Irish association for cancer research

Vol. 164 No. 3

IRISH ASSOCIATION FOR CANCER RESEARCH Proceed ings of Annual Scientif ic Meet ing held at Kinsale on April 15-16th, 1994.

THE OCCURRENCE AND SIGNIFICANCE OF LEUKAEMIC TRANSFORMATION OF DONOR CELLS FOLLOWING ALLOGENEIC BONE MARROW TRANSPLANTATION (BMT)

M. Lawler 1'2, A. LocasciullP, A. Bacigalupo 4, P. Humphries 1, P. Ljungman 5, S. R. McCann 2.

Genetics~/Haematology 2, St. James's Hospital and Trinity College, Dublin; Ospedale San Geraldo Monza 3, Ospedale San Martino, Genoa 4, Italy; Univesity Hospital, Huddinge, Sweden 5.

While BMT is a successful therapy for haematological malignancies, the problem of leukaemia relapse remains and can be influenced by a variety of factors. Leukaemic relapse, normally occurs due to re-emergence of leukaemic recipient cells. However, there have been rare reports of leukaemia relapse occurring in donor cells. Many of these cases have not been proven using molecular techniques which provide better evidence of leukaemic transformation, especially in conjunction with cytogenetic data. We wish to report on 4 cases of donor leukaemia which we have documented by PCR. Two cases involved leukaemic transformation in patients transplanted for severe aplast ic anaemia (SAA). In both cases leukaemic transformation occurred in ihe first year post transplant. PCR of short tandem repeat sequences (STRs) indicated that the leukaemic clone had arisen in donor haemopoietic cells. One patient had a 9:11 translocation which is diagnostic of acute myeloid leukaemia (AML) FAB type M4" in his donor cells. This malignant clone arose within the first year post BMT. The second patient developed an undifferentiated leukaemia at day 180 following a second BMT from the same donor. In this case, conditioning for the second BMT included the use of total abdominal i r rad ia t ion (TAI). We have also conf i rmed a previously reported case of leukaemic transformation in a patient transplanted for chronic myeloid leukaemia in 1984 using PCR of a rch iva l mate r ia l . Recent ly , a fourth ease has been documented involving a patient with acute lymphoblast ic leukaemia. In addition, a patient who was transplanted for AML 5 years ago recently developed a non-random chromosomal abnormality. The rapid evolution of leukaemia in donor cells may reflect an acute transforming mechanism perhaps through a viral route. In this regard we have recently studied a patient transplanted for acute T cell leukaemia. The causative agent is the human T lymphotropic virus (HTLV 1). While the p~tient engrafted by PCR and erythrocyte antigen analysis, PCR indicated viral transfection of donor lymphocytes by the HTLV 1 virus. This observation may provide a clue to the mechanism for the phenomenon of donor leukaemia and help in a fuller understanding of the leukaemogenic process.

c-ERBB-2 ONCOPROTEIN EXPRESSION IN NONINVASIVE BREAST CANCER

N. Nolan, E. W. McDermott, J. R. Reynolds, A. McCann, R. Rafferty, P. Sweeney, D. Carney, N. J. O'Higgins,

M. J. Duffy. Departments of Surgery and Nuclear Medicine, St. Vincent's

Hospital and Department of Medical Oncology, Mater Misericordiae Hospital, Dublin.

The c-erbB-2 oncogene encodes a protein which is a putative growth factor receptor. While overexpression of c-erbB-2

219

oncoprotein in invasive breast cancer is usually an indicator of poor outcome, the biological s ignif icance of c-erbB-2 in noninvasive breast carcinoma has yet to be determined.

The purpose of this study was to address the relationship be tween c -e rbB-2 oncopro te in express ion and c l in ico - pathological parameters in noninvasive breast carcinoma.

Sixty nine patients with noninvasive breast cancer were studied. Eleven patients had lobular carcinoma in situ (LCIS) while 58 patients had ductal carcinoma in situ (DCIS). Of the 58 patients with DCIS, 36 had their disease initially detected clinically While 22 had mammographically-detectable d i sease only. Pa ra f f i n - f i xed sec t ions were s t a ined immunohistochemically with antiserum to c-erbB-2 oncoprotein.

c-erbB-2 oncoprotein was not detected in any of the patients with LCIS. In patients with DCIS, c-erbB-2 oncoprotein was expressed in 23 cases (40%) and was significantly associated with tumour necrosis (p<0.001) and the comedo subtype (p<0.02). Elevated levels of c-erbB-2 oncoprotein were found in 22/36 (61%) of patients presenting with clinical disease but in only 1/22 (4.5%) patients presenting with mammographicaUy- detectable disease (p<0.01).

Conclusion: This study supports the hypothesis that clinically detected and mammographically detected DCIS are biologically different diseases. The study also demonstra tes that the molecular pathogenesis of DCIS differs from that of LCIS.

IL-2 INDUCED LAK KILLING OF TUMOUR CELLS BY APOPTOTIC AND NECROTIC MECHANISMS IS

REGULATED BY IL-4

C. Gardiner, D. J. Reen. Children's Research Centre, Our Lady's Hospital for Sick

Children, Dublin 12.

LAK cells are reported to kill their targets by both necrotic and apoptot ic mechanisms. Various model systems have provided insights into some of the regulatory mechanisms controlling LAK cell mediated killing of tumour cells. Newborn NK cells provide an interesting effector model system since newborn NK cell activity is reported to be low compared to adult cells while LAK activity is high based on the 51Cr-release assay. The apoptotic potential of newborn NK cells is unknown.

In this study, parallel cytotoxicity assays were set up using a number of tumour target cell lines to measure effector induced killing by apoptotic and necrotic mechanisms. The effect of IL-4 on IL-2 induced LAK activity was investigated using both adult and newborn effector cells. Peripheral blood mononuclear cells and cord blood mononuclear cells were isolated by Ficoll- hypaque density gradient centr i fugat ion from adults and neonates respectively. Cells were cultured in IL-2 (500u/ml) supplemented medium, in the presence or absence of IL-4 (100u/ ml). Killing by necrosis was measured using a 4hr 5~Cr-release assay using the Daudi cell line as targets. Apoptosis of the YAC- 1 target cell line was measured using a modified cytotoxicity assay involving release of fragmented ~25I-deoxy-uridine labelled DNA.

Newborn cells exhibited higher LAK activity than their adult counterparts in the 4hr ~Cr-release assay. In the apoptosis based

220 Irish Association for Cancer Research I.J.M.S. July, August, September, 1995

assay, neonates also had higher activity at all effector:target (E:T) ratios examined arid this difference was signif icant particularly at lower E:T ratios (p<0.0014 at 6.25:1). Addition of IL-4 in the IL-2 dependent LAK assay reduced adult cytotoxicity significantly in both necrosis and apoptosis assays (from 66.2+25.0% to 51.6+30.7%, p<0.05 at an E:T of 12.5:1 in necrosis assay and from 49.1 + 18.9% to 23.8+39.0%, p<0.02 at an E:T of 12.5:1 in the apoptosis assay).

In the neonatal group, IL-4 appeared to have a differential regula tory effect on necrot ic and apoptot ic cy to tox ic i ty mechanisms. Using the StCr-release assay (n=8), IL-4 did not reduce IL-2 induced cytotoxicity. However, when using the ~25I-UdR release assay to measure apoptosis, IL-4 significantly reduced the IL-2 response in all cases (n=10) (p<0.01) for all E:T ratios examined).

In this study, adult LAK cells gave similar responses in apoptotic and necrotic based assay systems. However, in the case of the neonate, the apoptos is assay demonsti 'ates a differential sensitivity of newborn LAK cells to IL-4 and serves to highlight the importance of studying a model system reflecting physiological events likely to occur in vivo.

MODULATION OF PROTEIN KINASE C ISOZYMES BY TUMOR NECROSIS FACTOR IN L929 CELLS

M. A. O'Connell , D. Kelleher, N. Hall, L. A. J. O'Neil l*, A. Long.

Department of Clinical Medicine and *Department of Biochemistry, Trinity College, Dublin.

Tumour necrosis factor (TNF) is a potent cytokine which has antitumor effects in animal models and is cytotoxic in vitro to certain transformed tumor cell lines. TNF has been shown to activate protein kinase C (PKC). We have previously reported that staurosporine, a potent inhibitor of PKC, enhances TNF- mediated killing of a murine fibrosarcoma, the L929 cell line. PKC consists of a family of ten isoforms, classified according to differences in enzymatic properties. The aim of this study was to determine which PKC isoforms are involved in TNF- mediated cytotoxicity of these cells.

L929 cells (1 x 106/ml) were incubated alone or in the presence of 3ng/ml TNF for 5 min or 30 min. Total, cytoplasmic and membrane extracts were prepared and run on SDS-PAGE and determination of PKC isoforms carried out by Western blotting. A non-radioac t ive PKC act iv i ty assay was performed to de t e rmine ac t iv i ty of c a l c i u m - d e p e n d e n t and ca lc ium- independent isoforms of PKC.

The PKC isoforms ix, g, 8, e and ~ are present in L929 cells. 7, however , was not de tec ted . The PKC ac t iv i ty assay demonstrated that there was significant calcium-dependent and calcium-independent activity in resting L929 cells. On activation with TNF, calcium-independent but not calcium-dependent activity increased in the cytoplasm and there was a small increase in both calcium-dependent and -independent activity in the membrane . We subsequent ly examined t rans loca t ion of i n d i v i d u a l PKC i s o e n z y m e s to the p lasma membrane . Examination of cytoplasmic and membrane extracts revealed that the classical isoforms ct and g, are present in resting cytoplasm but do not translocate to the membrane after 30 min st imulation with TNF. e is present in the cytoplasm and membrane of resting cells, with a higher molecular weight form in the membrane; this isoform then appears as a doublet on stimulation with TNF for 30rain. ~ is strongly present in the

cytoplasm of rest ing and TNF-s t imula ted cells: a higher molecular weight species was induced in the plasma membrane by TNF.

These results demonstrate that TNF modulates PKC activity in L929 cells. Changes in activity are most l ikely due to mod i f i ca t ion o f i nd iv idua l i s o z y m e s , p o s s i b l y by phosphorylation, rather than translocation.

THE RELATIONSHIP BETWEEN DIFFERENTIATION STATUS OF HL-60 CELLS AND THE INDUCTION OF

APOPTOSIS

J. V. McCarthy, R. S. Fernandes, T. G. Cotter. Biology Department, St. Patrick's College, Maynooth, Co.

Kildare.

The HL-60 human promyelocytic leukaemia cell line will differentiate towards granulocytes when treated with retinoic acid (RA) over a five day period. During this differentiation period there is a marked decline in expression of c-myc and bcl-2 oncogenes. We have recently demonstrated (Science 257:212, 1992) a link between apoptosis and c-myc expression in T-cells. In this study, we sought to determine whether there was a l ink be tween express ion of these oncogenes and susceptibility to apoptosis.

Using three chemotherapeutic agents, the susceptibility of RA treated HL-60 cell cultures to apoptosis was monitored by morphological means and flow cytometry. HL-60 cells at apoptosis were monitored by morphological means and flow cytometry. HL-60 cells at various stages of differentiation were treated with either actinomycin D (5mg/ml), camptothecin (5mg/ ml) or etoposide (25mg/ml) for a 4 hour period. As cells with morphological characteristics of myelocytes became more predominant in the population, there was an increased resistance to the induction of apoptosis. On reaching terminal maturation HL-60 derived granulocytes rapidly underwent apoptosis induced by the above agents.

Flow immunocytometry revealed reduced levels of c-Myc and Bcl-2 oncoproteins in differentiating ceils, particularly in the myelocyte stage of the development. Together these results demonstrate that the differentiation state of a cell and expression of genes such as c-myc and bcl-2 determine how readily a cell undergoes apoptosis.

Supported by Cancer Research Advancementt Board and the Health Research Board of Ireland.

PROGNOSTIC SIGNIFICANCE OF RETINOBLASTOMA GENE PRODUCT EXPRESSION IN COLORECTAL CANCER

E. Ryan, A. Kitching, E MacMathuna, E. Mulligan 2, R. Merriman, R Dervan l, P Kelly 1, T. E Gorey 2, J. R. Lennon,

J. Crowe. Gastrointestinal Unit, Departments of Pathology ~ and

Surgery 2, Mater Misericordiae Hospital, University College Dublin.

Loss or inactivation of the retinoblastoma (Rb) gene has been demonstrated in several non-GI human malignancies. Recently, altered Rb gene expression has been reported in colorectal cancer (CRC) but the prognostic significance of this finding has not been determined. The aim of this study therefore was to correlate Rb gene product expression with longterm survival in CRC.

CRC samples from 76 patients collected over 3 years (1984-


86) were analysed for Rb gene product nuclear prote in expression by immunohistochemistry using the monoclonal antibody PMG3.245. Unlabelled CRC tissue and bladder cancer tissue were used as negative and positive controls, respectively. Prognostic significance was established from Kaplan-Meier curves which were generated from actuarial disease free survival, with differences in survival being compared by means of logrank analysis.

'Positive Rb expression of >80% in tumour cells was observed in 33/76 (44%) patients. Heterogenous Rb expression (20-80%) was seen in 39/76 (51%) patients, while no Rb expression occurred in the remaining 4/76 (5%). Rb expression of >80% correlated closely with poor survival (p = 0.03).

These findings suggest that the Rb gene is of prognostic significance in CRC and has an oncogenic-like function, in contrast to its reported tumour suppression activity in other human malignancies.

PREDICTABLE OUTCOME FOR DUKES B COLORECTAL TUMOURS EXHIBITING C-KI-RAS

ACTIVATION AND P53 IMMUNOSTAINING

M. A. Bennett, E. W. Kay, B. Curran, D. P. O'Donoghue, M. Leader, D. T. Croke.

Departments of Biochemistry and Pathology, Royal College of Surgeons in Ireland, Dublin 2 and Gastroenterology

Depai'tment, St. Vincent's Hospital, Dublin 4.

The Dukes staging system is currently the single most reliable determinant of prognosis for colorectal carcinoma. The aim of our study was to develop molecular assays for the detection of genetic aberrat ions in tumours to enhance the prognostic informativity of the Dukes system. One hundred and seventy- one in series Dukes B turnouts were analysed for a range of genetic and cellular phenomena. Mutational activation of the c-Ki-ras oncogene at codon 12 was assessed by a PCR-based method. Immunohistochemistry was used in the assessment of mutation at the p53 tumour suppressor gene locus. The presence of c-Ki-ras activating mutation at c0don 12 was associated with small tumours (p=0.003). Tumours located in the rectum were more likely to exhibit the activating c-Ki-ras mutation (p=0.006). Kaplan-Meier survival curves were constructed. Individuals whose tumours exhibited c-Ki-ras mutation tended to have an increased survival (p=ns) and those exhibiting strong p53 immunostaining in the cytoplasm exhibited a decreased survival (p=0.008). Cox regression analysis was used to generate a model (incorporating both c-Ki-ras mutation and p53 immunostaining) which is predictive of survival in Dukes B colorectal tumours. It is possible that the occurrence of specific phenomena in colorectal tumours is responsible for conferring a predictable phenotype on the tumour. Analysis of multiple genetic variables in colorectal tumours may result in the development of a more accurate prognostic system for the assessment of colorectal tumours.

AN INVESTIGATION OF THE EFFECT OF ALTERED THYMIDINE KINASE 1 ACTIVITY ON UV INDUCED DNA REPAIR IN HUMAN BREAST CANCER CELLS (MCF-7)

J. M. O'Connor , V. J. McKelvey-Mart in , P. G. McKenna. Department of Biological and Biomedical Sciences, University

of Ulster, Coleraine, BT52 ISA, Northern Ireland.

