., if they were involved with in situ disease, the LR rate was 21.2% and if they were involved with invasive disease, the LR rate was 32.4%. The overall LR rate was 8.3%. A similar analysis has now been performed for 98 cases of pure invasive Iobular carcinoma followed up for 5 years. Twenty-three patients (23.5%) had positive margins (cf. 13.3% in ductal carcinoma - Chi 2 P=0.008). Six

588

Abstracts

of these patients had LCIS in the margins and none of these developed LR. Seventeen patients had invasivc lobular carcinoma at the margins; 4 of these patients developed LR (23.5%) (cr. 32.4%0 in ductal - Chi = P = 0.47). Seveatyfive patients had negative margins and only 4 (5.6"/,) developed LR (cf. 5.2% in ductal). Overall the LR rate for Iobular carcinoma was 8.2% (cf. 8.3%0in ductal - Chi 2 P=0.95). These results suggest that marginal involvement is more common following breast conservation for invasive Iobular carcinoma. However, in this series, local recurrence rates following breast conservation for Iobular carcinoma are no greater than that seen with invasive ductal carcinoma.

physical morbidity (infection, seroma, shoulder movement) and psychological morbidity (Rotterdam Symptom Questionnaire, Checklist of Concerns, Hospital Anxiety and Depression Status) compared For each discharge policy 'pre-op and at I and 3 months post-op. To date 100 women have been randomized into the trial, 20% undergoing mastectomy and axillary node clearance and the remainder breast conservation surgery. Women discharged early had less wound pain and greater shoulder movement at one month post-op than standard discharge patients and were less likely to consult their general practitioner post-operatively (P<0.02). At one mouth a significantly lower Rotterdam Symptom Score was seen in patients undergoing early compared to standard discharge (P~g0.03) and generally psychological morbidity was lower in patients discharged early.

n

The accuracy of one-stop diagnosis for 1110 patients presenting to a symptomatic breast clinic

J. A. JibriP, E. L. Tahirt, J. Squair2, S. D. I-leysI, A. K. Ah-SeeI, G. Needham~, H. Deans3, F. GUberP, M. MeKean4, L. SmarP, O. Eremin ~

Professorial Sargical Unit I, Department of Public Health ~. Radiology~ and Cytolog), 4. University of Aberdeen and Aberdeen Royal Hospitals NHS Trust Background: To'minimize delay and anxiety and reduce unnecessary open biopsies, triple assessment with immediate reporting has been used in our symptomatic breast clinic since 1991. The effectiveneSs and reliability of the process .requires careful evaluation. Aim: The aim of this study was to establish the accuracy of the diagnostic modalities used and the el~cacy of this one-stop diagnostic policy. Methods: The data on I I l0 patients, newly presented to the symptomatic breast clinic between January and July 1993, were analysed and the subsequent 3 year follow-up and outcome established. Sensitivity, specificity and predictive values were determined using standard (SPSS for Windows) statistical analysis. Results: Fine needle aspiration cytology (FNAC) provided the highest (97.3%) overall prediction or benign and malignant lesions, with a sensitivity of 93.5% and specificity of 98.1%o. Uhrasonography provided 97.0% overall prediction with a sensitivity of 88.9°/. and specificity of 97.4%. Mammography provided 96.4% overall prediction with a sensitivity of 93.2"/,, and a specificity of 96.7%. When the mammogram or ultrasound scan were reported benign, the false negative rate was 0%. The false positive and false negative rates for FNAC were 2.4% and 1.4%, respectively. Following assessment, a diagnosis was made in 96% of patients; 62% were discharged at the first clinic visit. Conclusion: The 'one-stop' Symptomatic Breast Clinic provides a quick, accurate, and effective means of establishing a diagnosis.

A randomized controlled trial of early versus standard discharge after breast cancer surgery N. J. Bundred, Jill Reynolds, D. Allen, J. Rees, Jill Grimshaw, L. Barr, A. D. Baildam, P. Maguire

University Hospital of South Manchester and Christie Haspital. Manchester Early discharge after breast cancer surgery would reduce hospital costs and stay but its effect on physical and psychological morbidity is unknown. We have performed a randomized controlled trial comparing early discharge at two days post-op with a standard discharge policy. Patients have had

Median hospital stay (range) Restricted ann movement Rotterdam Score at I month Wound pain at 3 months

Early 50

Standard 50

2 (2-8) 89% 10.4 (0-54) 7 (15%o)

5 (3-13) 63% 14 02-51) 13 (33%)

(P_<0.01) (P ~;0.01 ) (P=0.03) <0.05

Early discharge alter breast cancer surgery is feasible and does nut increase physical or psychological morbidity.

Patient satisfaction and morbidity with tissue expansion breast reconstruction undertaken by breast surgeons M. G. Berry, R. A. M. AI-Mufti, S. Denton, M. Sullivan, A. Vaus, R. Carpenter

The Breast Unit, Royal Hospitals NHS TrlLVl. St. Barlholomew:v Hospital. London ECIA 7BE Immediate breast reconstruction remains a procedure offered to a minority of women in the UK despite the proven psychological benefits of reconstruction at the time of mastectomy. Traditionally performed months or years later by a Plastic Surgeon, currently there are a number of options within the scope of the interested Breast Surgeon which allow immediate reconstruction. One of the least complex is the Beckcr expander prosthesis which acts initially as a tissue expander, but remains in situ and on removal of the filling port, becomes the pennament implant. We have evaluated the first 100 patients, median age 52 years (range 25-74). to have this procedure at our Breast Unit. Clinical outcome and patient satisfaction were assessed, the latter using a postal questionnaire. The questionnaire response rate was 84%,,. Patient satisfaction was high with 84%°of respondents expressing satisfaction with the final result. A more detailed visual analogue scale (range 1-10) produced a median grade of 8. The largest cause of morbidity was infection requiring removal of the prosthesis in 14%. 28% have undergone revision surgery in the form of capsule release (capsulotomy) with no statistical relationship to radio- or chemotherapy. Contralateral surgery has been undertaken in women to achieve symmetry either at the time of surgery or subsequently in 8%,,. During the four-year study period 7% have died as a result of their disease which highlights one of the benefits of such a simple technique. Where necessary more complex revision surgery can be undertaken at a later date as in two of our patients. In summary, this is a technique which is able to produce results with a very high degree of satisfaction in the majority of patients.

Abstracts

589

Gastro-intestinal II

A phase !1 study of the oral matrix metalloproteinase inhibitor, marimastat, in patients with inoperable gastric cancer Gillian Tierney, S. L. Parsons, N. R. Griffin, Susan Watson, R..l.C. Steele

Deptlrtnwnt of Surgery Universit), Hospital. Nottingham Introduction: The MMPs are a family of proteolytic enzymes involved in turnover of the extracellular matrix lmd have been implicated in the process of tumour growth and metastasis. Marimastat, an orally available MMP inhibitor binds to the active site of the MMP molecule rendering it inactive. Aims: To confirm the safety of a four week course of marimastat. To assess the turnouts at endoscopy and examine biopsies histologically. Using zymography, to quantify turnout MMPs prior to and after treatment. Methods: Twenty-five patients with advanced gastric adenocareinoma underwent pre-dose endoscopy and biopsy of the tumour. They received marimastat al a dose of 50rag BD (first 6 patientsl or 25rag OD (all subsequent patients). Endoscopy was performed at day 28. Patients with a response to the treatment or static disease in the absence of side-effects were selected to continue. Biopsies were sent for histology and gelatin zymography. Results: Both doses gave adequate plasma drug levels (mean trough level: 260 l.tg/I on 50 mg BD, 50 lag/I on 25 mg OD). Fifteen patients had continued use of the drug, nine on the basis of response (defined as decreased tumour vascularity, evidence of stroma formation or decreased size). The side-effects were musculoskeletal; arose after 28 days of Urcatment, appeared doserelated and resolved after a treatment break. There was no difference in the zymography profile after treatment. Discussion: This study has demonstrated good oral bioavailability of marimastal. Side-effects appear dose-related and reversible. These effects may be due to inhibition ofcollagcnase in peri-articular tissues. A prospective r;mdomized phtcebo controlled study of this treatment is currently underway. M M P = matrix metalloproteinase.

Expression of endothelin-1 in 98 patients with colorectal cancer E. H. Asham, Marilena Loizidou, S. Lakhani**, K. Miller**, G. Burnstock*, P. B. Boulos, I. Taylor

Departments of Surgery Anatomy* and Histopathology**. University College London Medical School. Lomhm Endothelin-I (E'T-I) is a potent vasoactive 21-aminoacid peptide first isolated and sequenced from endothelial cells. It has a multifunctional role in a variety of tissues and cells. Recent studies suggest that ET-I has both mitogenic and angiogenic properties in many tumours. We have studied the expression of ET-I in resected eolorectal cancer from 98 patients. Sections (6 ram) of paralfin wax embedded specimens were stained for ET-I using an indirect peroxidase- anti-peroxidase immunohistochemical method, with Diaminobenzidine as the chromogen. Antigen unmasking was attempted using a variety of methods and was successful after microwaving. Intensity of staining (0. +1, +2, +3) and number of cells stained (0, +1<25%, + 2 = 2 5 - 5 0 % and +3>50%) were scored by 3 independent observers as Cover 10-20 fields per slide). Information on Dukes' stage and survival was collected. There were 9 (2%) Dukes' A, 62 (63.26%) Dukes' B and 27 (27.55%) Dukes' C patients. ET-I staining was graded as follows: Intensity 0 + I +2 +3

7 (7.14%) 43 (43.87%) 42 (42.85%) 6t (6,12%)

% of cells stained 7 4 31 56

(7.14%). (4.08%) (31.63"/0) (57.14%)

There was no correlation between either the intensity of staining or the % of stained cells and Dukes' stage or survival. In conclusion, ET-I was strongly expressed in cancer cells and the surrounding stroma. It may act in an autocrine/paracrine fashion to promote cellular growth and proliferation. ET- I = Endothelin- I.

Reduction in membranous expression of ~.:catenin predicts poor survival in gastric cancer S. Ramesh, P. McCufloch

Department of Surgery Universit.t,of Liverpool. Li~'erpool [~-catenin. a component of the E-cadherin-catenin cell-adhesion complex, also plays a separate intracellular signalling role, interacting with APC protein. Intraecllular accumulation of I~-catenin is common in colorectal neoplasia. [~-catenin abnormalities are associated with poor survival in gastric cancer, but previous studies do not differentiate between membraneassociated and intracellular 13-catenin. In this study we aimed to determine which type of I$-catenin expression abnornmlities correlate with survival in gastric cancer. Immunoperoxidase staining of paraffin-embedded sections from 40 gastric cancers was performed for E-cadherin. a- and ~-catenins using microwave unmasking and a Biotin-Avidin technique. Clinical data were obtained from case records. Abnormalities of intracellular 13-catenin staining were rare in gastric cancer (M40, 5%). Reduced membranous expression occurred in 10/I 2 (83%) diffuse and 8/28 (29%) intestinal turnouts (P=0.0014). It was associated with poor differentiation (P=0.0015) and short survival (P=0.032), but not with age, sex, tumour size or nodal status. Reduced E-cadherin membrane expression was associated with lymph node metastasis (P=0.02). Neither E-cadhedrin or et-catenin expression correlated with survival. Reduced membranous expression of 13--cateninpredicts poor prognosis in gastric cancer, whilst ectopic intracellular expression is rare. The apparent differences in ~-eatenin expression from those found in colon cancer merit further study.

