PROCEEDINGS O,F THE IRISH ASSOCIATION FOR
CANCER RESEARCH MEETING
University College, Galway, 21st September, 1984 TRENDS IN. COLORECTAL CANCER IN THE REPUBLIC OF IRELAND 1954-'81 J. P. Corridan. Department of Social and Preventive Medicine, University College, Cork. M~. A. Moran. Department of Statistics, University College, Cork. The statement "that for many of the common types of cancer the trends in death certification rates among people under 65 years of age may yield a much more reliable indication of the real trends in cancer onset rates than can the superficially more attractive study of any of the currently available data on registered incidence r a t e s " - - has been applied to the Irish data from 1954-'81. Over this period the rates for males and females are generally unchanged and closely similar. This finding appears to cast doubt on the generally current view that this cancer has reached epidemic proportions in this country in recent years. A STUDY OF THE WIDE RANGE OF PROBLEMS EXPERIENCED BY PATIENTS WITH MALIGNANT DISEASE AND THEIR RELATIVES B. Herity, N. Hilliard, M. Moriarty, J. Fennelly, M. M. McCambridge and M. Casey. St. Luke's Hospital, Dublin. The Study Population comprises 200 cancer patients and a responsible relative for each one in relation to total care during the illness. 50 patients were newly diagnosed. 100 patients were at an intermediate stage of their disease. 50 patients were terminally ill. A precoded questionnaire was administered to the patients and relatives which obtained information on : Diagnosis Psychological and Social Effects Symptoms and Therapy Communications Hospital care After care A preliminary report on this Study will be presented. STUDY ON THE SUBJECTIVE HEALTH OF CANCER PATIENTS M. Quigley, M. Moriarty and S. Hunt. Department of Radiotherapy, St. Luke's Hospital, Dublin. The aim of the study is to assess the usefulness of a Subjective Health Questionnaire in Oncology compared with ordinary clinical assessment and performance status. Two specific
440
clinical situations were chosen to elucidate this : 1. Patients undergoing radiation. 2. Patients with cancer (residual) on follow-up. We had one hundred patients in the study divided into four categories : Group A consisted of new patients receiving radiation. Group B were new patients not receiving radiation (control skin cancer group). Group C were patients with residual or recurrent disease. The patients were asked to complete three Health Profile Quesionnaires based on the Nottingham Health Profile dealing with problems in daily life. Results were scored on a weighting basis. Preliminary results show that the Health Profile picks up individuals with a high risk of psychological distress. We are checking the relationship of : a) Specific tumours and levels of stress. b) The relationship of scores and the clinical staging of tumours. c) The relationship between scores and outcome (survival and quality of life). THE CHARACTERISATION OF MELANOMA TUMOUR TYPE IN NORTHERN IRELAND Lynn Gordon and W. S. Lowry. Department of Oncology, The Queen's University of Belfast. Two hundred and forty cases of cutaneous malignant melanoma occurred in Northern Ireland over a five year period. These cases have been histopathologically reviewed and classified. This study examines the distribution of tumour types in the population. Forty-two per cent were nodular melanoma, 27% superficial spreading melanoma, 20% lentigo maligna melanoma, and 11% acral lentiginous melanoma. Other variables such as sex, age, anatomical site, thickness of lesion, tumour profile, ulceration, pigmentation and predominant cell type were also examined in relation to tumour type. Some aetiological and biological factors are considered as they relate to this classification. ANALYSIS OF SURGICAL MANAGEMENT OF SKIN CANCER TREATED AT ST. LUKE'S HOSPITAL, DUBLIN: 10 YEAR REPORT M. Moriarty, J. Prendiville, H. Raftery, P. Blake, B. Herity, A. Robin,son, R. C onroy and M. Pomeroy. St. Luke's Hospital, Dublin. Cancer of the skin is the most common type of malignancy in Ireland, accounting for approximately 30% of all new cases. The vast majority