Cytosolic thymidine kinase (TK1) i s a cell cycle regulated

enzyme having its highest activity during DNA synthesis. Deficiency of TK1 in mouse erythroleukaemia and human lymphoblastoid cells has been shown to result in increased sensitivity to a variety of mutagen treatments. Further research has indicated that TK1 may exert its influence by helping to maintain the correct balance of nucleotide pools for accurate DNA excision repair processes to occur. Significant elevation of TK1 activity has been reported in breast carcinoma relative to fibroadenoma and normal breast tissue. This correlated positively with cancer recurrence. Supplementation of MCF-7 medium with tamoxifen or estradiol 17B has previously been shown to resul t in decreased and increased TK act ivi ty respectively. The purpose of this study was to determine the effect of altered TK1 activity on UV induced DNA repair in MCF-7 cells. DNA damage and repair breaks were quantified using the single cell gel electrophoresis (Comet) assay. UV induced DNA repair incisions were accumulated in the Comet assay using aphidicolin. Cells were treated with hormone; exposed to UV irradiation and subsequently allowed to repair for five min. Control cultures were processed in parallel. Neither tamoxifen nor estradiol 17g treatment resulted in significantly altered levels of UV induced DNA repair incisions. Treatment with estradio117B alone, however, did result in significant DNA damage in the absence of UV treatment. Colony forming assays and cytogenetic analyses are currently underway to determine the biological significance, if any, of altered TK activity in these cells.

HEPATITIS C INFECTION IN LONG TERM SURVIVORS OF BONE MARROW TRANSPLANTATION (BMT)

J. M. O'Riordan l, A. Tobin 2, M. O'Mahoney 3, S. R. McCann ~.

Departments of Haematology i and Medicine 2, St. James's Hospital and the Virus Reference Lab3., University College

Dublin.

Hepatitis C virus (HCV) is the cause of over 90% of post transfusion hepati t is . We studied the prevalence of HCV infection in 73 long term survivors of BMT (range 1-9 years), transplanted for leukaemia (n=60), aplastic anaemia (n=8), lymphoma (n=4) and Diamond Blackfan Syndrome (n=l). Sixty- six of the patients were recipients of allogeneic BMT while seven patients received autologous grafts. Sixty-four patients were transplanted prior to HCV antibody screening of blood donors. Ini t ial screening was by 2nd generat ion enzyme immunoassay (EIA Ortho). Positive sera were additionally tested by one of two EIA's (Abbott or UBI). A 2nd generat ion recombinant immunoblot assay (Chiron RIBA) which detects reactivity against c100-3, 5-1-1, c33c and c22-3 HCV antigens was used as a confirmatory test. Reverse transcription PeR (RT PCR) for HCV RNA was performed using nested primers and results were assessed after ethidium bromide staining of agarose gels. Serial liver biochemistry was reviewed and liver biopsy performed in selected cases. Initial screening was positive in 15/73 (20.5%) patients, of whom 11 were positive by additional EIA (9 of 11 Abbott, 2 of 4 UBI). RIBA-2 was positive in 9 of 10 sera which were positive by both EIA's and was indeterminate in the tenth. This patient was HCV PCR positive. Positive RIBA- 2 was associated with abnormal liver function tests (LFTs) in all but one patient. HCV PCR was positive in all 4 RIBA-2 positive patients tested and 3 patients are currently undergoing therapy with interferon alpha. RIBA-2 was indeterminate in all 4 patients positive by only a single EtA ((Ortho.). In 3 of

Vol. 164 223 No. 3

these 4 cases, LFTs were abnormal; one of these patients had clear evidence of hepatitis on biochemistry and liver biopsy and was PCR positive. Thus 20% of a cohort of long-term survivors of BMT have evidence of HCV exposure, of whom the majority have evidence of ongoing infection which has serious implications for their long term health. Nocases of HCV seroeonversion were seen in the 9 patients transplanted since the introduction of blood screening.

ASSESSING THE IMPACT OF BONE MARROW TRANSPLANTATION ON PATIENTS AND FAMILIES

F. M. Keogh, J. O'Riordan, S. R. McCan#, C. McNamara z. BMT Unit, St. James's HospitaP, Dublin 8 and Trinity

College 2, Dublin 2.

Bone marrow transplantation (BMT) has become a standard therapy for a variety of malignant haematological and genetic disorders achieving long term survival rates of 50-60%. This procedure is demanding for both patients and families and considerable psychological and social sequelae have b.een reportedL Using a prospective, repeated measures design this study investigates the psychosocial functioning of patients following BMT and the effect of this event on a close relative (not the donor). All patients u~dergoing BMT procedure in St. James's Hospital Unit for the twelve months from July 1993 are included in the study. Patients and relatives are interviewed on four occasions: I week pre-transplant and 3, 6 and 12 months post-transplant. The patient completes the Hogpital Anxiety and Depression Scale (HADS) (Zigmond and Snaith, 1983, Acta Psychiat. Scand. 67, 361) and the Rotterdam Symptom Checklist (RSCL) 2 and the relative completes the CHQ 12 (Goldberg 1978). Pre-transplant data reveal a considerable level of distress among patients (n = 14); HADS scores suggest 9 cases of anxiety and 2 cases of depression. The mean GHQ score for relatives (n=12) was 14.5 (sd=4.1) with at least 6 cases, suggesting a high level of strain ord is t ress among relatives. Preliminary data confirm a high level of anxiety in both patients and relatives before BMT which seems to be related to concerns as to how other family members are coping and the extra burden the family has to deal with. Support which focuses on these particular sources of anxiety would be beneficial to these patients and their families at this particular time.

References 1. Andrykowski et al. Bone-Marrow Transplantation, 1989: 4, 505. 2. de Haes et al. Br. J. Cancer, 1990: 62, 1034.

INTRACRANIAL RELAPSE IN HODGKIN'S DISEASE

P, McEneaney l, E A. Daly t, M. Farrell 2, S. Young 3. Department of Clinical Haematology and Oncology ~, St.

James's Hospital, Dublin 8 and Departments of Pathology 2 and Neurosurgery 2, Beaumont Hospital, Dublin 9.

Intracranial Hodgkin's Disease (H.D.) is very rare. The brain is a most unusual site of primary presentation of H.D. When involved it is usually in cases of relapse and is frequently associated with systemic relapse. In his review of the literature Sapozink (Cancer 52: 1301-7, 1983) found no subgroup of patients at high risk of intracranial relapse. Median survival from intracranial presentation was 4.5 months. This report represents a typical case of intracranial HD. A 37 year old man

with Stage IIIB NS Type 1 HD was treated with combination chemotherapy. He attained a remission lasting 10 months. He re-presented with B symptoms but no c l in ica l ly evident adenopathy. CT scan revealed enlarged deep inguinal nodes bilaterally. He was commenced on the VAPEC-B regimen with a plan for subsequent high dose chemotherapy and stem cell reconstitution. At the end of this eight week course, he developed a cholestatic hepatitis - probably drug-induced and related to ketoconazole taken as prophylaxis against fungal infection. Liver function tests continued to deteriorate for 2 months and then there was spontaneous improvement. At this stage he complained of right frontal headache with photophobia. This became more severe and was assoc ia ted with focal seizures . CT and subsequent MRI of his brain revealed a large right temporal l es ion with mass effect . He improved on high dose dexamethasone then underwent c ran io tomy and anter ior temporal lobectomy. His to logica l examinat ion confirmed cerebral HD with an inflammatory reaction in the meninges. Two weeks after surgery he again developed B symptoms. Further CT examination demonstrated new nodes at the aortic bifurcation. This patient has had cranial radiotherapy and is currently receiving further chemotherapy. His altered liver function persists but is improving and his fevers have resolved. He has no significant neurological deficit.

UROKINASE-TYPE PLASMINOGEN ACTIVATOR (uPA) AS A PROGNOSTIC INDICATOR IN COLORECTAL

CANCER

D. Gibbons, P. McCarthy, H. Mulcahy, M. J. Duffy, N. A. Parfrey, D. P. O'Donoghue, K. Sheahan.

Department of Pathology, Gastroenterology and Liver Unit and Department of Nuclear Medicine, St. "Vincerit's

Hospital, Dublin.

Urokinase-type plasminogen activator (uPA) is a serine protease implicated in the breakdown of the extracellular matrix, and may be involved in the development and progression of human cancers . The p rognos t i c s i gn i f i cance of uPA measurements has not been determined in large bowel cancer. The aim of this study was to correlate uPA imunohistochemical staining with multiple clinical and pathological features, and survival in patients with non-metastat ic colorectal cancer extending beyond the bowel wall (Dukes' B). 70 patients admitted to a single institution with Dukes' B colorectal cancer were studied. Mean follow-up time was 7.1 years, (range 4.9- 10). Formalin-fixed paraffin-embedded sections were stained for immunohistochemistry using a monoclonal antibody against the B-chain of uPA (American Diagnostica Inc., CT). Epithelial and s t romal pos i t iv i ty were scored independen t ly by a pathologist blinded to clinical details and patient outcome. Epithelial staining was graded as:- 1) <2%; 2) -20%; 3) 21- 50%; 4) >50%. Stromal staining was graded:- 1) few cells; 2) focal cellular aggregates; 3) multifocal aggregates; 4) diffuse. Grade 1 epithelial uPA reactivity was found in 0 cases, grade 2 in 5 (7%), grade 3 in 8 (12%) and grade 4 in 57 (81%). Grade 1 uPA stromal staining was seen in 16 patients (23%), grade 2 in 22 (31%), grade 3 in 19 (27%) and grade 4 in 13 (19%). A positive correlation between both forms of staining was seen (p = 0.036). No significant association was found between epithelial or stromal reactivity and patient age, sex, tumour site, tumour size, histological type, tumour grade, vascular invasion

224 Irish Association for Cancer Research

or perineur~l invasion, High levels of epithelial uPA (grade 4) correlated with turnout necrosis (p = 0.03). Five year survival estimated by the Kaplan-Meier lifetable method was 81% for patients with grade 1, 2 and 3 epithelial uPA positivity (n = 13) versus 58% for those with grade 4 (n = 57) (Logrank analysis: p = 0.01).

Stromal reactivity was not significantly related to survival. Regression analysis identified epithelial uPA reactivity as an independent prognostic factor within the Dukes' B group (relative risk 4.25: p = 0.04). Thus, epithelial uPA reactivity may be a useful marker of turnout aggressiveness in Dukes' B cancer .

USE OF FOUR DIFFERENT ANTIBODIES TO STAIN FOR P53 PROTEINS IN BREAST AND CERVICAL

CARCINOMAS

H. Lambkin ~,2, C. Mothersill 1, D. Chin e, M. J. Duffy 2, K. Sheehan 2, P. Kelehan 3, N. Parfrey z.

~Radiation Research Laboratory, DIT, Kevin Street, Dublin 8; 2Department of Surgery, Nuclear Medicine and Pathology,

St. Vincent' s Hospital, Elm Park, Dublin 4; 3Pathology Department, National Maternity Hospital, Holies Street,

Dublin 2.

Increased expression of p53 proteins is a common event in human cancers and is usually due to missense mutations in the p53 gene. A variety of antibodies reacting with different epitopes of the p53 protein have been used to study p53 overexpression. The use of different a, a tibodies and immunohistochemical detection methods has led to conflicting reports on the frequency of p53 overexpression in human cancers. The aim of this work was to compare p53 protein expression using four antibodies - p53-CMI, p53-1801, p53-DO7 and p53-240 (Novacastra Laboratories). Twenty breast adenocarcinomas evaluated for missense mutation in exons 5-8 by single strand conformation polymorphism (SSCP) and sixty uterine cervical carcinomas were immunostained with the antibodies. Immunostaining was performed with and without microwave antigen retrieval in citrate buffer. Agreement between SSCP (+) findings and immunostaining in the breast carcinomas was found using p53- 240 (4/10 cases) and p53-1801 (5/10 cases, no section pretreatment); for these antibodies only 1/10 SSCP (-) cases was positive, p53-CMI and P53-DO7 immunostained positively in 6/10 and 4/10 SSCP (+) cases but also in 4/10 and 3/10 SSCP (-) cases. Approx. 8-15/60 cervical carcinomas immunostained Positively for p53 proteins with the various antibodies. Tissue fixation was an important factor influencing immunostaining. Microwaving improved immunostaining intensity and doubled the number of immunoposit ive cases. Microwaving also produced positive immunostaining in non-malignant epithelia.

INVESTIGATION OF THE INCIDENCE OF HEREDITARY CANCER SYNDROMES AND THE EXPRESSION OF p53

GERMLINE MUTATIONS IN THESE KINDREDS.

M. Morrin, E Khan, R Delaney. Colorectal Research Unit, Limerick Regional Hospital, and

University of Limerick.

The clustering of cancer in certain families is well recognised. This may be due to chance and/or other environmental exposures. However, there is increasing awareness of the

I.J.M.S. July, August, September, 1995

variability defined hereditary cancer syndromes which are estimated to account for approximately 10% of the total colorectal cancer burden. Recent work has indicated that mutations in the p53 tumour suppressor gene can occur not only at the somatic level but also in the germline, thereby predisposing affected individuals to development of cancer. We are investigating the incidence of hereditary cancer syndromes in this region and the expression of p53 germline mutations in these kindreds. To date we have 22 families participating in the study, With a diverse range and frequency of cancer. Four of the families each have two or more first degree relatives with colorectal cancer and can thus be categorised as having familial cancer syndrome. Other families have a high frequency of cancer but with diverse primary locations. Blood samples have been collected from 66 members of the different families and the extracted DNA from 61 of these examined using polymerase chain reaction -single strand conformat ion polymorphism (PCR-SSCP) analysis for p53 germline mutations. Exons 5, 6, 7 and 8 of the p53 gene have been amplified, denatured to single-stranded DNA and separated by polyacrylamide gel electrophoresis under non-denaturing conditions. Mobility shifts have been detected in exons 5, 6 and 7 in 7 individuals from the cancer families. These DNA samples are being sequenced to determine whether these mobility shifts are caused by polymorphisms or germline mutations in the p53 gene.

SYNDROMES AMONG PREMENOPAUSAL WOMEN WITH INHERITED PREDISPOSITIONS TO BREAST CANCER - IMPLICATIONS FOR SCREENING AND

MANAGEMENT

D. M. Rowan, W. J. Orminston, P. P. Donnellan, P. A. Daly. Department of Clinical Haematology and Oncology, St.

James's Hospital and Trinity College, Dublin.

A number of clinical syndromes with an autosomal dominant inheritance pattern associated with predisposition to the development of breast cancer are now recognised. They include site specific hereditary breast cancer, breast-ovarian cancer, Li- Fraument syndrome, Lynch syndrome II (Cancer Family Syndrome), Gorlin's syndrome, Cowden's syndrome, Muir-Torre syndrome and heterozygosity for ataxia-telangiectasia. Among 24 patients with premenopausal breast cancer we have identified 35 who appear to have inherited a predisposition to the development of the disease and who fulfil the criteria for hereditary breast cancer t. By examining further the spectrum of cancers and other clinical manifestations in individual patients and their families, we sought to define the nature of the predisposition and to place the patient/family where possible within one of the defined clinical syn,dromes. This proved possible in 22 cases. Of these 4 were consistent with familial breast-ovarian cancer, one of whom also showed ~features of the Muir-Torre syndrome. The other 18 cases were consistent with site-specific hereditary breast cancer. With the other 13 unclassified cases, work is ongoing towards detecting clinical associations and cancer clusters so that they can be better categorised and possible mutations detected which are responsible for their predisposition to cancer. In one of these families, we have extended the pedigree to include greater than 510 members, spanning five generations. We feel this family represents a new cancer family syndrome characterised by a

Vol. 164 225 No. 3

high incidence of breast cancer, including male breast cancer, and an increased incidence of other common cancers, especially lung, melanoma, penis, bladder, leukaemia, larynx, ovary, oesophagus, pancreas and Hodgkin's disease. Similar families have been identified by other investigators and are all the subject of a report in press. Linkage to any of the known loci for breast cancer predisposition has not been established for these families. Among the 35 families, the four breast-ovarian families are likely to have linkage to BRCA 1 locus,on 17q. Since genetic counselling based on BRCA 1 linkage is now being offered in other centres, our goal is to work towards this service for our patients and their relatives. Reference t. Lynch. Cancer Genetics & Cytogenetics 1986" 22: 4: 369-37.