Long term perfusion of composite free flaps I. H. McVicar, Sheila E. Fisher, M. L. Wastie, A. C. Perkins, R. M. Vincent

Maxi/lofi:cial Unit and Department of Medical Physics. Queen:v Medical Centre, Notlinghatn Bone scans are used routinely to help in determining whether the mandible may be invaded by tumour. They are also used to confirm the viability of bony free flaps used to repair defects following ablative tumour surgery. Short term studies have shown that a viable bone free flap appears as a 'hot' bone scan. It is not known whether the bone scan remains 'hot' over a longer period. Aim: The aim of this study was to establish whether the bone scan remains 'hot' after a period of at least one year. What happens to the appearance of the bone scan as these flaps mature has been unknown. This has led to the inability to interpret later bone scans particularly when recurrence is suspected or new primary disease presents. The practical significance of this study is to avoid unnecessary morbidity and needless sacrifice of free flaps when later surgery is necessary for recurrence or new primary disease. Nine patients were entered into an ethically approved trial. Technetium bone scans were performed in all cases at least one year following surgery. Results: There is no increase in bony uptake in free flaps as measured by Technetium bone scans after at least one year. Conclusions: The activity of the bone in the composite free flap as measured by Technetium bone scans returns to normal levels within one year.

Abstracts

590

Head and Neck

Ablation of basal call carcinomas with an optomeehanically flash scanned carbon dioxide laser. Which turnouts are suitable?

CYP2D6 = Cytochrome 1'450 2D6; EM = extensive metabolizers; PM = poor metabolisers: PCR =polymerase chain reaction

N. M. Horlock, A. O. Grobbelaar, D. T. Gault RAFT brstitute for Plastic Surgery Molmt I"ernon Hospital. Northwood Introduction: Carbon dioxide laser ablation has been advocated for the treatment of basal cell carcinomas. However, the ability of this method to completely ablate basal cell carcinomas has not been demonstrated. Furthermore the selection of patients most suitable for this treatment modality has not been determined. Method: A treat and excise protocol was utilized. The basal cell carcinomas were biopsied, then ablated with the CO2 laser (Sharplanl with a optomechanical flash scanner (Swiftlase), followed immediately by surgical excision. Histological examination of the excision specimen was performed. Data was collected for tumour type, ablation margin (complete or incomplete), for ablation depth and for laser and excision times. Results: Fifty-two basal cell carcinomas, ranging from 4-35 mm, were ablated. Clinically there were 22 superficial, 27 nodular and 2 infiltrative types. Complete ablation at the deep margin was associated with ablation depth ( P = 0.006) and with turnout type (P = 0.01 ). All tumours of superficial subtype could be reliably completely ablated provided they were lasered to a depth of the middle dermis or deeper. In contrast nodular tumours could not reliably be ablated by this method at any depth. A small subset of nodular tumours less than 10 mm diameter, however, were all completely ablated provided they were lasered to a depth of the lower dermis or deeper. Laser treatment was significantly faster than excisional surgery (mean 3.7 minutes compared to a mean of 17 minutes). Conclusion: This fast treatment modality requires careful patient selection with precise control of the albation depth. Patients with multiple superficial basal cell carcinomas are the most suitable.

Free tissue transfer in patients over 75 with head and neck malignancy A. M. Richards, R. P. Cole Odstock Centre fi~r Borns. Plastic aml Maxilh,-fiwial Surger.t; Salisl,ury District Haspital. Salisbury Radical excision and reconstruction, often with free tissue transfer, has been shown to offer the best hope of cure for advanced head and neck malignancy. Despite the fact that these patients are often elderly, with significant preoperative morbidity tbere have been few. if any, reports of tile success of excision and free tissue reconstruction in patients over 75 years old. We therefore performed a consecutive review of 12 patients over 75 undergoing such surgery in this Unit over the past three years in an effort to clarify indications, success rate and complications. The age of the patients ranged from 75 to 88 years (mean 81). All of the flaps survived. Two (16%) required early re-exploration. Six of the twelve (50%) patients remain tumour free an average of 18.3 months after surgery (range 1-33 months). One died of causes unrelated to his tumour 14 months after surgery. O f the remaining five. two died in the early post-operative period, two of recurrent tumour or a second primary and one remains alive 8 months post-surgery despite local recurrence. We discuss the indications, complications and outcomes of surgery in these cases and discuss patient selection and measures which may help reduce peri-operative morbidity and mortality in these elderly patients undergoing radical surgery.

Analysis o f the p l 6 gene in o r a l s q u a m o u s cell c a r c i n o m a

CYP2D6 polymorphisms are associated with an increased risk of oral squamous cell carcinoma S. F. Worrall Department of Surgery Universit.v of Keele School of Postgroduate Medicine. Stoke-on- Treat Oral squamous cell carcinoma is the end result of a multi-phase process in which carcinogen induced genetic changes produce unrestrained cellular growth. The first step in this process involves the covalent binding of xenobiotie compounds to cellular DNA. To protect themselves from xenobiotic damage higher organisms have developed complex and complementary enzyme systems capable of detoxifying and eliminating these harmful compounds. The cytochrome I)450 mono-oxygenases are responsible for the phase I metabolism of numerous electrophiles including those present in tobacco products which are the major xenobioties involved in oral carcinogenesis. Variation in detoxification enzyme activity has been shown to influence susceptibility to various diseases including many cancers. CYP2D6 has variable activity throughout the population due to allele polymorphism at its gene locus on 22ql3.1. Homozygous 'wild type' individuals are termed EM while individuals homozygous for a mutated allele are termed PM. This study investigated whether CYP2D6 polymorphism carries an increased risk of oral cancer. Following ethical committee approval, 5 ul of venous blood was drawn from 105 patients with a histologically confirmed diagnosis of oral squamous cell carcinoma. Leukocyte DNA was extracted by phenol/chloroform precipatadon and amplified using standard PCR techniques. The PCR products were digested with the restriction enzymes Hpall and Bstnl. The enzyme products were separated by agarose gel electrophoresis and photographed under ultra violet light. 85°/, of samples were fully informative for the CYP2D6 polymorphism. The allele frequency in the 89 cases was compared to the allele frequency in 467 cancer free local controls. The cases had a significantly higher PM frequency (P=0.03). Data were also analysed to assess for the effects of age, tobacco and alcohol consumption. Patients below 62 years at diagnosis had an EM frequency higher than controls (P=0.012) while those aged over 62 years had a PM frequency higher than controls (P=0.004). There were also differences in allele frequencies in patients who were non-drinkers and nonsmokers compared to controls (P=0.09) and in patients who were heavy drinkers and heavy smokers compared to controls (P=0.026). These results suggest that CYP2D6 polymorphisms are associated with an increased oral cancer risk and that their effects vary with the level of exposure to xenobiotics in tobacco and alcohol products.

N. S. Thakker, Chu Lee Wu, L. Roz, P. Sloan, A. P. Read, S. Porter, C. Scull),, P. Speight

Medical Genetics and Oral Patholog)qOral Mediciae and Oral Palholo~,:v Univer.rit), of Manchester. Manchester. Eastman Dental hlstitnte. London The p l 6 protein is a specific inhibitor of the cyclin dependent kinases CDK4 and 6 and its inactivation leads to failure of cells to undergo cell cycle arrest in GI. The inactivation of the p l 6 gcnc on chromosome 9p21 has been implicated in the devclopmcnt of a variety of malignant tumours including head and neck squamous cell carcinomas (HNSCC). Cytogenetic abnormalities involving chromosome 9p, are one of the commonest aberrations in H NSCC and losss of heterozygosity for polymorphic markers at the pl6 locus has been observed in between up to 75% of HNSCCs. More recently, inactivation of pl6 by methylation and homozygous deletions has been reported in up to 83% of HNSCCs. We report here analyses of the pl6 gcnc specificany in oral and oropharyngeal squamous cell carcinomas (OSCCs). Thirty six formalin-fixed paraffin embedded tumours were tested for LOH on chromosome 9p using up to 9 STRPs. 78% (28136) of the turnouts showed LOH at one or more loci. Eighteen turnouts in total showed interstitial deletions. These appeared to define 2 discrete areas of deletions: one ceq.U'omeric and the other telomeric to the pl6 locus. Nine of these 18 tumours had retention of hcterozygosity at the loci encompassing p l6 gene (1FN D8S 171 ) accompanied by LOH at the next informative flanking markers both proximally and distally. Such a pattern could represent homozygous deletions at the pl6 locus with an apparent retention of heterozygosity due to amplification of stromal contamination in absence of tumour alleles or it could represent novel tumour suppressor gene loci flanking the pl6 gene. Thirty primary tumours and 7 cell lines were screened for mutations in all exons of the pl6 gene using SSCP-heteroduplex analysis. Two primary turnouts had a nonsense mutation in codon 58 (RI72X). The same mutation was found in 2 cell lines (HI03, H357). A third cell line (HI57) had a nonsense mutation at codon 80 (R238X). Thirty primary turnouts and 7 cell lines were investigated for mcthylation of 5'CpG island of the p l6 gene. De novo methylation was observed in 7/30 (23%) primary tumours and 3/7 (43%) cell lines. The pl6 mutations and promoter methylation were accompanied by LOH in some cases. However, none of the samples showed inactivation of one pl6 allele by mutation and the other by methylation. These findings suggest that these pl6 inactivation events may be mutually exclusive. In conclusion, we have demonstrated inactivation of p l6 by either methylation or mutation in approximately 30% of OSCCs. In addition, if the pattern of LOH in these tumours is indicative of homozygous deletions then at least additional 25% of the tumours show homozygous deletions.

Abstracts H N S C C = H e a d and neck squamous cell carcinomas; O S C C = o r a l / oropharyngeal squumous cell carcinomas; S T R P = s h o r t tandem repeat polymorphisms.

The midfacial degloving approach to sinonasal tumours R. W. Clarke, Gady Har El Department of Otorhinolaryngology. State University of New York The midfacial degloving approach (MFD) provides wide access to the nose siuus's nasopharynx and skull base. It avoids facial scars and fitcilitates

591

bilateral surgery. It also avoids the risk of post-operative palatal dysfunction. Fifty two such procedures have been performed at the SUNY O R L " • Department in an eight year period. These include 42 patients with midfaeial and skull base tumours including inverted papilloma, carcinoma, angiofibroma and esthesioncuroblastoma. The age range wits 4-82 years. In all cases adequate exposure was obtained to permit good local clearance. The main complication was paraesthesia in the distribution of the infraorbital nerve, although usually short-lived, this persisted for one year in three patients. There was one case of symptomatic posp-operative nasal stenosis. The M FD approach is now the procedure of choice for angiofibroma and the the en-bloc removal of turnouts of the antro-ethmoidal region when full craniofacial resection is not required.