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are cured by radiotherapeutic or surgical techniques. The choice between these two modalities is usually dependent on the age of the patient, the size and site of the tumour, histology and vacularity of the underlying tissue. The surgical management of skin cancer at St. Lukes's Hospital during the period 1965-1974 was evaluated. A total of 1,140 lesions were present in 1,025 subjects, male: female ratio being 7.3. The majority of patients (54%) were over 65 years of age. Of significance, the surgical procedures were usually performed under local anaesthesia, excision (54%) being the most frequent. Fifty-two per cent were squamous cell carcinomas, 9.2% basal cell carcinomas and 4.3% melanomas with over 66% of the lesions occurring in the head and neck region. The relationship of site, histology and procedure to treatment failures is discussed. The implications of the findings for surgical management and for follow-up is considered. STUDIES OF THE EPIDEMIOLOGY OF CARCINOMA OF THE UTERINE CERVIX IN IRELAND Bernadette Herityl, 3, j . F. Murphy2 and Leslie Dalyl. 1Department of Community Medicine and Epidemiology, University College, Dublin 2. 2National Maternity Hospital, Holies Street, Dublin 2. 3St. Luke's Hospital, Rathgar, Dublin 6. Two case-control studies of the epidemiology of carcinoma of the uterine cervix in Ireland were underaken. One hundred women with cervical intraepithelial neoplasia grade 3 (CIN) and 100 women with invasive squamous-cell carcinoma (SCC) together with 100 age-matched controls for each group were interviewed concerning their socio-demographic, sexual, marital, reproductive and previous medical histories. Behavioural riskfactors such as early age at first coitus, marriage and pregnancy, and multiple partners were significantly more comon among both sets of cases than controls. Social deprivation and low uptake of medical care facilities was seen among the SCC cases only. It is possible that although changes in sexual mores may have increased the prevalence of behavioural risk factors in Ireland, better education and easier access to diagnostic and therapeutic services may ensure that many of those affected will be identified at the preinvasive stage of their disease CHEMOTHERAPY OF EPITHELIAL OVARIAN CANCER R. J. Atkinson. Department of Oncology, Queen's University, Belfast. Neoplasms of the ovary continue to cause more deaths than the sum caused by the other turnours of the female genital tract. A review of patients attending the Gynae/
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Oncology Unit of the Belfast City Hospital was undertaken for the years 1980-84. Debulking surgery was followed by chemotherapy. Forty-two patients were treated with a combination of CIS Platinum/Chlorambucil. In 1982 entry to the current MRC Late Ovarian Trial was commenced. The chemotherapy regimens used will be described and a recent analysis of survival figures given. AN ANALYSIS OF 45 CASES OF MALIGNANT TESTICULAR GERM CELL TUMOURS TREATED AT A SINGLE CENTRE BETWEEN 1971-1984 M. Pomeroy, D. Kelly, M. McCabe, J. J. Fennelly, M. Moriarty, G. Duffy, F. Duff, A. Robinson and J. Duffy. St. Vincent's and St. Luke's Hospitals, Dublin. Maligant testicular tumours, though rare, are of importance because of: 1. Their occurrence in the younger age group. 2. The availability of sensitive and relatively specific tumour markers. 3. Advances in their treatment in the past decade. We studied~ 45 such patients treated at St. Vincent's Hospital 1971-1984 with a mean follow up time of three years. Mean age at diagnosis was 27 years for teratoma and 39 years for seminoma. The well known association with undescended testis was confirmed. Histopathology was classified according to the British Testicular Tumour panel classification, and pathologic extent of primary tumour was noted. All patients had a h istologic diagnosis, and when these became available, serum alphafoetoprotein, beta H.C.G. and immunohistochemistry were performed. After full investigation all patients were allocated' to stage according to the Royal Marsden staging classification and treatment selected accordingly. Multimodality therapy of these tumours have long been practised though the relative roles of surgery, radiotherapy and. chemotherapy have evolved and changed over this period. Major progress in chemotherapy was shown by the Einhorn regime, whereas more recent combinations, e.g. Charing Cross regime, have been designed for the treatment of advanced bulky metastatic disease. In this series 2 year disease free survival was significantly related to :. 1. Stage at diagnosis. 2. Bulk of involved paraortic nodes for stage II, 111, IV. 3. Treatment. Factors unrelated 'to 2 year disease free survival in this series include: 1. Pathologic extent of primary tumour. 2. Histopathologic subtype of teratoma (when standardised for stage).