MAMMOGRAPHY IN THE DIAGNOSIS OF IMPALPABLE BREAST CANCER IN A BREAST CLINIC POPULATION

A. Khalid~ M. Kerin, D. M. O'Hanlon, P. Kent, P. McCarthy ~, H. E Given.

The National Breast Cancer Research Institute and Departments of Surgery and Radiology ~, University College

Hospital, Galway.

The eff icacy of screening mammography in the early detection of breast carcinoma is accepted. In this study we have prospect ive ly assessed the role of mammography in the assessment of 405 patients ~vithout suspicious lumps who presented to a breast clinic over a 12 month period. All patients with clinically or cytologically suspicious lumps were excluded from the study and proceeded to excision biopsy. The indications for referral were non-specific nodulariy 130, breast pain I 12, nipple discharge 72, positive family history 412, non specific 50. Patients ranged in age from 35 - 80 years with a mean of 52 years. Cranio-caudal and medio-lateral views were obtained in all cases. Age Number of patients Cancers (%) 35-39 24 0 40-49 166 5 (3%) 50-59 131 6 (4.6%) >60 84 7(8.3%)

Eighteen (4.5%) patients had malignancy. Fourteen of these had intraductal carcinoma on histological assessment. This study confirms the higher incidence of malignancy in symptomatic patients presenting to a breast clinic. Mammography is a very important diagnostic aid and should be performed in all patients over 40 who present to a breast clinic.

MOLECULAR GENETIC STUDIES OF PAEDIATRIC MALIGNANT LIVER TUMOURS

S. M. Kennedy I, G. McGeoch 2, N. K. Spurr 2. Department of Pathology, Royal Victoria Eye and Ear

HospitaP, Dublin and I.C.R.F3, London.

Cellular and molecular genetic studies were performed on childhood hepatocellular carcinoma (HCC), hepatoblastomas (HBA) and embryonal sarcoma (ES).

Histopathological , immunohistochemical and molecular methods. The turnouts were probed at the DNA level using known oneogenes as probes and allelic deletion studies were performed. Ploidy was assessed by image analysis.

Morphologic evidence of antecedent liver disease was found in 50% of the HCCs. None of the HBAs had signif icant morphologic abnormalities in the non-neoplastic liver. 2/7 tumours had aneuploid DNA content. All of the cases had a high proliferating fraction with the monoclonal antibody PC 10 raised against proliferating cell nuclear antigen. Amplification of the oncogene c-myc was found in five cases, including two ES, two HCC and a HBA. Amplification of the epidermal growth factor receptor was found in two HBAs and a dysplastic nodule from a hepatectomy specimen affected by H'I~I. Mutational analysis of exons four to nine of the p53 gene by means of gene amplification, using the polymerase chain reaction and direct sequencing, revealed no mutations. The p53 protein was over expressed in a major i ty of tumours (67%). Loss of heterozygosity was found on chromosome 16,4,1.

Conclusions: p53 protein overexpression has no correlation with gene mutations in the species conserved regions of the gene. Loss of he te rozygos i ty on chromosome 16 in 3/6 informative cases suggests that chromosome 16 may harbour a tumour suppressor gene important in the development of HCC but not HBA or ES.

CHALLENGES FOR IMMUNOTHERAPY OF TUMOURS - ANTI-TUMOUR IMMUNE RESPONSES SELECT FOR

VARIANTS OF LOW ANTIGENICITY FROM ESTABLISHED VASCULARISED TUMOURS

J. Barrett 1, G. O'Sull ivan 2, J. K. Collins 3. Departments of Microbiology ~, Surgery 2, and Medicine ~,

University College and Mercy Hospital, Cork.

Successful immunotherapy o f es tab l i shed turnouts is observed in some experimental systems where the turnouts are inherently immunogenic. However, this is not a real challenge as the major i ty of spontaneous tumours are inheren t ly immunogenic. The challenge of immunotherapy therefore is to increase immunogenicity or to increase the number of committed anti-tumour effector cells to augment detection and effect turnout elimination. We have developed a novel tumour model to evaluate the efficacy of anti-tumour immune responses. We have used SV40 t ransformed SV3T3 cel ls . These cel ls carry intranuelear T-antigen and surface T-antigen which is viral coded. This surface T-antigen is a strong immunogen which evokes a T cell response, mediates elimination of tumour cells and is thus called tumour specific transplantation antigen (TSTA). We have isolated a T-antigen negatiye varient of SV3T3 called JBS. These ceils are transformed, weakly immunogenic, MHC res t r i c t ed yet capab le of induc ing tumours in immunocompetent BalbC mice. We induced turnouts with mixtures of JBS and SV3T3 cells in syngeneic BalbC mice. Tumours developed with JBS and SV3T3 cells which became infiltrated with immune effector cells. This resulted in apparent total regression of established tumours. However, regression was only temporary in greater than 80% of animals with regrowth of tumours containing only JBS cells. Interestingly, a small percentage of animals had no regrowth and were refractory or immune to subsequent challenge with tumourigenic doses of JBS cells. In conclusion, we have shown that immune responses can mediate regression of established vascularised turnouts containing strongly and weakly immunogenic cells. However, this is not curative in the majority of eases and results in the selective regrowth of weakly antigenic cells forming tumours in immunocompetent animals.


DETECTION OF MUTATIONS IN p53 GENE IN BREAST CANCER BY SINGLE STRANDED CONFORMATION

POLYMORPHISMS (SSCP)

D. Chin, M. J. Duffy, T. Willcocks, P. McCarthy, S. Kennedy, J. Dolan, W. Gallagher, E. McDermott,

N. O'Higgins, N. Parfrey. Departments of Surgery, Nuclear Medicine and Pathology,

St. Vincent's Hospital, Dublin.

p53 is the most frequently mutated gene so far described in human malignancies. Alterations in this gene are thus likely to be key events in the genesis of many human cancers. The aim of this investigation was to study the distribution of mutations in the p53 gene in human breast cancer using single stranded conformation polymorphism (SSCP). DNA was extracted from the breast tumour using chloroform-phenol. Exons 5, 6, 7 and 8 of the p53 gene were amplif ied using polymerase chain reaction (PCR). The amplified products were then separated on non-dena tu r ing p o l y a c r y l a m i d e gel e lec t rophores i s . Electrophoresis was carried out both with and without 5% glycerol at 4~ and at room temperature with glycerol. Samples with altered mobility by electrophoresis were considered to contain a mutation. Using this approach, mutations in the p53 gene were found in 37/91 (40.6%) of samples. Five mutations were found in exon 5, 17 mutations in exon 6, 20 mutations in exon 7 and 5 mutations in exon 8. Six samples contained mutations in 2 different exons, 3 samples contained mutations in 3 different exons, while 1 sample has mutations in all 4 exons. No significant correlation was found between mutations detected by SSCP and increased p53 protein as detected using CM-1 antibody (Novocastra Laboratories). Similarly, no correlation was found between the presence of mutation and either tumour size, nodal status or oestrogen receptor status. We conclude that mutations in the p53 gene is a common event in human breast cancer. The significance of this in breast cancer genesis however, remains to be established.

IDENTIFICATION OF A POLYMORPHIC MONONUCLEOTIDE REPEAT (MICROSATELLITE) IN

THE HUMAN PROHIBITIN (PHB) GENE BY DENATURING GRADIENT GEL ELECTROPHORESIS

R. Hagan, R. McManus, W. Ormiston, P. Daly. Department of Haematology/Oncology, Trinity College and

St. James's Hospital, Dublin.

The human prohibitin (PHB) gene, located on chromosome 17q21, is a putative tumour suppressor gene associated with some sporadic breast cancers. Analysis of germline mutations in the PHB by denaturing gradient gel electrophoresis (DGGE) was undertaken to further determine its involvement in breast cancer. DGGE depends upon the differential electrophoretic migration of wild-type and mutant DNA for the detection of mutations. As the double stranded product enters a critical concentration of denaturant, domains within the fragment melt, thus producing partially denatured DNA. This causes an abrupt decrease in mobility of the fragment through the gel and is capable of detecting single base substitutions. With heteroduplex formation the rate of detection of base differences approaches 100%,

Previous mutational analyses of PHB ~ by RNase protection assay and sequencing identified 5 somatic mutations among 120 primary breast cancers. 3 of these were in the highly


conserved exon 4, and a fourth in a flanking intron. In our study genomic DNA was extracted from PBLs of patients with primary breast cancer and, in families where there was a history of the disease, from relatives of the proband. PCR products of exon 4 were amplified using primers in the introns flanking exon 4, creating a 377 bp fragment. The first 80 bases of this fragment represent one melting domain (wholly intron sequence), as calculated by the computer programme MELT. From a sample of 20 unre l a t ed ind iv idua l s , wi th in our popu la t ion , 4 polymorphisms were identified by parallel DGGE. From the known base sequence, the most probable region for base differences to occur was a poly (A) stretch of 25 bases interrupted after 15 bases by a C residue. To focus on possible length variations within the microsatellite, one nested primer was designed which amplified an 84bp region and the products were run on an 8% denaturing polyacrylamide gel. 4 alleles of this microsatellite, 72, 74, 84 and 85bp, are present within our sample, indicating that this locus may be useful in genetic mapping. Each of these alleles has been detected on the denaturing gradient gels. The resolution, however, of the microsatellites sizes is much poorer than that of the DGGE, as shadow bands made interpretation of the allele size very difficult. For this polymorphic mononucleotide repeat DGGE is more appropriate than microsatell i te sizing for detecting alleles differing by only a single base pair. In conclusion, DGGE may offer a more sensitive and nonradioactive technique for sizing certain microsatellites than the procedures currently being used.

Reference 1. Sato, T., Sakamoto, T. Takita, K., Saito H, Okui K, Nakamura, Y. The human prohibitin (PHB) gene family and its somatic mutations in human tumors. Genomics 1993: 17, 762-764.

LINEAGE PROMISCUITY IN MALIGNANT LYMPHOMAS

O. Sheils, M. McDermott, D. S. O'Briain. Histopathology Department, St, James's Hospital, and

Trinity College' Medical School, Dublin.

The polymerase chain reaction (PCR) has been used for some years in the diagnosis and characterisation of B-cell lymphoma, but suitable T-cell primers have not been validated in general use. In a preliminary study, we used the PCR technique with 3 sets of primers to identify rearrangements in B-cells (primers representing the V and J regions of the immunoglobulin heavy chain gene and a pair corresponding to opposing sides of the BCL2 t14:18 chromosome translocation), and T-cells (primers representing the V and J regions of the TCR y chain gene). We analysed formalin fixed, paraffin embedded material from 24 biopsies taken from 18 patients, including 6 benign lesions and 12 mal ignan t l ymphomas which were c l a s s i f i ed by immunocytochemistry as B-cell lymphomas (9 biopsies) and T-cell lymphomas (9 biopsies), and T-cell lymphomas (9 biopsies). The results are summarised in the following table.

PCR Positive Results No. of biopsies Ig heavy chain BCL t(14:18) TCRv

B cell lymphoma 9 6 3 3 T-cell lymphoma 9 2 1 8 Benign 6 0 0 0

No benign lesion produced a false positive result, and the T- cell assay identified 8 of 9 immunohistochemically diagnosed T-cell lymphomas. This study indicates that it is feasible to identify T-cell lymphomas by PCR. The finding that 4 cases

Vol. 164 227 No. 3

were positive for both B and T-cell assays is noteworthy, as it raises the possibility that lineage promiscuity - a phenomenon well recognised in leukaemias where expression of two distinct lineages such as lymphoid and myeloid may occur in the same cell - may also occur in lymphomas. Without the use of a PCR panel such cases might be misclassified. Less likely explanations for this finding are that it represents a technical anomaly or combined B and T-cell tumours. Co-expression of B and T-cell genotypic markers may occur in a s ignif icant number of lymphomas and are therefore a potential source of diagnostic error.

LAPAROSCOPIC OVARIAN ABLATION IN PATIENTS WITH BREAST CANCER

E Kent, D. M. O'Hanlon, M. Kerin, D. Maher, H. E Given. National Breast Cancer Research Institute and Department

of Surgery, University College, Galway.

Ovarian ablat ion Offers a significant advantage in both survival and disease free interval in premenopausal patients with breast cancer. (}urrently there are four techniques available for ovarian abla t ion - radiat ion, open surgery, hormo'nal manipulation and possibly chemotherapy - each with significant d i sadvan tages . The novel t echn ique of l apa ro sc op i c oophorectomy has been performed on forty-five patients in this unit. The procedure was perfolmed using standard laparoscopic equipment including one 10mm and two 12mm ports, a 10mm laparoscope and an Endo-GIA (Autosuture Ltd.) for haemostasis! division of the ovarian pedicle. The mean age of the patients was 42 years ranging from 33 to 50 years. The technique was used in an adjuvant setting in forty-three patients and in two following disease recurrence. Operative time ranged from 12 to 85 min with a mean of 33 min. This decreased with experience. Conversion to open oophorectomy was required in one patient, the first. This patient also developed a wound infection. One patient required transfusion after a bleed from a port site. The mean post-operative hospital stay was 2.3 days. The technique described is technically straightforward to perform and has minor complications. It has distinct advantages over ovarian i r r ad ia t ion , open surgery , endocr ine manipulat io.n and chemotherapy and should be considered as adjuvant treatment in all premenopausal patients with breast cancer.

ANGIOGENESIS AND BREAST CANCER: RELATION TO PROGNOSIS AND COMPARISON OF

PATHOLOGICAL AND RADIOLOGICAL ASSESSMENT

P. Costello, A. McCann, E Flanagan, J. Stack, J. Ennis, P. Dervan.

Departments of Pathology and Radiology, University College, Dublin and Mater Misericordiae Hospital, Dublin.

Assessment of tumour vascularity has been reported to be of prognostic value in breast carcinoma. We present the results of a study examin ing its ut i l i ty both in a re t rospect ive pathological and prospect ive combined pathological and radiological study.

Vascularity was assessed by counting microvessel (MV) dens i ty in "ho t spo t s " of tumour ang iogenes i s af ter immunohistochemistry for FVIII-RA to highlight endothelial cells. Paraffin embedded tissues were used for retrospective

analysis of 87 lymph node negative patients for whom a follow- up of 2-10 years was available.

A further group of patients were assessed pre-operatively using gadolinium enhanced magnetic resonance imaging. Enhancement could be plotted as a curve which showed characteristic features for malignant tumours. MV density was then assessed after excision of the lesion.

Comparison of MV density with property of the enhancement curve ca l led g, which cor responded to the slope of the enhancement curve, showed a positive correlation. Analysis of the retrospective group did not reveal any difference in disease free or overall survival between low and high MV density groups.

We conclude that high MV counts do not reliably identify patients with a poorer prognosis in lymph node negative breast cancer. The importance of angiogenesis in the progression of cancer may be due to properties of the vessels rather than their density.

EVALUATION OF A PROLIFERATION MARKER TPS IN BREAST CARCINOMA

D. M. O'Hanlon, M. Kerin, P. Kent, D. Maher, H. Grimes ~, H. F. Given.

National Breast Cancer Research Institute and Departments of Surgery and Biochemistry l, University College, Galway.

The role of tumour markers in breast cancer diagnosis and follow-up is controversial. TPS (Tissue polypeptide specific antigen) formed and released during the late S and G2 phase of the cell cycle is a marker of cell proliferation. This marker was assessed in 97 patients with breast carc inoma using the monoclonal antibody M3 against the specific epitope of TPS and compared to 43 patients with benign breast disease. The mean level in patients with benign breast disease [55.7(5.9)] was similar to pre-operative levels in patients with operable breast carcinoma [71.7(11.3), P=ns]. TPS was measured during follow-up in 80 patients. Forty-nine patients have not developed recurrence and TPS levels were not significantly different from the operable group [81.2(9.1), P=ns]. In the 17 patients with loco-regional recurrence, TPS was elevated [112.9(35.8)]. In the 14 patients who progressed to systemic metastases, TPS levels increased s ign i f i can t ly to a mean of 569(149.7) (P<0.0.05), Mann Whitney U) demonstrating a substantially elevated level in 8 of the 14 patients.