Posters

MRI in the detection o f breast cancer multicentricity M. Douek ~, J. Vaidya ~, S. R. Lakhani2, K. Blanclfard "~,M. A. HalI-CraggsJ, 'l\ DavidsonI, M. BaumI, I. Taylor t Departments t~ ISurger.l~ 'Hist~puthology and ~RadioloKI, UniversiO, College London Medical School. London WCI N 8/I/I The significance of small multicenlric loci in breast cancer has been questioned. Whilst mammograms rely on microcalcifications for detecting cancers, contrast-enhanced MRI relies on vascularity and vascular permeability. We propose therefore that MRI would detect only clinically important multicentric loci. In this study we compared the pre-operative detection of multicentric loci by contrast-enhanced MRI with standard radiological-histological examination of the resected specimen. Method: Ten patients with newly diagnosed breast cancer underwent preoperative contrast-enhanced breast MRI using a transverse TI-weighted thrce dimensiomd (3D) FLASH sequence. After surgical excision the specimens were fixed and cut in the same plane as the MRI. The pathologist sampled any identifiable lesion for histological analysis and subsequently a mammogram was performed on the specimen slices. Two observers identified calcifications and these were then sampled and paraffin blocked. One section was cut from eat.'h block, stained and examined by microscopy. The remaining blocks were x-rayed and any calcifications led to further sectioning. MR images were reviewed independently a,~d findings compared with histology. Results: On mammography of tumour slices, 71 suspicious areas were identified and sampled. Histologically. 12 areas of DCIS or invasive cancer were identified l - 4 c m from the primary tumour. Two turnouts showed diffuse patchy enhancement which on histology was DCIS. There was concordance between the magnitude of satellite enhancement loci and the number of histologically abnormal samples. Conclusion: These preliminary findings suggest that MRI is highly sensitive for showing multifocal invasive or in-situ tumours but that the specificity for the diagnosis of malignant change in these loci may be low.

Expression o f antimetastatic oncogene nm23 in colorectal carcinoma definition with P.C.R. D. P. Mandrekas, H. Karageorgos, A. Macheras, T. H. Liakus, A. Karameris, M. N. Seehas

t

3d Department of Surgery UniversiO, of .4thens, Medical School-Greece Sotira Hospital The gene nm23 is located in the chromosome 17,q2 I 1.3-22, and its expression is associated with low metastatic potential. The aim of the study was to demonstrate the value of rim23 as a metastatic potential indicator. For this purpose selected cases of colorectal carcinomas (blood serum and tissue samples from paraffin blocks were obtained) were divided in two groups: five cases with low infiltrating tumors and five cases

of tumors with local and distant invasion. Wc measured I) the expression of nm23 monoclonal antigen in paraffin blocks using immunohistochemical methods and 2) the transcriptional levels of antigen in blood serum samples using molecular biology techniques. The resulting analysis demonstrated an increased expression of nin23 (in both tissue and blood serum samples) in the cases with low infiltrating tumors and absence of any expression in tumors with local or distant metastasis. Low differential carcinomas had no detectable expression of the oncogene nm23, while the physiological colorectal mucosa (obtained from the same paraffin blocks) expressed low levels of the nm23. In conclusion, it seems that there is a strong correlation between the expression of the nm23 and the metastatic potential in eolorectal carcinoma and probably measurement of nm23 levels may be an important indicator of the biological behaviour of this type of tumor.

Assessment o f cellular m a r k e r s in o r a l t u m o u r s

D. Srinivasan, Sheila E. Fisher, Rashmi Seth, D. Jenkins, N. R. Grifliths Queen's Medical Centre. UniversiO, Hospital NHS Trust. Nott#~ghmn Aim: To assess markers of apoptosis and cellular proliferation and to determine whether these are linked to prognosis in a highly selected group of patients. Method: The patient groups were selected from our clinical database and included: a) the five most clinically indolent tumours. b) the five most clinically aggressive tumours, c) a group of four turnouts, where a good long term clinical outcome was noted despite extensive nodal involvement. Material was tested using bel 2 as a marker of apoptosis, mib I as a marker of cellular proliferation and also p53. Slides were prepared from archived material and immunohistocytochemistry staining performed by the streptavidin biotin complex method on a DATO Techmate 500 processor. The specimens were also tested for high risk HPV sub-types by PCR. Results: a) None of the turnouts were positive for bel 2. b) There was no correlation between clinical aggressiveness and cellular proliferation as measured by mib I. e) P53 likewise did not correlate with tumour aggressiveness. d) HPV positive turnouts were only seen in the indolent group. Conclusions: Using standard markers, no relationship between apoptosis and cellular proliferation and tumour behaviour has been demonstrated in this small series. However, only tumours with an indolent clinical behaviour were positive for HPV and this possible relationship merits a larger study.

592

Abstracts

Enhanced activity of matrix metalloproteinase 2 and its activated form in human colorectal cancer C. Keh, T. lsmail, Phillipa G. DeTakats, A. Martin, M. J. O. Wakelam, D. J. Kerr CRC Institute for Cancer Stadies and Department of Sargery University Hospital, Birminghmn Background: Matrix metalloproteinase 2 (M MP-2) is a member of the matrixdegrading metalloproteinases family. MMP-2 degrades extracellular matrix in tissue surrounding the tumour and are believed to play a critical role in cancer invasion and metastasis. The aim of this study was to examine the activity of M M P-2 in colorectal cancer and its relation to the different stages and grades of the disease. Methods: Fifty-nine paired tumour and its corresponding normal mucosa were taken. Gelatin zymography were performed and the amount of type IV collagcnase activity were quantitated by computer assisted densitometry, Results: The activities of both latent and active MMP-2 are significantly increased in tamour. In tumour a higher proportion of the MMP-2 are activated. There is an increasing trend with more advanced and aggressive disease. • Discussion: The findings imply that MMP-2 activities are enhanced in colerectal cancer. The activation of M M P-2 may be an important therapeutic target in the future.

Breast fine-needle aspirations (FNA) by GPs - are they useful? Sarah Prance, Linda Campbell, C. Tea.~lale, R. M. Watkins

•Derriford Itospitol. Plymouth PL6 8DH Recent guidelines issued by the NHS Breast Screening Programme indicate that only if patients have multiple recurrent breast cysts, and the GP has the necessary skills, is aspiration by the GP acceptable. This study was designed to assess the accuracy of FNA by GPs prior to referral breast clinic. It has also examined whether or not each FNA was appropriate. A retrospective review of 75 patients attending the breast clinic between May 1994 and July 1997 was performed. The case notes of those patients whose referral letter stated that they had recently undergone FNA by their GP were studied. Of 15 patients with cysts aspirated by their GP, cyst fluid was sent for cytological examination in II (73%) cases. All specimens were either CI or C2. SLx (40%) patients needed further aspiration in the clinic. Fifteen patients with a final diagnosis of carcinoma had undergone FNA by their GP. No cytology specimen was sent in 8 (53%) cases and two specimens were inadequate (CI). Of the five adequate specimens none proved diagnostic of carcinoma. Twenty patients with benign breast change and no discrete mass had undergone FNA by their GP. Twelve (60%) specimens were not sent for cytology. Only two specimens reported benign cells with adequate cellularity. This study indicates that the accuracy of FNA cytology by GPs is low, cyst fluid is often sent unnecessarily and FNA is often performed inappropriately.

Vital tissue staining of oral epithelial dysplasia

Pre-operative radiotherapy - The early North Trent experience using the Uppsala regime

C. J. Kerawala, I. C. Martin, M. Reed Department of Oral and Facial Sargery. Snmterland Royal Hospital

A. Raza*, R. Keadal*, D. Radstonet, J. Bolgert, A. Shorthoes~ M. Reed¶ G. Jaeabs:[:, K. B. Hosie*

Introduction: The detection of pre-malignant lesions of the oral mucosa allows for early treatment which potentially prevents progression to invasive carcinoma. Although a number of techniques have been developed to enhance the detection of epithelial dysplasia, clinical examination remains the gold standard. Toluidine blue vital staining has been advocated as a simple, inexpensive and sensitive chair-side test with high sensitivity. However, the ajority of clinical series investigating its efficiency have relied on clinically normal mucosa and/or tissue which has stained. Method: In order to quantify the true sensitivity of toluidine blue in the assessment of malignant and pre-malignant lesions of the present study therefore examined both clinically normal and abnormal mucosa and compared it to the presence or absence of staining. Results: Whilst the sensitivity of toluidine blue was absolute with respect to invasive carcinoma, false negative rates of 43% and 58% respectively were obtained for carcinoma in-situ and mild/moderate dysplasia. Conclusion: These results suggest restricting the role of vital staining to selective use in either high risk patients or those with suspicious oral lesions.

Pyridinoline and Deoxypiridinoline excretion in patients with breast cancer

J. Winstanley, A. Modi, W. Fraser Departments of Surgery and Clinical Chemistry. Royal Liverpool University Hospital Liverpool U.K. Bone is one of the most frequent sites of metastasis in patients with breast cancer. The current gold standard for diagnosis is an isotope bone scan, however these are costly, time consuming and not always easy to interpret in the presence of significant degenerative disease. Therefore, there is a need for other more sensitive indicators of boney metastasis. The breakdown of bone matrix associated with metastases releases two components of collagen, pyridinoline and deoxypyridinoline both of which are measurable in urine, as such it is postulated that elevated levels of these may be a useful screening tool for boney metastases. We have collected urine from patients coming for routine follow-up of breast cancer to determine the value of this test as a screening tool for boney metastasis. Urine samples were collected from 80 patients undergoing routine followup for breast cancer. Of the 80 specimens 23 had elevated levels ofpyridinoline/ deoxypyridinoline. Of these 23 patients with elevated levels 5 had bone SCan metastases; the mean level of pyridinoline deoxypyridoline in the urine of these 5 patients was significantly higher (Mann-Whitney P =0.001) than in the patients without metastases. , The results suggest that this test may be a useful means of assessing patients for the likely presence of skeletal metastases and avoid unnecessary bone scans.

Departments of Surgery, *Northern General Hospital. ¶Hallamshire tlospitaL ~Doncaster Royal Infirmary and Ontology t Weston Park ltospitaL SheffieM A recent study using adjuvant pre-operative radiotherapy of 25 Gray given as 5 fractions over 5 days immediately pre-operatively has shown a significant reduction in local recurrence with an acceptable peri-operative morbidity. We have audited the peri-operative mortality and morbidity following adoption of this regime for selected patients by specialist eolorectal surgeons in North Trent. Uppsala

30 Day Mortality Anastomic Leak Rate Perineal Wound Breakdown No Complications

North Trent

PreOperative DXT

No DXT

2% 11%

2% 8%

20% 56%

66%

Preoperative DXT

No DXT

(n=28)

I if'/,,

13.6% 21% (3/14)

5.1% 7.4%

38% (5/13) 36%

<10%? ?