442 3. Serum marker po sitivity (when standardised for stage). However, preoperative marker status, both alphafoetoprotein and beta HCG for teratoma, and' beta HCG for seminoma was significantly related to stage at diagnosis. More aggressive treatment of advanced bulky disease has lead to improved response rates which it is hoped will lead to, improved survival. In. all other groups there is likely to be increased emphasis on reduction of treatment related morbidity. BONE MARROW RECONSTRUCTION FOLLOWING ALLOGENEIC BONE MARROW TRANSPLANTATION--AN IN VITRO STUDY Maura Reynolds and S. R. McCann. St. James's Hospital and Trinity College Dublin.
I.J.M.S. November. 1985 stromal cells in haemopoietic reconstitution will be evaluated. IMMUNOCOMPETENCE BEFORE AND AFTER OESOPHAGECTOMY FOR CANCER P. Keeling, P. J. Byrne, K. Mealy, A. Whelan, C. Feighery, T. P. J. Hennessy. Depts. of Surgery and Immunology, Trinity College and St. James's Hospital, Dublin. Depression o.f the immune system may predispose patients to post-operative infections, particularly following oesophagectomy for oesophageal carcinoma. The aims of this study were (a) to determine the incidence of pro-operative depression of the immune system and (b) to compare these results with the number of infective complications following oesophagectomy in 20 patients with oesophageal carcinoma. Healthy subjects were tested in parallel. In vivo cellular immune response was tested with 7 recall antigens with one control substance. In vitro non-specific tests included concanavalin A and Phytohaemagglutinin, pro-operatively, 7, 14 and 42 days following surgery. At similar time intervals, absolute lymphocyte counts were measured'. Peripheral blood mononuclear cell populations were identified and counted using 5 apecific monoclonal antibodies. The results of this study demonstrate that pro-operative anergy and depression of the cellular immune system is associated with postoperative infective complications (9 out of 10 infective episodes). One week following surgery the immune system was suppressed in all 20 patients. However, in those patients with postoperative infective complications, the results of the in vitro testa of cellular functions were separated clearly, P<0.001. Pro-operative depression of the non-specific cellular immune system is strongly associated with post-operative infective complications.
AIIogeneic bone marrow transplantation provides an ideal model to study haemopo,ietic of reconstitution following oblative chemoradiotherapy. Sequential studies were carried out on two patients following allogeneic sibling bone marrow transplantation. Both patients had received prior chemotherapy and, were in remission from acute leukaemia (AML AND ALL). The conditioning regimen consisted of oral Busulphan in a total dose of 16 mgs per kg given~ daily on four consecutive days followed by Cyclophosphamide 50 m.gs per kg i.v. daily for four consecutive days. This was followed by an infusion of 2 x 108/kg nucleated bone marrow cells from a HLA compatable MLC non-reactive sibling donor. In both cases the recipients were female and the donors were male. Marrow was aspirated under local anaesthesia from the posterior lilac crest from the bone marrow recipient at weekly intervals following transplantation. The mononuclear cell fraction was separated by ficoll hypaque density gradient centrifugation. Nucleated cells at a concentration of 2 x 10s were cultured for the proliferation of erythroid (CFU-E and BFU-E) progenitors in the NEUTROPHIL FUNCTION AND POSTpresence of sheep erythro-ponetin and myeloid OPERATIVE SEPSIS IN OESOPHAGEAL (CFU-G,M) progenitors in the presence of placCARCINOMA enta conditioned medium. Cells were cultured in methyl cellulose in a concentration of 5% CO= K. Mealy, P. Keeling, P. J. Byrne, A. Whelan, C. for periods of 7 or 14 days. Stromal cell proFeighery and T. P. J. Hennessy, Departments of genitors (CFU-STROM) were also evaluated' using Surgery and Immunology, Trinity Sollege and St. a short-term methyl cellulose technique. James's Hospital, Dublin. Evidence of bone marrow engraftment consisted of rising peripheral blood counts, normal Sepsis and anastomotic breakdown remain the bone marrow morphology, change in red cell two most important post-operative complications genotyping and' karyotype analysis. The relation- in oesophageal carcinoma surgery. In recent ship between the sequential studies of bone mar- years emphasis has been placed on the role of row haemopoietic progenitors and other evidence cellular immunity in relation te prognosis after of haemopoietic engraftment will be presented. major surgery. However, the role of the neutroThe origin of the haemopoietic stromal cells folphil has virtually been ignored. lowing allogeneic bone marrow transplantation Using Chemiluminescence (CL) a sensitive will be discussed and the possible role of marrow technique which quantifies the amount of energy
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released during phagocytosis, we studied neutrophil activation in 15 oesophageal carcinoma patients, undergoing resection. CL Volts Septic N=8 Non-septic N=7 P Values