The median follow up was 10 month's. TPS levels were elevated in 3 patients with loco-regional recurrence, all of these patients has rapid disease progression compared with 5/14 patients with normal TPS levels. In patients with metastatic disease 7/8 patients with elevated levels had disease progression. This study demonstrates that this marker is elevated in a significant proportion of patients with metastatic disease. In addition, it identifies patients at high risk of disease progression.

TPS levels given as mean (SEM) and in U/1.

BREAST ENLARGEMENT IN THE MALE OVER 40; A DIAGNOSTIC DILEMMA

D. M. O'Hanlon, M. Kerin, P. Kent, D. Maher, H. F. Given. National Breast Cancer Research Institute and Department

of Surgery, University College Hospital, Galway.

Breast enlargement is a common condition in males with advancing age. It is important to differentiate gynecomastia from


carcinoma. Over a 14 year period, 67 patients over the age of forty were operated on for breast masses (11 carcinoma; 49 gynecomastia; 6 lipoma and I lymphoma) in this unit. Presenting complaint, past medical Hx (history), family Hx, social Hx, drug Hx and physical examination were compared in patients with gynecomastia and carcinoma and the results are summarised in the table.

Pathology (number of patients) Carcinoma (1~ Gynecomastia (49) P value**

Mean (range) age (years) 66(44-84) 63 2(40-91) History lenght (range mrs) 33(6-60) 3 . 4 ( 1 - 1 2 ) P<0.005 Breast pain 2(18.2%) 10(20.4%) P = as PHx carcinoma 2(18.2%) 0 P <0.05 FHx breast carcinoma 4(36.4%) 7(14.3%) P= ns DHx* 3 (27.3%) 14(28.6%) P = ns Cigarettes 2(18.2%) 21(42.9%) P = ns Alcohol consumption 6(54.5%) 33(67%) P = ns" Scrotal pathology 0 7(14.3%) P = ns *Drugs known or suspected to cause gynecomastia. ** Statistics: Mann Whitney U, Chi square with Yates connection.

All patients presented with masses. "In the cancer patients, only patients with ulcerating lesions presented with pain. In conclusion carcinoma must be excluded in patients presenting with unilateral breast masses in particular patients with painless masses. Clues to the diagnosis are provided by a past history of carcinoma or a family history of breast carcinoma. Masses in patients on drugs known to cause gynecomastia should not be dismissed as being solely due to the drug.

A S S E S S M E N T OF p53 PROTEIN EXPRESSION BY MICROWAVE IRRADIATION IN 150 THYROID NEOPLASMS

A. Yanni, M. Harrison, P. Dervan, Departments of Pathology, University College Dublin and

Mater Misericordiae Hospital, Dublin.

Recent studies have yielded poor p53 immunohistochemical pos i t iv i ty in benign thyroid adenomas. In general , only malignant neoplasms of the thyroid are p53 positive. We examined p53 nuclear immunohistochemical staining patterns in 42 (papillary, follicular and anaplastic) thyroid carcinomas, and 102 (microfoll icular, macrofollicular, normofollicular, Hurthie cell, cellular and atypical) adenomas, for p53 gene product accumulation using mouse monoclonal antibody DO7 and 20 minute microwave irradiation of formalin-fixed paraffin- embedded tissues. Overall percentage of nuclear positivity in each tumour category was obtained by counting 1,200 cells on each section. Percentage of positively-staining nuclei in each case was graded as I (1-30% of total nuclei staining), II (31- 60%) and III (61-100%). Furthermore, we assessed the pattern of nuclear staining intensity as weak (1+), moderate (2+) or strong (3+). Our data revealed, contrary to previous studies, that a large number of adenomas (63%) stained positively compared to 69% of malignant cases. We also found that the percentage of grade III staining in the adenomas was 55 and in the carcinomas 79. High intensity (3+) homogenous p53 nuclear reactivity showed a distinct pattern in individual subgroups with adenomas collectively showing 7% of nuclei with strong staining and carcinomas with 22%. Interestingly, no adjacent normal thyroid tissue showed immunopositivity. Our study clearly shows a very signif icant proport ion of thyroid adenomas exhibiting accumulation of nuclear p53 gene product with distinct patterns in the percentage and intensity of cells staining,

with in both groups most cases showing a 10-fold yield in immunoreactivity after microwave irradiation. The significance of p53 staining within adenoma subgroups is unclear at this stage. The possibility that it represents part of a spectrum of premalignant change needs further study.

THE CLINICAL, PATHOLOGICAL AND MOLECULAR PROPERTIES OF OVARIAN CANCER

W. S. Lowry, S. E. H. Russell, R. J. Atkinson, P. White, I. Hickey, D. W. Bell, D. Biggart.

The Queen's University of Belfast.

The clinical and pathological features of tumours are now being described in terms of their molecular properties. It is important to distinguish between the various parameters in interpreting their biological significance. Analysis of over 100 ovarian turnout specimens reveal that histopathological grade is directly related to the underlying genetic alternations in ovarian cancer. It accounts almost entirely for the apparent genetic relationship to clinical stage and turnout type. The biological significance of grade as a major factor in determining the degree of malignancy is thus confirmed at the molecular level.

PROLIFERATING CELL NUCLEAR ANTIGEN (PCNA) IN DUKE'S B COLORECTAL CANCER

D. Gibbons, R McCarthy, H. Mulcahy, J. Doyle, N. Parfrey, D. E O'Donoghue, K. Sheahan.

Departments of Pathology and Gastroenterology and Liver Unit, St, Vincent's Hospital University College Dublin.

Colorectal cancer is conventionally staged on the depth of penetration and the nodal metastatic status of the primary tumour. Outcome within the Dukes' B stage is especial ly variable, indicat ing that other tumour factors are also of prognos t ic impor tance . PCNA i s a 36kd pro te in which accumulates in the late G I and early S phase of cycling cells and is a marker of cellular proliferation. The aim of this study was to correlate PCNA expression with clinical and pathological features, and survival in patients with Dukes' B colorectal cancer. 159 patients admitted to a single institution'with Dukes' B colorectal cancer were studied; M:F 51:45 mean age 67 (range 26-86). Mean follow up 6. I years. Paraffin embedded sections were stained for immunohistochemistry with a monoclonal antibody PC10 against PCNA (Dako) using the avidin-biotin complex technique. Cases were scored by a pathologist blinded to clinical details and patient outcome. 1000 cells were counted to determine the PCNA 9 index. Heterogeneity was reflected in the PCNA score (index x % tumour showing intense staining). The median tumour PCNA index was 80 (mean 79; range 27- 98) and the median PCNA score was 49.4 (mean 49.5; range 4.8-90.3). No significant correlations were found between PCNA index or score.and patient age, sex, tumour site, tumour size, tumour differentiation, vascular invasion or perineural invasion. Tumours displaying extensive necrosis had a higher mean PCNA index (p<0.001) and score (p<0.001) than those with absent or minimal necrosis. Survival was similar in patients whose tumours were categorised as above or below the median PCNA value (Logrank analysis: PCNA index, p = 0. I9; PCNA score, p = 0.48). PCNA expression does not assist in selecting a subgroup of Dukes' B patients with a poor prognosis.

Vol. 164 229 No. 3

APOPTOTIC INDICES VARY IN DIFFERENT HUMAN TUMOR TYPES

M. J. Staunton, E. F. Gaffney. St. James's Hospital and Trinity College, Dublin, Ireland.

Apoptosis is the genetically-mediated mechanism controlling deletion of individual ceils in normal and diseased tissues. There is very little data on the degree of apoptosis present in different human tumour types. We initially evaluated H- & E-stained paraffin sections of 102 malignant tumors (58 types) for apoptotic cells and apoptotic bodies ~, using the 40X objective with a calibrated eye-piece, and counting approximately 3,000 tumor cells per case, avoiding necrotic zones. The percentage of apoptotic cells/bodies in the total number of tumor cells examined was designated as the apoptotic index (AI) for each case. Although there was a wide range in AI for different tumor types - from 0 in follicular carcinoma of thyroid to 27% in small cell carcinoma of lung - 52 tumors had AI<2% and 79 had AI<5%. The AI in 30 add i t iona l tumors (3 types) was subsequently determined, and 12/12 colonic adenocarcinomas had AI of 1.5%-5.7%, 11/12 malignant melanomas (including primary, recurrent and metastatic tumors) had AI of 0-1.4%, and 11/12 immunoblastic lymphomas had AI of 0.4%-5.7%. Each of the 132 tumors had a consistent overall AI, and, in general, apoptotic nuclear material was more prominent than mitoses.

These resul ts suggest that many tumor types have a characteristic AI, that reflects innate tumor cell susceptibility to undergo apoptosis. However, much additional data is needed to determine whether significant variations i~ AI correlate with a l te red p r o l i f e r a t i v e ind ices , aber ran t oncogene / tumor suppressor gene expression, and standard clinicopathologic variables.

Reference 1. Kerr et al. Br. J. Cancer 1973; 26: 239.

AN INTERIM REPORT ON SOFT TISSUE AND VISCERAL SARCOMAS IN IRISH PATIENTS

E. F. Gaffney, P. A. Dervan, M. M. McCabe, K. Sheahan, E. W. Kay, M. Leader, J. Doyle, P. A. Daly, J. J. Fennelly, D. N. Carney, M. O'Reilly, J. N. McMahon, M. Moriarty,

B. Hurson. St. James's and Federated Dublin Voluntary Hospitals and St. Vincent's, Mater Misericordiae, Beaumont, St. Luke's

and Cappagh Hospitals, Dublin.

The Dublin Soft Tissue Sarcoma Panel was established in 1989 with a view to achieving a unified approach to diagnosis and management of soft tissue and visceral sarcomas. This interim report presents data on 265 prospectively-evaluated patients and on a separate retrospective series of 126 patients. The patients in the prospective series were treated by 93 different surgical and medical specialists. Tumours presented in all ana tomic s i tes and ranged in s ize f rom 0.2 to 60 cm. Leiomyosarcoma was the commonest tumour type. Eight-nine tumours were inoperable at clinical presentation. There was a consensus panel diagnosis in over 90%, non-neoplastic reactive lesions and primitive round cell tumours being the most difficult cases diagnostically. Management, including onward referral for chemotherapy or radiation therapy, was inconsistent. The 2-year survival figures were: 43% (1989-1991) and 37% (1980- 1988).

The findings of this group, facilitated by the Panel review process and the cooperation of numerous specialists involved in patient management, will provide a basis for the evaluation of coherent investigation and treatment strategies to improve the prognosis for Irish sarcoma patients.

Supported by the Irish Cancer Society and National Cancer Registry (Ireland).

ABBERANT bcl-2 EXPRESSION IN NON-SMALL CELL LUNG CARCINOMA: CORRELATION WITH APOPTOSIS, Ki-67 IMMUNOSTAINING AND

PROGNOSIS

A. J. O'Neill , M. J. Staunton, E. F. Gaffney. Department of Histopathology, St. James's Hospital and

Trinity College Dublin.

The proto-oncogene bcl-2 inhibits apoptosis without affecting cell proliferation and thus favours slow tumour growth and relatively non-aggressive biological behaviour. The bcl-2 protein is abnormally expressed in certain slow-growing tumours, such as follicular lymphoma. Recently, it was reported I that bcl-2 immunoreactivity is a strong predictor of survival in patients with non-small lung carcinoma (NSCLC). We examined bcl-2 expression apoptosis (by H & E and by in s i tu end-labelling) and proliferation indices, using an antibody to Ki-67 following microwave-stimulated antigen retrieval, in paraffin sections of 51 non-small cell lung carcinomas. Clinical follow-up was obtained from hospital charts and from the patients ' GPs. Thirteen squamous cell carcinomas (SqCC) stained for bcl-2. All bcl-2 positive SqCC has apoptotic indices (AI) of <3.8% and corresponding ISEL staining. However, 24/28 bcl-2 negative SqCC also had AI <3.8%. All I0 adenocarcinomas were bcl-2 negative. Proliferative indices (Ki-67) showed a wide range (10-70%) in both bcl-2 positive and bcl-2 negative tumours. Clinical follow-up (mean = 459 days) was ax~ailable for 35 patients with SqCC: 3/18 living patients and 6/17 who died had bcl-2 positive tumours.

Our data suggests that in NSCLC bcl-2 immunoreactivity does not correlate with reduced apoptosis, or with a favourable outcome, and that there is no basis for its use as a "prognostic marker".

Supported by: I.A.C.R. (Studentship to AJO'N) & C.R.A.B.

Reference 1. N. Engl. J. Med. 1993; 329: 690-4.

DUCTAL CARCINOMA I N S I T U - AN INTERPHASE CYTOGENETIC STUDY

M. Harrison, H. Magee, J. O'Loughlin, D. N. Carney, R A. Dervan.

Departments of Pathology, University College Dublin and Mater Misericordiae Hospital, Dublin.

Invasive breast carcinomas frequently show karyotypic abnormalities, commonly involving chromosome 1. Cases of DCIS rarely have fresh tissue available for karyotyping, however an in s i tu hybridisation technique I applicable to interphase nuclei, allows numerical chromosomal abnormalit ies to be detected in paraffin sections.

The purpose of this study was to assess the frequency of chromosome 1 aneusomy in DCIS, in interphase nuclei and to compare the findings to nuclear grading.

230 Ir ish Assoc ia t ion f o r Cancer Research I.J.M.S. July, August, September, 1995

17 cases of pure DCIS and 6 predominant DCIS cases (less than a 5% invasive component) identified in the first round of a mammographic screening programme were studied. Non- isotopic in situ hybridisation, using a centromeric probe for Chromosome 1 was applied to these cases.

66% of DCIS showed aneusomy for chromosome 1. This was assoc ia ted with high nuclear grading. The invas ive component, where present, also showed aneusomy. In one ease the adjacent normal breast epithelium also showed aneusomy. Adjacent epithelium in all other cases was normal.

This study has confirmed the presence of chromosome 1 aneusomy in DCIS, in interphase nuclei. Nuclear grading is shown to be associated with the presence of aneusomy.

Further application of this technique may be helpful in identifying numerical abnormalities of other chromosomes and clarifying whether nuclear morphology reflects these cytogenetic abnormalities.

Reference 1. Hopman, A. H. N., Ramaekers, F. C. S., Vooije, G. F. Interphase cytogeneties of solid tumours. In: In situ hybridisation. Principles and Practice, edited by Folak, J. M. and McGee, J. O'D., Oxford, New York, Tokyo; Oxford University Press, 1990, p. 165.

FOLLOW-UP OF PREMENOPAUSAL WOMEN POST ADJUVANT THERAPY FOR BREAST CANCER - WHAT

ARE THE GOALS?

P. McEneaney, P. A. Daly. Department of Clinical Haematology and Ontology, St.

James's Hospital, Dublin 8.