Although these early results represent a small number of patients, the high peri-operative morbidity and mortality suggest that pre-operative radiotherapy should be introduced with caution and the outcomes should be carefully audited.

J u s t a m a t t e r of routine? Patient's perceptions of follow-up after treatment for breast cancer E. L. Peanery, G. Guli, J. Manett, I. Smith

Royal Marsden NItS Trust, Rehab Department, Folhmn Road, Loadon SW3 6JJ Early detection and longer survival have led to an increasing number of patients requiring follow-up after treatment for breast cancer. This constitutes a significant workload in breast clinics that is time consuming and involves costly investigations with undefined efficacy. An extensive review of the literature exposes the lack of consensus regarding frequency and duration 6f clinical follow-up and which investigations should be performed. There is no evidence that early detection of recurrence is associated with a more favuurable outcome and there is little data on the effects of the current system on health related quality of life. Breast cancer is a chronic disease and patients have multiple needs which change over time and which may not be met during routine follow-up. The current system of follow-up is traditionally routinized and lacks an individualised approach. Opportunities

Abstracts for change lie in discovering what patients' perceptions and needs are and formulating an intervention to address those needs. The aim of this preliminary study was to ascertain patients' perceptions of routine follow-up after completion of treatment for breast cancer. Patients (n=24) were recruited using systematic, stratified sampling. Data were collected using semi-structured taped interviews to allow for exploration and in depth coverage of defined parameters. The tapes were analysed and coded to ascertain predominant themes. Patients reported that examinations were hurried and investigations ware not reassuring. Poor continuity was deemed unacceptable by 92% of the participants. The majority of patients felt uncomfortable expressing emotional concerns or asking questions. Tbree-qu:trters of the sample would prefer to receive all or part of their follow-up from a breast care nurse. These results identify areas for future consideration to include improving patient satisfaction with follow-up services, ensuring continuity of care and more time for patients' individual needs to be met. This in turn ensures a greater focus on psychological, emotional and informational nceds, thus enhancing quality of life for individual patients and promoting an interdisciplinary approach to patient care. Existing practice needs to be modified in light of resource implications and cost etficiency. These results now justify a randomized, patient-focused, nurse-led intervention, which will be formerly, prospectively evaluated using comparisons of outcome of patients randomized to this or conventional medical follow-up.

M e r k e l cell carcinoma in the elderly S. S. Mudan, P. C. Weaver

Queen Alexandra Hospital. Coshmn. Hmnpshire Introduction: Merkel cell tumours are rare neuroendocrine lesions of the skin. The prognosis is thought to be particularly poor. We have examined this question in patients over 70 years of age. Method: The prospective data base from our multidisciplinary oncology clinic was examined and 13 cases of Merkel cell tumours arising in patients over 70years at presentation were identified. Demographic and tumours related factors were subjected to tumour-specific survival analysis using the Kaplan-Meier method and the log-rank test. Results: The male female ratio was 3:10 and the median age was 79 year (range 70-93). Ten cases arose on the face, two on the thorax and one on the thigh. The symptoms ranged from ulcer, bleeding to rapidly enlarging swelling• The median size at presentation was 2.4 cm. All patients underwent some form of excision biopsy. Five patients had positive pathologic margins and received adjuvant radiotherapy. Four patients remain free of disease, four arc dead of disease, four dead of other causes and one has recurrent disease for median institutional follow-up tine of 30 months. Three local recurrences were seen and these occurred only in cases of positive margins and despite adjuvant radiotherapy. Conclusion: In the elderly population Merkel cell tumours still carry a poor prognosis. Successful treatment rests with complete surgical and pathologic removal as despite radiotherapy there was a high failure rate in incompletely excised lesions.

A colour substractin pulse sequence for magnetic resonance guided interstitial thermoablation - correlation with temperature and lesion" • size S. Gould, G. Lamb, N. Vaughan, W. Gedruyc, R. Goldin, A. Mansfield, A. Darzi

Academic Surgical Unit, Imperial College of Medicine at St. Mary's and Centre fiJr Biological and Medical Systems. Imperial College, London, UK Background: Interstitial laser thermotherapy (ILT) has been proposed as a minimally invasivc treatment for malignancies in solid organs. Magnetic resonance imaging (MR) is the most sensitive modality for monitoring this therapy. However dependence on interpretation of greyscale changes and variable tissue TI times make it less than ideal. A prototype real-time pulse sequence (General Electric (GE), USA) that assigns a colour spectrum to greyseale changes will potentially increase accuracy of MR guided thermal surgery. However the spectrum must be calibrated with temperature and image-predicted necrosis correlated with histological effect to allow its' clinical use. Materials and Methods: Porcine livers were placed within a 0.5T GE SPI0 Interventional MR Unit. A 600 p.m Nd YAG laser fibre (L = 1064 nm) with diffuser tip was placed in the liver parenehyma with an MR compatible thermocoupie. A sagittal MR image containing the fibre tip was chosen as the template. A 3cm region of interest (ROI) wits drawn centred on the fibre. Thermal lesions were produced (5W, duration 20 rains) with real-time colour-subtractinn MR monitoring throughout (FGR60, acquisition time 4 s). At minute intervals the pixel intensity value, temperature and colour at the laser tip were measured. Twenty burns were produced. The pixel intensity measurements were expressed as a percentage of the mean pixel intensity of the RO1 to standardize measurements. Using the colour representing the temperature'at which tissue necrosis would be expected to occur, predicted maximum lesion size was measured from the images and compared with histological assessment. Results: There was a linear relationship between temperathre and percentage pixel change (r-'=0.84). Six discrete colours were determined in the spectrum and all were significantly different from each other in terms of mean percentage pixel change (P<0.01) and mean temperature (P<0.01 except between orange and yellow, P=0.037). Green had a mean temperature of 55.6 (+5) °C, and thus predicted necrosis. Image-predicted maximum lesion size correlated closely with histology (r2=0.93). Conclusion: The colour changes produced by this unique pulse sequence have been calibrated with tissue temperature in vitro. The colour representing a temperature above which necrosis is known to occur can be used to accurately predict lesion size. This will potentially allow greater accuracy and safety for MR monitoring of ILT in vil,o.

O c u l a r toxicity from standard dose adjuvant tamoxifen therapy P. Dulley, A. Patel, M. W. E. Morgan, F. Palazzo, G. Body, S. Wijeyekoon, I. Faweett

Breast Unit, St. Margaret's Hospital, Epping, Essex CMI6 6TN Since the first reported case of Tamoxifen associated ocular toxicity, there have been 21 cases reported as single case reports. In addition there have been a few cross-sectinnal and prospective studies, but the incidence has varied from nil to 6.3% (Table). First Author/Year

Preservation of pectoralis minor muscle in axillary clearance for breast cancer: influence on node count

593

No of Age Patients (Yrs) Mean

Beck & Mills/1979 19 Vinding/1983 17 Longstaff/1989 79 Pavlidis/1992 63 Heier/1994 135

68 67.5 68 58 65

Daily Dose (rag)

Total Dose (g) Mean

20-40 20-30 20-60 20 20

NR 10.6 24.3 14.4 17.2

Duration Ocular of Toxic Therapy (Mths) Mean 16 16 27 25 28

0 2 0 4 2

P. Markandoo, !. S. Fentiman

Hedley Atkins Breast Unit. Guys Itospital, London There has been a gradual shift from radical surgery for breast cancer. As part of this pectoralis minor muscle is more frequently preservfd in women undergoing axillary clearance as part of either breast conservation or mastectomy. A review has been conducted of 578 patients who underwent axillary clearance, 276 of whom had excision of pectoralis minor and 302 had the muscle preserved. The mean number of nodes retrieved in those who had pectoralis minor excised was 25, (range 8-50) compared with 24.5 (range 9-68) in the pectoralis minor reservation group. These results suggest that retention of the pcctoralis minor muscle does not result in any significant diminution in number of nodes excised, and thus is unlikely to lead either to understaging of disease or increased risk of axiIlary recurrence.

In view of the variance in the above series we decided to undertake a study at our institution. We have now studied 710 patients on standard dose adjuvant Tamoxifen therapy for breast cancer for between six and 126 months. All these patients were assessed for signs of ocular toxicity with the cornea and retinae being examined for characteristic keratopathy/retinopathy. Many aspects of visual function, including visual acuity and central visual field analysis were tested. In our study ocular toxicity was found in 5.5% of patients: keratopathy in 2.75% and retinopthy in 2.75%., The cumulative dose has varied from 3.6 g to 75 g and so probably the ocular toxicity is not dose related. The case reports in the literature have largely been in symptomatic patients, but our results in this study suggest that signs of ocular toxicity can be found

594

Abstracts

while patients still retain good vision and are asymptomatic. This suggests a need for a baseline study prior to commencing Tamoxifen therapy and subsequent follow-ups at yearly intervals to accurately assess whether Tamoxifen toxicity is clinically significant.

This limited study indicates that telomerase reactivation is associated with acquisition ofinvasive m:dignancy in the human breast and seems to correlate with malignant progression of breast cancer.

Design and implementation of the new Liverpool head and neck

Expression of membrane-types-1 and -2 matrix metalloproteinases and matrix metalloproteinase-2 in gastric cancer

database

Gillian Tierney, Hilary Collins, Susan Watson

N. J. P. Beasley, K. Mistry, L. Williams, A. S. Jones

Department of Surgery. Univer.riO,of Nottingham

Department of Otorhinolaryngology, Universio, of Liverpool. Liverpool

Background: The MMPs are a family of proteolytic enzymes involved in turnover of the extracellular matrix and have been implicated in the process of turnout growth and metastasis. These enzymes are secreted as inactive proenzymes requiting cleavage for activation. MMP-2 is strongly associated with the malignant phenotype in gastric cancer. This enzyme, in contrast with other MMPs is not activated by proteases, but has been shown to be activated by membrane-bound MMPs (MT-MMPs). Aims: To quantify relative m R N A levels for MMP-2 and MT-MMPs-I and -2 in gastric tumour tissue and adjacent normal mucosa. To assess the extent of co-localization of these enzymes. Methods: Gastric carcinoma tissue and normal mudosa were obtained from twenty resections, mRNA was extracted from all samples and reverse transcribed to eDNA. PCR was performed incorporating gene specific external standards to compete with the sample eDNA. Results were normalized to G A P D H . Results: m R N A for MMP-2 was detected in 15120 carcinomas and 2/ 20 normal tissue samples, mRNA for MT-MMP-I was detected in 14/20 carcinomas and 5•20 normal tissue samples, m R N A for MT-MMP-2 was detected in only 3 samples, all tumour.