Pre-op 17+34
Post-op, 1 week 156
+24
156_+23.4 337.5+83 NS
P<0.05
Post-op, 2 weeks 134_+26 167_+42.4 NS
Values=Mean_+1 SE The results show that in those patients who undergo surgery without complications, a significant rise in neutrophil activation occurs in the immediate post-operative period (P<0.05). This had returned to normal by 2 weeks. In contrast to this in those patients who develop septic complications, no such increase occurs. Pre-operative function in these patients is slightly decreased, although not significantly so, in comparison with the non-septic group. The results demonstrate that although initially normal neutrophil activation fails to increase in the post-operative period in those who develop sepsis, suggesting that neutrophil function may be of prognostic value in the post-operative period in oesophageal carcinoma patients. PEROXIDASE ACTIVITY, A POSSIBLE PROGNOSTIC MARKER IN BREAST CARCINOMA M. J. Duffy, M. O'Connell, B. Cantwell, J. Fennelly, L. O'Siorain and R. Conroy. Departments of Nuclear and Medical Oncology, St. Vincent's Hospital, Dublin 4. Peroxidase activity is massively induced in the immature rat uterus following estradiol administration. If a similar induction occurred in breast carcinomas peroxidase could be a marker for a functional estradiol receptor (ER) and thus be of great value in predicting hormone-dependent tumours. Although peroxidase activity has been reported to be higher in some hormone-dependent animal breast tumours than in hormone-independent tumours, no correlation has been found between ER and peroxidase activity in human breast carcinomas. Thus peroxidase is unlikely to be a marker for predicting hormone-dependent human breast tumours. On the other hand peroxidase activiey may be a prognostic marker in breast cancer. In this investigation, patients whose tumours possessed peroxidase activity had both shorter disease free interval and shorter survival than patients with peroxidase-negative tumours. These differences were statistically significant; for disease free in-
443
terval P=0.0475; for survival P=0.0165. Patients with ER~positive tumours also had a longer disease-free interval and longer survival than ERnegative tumours. However since ER were mostly confined to low grade and low stage tumours, this protein may not be a totally independent prognostic marker. In contrast, peroxidase activity showed no relatioiship to tumour stage or grade. It may however be a measure of lypmocyte infiltration of the tumour (Duffy ea al, Eur J. Cancer, 18, 453, 1982). Conclusion: although peroxidase activity is unlikely to be a marker for hormone-dependent breast turnouts, its presence in these tumours is a bad prognostic feature and its absence suggests a good prognosis.
STUDIES ON CATHEPSIN B IN BREAST CANCER M. J. Duffy, J. Simon, M. G. Harrington, LI O'Siorain and S. O'Briain. Department of Nuclear Medicine, St. Vincent's Hospital, Dublin 4; Department of Biochemistry, University College, Dublin 4 and Department of Pathology, St. James's Hospital, Dublin 8. Cathepsin B (CB) is a thiol-dependent protease found in lysosomes. Although its physiological function is unknown, considerable evidence suggests that it plays a role in tumour invasion and metastasis. The main aim of this investigation was to study the distribution and properties of a CB-like enzyme in breast tumours. Ultimately it is hoped to show whether the CB-like enzyme is a marker for metastasis in breast cancer. CB-like activity was assayed using CBZ-arg-argB-methoxy-naphthylamide as substrate. Using this substrate, hydrolase activity was stimulated by both DTT and cysteine. Activity was inhibited by both iodoacetamide and leupeptin but not significantly by serine protase inhibitors such as PMSF, SBTI and aprotinin. Using differential centrifugation, CB-like activity was mostly confined to the lysosomal-mitochondrial fraction. Thus CBZ-arg-arg-B-methoxy-napthylamide appears to be a suitable substrate for assaying CB in mammary carcinomas. In contrast, .hydrolase activity using 2 other substrates, namely, CB-argnitroanilide and N-benzoyl-phenylalanyl-valyl-argnitroanilide was not stimulated by reducing agents. Furthermore using both these substrates, activity was mostly found in the cytosol fraction rather than in the lysosomes. Thus these substrates which have previously been used to measure CB in normdl tissues are unsuitable for assaying CB in malignant breast tissue. Using CBZ-arg-arg-B-methox.y-naphthylamide as substrate, CB-like activity was found in 61% of 121 primary breast carcinomas, 75% of 8 axillary node metastases, 50% of 10 chest wall recurrences and in only 14% of 14 benign breast
444 tumours. The proportion of CB-positive primary tumours tended to decrease as both t umour stage and number of axillary nodes involved increased. Thus in contrast to what might be expected if CB played a role in tumour spread, our prelominary data suggests that CB is mostly confined to carcinomas with good prognosis. Presently the relationship between CB-like activity and survival of patients with breast cancer is being investigated.