It is standard practice to follow-up patients treated for breast cancer. The usual aims are the early detection of recurrence of breast cancer and other primary malignancies. Early diagnosis of local relapse may occasionally lead to curative treatment. In terms of psychological support, breast clinics are highly rated for providing reassurance and reducing anxiety. Follow-up visits in our unit consist of clinical evaluation, full blood.count, biochemical profile and annual chest radiographs. We studied the outcome of this evaluation in the detection of disease relapse and other abnormalities in 100 premenopausal patients following adjuvant therapy for early stage breast cancer. The duration of follow-up ranged from 1 to 148 months (mean 40 months). To December 1993, 30 patients had relapsed. Of these nine (30%) recurred locally, six (20%) metastasised to bone, five (17%) to liver, four (13%) had pulmonary involvement and three (30%) had intracranial metastases. Two patients (7%) had involvement of supraclavicular nodes and one patient (3%) has extensive chest wall relapse. All of these except three cases of liver and two of pulmonary metastases were suspected clinically. These five exceptional cases were investigated on the basis of abnormal liver function tests or chest radiographs. Surprisingly only two cases of skeletal metastases were associated with abnormal biochemistry - both of these patients had fractures. Other abnormalities detected included two cases of hypothyroidism and one of hyperthyroidism. There was also one case each of refractory anaemia with excess blasts, acute myeloid leukaemia, alcoholic liver disease and gallstones. Our study agrees with previous data which show that clinical evaluation leads to diagnosis of recurrence in over 80% of cases. Chest radiographs and laboratory investigations did not aid in the detection of curable re lapse . The main r o l e s for f o l l o w - u p in these

circumstances would appear to be psychological support, the detection of disease relapse requiring palliative care and the diagnosis of second malignancies and other medical conditions.

MANAGEMENT OF METASTATIC MALIGNANT TUMOURS WITHOUT HORMONAL MANIFESTATIONS

IN PATIENTS WITH MULTIPLE ENDOCRINE NEOPLASIA TYPE 1 (MEN-l) SYNDROME

P. Cremin, W. Orminston, P. Duly, D. S. O'Briain. Departments of Clinical Haematology and Ontology and

Histopathology, St. James's Hospital, Dublin 8.

In descriptions of patients with MEN-1, attention is primarily focused on the range of neoplasia within individuals and families and associated hormone production. Little attention is paid to the late metastatic effects of malignant tumours occurring in the context of MEN-I and the potential for palliation with combination chemotherapy. Three individuals from three different families with MEN-1 have been treated during the past 10 years for malignant tumours in whom widely metastatic disease without associated hormonal effects posed a clinical problem. Patient (A) had an atypical tumour metastatic to mediastinal and cervical glands with development of superior vena caval obstruction. The others (B & C) had pancreatic islet cell turnouts metastatic to liver, lymph nodes, bone and central nervous system (CNS). The clinical behaviour of disease in these two patients suggested progressive dedifferentiation of disease presumably due to accumulation of further genetic damage. All three patients were treated with combination chemotherapy and radiation therapy. Patients (A) and (B) are alive 5 years and 16 months from presentation respectively. Patient (C) lived for 4 years from diagnosis. Two patients (A & B) had radiation therapy. All three patients received chemotherapy, including streptozocin, 5FU, cyclophosphamide, epirubicin, VP-16, carboplatin, DTIC, doxorubicin, methotrexate, bleomycin, vincristine, cisplatin in various combinations. These patients presented with widely metastatic and advancing disease, yet successful palliation was achieved in all cases.

GM-GSF ENHANCES MONOCYTE MEDIATED CYTOTOXICITY THROUGH FREE RADICAL AND TNF-

INDEPENDENT MECHANISMS

J. McCarthy, E Redmond, S. Duggan, S. Rea, D. Bouchier-Hayes, J. O'Donnell .

Departments of Haematology and Surgery, Beaumont Hospital, Dublin 9.

The acute phase cytokines, IFN-)' and IL-6 have Mo-mediated antineoplastic effects that are limited by systemic toxicity. The haemopo ie t i c g rowth fac tor G M - C S F may also have antineoplastic effects mediated thorough Mo but mechanisms are unclear.

The aim of th is study was to compare the tumoricidal role of GM-GSF with IFN-~/ (gamma) and IL-6 against a Mo- sensitive tumor target and elucidate the cytotoxic mechanisms.

Human Mo were obtained from healthy donors, separated by density gradient, adhered, and studied as follows: cytotoxicity against the cell line WEHI 164 sub-clone 13 was assessed using a standard chromium release assay. TNFa, a cytotoxic and in f l ammatory med ia to r was measured us ing an ELISA

Vol. 164 231 No. 3

technique. Superoxide anion (02-), another cytotoxic and inflammatory product, was also measured spectrophotometrically.

Cytotoxicity TNFtx 02 - % WEHI killing pg/ml nmols/105Mo

Control 24-1-10 1.4:t :0.3 0.065+0.066 GM-CSF 405:8** 0.6+0.5* 0.022+0.02 IL6 395:15" 4.2+0.6** 0.069+0.044 IFNr 475:23* 2.7+1.3 0.225:0.018*

D a t a = m e a n + sem *p<0.05 **p<0.005

CM-CSF significantly enhanced Mo-mediated antitumour cytotoxicity, without a concomitant increase in the inflammatory mediators TNF or 02-, IFNy enhanced cytotoxicity predominantly through enhancement of the free radical 02-, while IL-6 increased cytoxicity through enhancement of the proinflammatory cytokine TNF. These results demonst ra te GM-CSF has potent Mo stimulatory properties for anti-tumor function without stimulating pro-inflammatory mediator release. Thus, GM-CSF may prove beneficial as an adjuvant to chemotherapy both in terms of haem0topoiesis-and as a non-toxic anti-neoplastic agent.

adenocarcinoma, including breast, ovarian, gastric and lung cancer. Previous methods to detect this protein have used immunohistochemistry, a procedure that is both subjective and difficult to quantitate. Here we describe the use of an ELISA (Ciba Corning Diagnostics Corp.) to measure c-erbB-2 protein levels in breast cancer extracts. Using the kit control, inter- assay CV was 7.1% (mean, 22 units/ml; range 19-24 units/ml; n=10). Using a cut-off point of 100 units/ml, quantitative concordance between the ELISA and immunohistochemistry was found in all 25 samples. In the breast cancers, c-erbB-2 levels correlated significantly with EGRF (r=0.30, p=0.0046, n=90), and urokinase plasminogen activator (r=0.221, p-0.0036, n=174). However , c - e rbB-2 leve ls , showed an inverse relationship with oestrogen receptors (r=0.211, p=0.014, n=137) and no significant correlation with either tumour size or nodal metastases. Patients with tumours containing high levels of c- erbB-2 oncoprotein had a significant shorter disease-free interval than pa t ien t s with low leve ls of the oncop ro t e in (chi square=7.86, p<0.01). We conclude that measurement of c-erbB- 2 oncoprotein by ELISA is simple, quantitative, reproducible and provides significant prognostic information in breast cancer.

EVALUATION OF A NEW ELISA KIT FOR ASSAY OF UROKINASE PLASMINOGEN ACTIVATOR IN BREAST

CANCER

C. Duggan, E. McDermott~ N. O'Higgins, J. J. Fennelly, J. Crown, M. J. Duffy.

Departments of Nuclear Medicine, Surgery and Oncology, St. Vincent's Hospital, Dublin 4.

Urokinase plasminogen activator (uPA) is a serine protease involved in cancer invasion and metastasis. High levels of this protease have been shown to correlate with poor prognosis in a number of mal ignancies , including breast , bladder , lung, colorectal and gastric cancers. The aim of this investigation was to evaluate a new ELISA kit (American Diagnostica Inc.) for quantitation of uPA. This ELISA detects precursors uPA, mature uPA and also uPA bound to its receptor and its inhibitors, PAI- l and PAI-2. Extraction of low speed (2000g) breast tumour cytosols with 1% Tri ton X-100 increased uPA levels by approximately 2-fold. Levels of uPA in malignant breast tumours were significantly higher than in benign samples (p-0.001). However, levels in primary carcinomas and metastatic cancers were not significantly different, uPA levels in primary cancers showed no significant correlation with tumour size, nodal status or oestrogen receptor status. However, uPA levels correlated significantly with its 2 main inhibitors PAl-1 (r=0.283, p=0.0001, n=291) and PAI-2 (r=0.293, p=0.0022, n=110). In addition,uPA correlated significantly with c-erbB-2 levels (r=0.221, p=0.0036, n=174) and cathepsin D levels (r=0.423, p=0.001, n=62).

We conclude that the new uPA ELISA kit is reliable and easy to use. It should permit the further evaluation of uPA as a prognostic marker in breast and other cancers.

ASSAY OF c-ErbB-20NCOPROTEIN IN BREAST CANCER BY ELISA

D. Bermingham, A. Nugent, E. McDermott, J. J. Fennelly, N. O'Higgins, J. Crown, M. J. Duffy.

Department of Nuclear Medicine, Surgery and Oncology, St. Vincent's Hospital, Dublin 4.

Increased levels of c-erbB-2 oncoprotein have been shown to be a p rognos t i c marker in, many d i f fe ren t types of

THE USE OF A NUCLEAR MARKER (Ki 67) AS A PROGNOSTIC INDICATOR IN PATIENTS WITH

PLASMA CELL DISORDERS

C. Fleming t, P. Crosby 1, S. Wolff 2, D. McCarthy ~. 1Department of Haematology, St. Vincent's Hospital, Dublin and 2Department of Haematology, Charing Cross Hospital,

London.

The proliferative activity of bone marrow plasma cells was evaluated by means of immunoenzymatic staining with the monoclonal antibody Ki-67 in 41 patients with monoclonal gammopathies. 18 patients had symptomatic multiple myeloma (MM) at time of testing. 18 were classified ias monoclonal gammopathy of uncertain s ignif icance (MGUS) and 5 as smouldering myeloma (SM). The percentage of bone marrow plasma cells in each of the patients' cytocentrifuge preparations was evaluated by two'staining methods: (a) a Wrights-Giemsa stain (Hematek) and (b) an immunoenzymatic stain with the monoclonal antibody CD 38, which is relatively specific for plasma cells. The percentage of plasma cells counted in the preparations stained by Wrights-Giemsa stain was compared with the percentage of CD 38 posi t ive cells using linear regression. These correlated closely (r=0.97 p <0.001). The Ki- 67 proliferation index was compared to the tritiated thymidine labelling index by means of a two dimensional dot plot and the results compared favourably. The Ki-67 proliferation index discriminated between MGUS and SM from MM. This is particularly useful clinically with regard to treatment.

AN INCREASED EXPRESSION OF p53 IMMUNOSTAINING IN SOFT TISSUE TUMOURS WITH

MICROWAVE ANTIGEN RETRIEVAL

C. Barry Walsh, E. W. Kay, M. Cassidy, S. Husain, M. Leader. Department of Pathology, Royal College of Surgeons in

Ireland, St. Stephen's Green, Dublin 2.

Current evidence suggests that malignant transformation is accompanied by several aberrations of oncogenes and tumour suppressor genes. P53 gene mutation and overexpression has emerged as one of the most promising candidates in this

232 Irish Association f o r Cancer Research I.J.M.S. July, August, September, 1995

multistep process of neoplasia. Whilst abnormalities of p53 expression are well described in a wide range of malignant epithelial and lymphoid tumours, there is a paucity of information regarding its role in the development of soft tissue turnouts.

The a ims of this s tudy were, f i rs t ly , to assess (by immunohistochemistry) the expression of p53 in diverse sarcoma groups and secondly to describe the effect, if any, of microwaving the tissue sections prior to staining. Formalin-fixed paraffin embedded sections of 53 sarcomas (16 MFH, 11 synovial sarcomas, 8 leiomyosarcomas, 8 liposarcomas, 6 fibrosarcomas and 4 neurofibrosarcomas) were immunostained and 15% showed nuclear staining with pos i t iv i ty detected predominantly in leiomyosarcomas and MFH. A smaller sample of 13 sarcomas were microwaved and results showed that 35% of cases were i m m u n o p o s i t i v e with p r e v i o u s l y nega t ive groups now demonstrating positivity. However, methodological difficulties were encountered with the use of microwaving. In summary, p53 overexpression is infrequent within most sarcoma groups but over expression is enhanced by the microwave technique.

THE FEASIBILITY AND VALUE OF PLOIDY IN CERVICAL SMEAR SAMPLES

C. Barry Walsh l, E. Kay ~, M. Thornhill l , D. Whelan ~, D. Barry 2, M. ~Turner 3, W. Prenderville 3, M. Leader ~.

ZDepartment of Pathology, Royal College of Surgeons in Ireland, Dublin 2, 2Department of Pathology, Letterkenny

General Hospital and 3Departmerit of Obstetrics and Gynaecology, The Coombe Hospital, Dublin 8.

In recent years results from DNA ploidy studies (DNA quantitation) performed on histologic samples using static and flow cytometry have shown that cervical intraepithelial neoplastic (CIN) lesions can be usefully subdivided into different groups on the basis of their DNA content. Despite these very promising results, the application of DNA quantitation to cervical smears as a means of fol lowing up CIN lesions has not achieved widespread clinical and laboratory use.

The aims of this study were to analyse and compare the DNA profile by image analysis of CIN 3 lesions on cervical smear and formalin fixed paraffin embedded tissue (FFPET) blocks and to evaluate the feasibility of applying this technique to smears as a routine in the laboratory. A total of 7 cervical smears and 17 cone b iopsy specimens were analysed. Al l 24 samples examined were non-diploid. Five (70%) smears were aneuploid and two (30%) tetraploid. Four (51%) biopsies were aneuploid and three (43%) tetraploid. The results of the smears and corresponding biopsies were concordant apart from one case. The remaining 10 cases of CIN 3 analysed on FFPET showed 5 aneuploid and 5 tetraploid lesions. We feel DNA quantitation is technically feasible in cervical smears and could be used to determine the DI and the presence or absence of aneuploidy in CIN 3 lesions.

AN ILLUSTRATION OF A PLOIDY FIELD CHANGE IN THE UTERINE CERVIX IN THE CASES OF CIN.

E. W. Kay ~, C. Barry Walsh ~, F. Murphy l, M. Turner 2, W. Prendiville 2, M. Leader ~.

~Department of Pathology, Royal College of Surgeons in Ireland, Dublin 2 and 2Department of Obstetrics and

Gynaecology, The Coombe Hospital, Dublin 8.

In the uterine cervix the behaviour of high grade lesions (CIN

2, CIN 3) is uncertain despite the finding of aneuploidy in the majority of these lesions. Recent studies in bladder carcinoma have suggested that the ploidy profile is abnormal in apparently normal mucosa adjacent to tumour and that this alteration offers reliable prognostic information for patients with high grade tumours. The aims of this study were to estimate the ploidy (by IA) of CIN 2/3 lesions and of apparently normal cervical squamous mucosa in cone biopsy samples in an attempt to eva lua te the p resence or absence of a n e u p l o i d y in morphologically normal mucosa and to compare the ploidy profile of normal mucosa with the adjacent lesion. 24 cases were studied, (CAS 200 IA). 67% (n=l 6) of the CIN 2/3 lesions were non-diploid. 83 % (n=20)) of normal mucosa was aneuploid. In the remaining 4 cases the normal mucosa was aneuploid despite a diploid turnout profile. We confirm that the majority of CIN 2/3 lesions are non-diploid. Furthermore, 84% of the morphologically normal mucosa demonstrated aneuploidy. This finding has not previously been recorded and its effect, if any, on the behavi0ur of the neighbouring CIN lesion requires further study.

SQUAMOUS CELL CARCINOMA IN RENAL ALLOGRAFT RECIPIENTS: A STUDY OF PATHOGENIC

FACTORS

G. Gibson, T. O'Grady, M. Carmody, J. Donohoe, J. Walshe, M. Leader, G. M. Murphy.

Beaumont Hospital, Dublin.

Squamous cell carcinoma (SCC) of the skin is the commonest malignancy occurring in renal al lograft recipients (RAR). Aetiological factors may include ultraviolet radiation (UVR), immunosuppression, tumour suppressor gene mutation and human papilloma viruses (HPV).