High quality clinical databases bring research closer to practice and audit. They generate large samples, give accurate information for clinical practice and audit and inspire research ideas (Black, N. (1997) BMJ 315: 381-2). The Liverpool Head and Neck Database was established in 1963 by Professor Stell. The programme was updated in 1977 to reflect improvements in computer technology but by today's standards it is diflicuh to use and idiosyncratic. We discuss the process of designing and implementing a new clinical database. Information flow within the departments of head and neck surgery, oncology and t~athology was assessed. From this a core dataset was established for all patients with head and neck cancer. If widely adopted it would allow exchange of information between departments despite differences in database programmes. Using a modular design it will be possible to modify the database to incorporate future needs.

p53 and cellular proliferation in pro-malignant disorders of the oesophagus Y. Mohsen, R. Conrad, M. Wiuslet

Deportment of Surgery Royal Free Hospital and School af Medicine, London Oesophagead carcinomas are classified into either squamous or adenocareinoma. There are epidemiological differences between both tumour types. Adenocarcinoma is frequently associated with Barrett's oesophagus while carcinoma in situ (CIS) is a precursor of invasive squamous carcinoma. T#e western world is now experiencing an increasing incidence of adenocarcinoma. Genetic or biochemical markers for early detecton and determination of prognosis are needed, p53 (tumour suppressor gene) appears to play an important role in the progression of malignancy. Aim: To examine the role of p53 and tumour proliferation as biomarkers in normal and premalignant oesophagcal lesions. Methods: 10 normal, 10 Basal cell hyperplasia (BCH), 19 Barrett's and 15 specimens of squamous carcinoma with areas of CIS were analysed immunohistochemicany for p53 and proliferation rate (MIB-I). A 3 layered immunostaining technique using monoclonal antibodies and microwave oven heating was utilized. Results: Normal non proliferating oesophageal mucosa rarely expressed p53. In BCH the proportion of cells expressing p53 was higher than the normal mucosa. In Barrett's disease the two types of mucosa stained differently. Columnar (metaplastic) cells stained for p53 except in one specimen (5.3%) while in 13 specimens (68.4%) squamous cells were negative for p53. The columnar cells had a much higher level of p53 expression and proliferation rate than the squamous cells. All areas of CIS were positive for p53, however in 3 cases the underlying tumours were p53 negative. Mean expression of p53 and cellular proliferation rates were higher in areas of CIS than underlying invasive tumours. Expression of p53 and proliferation rate correlated significantly in all the different conditions (P<0.01). Conclusion: p53 accumulated with increasing frequency and levels in Basal cell hyperplasia, Barrett's and carcinoma in situ. p53 accumulation appears to fulfil its proposed role as biomarker in oesophageal carcinogenesis.

Telomerase activity in human breast cancer K. Mokbel, R. Radbourne, S. Jezzard, M. Ghilchik, R. Newbold St. MorJ,'s Itospital (London). University of Brlmel (Middle.vex) Recent evidence suggests that telomerase reactivation may be necessary for cell immortalization and acquisition of malignancy. We examined telomerase activity in 22 breast lesions using a sensitive PCR-bascd assay. Telomerasc activity was detected in 70% (10114) of invasive breast cancers. None of the DCIS (4) and benign (4) breast lesions contained telomerasc activity. Moreover, all telomerase-negative invasive breast cancers (4) were lymphnode negative and < 2 c m in greatest dimension, 70% (7/10) of the teleomcrase-positive tnmours were larger than 2 cm in greatest dimension and had a histopathological grade 3. Lymph node metastases occurred in 44% of telomerasc-positive tumours.

mRNA MMP-2 MT-MMP-I MT-MMP-2

Tumour median

Normal median

P=

7.35 x 10-7 6.5 x 10-4 3 x 10-4

1.0 x 10-8 1.0 x 10-8

0.017 I).027

Relative amounts of each enzyme were compared using Wilcoxon statistics. Conclusions: Co-localizution of the m R N A for MMP-2 and its activator MT-MMP-I has been demonstrated. There is a significant difference between turnout ~,nd normal mucosa in the quantity of m R N A for these two enzymes demonstrated using competitive RT-PCR. Further studies using in-situ hybridization are planned to identify the cell type expressing this mRNA. M M P = m a t r i x metalloproteinase. MT-MMP=membranc-type MMP. PCR=polymerase chain reaction. GAPDH=glyceraldehyde-3 phosphate dehydrogenase.

Expression of cadherin--catenin complex in premalignant gastric mucosal lesions S. Ramesh, P. McCulloch

Department of Surgery. UniversiO, of Liverpool, Liverpool E-cadherin, 13-catenin and a-catenin are normally found at the cell membrane, forming the basis of intercellular adherens junctions. Weak membranous or strong cytoplasmic expression of these proteins is therefore abnormal, and can occur in gastric cancer. The aim of this study was to determine cadherin and catenin expression in pre-malignant gastric mucosal lesions. Paralb~ embedded material from endoscopic biopsies showing dysplasia (n = 17), intestinal metaplasia (IM, n = 17) or chronic atrophic gastritis (CAG, n=25) were stained for E-cadherin, a- and 13-catenins by immunohistochemical methods and compared with 20 histologically normal biopsies. Abnormal expression of the three proteins was noted as shown in the table.

E-cadherin (cytoplasmic) E-cadherin (membranous) a-catenin (cytoplasmic)

Expression

Dysplasia

I.M.

C.A.G.

Normal

strong weak/neg strong wcak/neg strong wealdneg

12* 5 9 8D I I* 5

7° 7 13 I 3 8

0 25 25 0 0 25

0 20 20 0 0 20

P= <0.02, * = <0.001, x:, Yates & Bonferroni corrected. Both dysplasia and IM showed significant cytoplasmic expression of Ecadhcrin; dysplasia also showed significant cytoplasmic expression of 0tcatcnin and reduced membranous E-cadherin expression.

Abstracts Abnormal expression of E-cadherin and 0t-catenin increases with increasingly severe pre-malignant change in gastric mueosa. Further studies are warranted to determine whether these changes define a subset at higher risk of malignant change.

Plasma VEGF level correlation with vascularity and tumour volume in patients with colorectal liver metastases M. M. Davies, Sonia ./onus, Suki Kaur, T. G. Allen-Mersh Department of Sargery. Imperia/ College School of Medicioe. Chelsea and I~Vestminster Hospital. London Circulating levels of vascular endothelial growth factor (VEGF) arc raised in advanced colorectal cancer. The extent to which this is a marker of increased vascularity or reflects increased tumour volume is not known. We measured plasma VEGF in CLM patients (who had previously undergone primary tumour resection) and 'no cancer" control patients using an ELISA technique. Liver metastasis volume was determined by CT scan. Metastasis vascularity was assessed by staining biopsies with anti-endothelial antibody (JC70) and vessel count was determined by microscopic graticule counting at × 200 magnifieation. There was no significant (P=0.09. M.W.U.) increase in plasma VEGF level in CLM patients (median 165.4 pg/ml i.q.r. 127.9-208.4 pg/ml) europa red with controls (median 125.8pg/ml i.q.r.58.2-235.9pg/ml). There were significant correlations (Spearman) between plasma VEGF level and both vascularity (r=0.66, P=0.03) and tumour volume (r=0.53, P=0.03). This study suggests a relationship between circulating VEGF and tumour vascularity in CLM patients but also suggests that plasma VEGF level is influenced by tumour volume. There were also detectable circulating levels of VEGF in 'no cancer' controls and it was only in patients with greater liver metastasis bulk that the control range of VEGF was exceeded. VEGF=Vascular Endothelial Growth Factor; CLM=Colorectal Liver Metastases; E L I S A = E n z y m e Linked Immunosorbant Assay; M.W.U.= Mann-Whitney U test.

H o w a c c u r a t e is R T - P C R in identification o f c i r c u l a t i n g colorectal t u m o u r cells?

R. Q. Wharton, S. K. Jonas, K. Feldmann, M. Henry, T. G. Allen-Mersh DtTxtrtment of Surgery Imperial Colk,ge School of Medicine. Chelsea and Westmflh~ter Hospital. Londoo. UK Background: RT-PCR is being used increasingly to detect circulating cancer cells. However the significance of positivity to a single eDNA sequence is not clear. In this study we compared circulating cell positivity to m R N A coding for two colorectal carcinoma-associated antigens - carcinoembryonic antigen (CEA) and cytokeratin 20 (CK20). Methods: Fourteen mls of peripheral venous blood were sampled from colorectal cancer patients and no-cancer control patients via a cannula, after discarding the first five mls. After the addition of erythrocyte lysis buffer, total RNA was extracted by conventional methods from the remaining cells, and subjecled to RT-PCR using specific primers for both CEA and CK20 transcripts. The PCR products were run on electrophoretic gels. The PCR product was confirmed by Southern blotting using an oligoprobe which lay between the two primers in eacb case. Results: Samples

n (patients}

CK20 & CEA both positive

CK20 positive CEA negative

XEA positive CK20 negative

cancer control

53 43

19 0

II I

6 0

Conclusions: There was agreement between assays in 68% of cancer patients. The low false positive rate in controls implies that single assay positives in cancer patients were true positives where m R N A of only one of the two antigens was above the detectable threshold. The results suggest that different RT-PCR assays identify the same circulating cells, but use of a single assay may underestimate prevalence.

595

Pelvic bypass surgery for extensive malignancy D. A. Griffiths, A. Sharif, R. Vowles • Department ofSargery, Yeavil District Ho.~7~ital, Ycovil. Somerset The surgical salvage management of malignant disease is an option that is becoming more common as the result of improvements in palliative care. radiotherapy and chemotherapy. The "frozen pelvis', whether as a result of residual turnout or side effects of therapy, is fortunately uncommon, however pelvic obstruction or fistulation produces severe symptoms. The authors describe this experience in the faecal and urinary diversion 9f Iburteen cases with widespread active and quiescent malignant pelvic disease. The patients ages ranged from 29 to 70 and their pelvices were affected by malignancy of the uterine cervix, vagina, prostate and rectum. Six patients had faecal diversion by means of colostomy two to six years before they required ileal conduit diversion. Seven patients had synchronous faecal and urinary diversion. One patient had a urine diversion for fistulae three years before a colostomy was required for obstruction. The survival varied from seven patients who survived for a year or less. Four patients survived between two to five years and three patients survived over eight years following diversion surgery. In conclusion, in selected cases, diversion of faeces and urine improved the quality and comfort of patient with extensive pelvic cancer.

Prognostic indicators in basal cell carcinoma - can they predict recurrent or horrifying tumours? N. M. Horlock, R. Sanders, G. D. Wilson RAFT Institute for Plastic Surgerl: Mmmt Vernon Hospital, Northwood Introduction: Local recurrence rates exceed 10"/o for basal cell carcinoma. Recurrent-recurrent rates may reach 40"/0. Horrifying tumours cause massive local tissue destruction, usually on the head and neck. Markers, to predict potentially aggressive tumours, may facilitate the choice of excision margin and follow up. Studies of cell proliferation, cell loss and of cell cycle control have proven benefit as prognostic markers in other tumours. Method: 164 tumours of mixed clinical type (non recurrent, recurrent and horrifying) were studied by immunohistochemical analysis of cell proliferation (Ki67). cell loss (Bcl2) and cell cycle control (p53). Histological parameters and excision margins were also recorded. Results: The biological markers did not predict clinical behaviour. Cell proliferation was related to histological parameters with infiltrative tumours being significantly more proliferative than nodular tumours. Recurrence was associated with incomplete excision. Horrific turnouts were most commonly related to post radiotherapy recurrence or late presentation. Conclusion: These studies are of interest in understanding the biological processes in this cancer. They do not predict clinical outcome because surgical factors such as incomplete excision and late presentation are the main determinants of local recurrence.