GROWTH IN CULTURE OF EPITHELIAL CELLS FROM NORMAL AND MALIGNANT HUMAN SKIN S. McDonnell, M. Moriarty and M. Clynes. School of Biological Sciences, NIHE, Glasnevin, Dublin 9 and St. Luke's Hospital, Rathgar, Dublin 6. Skin keratinocytes are being used as a model system to study culture conditions and growth control for human epithelial cells. Over the past year cultures have been initiated from 49 skin samples (33 tumour, 12 normal, 4 uncertain). Epithelial growth was observed in 32 samples; the remainder failed to grow or suffered rapid microbial contamination. Cultures were initiated from 1 mm 3 explants or as cell suspensions obtained following stirring at 37~ with 0.25% trypsin plus 0.04% EDTA. Disaggregated primary cultures, or subcultures from explant outgrowths required lethally-irradiated (600 rad) mouse 3T3 feeder cells for attachment and growth. Epithelial outgrowth from explants was most consistent when explants were placed with the dermis next to the culture surface. Fibroblast overgrowth was controlled by selective fibroblast detachment (0.02% EDA for 6 minutes at room temperature followed by 0.01% trypsin plus 0.02% EDTA for 6 minutes at 37~ monitored with an inverted microscope). A 1 : 1 (v/v) mixture of DMEM: Ham's F12 (plus 10% foetal calf serum and 0.4 p.g/ml hydrocortisone) proved superior to medium 199 or DMEM (both supplemented with 20% foetal calf serum and hydrocortisone as above). Addition of epidermal growth factor had no apparent effect. No permanent cell lines have been established as yet; it may be relevant that most of the permanent squamous cell carcinoma lines reported in the literature originated from soft tissue such as tongue, whereas the samples described here came from highly keratinised superficial areas. Tentative morphological identification of the cells as epithelial was confirmed using polyclonal and monoclonal antibodies to keratin, a component of the intermediate filaments of epithelial cells. The species of the cells was confirmed as human using agarose gel electrophoresis of lactate dehydrogenase isoenzymes.
I.J.M.S. No:ember. 1s CULTURE OF NORMAL AND TUMOUR CELLS OF HUMAN OESOPHAGEAL MUCOSA 1C. B. Seymour, 1C. Mothersill, 2p. Byrne, 2T. P. J. Hennessy and 1M. Moriarty. 1St. Luke's 'Hospital, Rathgar, Dublin 6; 2TCD Department of Surgery, St. James's Hospital. The establishment of model systems for the study of human cancer is of considerable importance. One of the most promising approaches to the development of model systems in the use of tissue cultures established from human surgical or biopsy specimens of the organ of interest. Workers at St. James's Hospital have been examining the development and aetiology of oesophageal cancer for several years and recently in association with the Cell Culture group at Saint Luke's Hospital are attempting to develop a culture system for human oesophageal mucosa from normal, malignant and transition areas cf tissue from patients undergoing surgery for squamous or adenocarcinoma of the oesophagus. Results so far from four patients indicate that cultures can be established following digestion of the specimen in trypsin/collagenase mixture or Ltsing the explant technique. Growth of tumour and transition samples in Basal Medium containing insulin, hydrocortisone and 20% foetal calf or lamb serum is very vigorous and most specimens are relatiyely free of fibroblast contamination. Normal .tissue is not as successful and cultures tend to be dominated by fibroblast like cells. Structural organisation of cells around explants has been observed and PAS positive material (possibly mucin) has been detected. The results suggest that it is relatively easy to