25 RAR with 112 squamou s cell carcinomas (SCC), 16 intraepidermal carcinomas (IEC)and 8 dysplastic lesions were s tudied for c l in i ca l , i m m u n o l o g i c a l and h i s t o l o g i c a l characteristics. All tumours occurred on sun exposed sites and were related to the degree and duration of immunosuppression. Tumours were commonly associated with outdoor occupation and viral warts. Histologically, 86% of tumours were well differentiated, 14% moderately differentiated and no metastic tumours occurred. A l l tumours and dysplastic lesions were stained with monoclonal antibodies (Mab) to p53" protein (DO 1, DO 7). Frozen sections from 8 tumours, 8 dysplastic lesions and 7 normal control skin samples were stained with a Mab (Leu 6) to the C D l a epi tope on Langerhans cel ls (LC). Epidermal LC numbers were counted per 100 basal cells and adjusted for a unit depth of 40urn. Peripheral blood lymphocyte counts and T cell subsets were studied in 9 patients. NK cells were counted using a Coulter clone Mab which recognises the CD56 ant igen. 52% of SCC expres sed p53 main ly in proliferating cells. 56% of SCC showed p53 expression in basal cells of adjacent uninvolved epidermis, p53 was exPressed in tumour or adjacent skin in 73%. 69% of IEC and 67% of dysplastic lesions expressed p53.14 lesions were DO7 positive and DO1 negative but all of those which were DO1 positive were also DO7 positive with similar staining pattern. All p53 posi t ive lesions s ta ined s t rongly with a Mab (PC10) to proliferating cell nuclear antigen (PCNA). Mean LC counts per 100 basal cells per 40um depth of epidermis were as follows: normal skin: 17.8 (R:15.4-19.4), tumours: 9.4 (R:4.8-15.6), dysplastic lesions: 2.8 (R:1.0-5.6). Of 9 patients who had peripheral lymphocyte studies, 7 had lymphopenia, 8 had

Vol. 164 233 No. 3

reduced B cells and 5 a reduced CD4 count. There was no correlation between NK cell numbers and tumour propensity. Reduced systemic and cutaneous immunosurveillance in RAR in conjunction with the ability of UVR to diminish LC numbers and induce mutant p53 expression in skin may be important factors in the pathogenesis of recurrent SCC.

c -ERBB-20NCOPROTEIN EXPRESSION IN NON- INVASIVE CARCINOMA OF BREAST

N. Nolan l, J. R. Reynolds 2, E. W. McDermott 2, R. Rafferty l, A. McCann 4, E Sweeney 2, D. Carney 4, N. J. O'Higgins 2,

M. J. Duffy 3. Departments of Pathology 1, Surgery 2 and Nuclear Medicine 3, St. Vincent's Hospital, Elm Park and Department of Medical

Oncology 4, Mater Hospital, Dublin.

The c-erbB-2 oncogene encodes a protein which is a putative growth factor receptor. While over-expression of c-erbB-2 oncoprotein in invasive breast cancer is usually an indicator of poor outcome, the biological significance of c-erbB-2 in noninvasive breast carcinoma has yet to be determined.

The purpose of this study was to address the relationship be tween c -e rbB-2 oncopro te in express ion and c l in ico- pathological parameters in non-invasive breast cancer.

Sixty-nine patients with in situ breast carcinoma were studied. Eleven patients had lobular carcinoma in situ (LCIS) while 58 had ductal carcinoma in situ. Of the 58 patients with DCIS, 36 had presented clinically while 22 had mammographically detected disease only. Formalin fixed and paraffin embedded sections were studied after immunohistochemical staining with antiserum to c- erbB-2 oncoprotein.

c-erbB-2 oncoprotein was not detected in any of the patients with LCIS. In patients with DCIS, c-erbB-2 oncoprotein was expressed in 23 cases (40%) and was significantly associated with tumour necrosis (p<0.001) and the comedo sub-type (p<0.02). Elevated levels of c-erbB-2 oncoprotein were found in 22/36 (61%) of patients presenting with clinical disease but in only 1/22 (4.5%) patients presenting with mammographically only detectable disease (p<0.01).

This study supports, the hypothesis that clinically detected and mammographically detected DCIS are biologically different diseases. The study also demonstrates that the molecular pathogenesis of DCIS differs from that of LCIS.

M.C.W.E. REVERSES IMPAIRED CELL-MEDIATED IMMUNITY IN BREAST CANCER

P. Kent, J. O'Donoghue, C. Curran, D. M. O'Hanlon, K. Kerin, D. Maher, H. F. Given.

National Breast Cancer Research Institute and Department of Surgery, University College, Galway.

Variability in the natural history of untreated breast cancer may be explained partially by altered immune response. The aim of this study was to assess cell-mediated immunity in a population of breast cancer patients and to assess the effect of a novel immunostimulant (MCWE - a purified cell wall extract from Myocabacterium phlei) on immune function. Lymphocytes, isolated from peripheral blood of 29 patients presenting with breast cancer and from 10 normal females were divided into two groups, controls and cells exposed to MCWE at a dose of

5ug/ml. Both groups of cells were pulsed with tritiated thymidine and the blastogenic response to the mitogen- phytohaemaglutinin (PHA) was measured using a scintillation counter. 6/29 (21%) of breast cancer patients had significantly reduced blastogenic response to PHA compared to normal subjects. This was not correlated to the stage of the disease. Exposure to MCWE significantly increased the blastogenic response in ceils from the immunosuppressed group from a mean value of 15,803 cpm, SEM (2784) for control cells to a mean value of 41,266 cpm, SEM (3675) for MCWE exposed cells, (p <0.01, students t test). The results of this study suggest that a subpopulation of patients with breast cancer have immunosuppression unrelated to tumour burden. The use of the novel immunos t imulan t MCWE stimulates the blastogenic response to PHA and may be of therapeutic benefit.

THE ROLE OF PROTEIN KINASE C IN THE REGULATION OF EXPRESSION OF CD44 SPLICE VARIANTS

A. Long, S. Ahern, D. Kelleher. Department of Clinical Medicine, Trinity College Medical

School, St. James's Hospital, Dublin 8.

CD44 is an adhesion molecule widely expressed on cells of the haematopoietic system. Multiple forms of the protein are generated by alternative splicing of the CD44 gene. Variants of CD44 have been shown to confer metastatic potential on tumour cells. Protein Kinase C (PKC) comprises a family of serine/threonine specific kinases with differing enzymatic properties and a complexity of substrates and functions. We have generated a cell line from the T cell lymphoma HuT 78 designated K-4. HuT 78 expresses PKC ~, B, e and ~ while K- 4 expresses PKC ~, e and ~ but not B. K4 cells express a high molecular weight form of CD44 (approx 130kD) in addition to the 85-90 kD form, the sole isoform expressed oa HuT 78. PKC activity was inhibited in HuT 78 cells by incubating with either, staurosporine or Ro 8220 (both potent PKC inhibitors) for 24, 48 and 72 hr. Cell extracts were analysed for CD44 expression by Western blot analysis. At 24 hours a high molecular weight form of CD44 (approx. 130kD) was detected in HuT 78 which was absent at 48 and 72 hr. Further Western blot analysis with anti-v6 specific antibody showed the v6-containing species in both cell lines to be 180-200kD. Flow cytometric analysis revealed that the expression of this exon is up-regulated on both HuT 78 and K-4 by activating the cells with the tumour promoter phorbol myrisate acetate (this directly activates PKC). Inhibition of PKC activity in HuT 78 cells by staurosporine does not result in the increased expression of v6 on these cells. Thus, expression of a 130 kD variant is negatively regulated by PKC. However, expression of v6, the exon implicated in metastasis in non- Hodgkin's lymphoma, is up-regulated by activation of PKC.

PATTERNS OF MIXED CHIMAERISM PREDICT OUTCOME AFTER UNRELATED DONOR BMT.

K. Molloy ~, N. Goulden 2, M. Lawler 1,3, P. Humphries 3, D. H. Pamphilon 4, S. R. McCannL

Departments of Haematology ~ and Genetics 3, Trinity College Dublin, Bristol Royal Hospital 2, Southmeade Hospital 4,

Bristol, England.

Although the majority of children with acute lymphoblastic


leukaemia (ALL) are cured fol lowing chemotherapy, the prognosis for patients in second or subsequent remissions (CR) remains poor. Allogeneic BMT is associated with a 40% disease free survival but is often limited due to lack of a HLA identical sibling donor. As HLA disparity significantly increases the risk of graft versus host disease (GVHD) and graft rejection, transplant centres have explored the use of matched unrelated volunteer donors (UVDs). This study was undertaken to investigate haemopoietic chimaerism following UVD BMT for ALL in 2nd or subsequent CR. Clinical outcome was unknown at t ime of study. Chimaer ism post BMT was assessed by amplification of polymorphic short tandem repeats using the po lymerase chain reac t ion (PCR). To date ch imaer i sm/ engraftment has been evaluated in 17 patients from 1-18 months post BMT. Five patients exhibited donor chimaerism at some point during the serial study. In two of these patients, donor chimaerism persisted at all timepoints post BMT and these patients remained in remission. In 3 patients donor chimaerism evolved to mixed chimaerism (MC) and was predictive of subsequent relapse. Five patients exhibited stable low level MC (level of recipient cells <1%) and all remain in remission. 7 patients showed initially high or progressive levels of recipient cells post BMT. 4/7 relapsed in the first 6 months post BMT while two patients rejected their grafts with high levels of recipient ceils and were given an autograft. Subsequent PCR revealed persistence of recipient cells. One patient showed an increas ing level of recipient cel ls by PCR analysis with subsequent emergence of a new clone of ceils without evidence of leukaemic blast cells and clearly merits close monitoring. I / 17 patients had severe GvHD and was shown to be a donor chimaera by PCR. In summary, a high incidence of mixed chimaerism was seen in this patient cohort. High levels of recipient ceils or a switch from donor to mixed chimaerism between 1 and 3 months or 3 and 6 months post BMT in patients in clinical remission were predictive of subsequent leukaemic relapse. Patients with donor chimaerisin or low level stable MC were long term survivors. Thus, chimaerism results were highly predictive of outcome in this patient group.

THE POTENTIAL OF VITAMIN A DERIVATIVES AS ANTI CANCER AGENTS

D. M. O'Hanlon, C. Curran, M. Kerin, P. Kent, D. Maher, H. F. Given.

National Breast Cancer Research Institute and Department. of Surgery, University College Hospital, Galway.

The retinoids, a class of natural and synthetic compounds structurally related to vitamin A, play an important role in cellular growth and differentiation. Three naturally occurring retinoids, retinol, alltrans retinoic acid and 13-cis retinoic acid and the synthetic compound fenretinide are potential modulators of epithelial cell proliferation. The aim of this present study was to assess their effect on growth of oestrogen receptor positive cell lines MCF-7 and ZR-75-1 in vitro. Growth curves were plotted for different concentrations of 4 different retinoids and compared to growth in the control cells. Dose response curves were plotted from this data and the concentration of retinoids at which 50% of growth inhibition occurred was calculated (Table).

Dose of different retinoids at which 50% growth inhibit ion occurred (in both MCF-7 and ZR-75-1 cell lines on day 6 and day 10 of growth curve) (Doses of retinoids in [dg/mi)

Retinoid MCF-7 MCF-7 ZR-75-1 ZR-75-1 Day 6 Day 10 Day 6 Day 10

Retinol 0.20 0.10 0.23 0.10 All trans RA 0.12 0.09 0.73 0.63 13-cis RA 0.14 0.10 0.52 0.61 Fenretinide 1.91 0.71 0.73 0.59

This study demonstrates that retinoids inhibit tumour growth at naturally occurring levels. The anticancer potential of different ret inoids on different cell lines varies and the synthet ic fenretinide is less effective than naturally occurring compounds with 50% growth inhibition requiring higher doses. This effect of retinoids has major implications for their use in an adjuvant setting.

SPONTANEOUS SECRETION OF TUMOR NECROSIS FACTOR BY A HUMAN T-CELL LYMPHOMA LINE

M. O'Connell, A. Long, L. A. J. O'NeilP, D. Kelleher. Department of Clinical Medicine and ~Department. of

Biochemistry, Trinity College, Dublin 2.

Tumor necrosis factor (TNF) is a potent cytokine produced by activated lymphocytes and other inflammatory cells. TNF has both cytotoxic and proliferative properties, which may be partially mediated by individual receptors. Protein kinase C (PKC) consists of a family of ten isoforms, which differ in their activation requirements. HUT78 is a T-cell lymphoma line which is resistant to the cytotoxic effects of TNF. The aim of this study was to determine if PKC was involved in TNF secretion by lymphocyes, using HUT78 and a PKC B-deficient clone derived from it, K-4. Proliferation of HuT and K-4 was assessed by the 3H-thymidine incorporat ion assay. TNF secret ion, ei ther unstimulated or in response to activating agents (10mg/ml PMA and/or 1/50 OKT3), was measured by both L929 bioassay and ELISA. TNF was present in the supernatants of resting HUT78, measured by both assays (L929 assay: HUT78, 136.6+64.3pg/ ml, x+SEM, mean of 3 expts). TNF levels were also present but at significantly increased levels in K-4 supernatants (L929 assay: K-4, 425. I+122. lpg/ml). Although stimulation with PMA and OKT3 led to increased TNF production by HUT78, there was no difference in production of TNF between resting and stimulated K-4. By contrast, interleukin 2 was not constitutively secreted in HUT78 or K-4 cells, although production could be stimulated by PMA and OKT3 in Hut78 cells, but not K-4. These data demonstrate constitutive secretion of high levels of TNFa in a T-cell lymphoma line resistant to cytotoxic effects of TNF. The mechanisms of autoprotection of HUT78 cells may provide important information regarding mechanisms of TNF-mediated cytotoxicity.

REGULATION OF CELLULAR AND VIRAL GENE EXPRESSION BY EPSTEIN-BARR VIRUS.

C. Power ~, A. Leroux 2, M.Perricaudet 2, D. Wails 1. 1The School of Biological Sciences, Dublin City University,

Dublin 9 and 2Institut Gustave Roussy, France.

The Epstein-Barr virus (EBV) is an oncogenic human herpesvirus that is linked with several cancers namely: African endemic Burkitt 's lymphoma and S.E. Asian Nasopharyngeal

Vol. 164 2 3 5 No. 3

Carcinoma. EBV is also associated with B-cell lymphomas in AIDS patients and patients undergoing immunosuppressive therapy. The ability of this virus to immortalise infected B- cel ls is an e x c e l l e n t mode l for s tudy ing ce l l growth- transformation and cancer. In this regard, it is a limited set of EBV latent proteins that are essential for B-cell transformation and immortalisation to occur. Our objective is to investigate the roles of several key EBV latent prot.eins (Epstein-Barr virus nuclear antigen 2(EBNA-2), EBNA-3A, EBNA-3B and EBNA- 3C) in the regulation of both cellular and viral gene expression. Our approach has been to position cellular/viral promoter sequences upstream of the chloramphenical acetyltransferase (CAT) or placental alkaline phosphatase (PAP) reporter genes. Promoter activities were then studied after electroporation of B-lymphocytes by assaying for reporter enzyme activity in cell extracts. The effect (if any) of each EBNA on promoter activity was assessed by co-transfect ion with plasmids capable of expressing individual EBNAs. The following is a summary of the results obtained: (a) EBNA-2 can stimulate the activities of several EBV and at least one cellular gene promoter (CD23 or the FcERII/IgE receptor); (b) in the case of one promoter we have mapped the sequence responsible for mediating EBNA- 2 trans-activation (EBNA-2 responsive element of E2RE) to an 81 base pair DNA fragment; (c) all EBNA-2 responsive promoters possess upstream DNA sequences which share significant homologies with the mapped E2RE; (d) EBNA-3A and EBNA-3C can individu~'lly repress the EBNA-2 mediated trans-activation of (at least) one such promoter. The functional antagonism between these latent EBV proteins has not been previously reported and is likely to be important in the overall viral strategy. The CD23 molecule has itself been shown to be involved in a pathway of autocrine growth stimulation and so it seems likely that EBNA-2 can induce B-cells to accumulate an IL-4 responsive surface antigen that is involved in cellular proliferation.

Supported by the Health Research Board (HRB).