Biological and physical observation on the freezing of tumour cells to - 4 0 ° C

S. A. EI-Shakhs t, Erica Beuson2, S. M. Shimi t, A. CuschierP IDepartawnl of Surger): Ninewells Hospital. 'Department of Mok,cular and Life Sciences. University of Abcrtay D~mdce. Scotlaod Introduction: The last few years have witnessed increasing interest of the use of freezing in treatment of liver tumours. However incomplete tumour ablation inevitably followed by tumour recurrence. Tumour recurrence is due to failure of reaching the lethal temperature at the tumour edge. The lethal effect of freezing is mainly due to intra-cellular ice crystal formation with its subsequent events of cellular membrane disruption. Aim: To determine the minimum final sub-zero temperature required to obtain complete growth inhibition of tumour cells. Material and Methods: The biological effect of freezing to different final sub-zero temperature on the growth kinetics of rat colonic carcinoma cell line DHD/KI2/TRb, was studied in experiment I using programmable freezer, The physical events during freezing of the same cells to different sub-zero temperatures (ice nucleation peaks), were studied using differential scanning calorimetry (DSC) in experiment 2. in experiment I, the growth kinetic curves were estimated over 7 days period in triplicates after freezing 3 pellets containing 6 × I 0 ~growing tumour cells to ( - 20°C, - 4 0 " C , - 60"C), then another group of same number of cells were frozen to ( -25*C, - 3 0 ° C , - 3 5 ° C , - 4 0 ° C ) . Finally a third group of same number of cells were frozen to ( - 3 5 ° C , -40*C) and left for three months of follow up. In experiment 2, the thermal events of freezing of growing rat colonic carcinoma cells and previously frozen cells to different final sub-zero temperatures were studied using DSC. Results: Complete growth inhibition was obtained only by freezing to

596

Abstracts

- 4 0 0 C or lower. The thermal scans for freezing growing tumour cells or cells frozen to higher than - 4 0 " C showed single peak of ice nucleation at -20"C_+ 3"C. Freezing of growing tumour cells to - 4 0 ° C or lower showed 2 peaks of ice nucleation at - 2 0 " C + 3 " C , and - 4 5 " C - + 5 ° C . Freezing of previously frozen cells to - 4 0 ° C showed 4 peaks for ice nucleation started at higher temperatures. Conclusion: Freezing to - 4 0 ° C or lower obtains complete growth inhibition, probably due to intracellular ice crystal formation with subsequent cellular membrane disruption. Freezing of liver tumours must be monitored by a thermocouple at the tumour edge to ensure reaching - 4 0 " C or lower.

We conclude that for most cases of breast cancer the proportion dying each year after diagnosis remains constant at a level determined by the stage of disease. This indicates a different biology of disease from other types of iameer. Thus any improvement in mortality may best be attained through altering the stage at presentation.

Increased tumour growth and inflammatiory cytokine response following intraperitoncal hypothermia from a pneumoperitoneum C. C. Nduka, M. I. Puttick, P. Coates, Lynette Yong, Averil Mansfield, A. Darzi

Efficacy o f l a p a r o s c o p i c u l t r a s o u n d in detecting liver m e t a s t a s e s d u r i n g colorectal c a n c e r s u r g e r y H. Kuumar, J. E. Hartley, K. Heer, G. S. Duthie, G. R. Avery*, J. R. T. Monson

The Universit), of Hull. Acodemic Surgical Unit, *Department of Radiology Castle Hill Hospital, Cottinghmn. HuH HUI6 5JQ. UK Introduction: The search for liver metastases prior to surgery forms an accepted part ofcolorcctal cancer surgical practice. Intraoperative USS with manual palpation of the liver is the gold standard for screening for liver metasta§es. Aim: The purpose of this study was to demonstnlte the efficacy of LUS in detecting liver metastases peroperatively in comparison to conventional modalities. Methods: A prospective controlled study was undertaken. Seventy-three consecutive patients undergoing laparoscopic eolorcctal resections for malignancy were recruited. Patients underwent preoperative liver USS and pcropcrative blinded LUS examination using a flexible-tipped 6.5 MHz curved array transducer, capable of scanning the liver to a depth of about 10cm. Results: Conventional USS was negative in all 73 cases. All patients have had MRI scans at appropriate intervals except two who died perioperatively and two who could not tolerate MRI scanning. Metastases were identified during initial peroperative laparoscopic inspection in one case and no additional lesions were found on LUS in this patient. On nine other occsions the LUS was abnormal. There were no false-negative LUS examinations. The presence of metastases were confirmed in 2 patients diagnosed to have metastases on LUS. O f the seven abnormal L:US scans, three patients with suspicious lesions on LUS were found to have metastases on MRI, in one it was deemed to be a cyst. Three remaining patients with suspicious lesions on LUS were confirmed, on serial MRI scans, to have benign lesions. Followup of all patients (median: 27.5 months; IQR: 18 m-38 m) has only revealed liver metastases in three farther patients at 12, 13 and 18 months. All three had normal MRI scans at the time of surgery. Conclusion: This study supports a role for LUS in the detection of liver metastases and in the selection of patients for further sophisticated imaging. LUS is cheaper and better than conventional USS and is as sensitive as MRI. LUS=Laparoseopic Ultrasound Scan; USS=Extracorporeal Ultrasound Scan; MR1 = Magnetic Resonance Imaging

Can the mortality of breast cancer be reduced?

Academic Surgicol Uait. hnperial CoUege School of Medicine at St. Mary,s. London. UK lntra-operative hypothermia is a significant source of surgical trauma with wide-ranging physiological and immunological sequelae. The intraperitoneal cytokine milieu created by the use of cold CO., gas to create a pncumoperitoneum may facilitatc tumour growth and disease recurrence. This study examined the effect of gas temperature during laparoscopy on local inflammatory cytokine response and tumour growth in an animal model. Inbred Wistar rats (n=28) were randomized to undergo insuffation with CO, at room temperature (~21°C) or normothermic pneumoperitoneum. The latter consisted of CO, insuffated at 37"C with additional radiant and forced-air warming post-operatively to maintain normothermia. At 4 hr and 24 hrs rats were sacrificed and peritoneal lavage performed to assess production of TNF-0t. In addition, peritoneal maerophages ( P M O ) were isolated and their activation was assessed in vitro, as shown by their TNFct release following endotoxin challenge. Peritoneal fluid and PMI~ supernatants were analysed by ELISA and data analysed by t-test. As a separate procedure 30WAG rats were randomized to undergo anaesthesia alone (n = 10) or to be insuffiated with CO, at room temperature or body temperature (n=20). The insuffation lasted for 30 minutes at 6 m m H g . During insuffation, l ml of CC531s tumour cells in suspension (I x 10S/ml) was sprayed into the peritoneal cavity. The control group was anaesthetized as above and injected with turnout cells without insuffation. At 3 weeks the rats were sacrificed and turnout spread assessed at autopsy using the modified Peritoneal Cancer Index (PCI) and tumour growth was compared weighing the excised nodules. Analysis was performed using nonparametric tests. Results lavage T N F - a (pg/ml) 4hr lavage T N F - a (pg/ml) 24hr P M O 4hr T N F - a (pg/ml) Mean total tumour weight PCI Score

warm CO.,

signif.

17.01 +_2.13 18.05_+2.76 362.1 -+64.75 0.043+_0.07 266

13.86_+ 1.08 11.79+2.69 512.0+_83.22 0.01 -+0.03 151

P=0.09 P<0.02 P<0.02 P=0.025 P>0.05

These data demonstrate that peritoneal insuffation enhances tumour growth and that a warmed pneumoperitoneum environment attenuates the growth of tumour. In addition, cold CO: caused a significant inflammatory cytokinc response with increased priming of peritoneal macrophages, In agreement with previous work, this study shows that pert-operative hypothermic stress in the form of cold gas insuffation is a significant source of surgical trauma which may be amenable to correction.

D. B. Kingsmore*, D. Hole**, C. R. Gillis**, W. D. George* *Department of Surger); Western Infirmary Glasgow,: ** H,'est of Scotland Cancer Surveillance Unit, Glasgow The mortality of breast cancer remains relatively unchanged by the therapeutic advances of the past twenty years. The aim of this study was to investigate the mortality curves of breast cancer and compare these with colorcctal cancer. All cases of breast aneer from 1980-96 were identified and the pathological stage derived. Cause of death was determined from the Registrar General. The yearly proportional mortality rate (YPMR) was calculated by dividing the number of women dying in each year after diagnosis was by the number of women entering that year. The YPMR due to breast caner was then analysed by turnout size and nodal status. From an existing database of long-term follow-up of colorcctal cancer with accurate stage, similar curves were drawn. The Y P M R remained almost constant over a ten year period of follow up for turnouts that were node negative, 1-3 nodes positive or less than 30 ram. This indicates a constant death due to breast cancer at a constant rate which does not decrease with time. For larger and 4 + nodes positive tumours there was an initial peak, in the Y P M R during the first three years after which the mortality rate levelled off to a constant rate. These curves are markedly different from those of eolorectal cancer, which show an exponential decay for each Dukes' stage.

cold CO:

m

P o s t - o p e r a t i v e t u m o u r g r o w t h is reduced a n d c e l l - m a t r i x adhesion is

altered by aministration of hyaluronate C. C. Nduka, M. !. Puttick, P. Coates, N. J. Taflinder, Averil Mansfield, A. Darzi

Academic Sugieal Unit. Imperial College School of Medicine tat St. Mary:~, London. UK Intra-peritoneal administration of various agents has been used in an attempt to prevent interaction between cancer cells and extra-cullular matrix (ECM) but with limited success. CD44 is a broadly distributed cell adhesion molecule which is important for cefi-cen and eefi-substrate interactions and has been implicated in the spread of epithelial cancers. The main ligand for CD44 is hyaluronate (HA), and coating the peritoneum with hyaluronate has been c:mployed clinically to prevent post-surgical abdominal adhesions. The aim of this study was to investigate the effect of HA on cancer cell interaction with ECM proteins in vitro and on tumour growth and intra-peritoneal adhesion formation in vivo using a CD44 positive cell line. In vitro study: 96-well plates coated with type IV collagen, gelatin, fibroncctin, or Matrigel were pro-treated for 30 minutes at 370C with phosphate-buffered saline (PBS) or HA solution (0.4% in PBS). Each well

Abstracts

597

seeding efficiency (%)

HA Control

collagen IV

gelatin

fibronectin

Matrigel

tumour score

adhesion score

30.7+6.6* 29.5_+ 1 3 . 9

22.8:1:2.1 '~ 34.3_+4.8

18.2__.3.5' 29.6-+ 13.2

26.4+12.4 '~ 67.4_+35.8

4.0+4.4 ~ 17.5,+7.8

1.6_+0.5° 3.0_+0.6

was then seeded with 1 × 104 CC531s colon cancer cells and incubated for I hour after which the seeding efficiency was measured spectrophotometrically. In J,iro study: Twenty-two WAG rats underwent caecal resection via a midline laparotomy and were randomized to receive hyaluronate solution or PBS. The peritoneal cavity was coated with 5mls of either solution immediately on entry into the abdomen. A suspension of 2 × 105 CC531 cells was then injected evenly into the peritoneal cavity which was coated once again prior to closure of the abdominal incision. Autopsy was performed at 3 weeks and the extent of turnout growth and intestinal adhesion formation rated using a standardized scoring system. Hyaluronate reduces the ability of cancer cells to adhere to major components of the ECM. Furthermore pre-coating the peritoneal cavity with hyaluronate solution resulted in significant reduction in intra-peritoncal adhesion formation and turnout growth.