establish predominantly epithelial cultures from tumour and transition areas of human oesophagus but that the present technique needs refinement before cultures of normal mucosal tissue can be established. EFFECT OF ANTIVlRAL AGENTS ON FILM SARCOMA S. M. Lavelle and M. Mac Iomhair. Department of Experimental Medicine, University College, Galway. The aetiology of film sarcoma is unknown. Johnson et al. (Cancer Res., 1983, 33, 3139), found virus-type structures in a proportion of film sarcomas in mice. We tested the anti-viral agents 5-iodo-2-deoxyuridine (IUDR 0.072 m g / filter) and methylglyoxal bis guanylhydrazone dihydrochloride (MGBG 0.01 mg/filter) on groups ol 50 female BALBc mice. The substances were soaked into 25 mm cellulose filters of 0.1 micron pore size, covered with a layer of 16 g/dl gelatine and embedded subcutaneously one to each mouse. Filters treated with saline were used in
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a control group of 50 mice. The mice were not grossly affected by the implants. At 84 weeks the tumour incidence was as follows :
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marginally so (p<0.2). The results suggest that a viral aetiology is not probable for film sorcoma. IUDR is also an inhibitor of polyamines. Its result
No. mice
No tumours
Weeks of life (W)
Weeks per tumour (W/T)
Saline
49
13
2900
223
MGBG
43
15
2378
159
IUDR
44
23
2511
109
In the dose used IUDR was markedly cocarcinogenic with the filter (p<0.02) and MGBG was
is thus paradoxical, since polyamines are themselves cocarinogenic with the filters.
SV40 TRANSFORMED MUTANT CV1 CELLS: ANALYSIS OF VIRUS-SPECIFIC PROTEINS D. O'Reilly, E. Mullins, S. Canty and J. K. Collins. Virology Unit, Microbiology Department, University College, Cork.
a polypeptide of 45,000 molecular weight which is the main structural protein component of the virion Balb C mice were immunised with SV40 virions purified from monkey kidney cell by ultracentrifugation. After one month the mice were boosted and the spleens removed three days later. Hybridomas were made using washed spleen cells and the myeloma line X-63 Ag8.6.5.3 using the polyethylene glycol technique. The fused mixture was plated in 24 wel trays with splenocyte feeders. Wels showing positive growth were screened using a B-galactosidase ELISA assay on SV40 infected monkey cell monolayers. Wels positive by ELISA were cloned by limiting dilutions using thymocytes as feeders. The following characteristics of anti-SV40 monoclonal antibodies have been determined (a) Neutralisation ability; (b) Antibody class and subclass; (c) Antigen specificity and (d) Ability to recognise both native and denatured antigen.
A mutant CVl cell line, designated u6, which resists SV40 infection has been isolated. Previous work has shown this mutant to be transformed, to express viral phosphoprotein large T antigen and two novel super T antigens constitutively. These cells have also been shown to express elevated levels of a cellular phosphoprotein, p53. The large T related antigens and p53 protein have been found associated in a high molecular weight complex. Sucrose gradient centrifugation dlemonstrates that the complex is larger in u6 cells than in infected wild-type CVI cells. The association is also shown to be considerably more stable in u6 cells than in infected wild-type cells. The role of differential phosphorylation of the large T related proteins and of the p53 protein in giving rise to the increased stability will be discussed. The DNA coding for the super T antigens is being cloned in a 31-based vector with a view to elucidating the role of the super T proteins in the transformation process.