THE MOLECULAR AND BIOCHEMICAL CHARACTERISATION OF THYMIDINE KINASE FROM

MAMMARY CARCINOMA

F. Britton, L. Brennan, Y. A. Barnett. Cancer and Ageing Research Group, Department of

Biological and Biomedical Sciences, University of Ulster, Coleraine.

A precondition of the chemotherapeutic treatment of human cancer is a highly selective interaction of a drug molecule with its biological target. One of these biological targets may be thymidine kinase 1 (TK1). In this investigation TK1 from established mammary carcinoma cell lines and carcinoma tissue will be characterised at the molecular and biochemical levels. DNA and RNA will be simultaneously extracted from carcinoma cells or tissues. Based on the sequence information of TK1, a PCR s t ra tegy has been deve loped which fac i l i t a tes the amplification of the translated and 3' untranslated ends of TK1. The RNA extracted from the carcinoma cells will be subjected to this PCR approach, which additionally allows the integration of the TK1 translated and untranslated regions into protein expression vectors. Sequence analysis o f the amplified regions will be performed to facilitate the detection of mutations in any biochemically altered TKls. An analysis of such sequence

data may shed light upon the molecular mechanisms leading to altered expression vectors. The resultant proteins will be purified. Biochemical analysis (to include kinetics and mass spectroscopy) will be performed on the purified TKls. Ultimately the biochemical characterisation of the TKls will allow the computer-aided design, based on a model for TK1, of specific inhibitory nucleoside analogues to the carcinoma-derived TKs, with obvious clinical relevance. The details of this investigation will be presented.

THE USE OF TWO DIMENSIONAL GEL ELECTROPHORESIS TO STUDY THE EFFECTS OF

CISPLATIN ON PROTEIN EXPRESSION LEVELS

B. Madden, L. P. G. Wakelin. St. Luke's Institute of Cancer Research, Department of

Pharmacology, U.C.D. Dublin 4.

Two-dimensional gel electrophoresis (2D-GE) allows the resolution of a complex mixture of proteins into a highly reproducible 2D map. The power of this technique lies in the fact that between 1,500 and 2,000 discrete protein spots can be resolved in a highly reproducible manner on a single map. Cisplatin, which is used in the clinic against testicular, ovarian and some head and neck cancers, is an example of an anti-turnout agent, whose principal biological target is DNA. Cisplatin preferentially attacks the N7 position of guanine and has the ability to form both inter- and intra-strand DNA crosslinks. The aim of this study is to look at the downstream effects of such DNA adduct formation, i.e. the ability of crosslinked DNA to act as a template for transcription and the ultimate effects on translation efficiency and hence protein expression levels within the cell. Two cell lines were used in this study, namely, K562, a human chronic myelogenous leukaemia, and HT29/219, a human colon carcinoma cell line. The cells were seeded in 24- plates and cisplatin was added during exponenti~al growth. Cells were counted using a Coulter Counter (ZM model) and the ICso values (cisplatin concentration at which drug-treated cell numbers were equivalent to 50% of the control cell values) obtained. These IC50 values were subsequently used to drug- treat cells during metabolic labelling with [35S]-methionine. Cel ls were labe l led in the p resence /absence of drug in methionine-depleted medium over a period of 16hr. Protein extracts were made and the incorporat ion level of [35S]- methionine into cellular protein was determined by carrying out a TCA-precipitation assay. These extracts were subsequently fractionated by 2D-GE and the protein maps obtained in both the presence/absence of cisplatin were compared. These studies demonstrate the usefulness of 2D-GE for analysing the effects of drug treatment on global protein expression levels within cells.

TARGETED DELIVERY OF BIOLOGICALLY ACTIVE REAGENTS TO CELLS BY LIPOSOMES

H. C. Loughrey and P. Corley. Department of Biochemistry', University College Galway.

L iposomes offer the a b i l i t y to protect and t ranspor t biologically active agents to destined target cells once made target specific by the attachment of appropriate proteins to the vesicle surface. To this end we have investigated the use of a number of cell surface molecules expressed on CEM cells (T-

236 Irish Association fo r Cancer Research I.J.M.S. July, August, September, 1995

lymphoblastic cell-line; multi-drug resistant phenotypes and the parent cell-population) and K562 cells (chronic myelocytic leukemic cell-line) as a means of delivering vesicle encapsulated contents to target cells. Cytoplasmic delivery is achieved by an endocytic mechanism. This was shown by (i) the delivery of the cy to tox ic drug metho t rexa te whose target is the cytoplasmic enzyme dihydrofolate reductase (as assayed by the MTT assay) and (ii) presence of intracellular fluorescence due to' the liposome encapsulated fluorescent molecule carboxy fluorescein (as viewed by confocal microscopy techniques). Presently the use of liposome encapsulated 8-hydroxy-l,3,6- pyrenetrisulfonate (HPTS), a pH sensitive fluorescent dye is under investigation as an approach to aid in optimising rapid cytoplasmic delivery of liposome contents from vesicles once exposed to the acidic environment in cells. This is important as optimal cellular delivery of vesicle entrapped biologically active agents such as cytokines, anti-sense reagents and DNA to cells by an endocytic pathway will depend on the ability of vesicles to deliver their load to the cytoplasm prior to exposure to degradative nucleases or the acidic environment encountered in the lysosome organelle in cells.

RESIDENT MACROPHAGES DO NOT REQUIRE DUAL SIGNAL STIMULATION FOR TUMOUR CELL LYSIS

S. Duggan, H. P. Redmond, J. McCarthy, R. W. G. Watson, D. Bouchier-Hayes.

Department of Surgery, Beaumont Hospital, Dublin 9.

Inflammatory MO require a dual signal stimulus to induce tumour cell killing. However, observations in this laboratory suggested that a single signal would suffice for activation of res ident MOs. The aim of this s tudy was to assess the requirement of single vs. dual signal stimulation for the induction of resident and inflammatory murine peritoneal MO cytotoxicity. Resident MOs (RMO) were harvested from control CD-I mice and inf lammatory MOs (IMO) were harvested 72 hr post injection of protease peptone, Cell populations were stimulated in vitro for 24 hr with LPS ( lug/ml) +IFN-r (gamma) (103U/ ml). Cytotoxicity was assessed using a Cr sl release assay~ TNF, 02- and cytosolic arginase activity were also assessed as markers of cytotoxic activity. Maturation/activation was assessed by CD1 lb cell surface expression.

Cytotoxieity 02- TNF Arginase % nmol/mg BSA pg/mg BSA mmol/mg BSA/hr

RMO IMO RMO IMO RMO IMO RMO IMO Control 27+I 334-1 0.0194.0 0.021-+0 4_+1 74-2 304.9 274-8 IFN-r 854-10' 414-2 0.034i'0" 0.022-+0 294-4* 10~2 83.6_+6* 43_+7 LPS 61_+9" 374-3 0.031:1:0" 0.021-+0 234-3* 84-3 614-11' 33_+6 LPS+IFN-r 714-5" 74+4* 0.028+0* 0.033-+0* 234-5* 30"J:5' 73_+6* 814-3"

Data --- Mean+SD Significance set at *P<0.05

RMOs stimulated with either IFN-r or LPS as single signals had enhanced cytotoxicity compared with same group controls and IMOs (p<O.05). These results correlated closely with TNF, 02- and arginase values obtained. Cell maturation/activation, as assessed by CD 1 lb expression, was 276_+6 MCF=RMO vs 65.5_+MCF=IMO. These results indicate that a single stimulatory signal is sufficient to enhance cytotoxicity by RMO, whereas a dual signal is a prerequisite for upregulation of tumouricidal ac t iv i tyby IMO. MO=macrophage, LPS=lipopolysaccharide, IFN-r=interferon-gamma, TNF=tumour necrosis factor, 02=superoxide anion, MCF=mean channel fluorescence.

THE INCREASING INCIDENCE OF PRIMARY GASTRIC LYMPHOMA: IS THERE A ROLE FOR HELICOBACTER

PYLORI?

I. Keogh, M. Kerin, D. O'Hanlon, S. Walsh ~, P. Kent, J. Callaghan ~, H. E Given.

Departments of Surgery and Pathology ~, University College Hospital, Galway.

Primary gastric lymphoma accounts for between 2 and 5% of gastric mal ignancies and differs grea t ly from gastr ic adenocarcinoma in terms of prognosis and response to radio- chemotherapy. The rarity of this tumour means that large series are unavailable for analysis outside of regional tumour registries. The aim of this study was to evaluate our experience with PGL in the light of recent suggestions that it is related to infection with helicobacter pylori (HP). Since 1974, twenty-seven newly diagnosed cases of PGL have been treated, accounting for 11% of all gastric neoplasms. Sixteen patients were male and the mean age was 64 (range 33-90 years). The incidence of primary gastric lymphoma as a proportion of the total number of gastric neoplasms increased from 4 out of 70 cases (5.7%) in the six year period 1976-1981 to 10 out of 76 o r 13.1% of cases in the six year period extending from 1988 to 1993 in which PGL accounted for 13 out of 96 or 13.5% of all gastric neoplasms. All blocks were restained for HP using haematoxylin and eosin and giemsa. The incidence of HP in the lymphoma group is similar to patients with gastric carcinoma and peptic ulceration. All 12 long term survivors have had surgical resection (mean follow up 50 months, range 12-152 months). While noting the surprisingly elevated incidence of PGL in our population, we are unable to attribute this increase to infection with HP.

IN VITRO CHARACTERISATION OF EARLY PASSAGE UVEAL MALIGNANT MELANOMA

M. McNamara t, S. M. Kennedy 1, A. Benedict-Smith 1, C. Barnes a, D. Neylon 2, M. Fenton ~.

~Resear.ch Department, Royal Victoria Eye and Ear Hospital, Dublin 2 and ~Dublin Institute of Technology, Dublin 8.

Object: Characterisation of early passage (2-5 passages) cell lines derived from uveal melanoma.

Pelleted cells from cell lines were embedded in agar and processed through paraff in , sub jec ted to cyt 'ologic and immunohistochemical analysis and compared to the primary nucleation specimen. Fixed cells were examined by electron microscopy.

All three cell lines were derived from primary turnouts with predominantly spindle B morphology. In culture, the cells transformed to predominantly epithelioid cell type. Cells had abundant eosinophilic cytoplasm, nuclear pseudoinclusions and mul t ip le nuc leo l i . Mi to t i c f igures were rare. Immunohistochemical analysis revealed diffuse positivity for S100 protein, HMB 45 and vimentin. Cells did not express cy tokera t in or actin. Ul t ras t ructura l ana lys i s conf i rmed melanocytic features. In culture, the cells had a chaotic growth pattern with focal development.

Conclusions: Early passage cultured uveal melanoma retains the cytologic and immunohistochemical characteristics of the primary tumour. Study of agar blocks of cultured cells provides a quick and easy method of c o n f i r m i n g ce l l type and immunophenotypic

voLt~ 237 No. 3

DESIGN AND SYNTHESIS OF DNA MINOR GROOVE BINDERS AS POTENTIAL ANTITUMOUR AGENTS

M. Searcey, C. M. Topham, L. G. Wakelin. Chemistry Group, St. Luke's Institute of Cancer Research,

Chemistry Department, University College, Belfield, Dublin 4.

Most antitumour agents currently in use in the clinic have DNA as their primary target. Compounds which bind in the minor grove of DNA, including the antibiotics distamycin A and netropsin and the synthetic dye Hoechst 33258, have potential as therapeutic agents, however those screened to date have not reached the clinic. Although these compounds tend to bind specifically to AT sequences, two different approaches have recen t ly demons t r a t ed that this code can be extended. Lexitropsins are analogues of the AT specific compounds in which the introduction of potential hydrogen bonding groups in the heterocyc!ic moieties (i.e. replacement of pyrrole with imidazole) is e.xpected to increase the acceptance of GC base pairs. Recent observations have shown that minor groove binders may bind both in AT and in the wider GC continuing sequences as dimers, each monomer perhaps forming hydrogen bonding interactions with the bases on one strand of DNA. We ,have designed and syn thes i seda potential minor groove binding co. mpound based upon the polyamide structure of the naturally occurring minor groove binders netropsin and distamycin, in which we have attempted to'retain the hydrophobic, hydrogen bonding and electrostatic interactions of these compounds. Due to the relatively simple structure, it may be possible to form dimers, as well as introduce different hydrogen bonding and hydrophob ic in t e rac t ions via s t r a igh t fo rward synthe t ic manipulation. Synthetic routes, molecular modelling and initial DNA binding studies will be presented.

AUTHENTIC PNA: PEPTIDE-BASED ANALOGUES OF DNA

N. M. Howarth, M. Searcey, C. M. Topham, L. P. G. Wakelin.

St. Luke's Institute for Cancer Research, Department of Chemistry, University College, Dublin.

Reagents that bind specifically to complementary nucleic acids are of major interest. Considerable attention has been focused on developing oligonucleotide surrogates that maintain Watson-Crick or Hoogsteen base pairing to RNA or DNA targets but which do not incorporate the usual phosphodiester linkages, due to their susceptibility to nucleases and incompatibility with passive membrane transport. One recent advance in this area is the polyamide nucleic acids (PNA) developed by Nielsen.

Our approach to this quest involves the use of a peptide backbone to connect the base-containing subunits. The main advantages of using a peptide backbone are that these analogues will be resistant to nucleases, can be made resistant to peptidases and as the backbone charge will be controllable, they may be made to exhibit improved transport properties. We have designed a peptide nucleic acid in which the amino acid moiety is derived from homoserine (the DNA base replacing the original hydroxyl group). These base amino acids are interspersed with glycine and the bases are connected to one another via a six atom linker as in DNA. A model of a 12-mer PNA:DNA duplex has been built using a combined energy minimisation and restrained molecular dynamics (MD) protocol. Unrestrained MD studies are in progress and the synthesis of these compounds is well advanced. Progress in synthesis and computation studies will be presented.

ESTRONE SULFAMATES: POTENT ESTRONE SULFATASE INHIBITORS AND POTENTIAL

THERAPEUTIC AGENTS IN BREAST CANCER

N. M. Howarth 1, A. Purohit 2, M. J. Reed 2, B. V. L. Potter ~. 1School of Pharmacy and Pharmacology, University of Bath,

England and 2Unit of Metabolic Medicine, St. Mary's Hospital, Medical Schg,ol, Imperial College of Scien6e,

Technology and Medicine, London.

Breast cancer is the most prevalent type of Cancer in Western countries. Approximately one-third of breast cancer tumors are hormone-dependent. There is considerable evidence to suggest that estrogens play a central role in supporting the growth of these tumors and that the hydrolysis of estrone sulfate to estrone by estrone sulfatase is an important source of these estrogens. Thus, the development of an efficient inhibitor of this enzyme would allow the importance of the estrone sulfatase pathway to be assessed. Such an inhibitor may ultimately be of therapeutic utility. We have designed, synthesised and evaluated a number of surrogates of estrone 3-O-sulfate to inhibit this enzyme. Among these, estrone sulfamates are emerging as important lead compounds in this area. Estrone 3 -0 sulfamate was found to have an IC50 value of 80 nM as an irreversible inhibitor of estrone sulfatase in placental microsomes. The synthesis and biological properties of these sulfamates will be discussed.

QUANTIFICATION OF THYMIDINE KINASE 1 GENE EXPRESSION BY IN-SITU HYBRIDIZATION IN A HUMAN BREAST CANCER CELL LINE (MCF-7)

J. M. O'Connor, V. J. McKelvey-Martin, P. G. McKenna. Department of Biological and Biomedical Sciences,

University of Ulster, Cromore Road, Coleraine BT52 ISA.