Are colonoscopic biopsies suitable for genetic analysis? Sioban B. SenGupta, C.-Y. Yiu, P. B. Boulos

Deparonent of Snrgery University College London Medical School. London Patients with HNPCC develop cancers that generally show microsatellile instability and are prone to synchronous and metaeh ronous colorectal cancers as well as extra-colonic cancers. HNPCC is caused by inherited mutations in one of the DNA mismatch repair genes. Screening several genes is required to identify the precise germline mutation involved, however microsatellite instability status is a useful marker for a defective mismatch repair system and may be of relevance in families that do not conform with the clinical criteria for HNPCC. Microsatellite instability has also been detected in patients at risk of metachronous colorectal cancers (SenGupta et al. BJS 1997, 8,1, 996-1000). Surgical treatment may therefore be influenced by preoperative knowledge of the genetic abnormality. This study aims to investigate the use of colonoscopic biopsy in genetic analysis. Using a colonoscopy forceps six specimens were taken and larger tissue specimens were obtained from 13 resected cancers and two villous adenomas and examined for microsatellite instability at D2SI23, DI8S58, BAT 25, BAT 26 and BAT40 by single stranded conformational polymorphism analysis. The APC gene was also investigated for mutations between codons 1264 to 1492. Although none of thcse tumours show microsatellite instability, the banding patterns of the colonoscopic biopsies and those obtained from resected specimens were completely consistent for each patient. In one tumour a somatic mutation of the APC gene was detected in all the specimens. These results suggest that colonoscopic biopsies can be used for genetic analysis of individual colorectal tumours to provide information for preoperative phmning. HNPCC = Hereditary non-polyposis colorectal cancer

hMSH2 and microsatellite instability in metachronons colorectal cancers O. Fajobi, Sioban B. SenGupta, C.-Y. Yiu, Virginia R. Sams, P. B. Boulos, Joy D. A. Delhanty Department of Snrgery University College London Medical School, London About 9°/,, of colorectal cancer patients develop further primary large bowel malignancies some years later. The risk factors for metachronous cancer include a history of previous colorectal cancer, synchronous polyps and inheritance of the HNPCC syndrome. Recently, replication errors in noncoding sequences of DNA called microsatellites have been shown to occur in greater frequency in metachronous than sporadic colorcetal cancers (SenGupta et al. BJS 1997, 84: 996-1000). RERs result as a consequence of defective mismatch repair. Five DNA mismatch repair genes have been identified, four of which are responsible for the manifestation of HNPCC. The RER observed in metaehronous colorectal cancers may be due to mutations in DNA mismatch repair genes. We studied the index and metachronous colorcetal cancers from 19 patients for evidence of germline and somatic mutations in hMSH2, one of the genes responsible for defective mismatch repair. DNA was extracted from cancer and normal tissue from paraffin wax embedded blocks. Polymerase chain

reaction amplification for each of the 16 exons of the hMSH2 gene was then attempted, but only exons 2-6, 9, I 1 and 16 gave products due to degradation of DNA from paraffin wax embedded blocks. Screening for mutations in each exon was carried out by single stranded conformation analysis. We found no evidence of involvement of this gene in the defective mismatch repair in the samples studied. HNPCC = Hereditary non-polyposis colorectal cancer; RERs = Replication errors

Fish-tail plasty as a method of treating post-mastectomy dog-ear Julie C. Doughty, W. D. George University Department of Sorger), IK,stera Infirntar); Dtanborton Road. Glasgow Gll 6NT Dog-car at the Intend end of a mastectomy wound is a common problem. especially in obese patients where mastectomy frequently leaves behind excess skin at the lower border oftbe axilla. Although an apparently minor problem, it often causes the patient great discomfort, the fold of the skin.rubbing against or overhanging the side panel of the bra. As there is a large fold of skin in this area simply excising the dog-car may result in an inability to close the wound or may produce a dog-ear more posteriorly. We describe a simple technique for excising this fold at the time of mastcetomy or performing as a secondary procedure. Under general anaesthesia the patient is positioned on her side with the arm on the affected side placed above her head. A W-Y plasty is performed; the skin is marked in the shape of a 'fish-taW and the 'fish-tail' excised with either knife or diathermy; caution is taken not to undermine and remove fat from the triangle of skin left behind. The resulting Y is then sutured using 3.0 vicryl. We have treated 20 patients using this technique, 5 as a primary procedure during mastectomy and 15 as a secondary procedure. In all 20 patients we have prevented or eliminated the dog-ear at the lateral end of the masteetomy wound. One obese patient developed a wound infection when the procedure was performed as part of the mastectomy but there have been no other complications and all patients have been extremely satisfied with the result. • We conclude that the 'fish-tail' plasty is a successful method of treating post-mastcetomy dog-car as either a primary or secondary procedure.

Hepatocyte growth factor (HGF) in the malignant and benign breast G. J. Byrne, Xiahong Lu, Fiona Knox, Judith A. Hoyland, A. Freemont, P. J. Stern, N. J. Bundred UniversiO' Hospital of South Manchester. Manchesler High tumour cytosol levels of HGF are reported to be associated with a poorer survival for breast cancer. HGF is reported to increase angiogenesis and is a motility factor. In order to determine the relationship between HGF and its receptor (HGFR) with angiogenesis and survival in breast cancer, in situ hybridization (for HGF mRNA) and immunohistochemistry (for HGFR expression) were performed on paraffin-embedded breast cancers (n = 115) and normal breast (n= 14). Specimens were scored 0 - 9 ) by estimating the intensity of staining and the number of cells stained and compared to the histological features of the tumours and mierovessel density (assessed by immunohistochemical staining of the CD31 antigen). All tumours expressed HGF mRNA and HGFR. Expression of HGF and HGFR correlated in benign epithelium (r=0.71, P<0.01) but not malignant epithelium (r=0,13). Malignant epithelium expre,ssed significantly higher HGF mRNA (mean score 6.13, range 2-9) and HGFR (mean score 5.58, range 1-9) than benign epithelium (mean scores 4.5 (P=0.05) and 2.2.5 (p<0.01) respectively). No relationship was found between HGF mRNA or HGFR expression and nodal status, tumour grade and tumour size. No correlation was observed between HGF and HGFR scores with microvesscl density. Levels of HGF mRNA in breast cancers did not correlate with angiogenesis or metastasis. The mechanism by which HGF production adversely influences prognosis requires further study and clarification. Statistics: t-test; Pearson correlation. HGF = Hepatocyte Growth Factor; HGFR = HGF Receptor

598

Abstracts

The prognostic significance of flap recurrence following mastectomy for b r e a s t c a n c e r G. J. Byrne, Christine Brooder, R. Swiodell, Linda Ashcroft, A. Howell, N.

J. Bundred UniversiO, Hospital of South Manchester, Manchester The management of flap recurrence (FR) after mastectomy for breast cancer remains controversial. Between 1976 and 1991, 5340 mastectomies were performed for breast cancer in one unit. An overall recurrence rate of 5.4% was observed with a mean follow up of 12.2 years (range 5-20) years. FR following mastectomy for T I - T 3 tumours without radiotherapy were identified (n=256) and were examined to determine the factors predicting survival after FR. Single spot recurrence was treated by simple excision whereas multiple spot and field change received radiotherapy and systemic hormone therapy or chemotherapy. Nodal status at presentation (P<0.05), time to recurrence and presence of concurrent disease (CCD) influenced survival. Five year survival for those women with CCD at the time of FR (n=45%) was poor when compared to survival in women without CCD (55'%) (P=0.0001), Early recurrence (within 12 months of primary surgery) correlated with poor survival (P<0.001) even allowing for CCD (P<0.00I). The type of recurrence (single spot, multiple spot or field change) did not influence survival and the frequency of CCD with FR did not depend on the time interval between diagnosis and recurrence. FR occurring in the first year following mastectomy for breast cancer predicts p~oor survival. All flap recurrences have a high chance of eoocurrent systemic disease and therefore all require systemic therapy. Statistics: Chi-squared test; Student's t-test. FR = Flap Recurrence; CCD =concurrent disease

Percutaneous insertion of long-term double lumen central venous catheter under local anaesthetic W. J. Farrington, 1". S. Bhatti, R. Guy, R. M. Watkius Department of General Surgery Derriford Hospital. Plymouth Recent trends in combination chemotherapy have led to an increased demand for more double lumen long-term central venous catheters. We present a series of 47 patients in whome double lumen central venous catheters were inserted over a two years period, using local adaesthetic. A 60 cm, 9 Fr Polyurethane long term central venous catheter with dacron cuff (Viggo-Sectramed, Cuff Cath T M Duo) was used. The subclavian vein was punctured percataneously. The catheter is tunnelled subcutaneously and is introduced into the vein using Desilet-Hoffman technique. The position was confirmed with conventional radiographs or fluoroscopy. The whole procedure was carried out under local anaesthetic. Forty-seven catheters have been inserted, all were for chemotherapy. Fortythree (91%) were inserted successfully under local anaesthetic. Forty-three (91%) were inserted pereutaneously, two (4.5%) required conversion to open technique and two (4.5"/0) inserted clectively by open method. The mean duration of the procedure was 36 minutes. Forty-three (01%) patients had no complication during insertion of the catheter. 3 small pneumothoraces occurred requiring no intervention, and there was one arterial puncture. Seventeen (36%) had long-term complications necessitating the removal of the catheter. The mean survival of the catheter was 80 days (range 1 to 243 days). Double lumen long term venous access can be safely achieved using percutaneous technique in majority of eases. This can be carried out successfully under local anaesthetic, saving time and resources.