PRODUCTION OF MONOCLONAL AN~TIBODIES TO STRUCTURAL ANTIGENS OF THE TUMOUR VIRUS SV40 J. Davis, J. Conroy and J. K. Collins. Virology Unit, Microbiology Department, University College, Cork. SV40 is a papova virus which induces tumours in experimental animals and transforms cells including human cells in culture. We have been studying tumour virus host cell interactions at a molecular level over a number of years. In order to develop a sensitive and specific assay system for late or structural gene expression, we decided to produce monoclonal antibodies to VP1
SERUM AND MONONUCLEAR LEUCOCYTE ASCORBIC ACID IN PEOPLE WITH MALIGNANCY IVt. B. Keatinge, P. B. Collins, J. P. Duignan, B. Dickson and A. H. Johnson. Department of Biochemistry, Royal College of Surgeons in Ireland, Dublin 2. Interest has recently been shown in the ascorbic acid levels in mononuclear leucocytes (MNL) and it is known that these cells, consisting of ]ymphocytes and monocytes, have the ability to concen.trate ascorbic acid from serum. Some reports also indicate that lymphocyte transformation is enhanced by in vivo or in vitro ascorbic acid supplementation., The aim of this study was to establish ascorbic acid levels in serum a.nd MNL and to relate these levels to the response of the lymphocytes to "lhe mitogen phytohaemagglutinin. The study was carried out on a group of patients ( n = 1 6 ) with solid tumours of the gastrointestinal tract, age
I.J.M.S. No:ember. 1~c85
446 range 48-83 years (mean 66 years). A healthy control group ( n = 1 1 ) was also studied, age range 21-38 years (mean 28 years). Serum ascorbic acid (p<0.001) and mitogen responsiveness (p < 0.001) were significantly greater in the controls as compared to the patients. There was a positive correlation between the MNL ascorbic acid and the mitogen responsiveness (r=0.76) in the controls. No such correlation existed in the patient group. Immune competence, as reflected by mitogenresponsiveness, is confirmed to be defective in our patient group. In the control group this mitogen-responsiveness has been shown to be related to the concentration of MNL ascorbic acid. It seems that the low serum ascorbic acid concentration in the patient group may in part be responsible for the depressed lymphocyte transformation. It is of concern that people suffering from malignancy which frequently requires surgical intervention are deficient in a vitamin known to be necessary for wound repair and whose possible role in immune function is currently under review.
COLONIC CELL CULTURE 1A. McCann, 1C. Seymour, 1C. Mothersill, 2A. Long, 1M. Moriarty and 2D. O'Donoghue. 1Saint Luke's Hospital, Rathgar, Dublin 6; 2Saint Vincent's Hospital, Elm Park, Dublin 4. Epithelial cell lines are by definition transformed to a certain extent. This means that the effect of carcinogens does not always correla'.e between cell lines and experience in vivo. For this reason it was decided to attempt to find a reproducible system for establishing primary cultures of colon (a major site of fatal cancer in Irish people) so that the effect of suspected dietary carcinogens, and of chemotherapeutic agents could' be ascertained in an in vitro situation as close to the in vivo reality as possible. Two basic methods for establishing the primary cell cultures have been tried. Where sample size permitted, usually with surgical specimens, the tissue was digested enzymatically to give a single cell suspension using a standard mixture containing neuraminidase, hyaluronidase and collagenase. Cells from the digest were harvested every 20 minutes to avoid damage to the protein components of the cell membrane caused by prolonged exposure to these enzymes. They were then washed and plated at approximately 106 cells /flask in 5 ml of Medium 199 containing 10% foetal calf serum and selected growth additives known to promote epithelial cell growth. Biopsy sized samples were set up as explants in 0.5-1 ml of the same medium. The critical size and depth of the biopsy necessary for the outgrowth of colonic epithelial cells in culture is being determined.