Angiotensin II receptor expression in breast cancer? Using RT-PCR A. J. Ogedegbe I'z, J. PuddefooP, G. P. VinsonI, R. Carpenter~ IDepartment of Biochemistry, Queen Mary and Westfield College, 2Breast Unit St. Bartholomew's Hospital, London ECI Angiotensin II (All) is the biologically active product of the renin angiotensin system. It is recognized as a potent vasoconstrictor and regulator of blood pressure, water and electrolyte balance, in part through its actions on aldosterone secretion and catecholamine release. Most of the known physiological actions of All are mediated via its type I (ATI) receptor. Since much evidence suggests that All regulates growth and development of tissues, and since the receptor is widely expressed in epithelial tissues, it may be an important factor in carcinoma. Using a monoclonal antibody to the ATI receptor, we have demonstrated the presence of the receptor in benign and malignant breast tiSsue. . To confirm our findings we have used RT-PCR to determine the expression of ATI receptor m R N A in breast cancer cell lines and primary breast cancer. Total RNA was reverse-transeribed from cell lines and fresh frozen human

breast tissue to generate eDNA. PCR amplification was performed using primers constructed from the published eDNA sequence library for the ATI .~ceptor. These primers were designed to amplify a 210 base pair fragment of the ATI receptor cDNA. ATI receptor was consistently expressed in MCF-7 and T47D breast cancer cell lines. The receptor was expressed in 14 of 14 primary breast cancer samples and one of 2 axillary lymphnode metastasis, and 2 of 2 normal breast samples. Angiotcnsin receptor expression in breast cancer has been confirmed. Quantitative variations in expression may explain differences in growth and development of breast cancer. RT-PCR = Reverse transcriptase polymerase chain reaction).

is there a therapeutic role for gamma linolenic acid in superficial bladder cancer? L. Solomon, A. Jennings, P. Sharpe, M. Loizidou, P. Bass, B. Birch, A. Cooper Department of Urolog): Southampton University Hospitals NHS Trust, Southamptml Introduction: G a m m a linolenic acid (GLA) is an effective cytotoxic agent when applied continuously to turnout cells in culture. In ~,ivostudies using parentcral GLA present problems of drug delivery and sequestration, but are avoided when drug delivery is topical. Intravesica[ (Topical) therapy is conventional treatment for superficial bladder cancer, but exposure is limited to 2 hours. Cytotoxicity over short exposure periods and bladder tolerance to GLA, have not been previously ew'duated. Methods: The transitional cancer cell lines, MGH-UI and RT112 were exposed to Meglumine-GLA (MeGLA) for between 30 minutes and 2 hours, and 1.95 pg-1 mg/ml. A tetrazolium assay was used to assess cylotoxicity. CIonogenic aSsays on suspended cells were also performed, hr vivo tolerance was studied using a rat bladder model. Results: Greater than 90"/,, inhibition was observed at 125 pg/ml (IC>90) and 2 hours drug exposure. Using shorter drug exposure times, higher drug concentrations were needed to induce the same effect. The sensitivity of cells in suspension to G L A - I mg/ml after 5 minutes, was also shown by the absence of any colony formation, hi vivo intravesical tolerance studies were performed. Rats given 2.5 mg/ml MeGLA intravcsieally for 2 hours remained well; bladder histology showed minimal changes. Conclusion: This study confirms that G L A retains its cytotoxicity at short exposure times and is also well tolerated by normal bladder mucosa. MeGLA is therefore a feasible intravesical agent for superficial bladder cancer, either as intravesical chemotherapy or intraoperative agent against cxfoliated cells. G L A = G a m m a linolenic acid; M e G L A = M e g l u m i n c gamma linolenic acid; IC>9fl= Drug concentration achieving over 90% growth inhibition.

The implications of multidrug resistance and serum contamination for the use of water as a tumoricidal agent in bladder cancer L. Solomon, A. Jennings, P. Sharpe, M. Loizidou, B. Birch, A. Cooper Department of Urology, Southamptm~ University Hospitals NHS Trust, Soutltampton Introduction: Water is widely used as a tumorieidal agent during transurethral resection of bladder turnout (TURBT). However, even with this treatment up to 85% of patients will have recurrent tumours, often during the first year. Two possible explanations for this may be: I) the presence of serous exudate in-the water from the operative site may diminish the osmotic effects of water and 2) the membrane changes associated with the multidrug resistant phcnotype, commonly found in newly diagnosed bladder cancers, may also affect tolerance to hypotonic shock. Method: The human urothdial cell lines MGHU-I and RTII2 and their drug resistant variants, were exposed to short pulses of water. A clonogenic assay was used to establish tumoricidal efficacy. These experiments were then repeated to assess the effect of added serum proteins on the test results. A colorimetrie general biomass assay was used. Albumin assays of the waste irrigant from Ifl T U R B T patients were obtained and the results used to select the levels of serum 'contamination' used in the study (I-20% serum). Results: In the multidrug resistant (MDR) clone of M G H - U I , colony counts were reduced to 80% of controls as compared to a reduction to 40°/,, in the parental cell line. Both the parental RT112 and its non-MDR cisplatin resistant clone reduced the colony counts to 25'/0 of controls. The addition o f 5% serum to water resulted in only a 5% reduction in the biomass. At higher levels of protein, there was no reduction in the biomass. Albumin estimation on waste T U R B T fluid revealed 5% or more serous 'contamination' in 80% of the patients. Conclusion: The tumoricidal efficacy of water is reduced in the presence of serous contamination. It is also less effective aga{nst MDR clones of cancer cells.

Abstracts TURBT=Transurethral resection of bladder tumour; MDR=Multidrug resistance.

Who will practise relaxation and guided imagery? L. G. WalkerZ, M. B. Walker z, K. Ogstonl, S. D. Heysz, A. K. Ah-Seez, A. W. Huteheona, T. K. Sarkar 4, O, Eremln2

BehaviouraI Oncology Unit~and Dgpartments of Surgerj ,z. Medical OncologjP and Radiotherapy4, UniversiO, of Aberdeen and Aberdeen Royal h~irmarj, NHS 7~ust Aim: The aim of the study wasto identify characteristics tlmt predict frequency of relaxation and guided imagery practice during primary chemotherapy. Methods: Ninety-six women with large (>4era) or locally advanced (T~, T~, T~N,) breast cancer were randomized to standard support with, or without, relaxation and guided imagery. Women kept detailed daily diary recordings. Patients received 6 cycles of primary CHOP chemotherapy every 3 weeks. Prior to chemotherapy, tlae Hospital Anxiety and Depression Scale (HADS), the Mood Rating Scale, the Eysenck PersonalitY Questionnaire (EPQ-R) and the Courtauld Emotional ContrOl Scale (CECS) were administered. Results: A!plm was set at. 0.05 (2,tailed), Relaxation frequency was significantly correlated with EPQ-L (conformity) (r=0.3fl), EPQ-N (emotional reactivity)(r= --0.34), EQO-P (tough mindedness) ( r = -0.34) and MRS Total (positive mood) (r=0,33). It was not significantly correlated with N stage ( r = - 0 . 0 1 ) , T stage (r=0.16), age (r=0:01) or ER status (-00). Self-rated imagery intensity was correlated highly with relaxation frequency (r=0,64), anxiety ( r = - 0 , 3 7 ) , EPQ-L,(r=0.34), and MRS total (r=0.38)~ It was not significantly correlated with N stage ( r = - 0 . 1 8 ) , T stage (r=0.03), age (r=--0.0,5) or ER status ( r = - 0 . 1 0 ) . Stepwlse linear regression revealed that EPQ-N and EPQ-P were independent (negative) predictors of rel~ixation frequency and I-IADS anxiety was an independent (negative) predictor of imagery ratings. Conclusions: If emotionally reactive and tough minded women are offered this treatment, they will require additional encouragmcnt to practice: The development of alternative ways of helping them to cope would be beneficial.

Breast cancer in women aged 35 years an d _under: prognosis and survival 1. C. Smith !, S, D. Heys t, H. AI--Sayer!, S. Payne z, I. D. Miller2, O. Eremin

Surgical Nutrition and Metabolism Unit. Departments of Surgery a and Patliolog)r~. University of Aberdeen Background: "I'lie relationship between age at diagnosis and survival in patients with breast cancer remains controversial. Some studies have demonstrated that patients aged 35 years and under have a poorer outcome than older women; others have htiled to show this.

599

Aims: The aims of this study were to identify prognostic factors in young. women, aged 35 years and under, with carcinoma of the breast. • Methods: 105 women, aged 35 years and under presenting with carcinoma of the breast, were identified. The details of clinical staging, local and distant disease recurrence and overall survival were obtained for all patients. Histological sections of tumour were examined for type, grade, size, presence of intraduct carcinoma, vascular invasion, lymph node status and oestrogen receptor (ER) status. Results: 91% of tumours were invasive duetal carcinomas and 73% were grade 3 tumours. 51% of patients had axillary lymph node involvement and 21% were ER negative. The overall 5 year survival was 64%. Predictors of a poorer survival (univariate analysis) were: inerensing tumour size ( P = 0.003), absence of EKs (P=0.03), vascular invasion (P=0.0001), axillary lymph node involvement IP=0.0001) and the presence of metastases ( P = 0.02). Multivariate analysis (Cox stepwise regression model) revealed that a high tumour grade, axillary lymph node involvement and the presence of metastases were independent predictors of a poorer survival. Conclusions: This study has identified that carcinoma of the breast in young pauents is most commonly invasive ductal carcinoma and of high tumour grade. Factors predicting survival and overall survival rates are comparable to those previously reported for older women with breast-cancer.

Visualization of idarubicin on sub-cellular fractions: is fluorescence quenching a factor? P. Sharpe, tVl. Hayes, L. Solomon, A. Jennings, A. Cooper, B. Birch.

Department of Urology. Southampton University Hospitals NI'IS Trust. Southampton Introduction: ldarubicin is a lipophilic anthracycline effective orally and against turnouts unresponsive to related drugs. We have described its apparent distribution in both parental and MDR bladder cancer cell lines as essentially cytoplasmic. This evoked debate over the role of fluorescence quenching of DNA-bound drug. The problem is here further addressed using three levels of sub-cellular fractionation. Methods: Intact parental and resistant MGH-UI ceils were exposed to ida and epi. Nuclei were stripped from ceils. Chromosomes were prepared from cells arrested in metaphase by colcemid and DNA comets were obtained eleetrophoretically from apoptotic cells. Fractions and gels were exposed to epirubicin or idarubicin and fluorescent confocal images obtained. Soluble DNA was complexed to drug and subjected to scanning spectrofluorimetry. Results: Live cells show nuclear localization with epi, nuclear sparing with ida. Stripped nuclei fluoresce strongly with both epi and only slightly less so with ida. Chromosomes fluoresce equally with both drugs. DNA comets are obtained with both drugs. Fluorescence quenching on free DNA is strong with both drugs and up to five times greater with ida, depending on concentration. Conclusions: Although ida fluorescence does show more quenching than epi, this is not great enough to prevent visualization of ida On chromosomal DNA at three levels of organization when deprived of the protection of the nuclear envelope. The fluorescence seen in whole cells reflects different handling of the more lipophilic drug. Ma = idarubicin: epi= epirubiein