Results so far suggest that successful cultures can be established from normal and tumour surgical samples using the explant technique and outgrowth of both epithelial and fibroblast cells have been observed. The findings are of consderable interest as other investigations have experienced difficulty in obtaining epithelial outgrowth from normal colonic mucosal tissue. ARE CLONOGENIC CELL SURVIVAL ASSAYS POSSIBLE WITH SOME PRIMARY CELLS ? S. Mulgrew, C. Seymour, C. Mothersill and M. Moriarty. Saint Luke's Hospital, Rathgar, Dublin 6. For many years it has been possible to grow fibroblasts in culture and several cell lines have been developed which allow accurate determinations of reproductive survival to be made using the 'clonogenic' technique of Puck and Marcus (J. Exp. Med. 1956. 103, 653-66). The technique involves seeding a small number of cells and scoring the number giving rise to discrete colonies at the end of a suitable incubation period as reproductively viable. However, despite extensive work using 'clonogenic' medium, differing media with various additives and several cloning techniques, some primary cultures never clone reliably and reproducibly, although a disparate monolayer can be produced and cell numbers would suggest a high survival of plated cells. Experiments~using cells cultured from human skin biopsies' and surgical specimens of thyroid gland suggest that little can be done to increase the cloning efficiency above 1% in untransformed thyroid cultures. Several approaches to the cloning of normal skin fibroblasts have failed. As all the initial work on clonogenic survival was performed on established cell lines, it has occurred to us that the ability of cells to form discrete colonies may be the first indication of cellular transformation. Alternatively, the original tissue used to establish a culture may contain only a small 'stem cell population' capable of undergoing cell division while other cells can live and possibly reorganise and differentiate but have lost the capacity to divide. The results suggest an urgent need to develop a method for determining reproductive survival in primary cultures and could suggest that survival assays such as those used for radiobiological experiments, which use clonogenic survival as the criterion for cell survival, may be inadequate to describe the events occurring in treated cell cult u res. THE RADIO-PROTECTIVE ROLE OF HYPERBARIC AIR T. J. Conere and S. W. Church. Department of Oncology, The Queen's University, Belfast. In a previous presentation (Conere et al in Proceedings of Irish Association for Cancer Re-
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search, Sept., 1983) some results from an earlier pilot study of the possible radio-protection offered by hyperbaric air were introduced. This present paper seeks to expand the theoretical basis for such protection as well as providing an update of the experimental data. The data now indicates that the ratio of surviving cells following irradiation to 10 Gy whilst at an air pressure of 0.3 x 16GPa (3 atmos) compared to cells irradiated to, 10 Gy under ambient conditions is 1.28. This ratio is statistically highly significant (P<0.005). It is suggested that such protection is related to the mechanism governing inert gas narcosis and anaesthesia.
THE KINETICS OF T H-LYMPHOCYTE SUBPOPULATION MIGRATION
THEORETICAL TARGETS IN CANCER RESEARCH Sidney Lowry. Deypartment of Oncology, Queen's University of Belfast. Theoretical and experimental research complement each other in the exact sciences. This approach is relatively new in the biological sciences t h e r e the construction of hypothesis and testing of theoretical models is a more recent phenomenon. It is proposed that cancer research, especially clinical research, is a strict scientific discipline and calls for a more rigorous approach to proceed on a sound rational basis. Multi-centre clinical trials in breast cancer provide one example of the problem. Therapeutic trials of surgery and radiotherapy often fail to recognise the variety of procedures and techniques that are available to treat different patients. Confounding the treatment modalities leads to confusion in assessing the final results. Again cancer chemotherapy has moved1 through different phases since it was first used 25 years ago. The early phase consisted of trial and error or serendipity. This was followed by a second phase o frational empiricism. Today we are looking hopefully towards a new phase of molecular intervention based on theoretical models of gene expression.
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B. Earls and R. J. Russell. Department of Microbology, Trinity College, University of Dublin. Locomotion of lymphocytes was first reported by Lewis and Webster (1921). Their directional locomotor properties was only demonstrated as recently as 1975 by Russell et al. The present study analysed this migration in vivo using a murine model over periods of up to eight weeks by implanting double-compartmented chambers intraperitoneally. The inner chamber was separated from the outer by a 0.22 #m micropore filter while the outer was accessible only via a 5 /zm poresize filter. When the inner chamber contained endotoxin-activated plasma incorporated in 1% agarose, a chemotactic gradient was established in vivo attracting motile leucocytes into the outer chamber where they were trapped for analysis. The non-phagocytic mononuclear cell infiltrate was characterised using subgroupspecific monoclonal antibodies. In this way it was shown that the T H subpopulation (Lyt 1) was the most actively responding cell type, entering the chamber to a level of over 50% of the lymphoid content by day seven postimplantation and thereafter declining to 17% by week five. Control chambers lackinf the chemotactic stimulus maintained a steady state of 46% T~{ cells throughout the same period althcugh the total cell infiltrate was very much less. On adding Ehrlich's ascites tumour cells to the inner chamber of the system a marked decrease in T~z migration occurred. Infiltration was maximal by day seven at a level of 20%, thereafter declinng to a level of 7% by week five. It is hypothesised that this effect was due to soluble inhibitory products from the tumour in the inner chamber. References Lewis, W. H. and Webster, L. T. 1921. J. Exp. Med. 33, 261-263. Russell, R. J., Wilkinson, P. C., Sless, F. and Parrott, D. M. V. 1975. Nature. London. 256, 646-648.