Thymidine kinase (TK) exists as two isozymes. TK2, of mitochondrial origin, has relatively constant levels throughout the cell cycle. The activity of TKI, the cytosolic isozyme, is regulated in a cell cycle dependent manner - being lowest in GI phase ceils and elevated during DNA synthesis. Significant elevation of TK activity has been reported in breast carcinoma relative to fibroadenoma and normal breast tissue; and this activity correlates positively with cancer recurrence. Numerous mechanisms at transcriptional and translational levels have been implicated in the control of TK activity. This communication presents a protocol for the detection of TKI mRNA in human breast cancer cells (MCF-7) by in situ hybridization fISH) using an anti-sense TKI mRNA probe. In order to investigate the method ISH was performed on MCF-7 cells grown in medium supplemented with Tamoxifen or Estradiol 17B which have been shown by Northern hybr idizat ion to cause decreases and increases respectively in mRNA levels relative to untreated control cells. Quantification of TKI mRNA hybridization was evaluated using an image analysis system and expected changes in mRNA levels were observed. Further studies will evaluate the use of TKI ISH data in diagnosis and in determining prognosis in breast cancer.


AN EVALUATION OF THE CONTENT OF MACROPHAGES IN SOLID TUMOURS OF THE HUMAN

COLON

W. J. Hatton, G. McKerr, D. Harvey 1, J. Carson ~, B. M. Hannigan.

Department of Biological and Biomedical Sciences, University of Ulster Coleraine, Northern Ireland and

1Histopathology Department, Northern Area Laboratory, Waveney Hospital, Ballymena, Northern Ireland.

The role of interactions between macrophages and tumour cells in determining the host tumour relationship is unclear. This study is providing, for the first time, a large body of unbiased data on macrophage numbers and their spat ia l distribution within excised human malignant colon tumours. Tumour samples are collected from the Waveney Hospital as formalin-fixed post-operative specimens. Samples of open colon wall are cut into arbitrary pieces of tissue at a known distance apart. The tissue slices are then blocked, processed and embedded. Every nth tissue section (from a random number generator) is stained for the presence of macrophages. Initially, 5 an t i -macrophage ant ibodies were evaluated. The most appropriate of these was found to be a monoclonal mouse anti- human macrophage antibody produced by Dako (Dako anti- CD68, M-M1). The possibility of cross reactivity with tumour f ibroblas ts was discounted. Volume-weighed numbers of macrophages within.~e tumours were obtained by the use of "Cavalieri 's principle" (for tumour volume) and "Disector" to give unbiased cell numbers. Data on a total of 10 tumour samples will be presented. Dual channel confocal microscopy is also being used to analyse optical sections in order that translational problems in section registration may be circumvented.

THE EFFECT OF CELL CYCLE STAGE ON BLEOMYCIN INDUCED DNA DAMAGE AND REPAIR CAPACITY IN FRIEND MOUSE ERYTHROLEUKAEMIA (FEL) CELLS

AS ASSESSED BY THE COMET ASSAY

P. J. McCarthy, V. J. McKelvey-Martin, P. G. McKenna. Department of Biological and Biomedical Sciences,

University of Ulster, Coleraine. Enhanced cromatid radiosensitivity has been observed in a

variety of cell types transformed in culture and in cells from individuals with cancer prone syndromes ~. It has been suggested that this may result from deficient DNA repair in the G2 phase of the cell cycle. In this study we examined the DNA damage and repair induced by bleomycin in the FEL cells in the Go/G v S and G2-M phases of the cell cycle. Cells were synchronised in Go/Gj and G2+M by centrifugedelutriation. S cells were obtained by growing Go/G~ cells for a further 5.5h. Cell cycle stage was confirmed using flow cytometry. Synchronised cells were treated with 5, 10 and 201xg/ml of bleomycin for lh at 4~ resuspended in fresh medium and harvested in the comet assay after 0. 30 and 60 min at 37~ DNA repair was observed in all treated samples after 30, or 60 min at 37~ No significant difference in the DNA damage nor in the DNA repair capacity was observed between the bleomycin treated Go/G v and S phase cells. G2/M cells however, showed significant DNA damage throughout the repair times as compared to the Go/G v S and S phase populations. These results were supported by the cloning assay for cell survival which indicated that there were no significant differences between the percentage of cells which

survived in the Go/G v S and G2+M phase, at all three doses of bleomycin used.

The financial support of the Ulster Cancer Foundation is gratefully acknowledged.

Reference 1. Sanford et ai. CRC Critical Reviews in Oncogenesis 1989: 1,323.

POST-TRANSLATIONAL REGULATION OF P- GLYCOPROTEIN EXPRESSION IN MAMMALIAN

TUMOUR CELLS EXPRESSING A DISTINCTIVE DRUG RESISTANCE PHENOTYPE FOLLOWING EXPOSURE

TO FRACTIONATED X-IRRADIATION

S. McClean, B. T. Hill. Imperial Cancer Research Fund, 44 Lincoln's Inn Fields,

London.

Human ovarian (SKOV-3) and Chinese hamster ovarian (CHO) tumour cells, following in vitro exposure to fractionated X-irradiation expressed a distinctive multiple drug resistance phenotype characterised by: (i) resistance to Vinca alkaloids and ep ipodophy l lo tox ins , while re ta in ing sens i t iv i ty to anthracyclines, and (ii) overexpression of P-glycoprotein (Pgp) in the absence of any alteration in Pgp mRNA. To establish the mechanism associated with this Pgp overexpression in these X-ray-pretreated sublines (SKOV-3//DXR-10 and CHO/DXR- 10), the half-life of Pgp was examined in [35S]-methionine- labelled cells after immunoprecipitation with two monoclonal antibodies to Pgp and compared to that of drug-selected 'classic' multidrug resistant (MDR) sublines. A reduced rate of Pgp turnover was identified in both human and hamster DXR-10 sublines, with a half-life of >40h, compared with 10- to 17h in drug-selected MDR sublines. These results provide one of the first detailed examples of Pgp regulation by post-translational increased stability and demonstrate that the development of drug resis tance fo l lowing X-ray-pre t rea tment may arise by a mechanism distinct from that resulting after drug selection.

SEQUENCE SELECTIVITY AND CROSS-LINKING EFFICIENCY OF DNA-DIRECTED ALKYLATING

AGENTS

S. McClean, C. Costelloe, W. A. Denny 1, L. P. G. Wakelin. St. Luke's Institute of Cancer Research, Rathgar, Dublin 6 and 1Car, cer Research Laboratory, University of Auckland,

School of Medicine, Auckland, New Zealand.

Alkylating agents derived from aniline mustard, such as melphalan, are widely used in the treatment of cancer. These compounds have no intrinsic specificity for DNA and can react with other cellular nucleophiles, e.g. water and proteins, leading to a loss in activity. In addition, they can form potentially mutagenic monoadducts with DNA at a frequency 20-fold greater than the formation of interstrand cross-links, the latter adducts being considered to be the lesions causing cell death. By targeting alkylating agents more effectively to DNA, these limitations may be overcome. A series of 4-anilinoquinoline- linked aniline mustards have been designed to target the alkylating moiety to the minor groove of DNA. Study of the sequence selectivities of the compounds on the SV40 early promoter fragment have shown that they preferentia!ly alkylate adenines in AT-rich regions, consistent with minor groove

Vol. 164 239 No. 3

binding. In particular, they bind to the adenines in 5' AGT or 5' ACT sequences and also tend to alkylate the most 3' adenine in a run of three As. The targeted compounds proved to be effective cross-linking agents. In an agarose gel assay, crosslinks were observed in linearised plasmid DNA at concentrations as lowas 0.11xM, a value over 10-fold lower than that required for melphalan under the same conditions. The formation of crosslinks was found to be considerably faster for these new agents than was achieved for melphalan. These studies demonstrate that by targeting alkylating agents more specifically to DNA, more effective drugs may be obtained for cancer chemotherapy.

EXPRESSION AND REGULATION OF THE MATRIX METALLOPROTEINASES MATRILYSIN AND

STROMEYLSIN-1 IN HUMAN TUMOUR CELL LINES

B. Fingleton, S: McDonnell. School of Biological Sciences, Dublin City University, Glasnevin, Dublin 9.

The processes of turnout invasion and subsequent metastasis are the most lethal aspects of cancer. Whilst many factors are involved, the matrix metalloproteinases (MMPs) have been implicated as key-rate limiting enzymes in the invasive process. The purpose of this study was to examine a range of human tumour cel l l ines for exp res s ion of two of these m e t a l l o p r o t e i n a s e s , m a t r i l y s i n and s t romelys in -1 . . A modification of the polymerase chain reaction, RT-PCR, was used to de tec t the p re sence of the mRNA for these metalloproteinases. Cell lines found to be expI'essing matrilysin and/or stromelysin-I include colon adenocarcinoma cell lines WiDR SW 620 and LoVo: the bladder carcinoma line T24 the leukaemic cell line K562 and a lymphoma-derived cell line DG- 75. We have also investigated the effects of various cytokines including IL1B, IL-2 and IL-6 on the expression of matr i lysin and stromelysin-I in these cell lines. This study is among one of the first reports of metalloproteinase expression in leukaemic and lymphomic cell lines.

TRANSLOCATIONS INVOLVING BAND 3q27 AND THE Ig GENE REGIONS IN NHL

M. Butler, N. Corbally, P, A. Dervan, D. N. Carney. Departments of Medical .Oncology and Pathology, Mater

Misericordiae Hospital and Department Pathology, University College, Dublin.

Non-random chromosomal abnormalities are found in up to 90% of patients with NHL and have been shown to play an impor tan t ro le in l y m p h o m a g e n e s i s by ac t iva t ing p ro tooncogenes . The most e x t e n s i v e l y s tud ied are t(14:18)(q32:q21) assoc ia ted with fo l l i cu la r lymphoma, t(8:14)(q24:q32) associated with Burkit t ' s lymphoma and t(ll:14)(q13:q32), associated with intermediate lymphocytic lymphoma. Recently, a translocation involving the band 3q27 and the sites of immunoglobultn genes has been described in a subset of patients with diffuse NHL. The name bcl-6 (B-cell lymphoma) has been proposed for the putat ive 0ncogene involved in these translocations, since it is ~ likely to play a role in the pathogenesis of certain B-cell tymphomas. The objective of this work was to determine the incidence of 3q27 breakpoints involving the bcl-6 gene in B-cell NHL using Southern blotting.

In this study, the incidence of the rearranged bcl-6 oncogene in 70 NHL patients presenting with high (n=36), intermediate (n=15) and low (n=19) grade lymphomas has been determined. DNA was extracted from snap frozen lymph node biopsy material and Southern blot t ing was performed fol lowing digestion with the restriction enzymes Xbal and BamH1. The blots were subsequently hybridized with a 4.0 Kb Sacl fragment of bcl-6 gene. Rearrangement of the bcl-6 gene was detected in 19% (7/36) high grade, 0% (0/15) intermediate grade and 5 % (1 / 19) low grade tumour samples. Of those case s presenting with bcl-6 rearrangements in the high grade category, one was histologically classified as a diffuse immunoblastic lymphoma, the remainder were of the diffuse large cell subtype. The patient in the low grade was a follicular mixed small and large cleaved cel l lymphoma. No t rans loca t ions were de tec ted in the intermediate grade category. These results correlate with recent findings of other workers showing an association of bcl-6 with di f fuse large cel l l ymphoma . The c l i n i c o p a t h o l o g i c a l significance of 3q27 breakpoints in relation to this frequent and aggressive lymphoma subtype, for which no specific molecular lesion has yet been ident i f ied , remains to be established.

Supported by C.R.A.B./Irish Cancer Society.

ALTERED CELL PROLIFERATION IN LEIOMYOSARCOMA IS NOT ASSOCIATED WITH

MUTATIONS IN p53 EXONS 5-8

J. N. McMahon*, J. F. Stephens, N. A. Parfrey, O. Shells, E. F. Gaffney.

St, James's Hospital and TCD and St. Vincent's Hospital and UCD (*Supported by H.R.B.).

The tumour suppressor gene p53 is an important regulator of cell growth and differentiat ion, functions that may be profoundly affected by al terat ions in its genomic DNA. Accumula t ion of mutant p53 pro te in has been de tec ted immunohistochemically in a variety of tumour types. However, p53 immunostaining may also detect wild type (normal) p53 protein or may fail to detect mutant p53 protein, because point mutation has resulted in insufficient protein stabilisation. Tumour DNA was extracted from 37 formalin fixed paraffin- e m b e d d e d soft t i ssue and v i sce ra l l e i o m y o s a r c o m a s , representing all grades. A nested PCR technique was used to amplify genomic DNA. Screening for p53 mutations in exons 5-8, a known p53 mutational "hot spot", was carried out by single conformation polymorphism (SSCP) analysis. Paraffin sections were also stained immunohistochemically for the cell proliferation marker Ki-67, using a microwave-st imulated antigen retrieval method suitable for paraffin embedded tissue. SSCP analysis of the p53 exons preferentially affected in human tumours showed abnormal electrophoretic mobility of exon 6 amplified DNA in 3 of 37 tum0urs. No abnormal bands were de t ec t ed in the r ema in ing tumours . In 5/20 tumours immunohistochemically stained for Ki-67, there was a high Ki- 67 labelling index (<50/10 high power fields), which correlated with high tumour grade. None of these 5 tumours showed p53 muta t ions on SSCP. These resu l t s i nd i ca t ed that in leiomyosarcoma altered proliferative indices are not associated with mutations in p53 exons 5-8, and furthermore, suggests that p53 mutation may not be a dominant influence in the commoner human types.


EVALUATION OF CELL CULTURE AND MOLECULAR METHODS IN THE ASSESSMENT OF MINIMAL

RESIDUAL DISEASE (MRD)

J. Doyle ~, G. Martin 3, A. McGirP, M. Lawler 1.2, E. Lawlor ~. Departments of Haematology~/Genetics 2, St. James's

Hospital and Trinity College Dublin and Hospital for Sick Children 3, Glasgow, Scotland.

�9 Treatment of acute lymphoblastic leukaemia (ALL) with high dose chemotherapy and/or BMT can lead to successful resolution of the disease. However, cytotoxic therapy may reduce the leukaemic burden without complete eradication of the leukaemic clone. Thus patients may exist in apparent remission but with small numbers of leukaemia cells. This state of minimalresidual disease (MRD) may be an important prognostic indicator of subsequent relapse. We have investigated the use of an in vitro culture assay system to grow leukaemic precursors from patients in clinical remission to detect evidence of MRD. Initial studies focused on the evaluation of two assay systems, the feeder cell assay and the B cell growth factor assay. Cells from 36 patients with B precursor ALL diagnosis were grown using either system but colonies with characteristic leukaemic blast cells were only detected for 50% of diagnostic samples. Remission samples showed a greater percentage of cells growing that were CD 22 § and showed morphological features of ALL blasts at diagnosis. However, the poor growth rate in diagnostic samples prompted us to change to a system with an allogenic stroma as a feeder layer to prevent apoptotic death of the ALL precursor blasts. FACs analysis has previously indicated that this system could maintain viable growth and use of the CD19 antibody allowed differentiation between stromal cells (predominantly CD19-) and B lineage leukaemic cells (CD19+). Molecular studies have also been initiated involving the immunoglobulin heavy gene which rearranges specifically to each B cell and so can be used as a marker of B lymphoproliferative disorders. PCR primers specific for each of a" family of variable regions (the Vh gene family) were used with a common joining region (Jh) primer to assess the Vh usage in our patient population. Samples from 22 patients were assessed and while some differences were seen compared to published data, they were not s t a t i s t i c a l l y s igni f icant �9 Use of some r a d i o a c t i v e immunoglobuin gene fingerprinting will allow evaluation, of MRD and provide molecular evidence of the leukaemic nature of the blast cells characterised in our in vitro culture system.

DISEASE FREE SURVIVAL FOLLOWING ALLOGENEIC BMT FOR CHRONIC MYELOID LEUKAEMIA (CML):

ASSESSMENT USING MOLECULAR AND CYTOGENETIC TECHNIQUES

N. Gardiner ~, S. R. McCann ~, S. Lynch 2, J. O'Riordan ~, P. Humphries 3, M. de Arce 3, M. Lawler ~.3.

Departments of Haematology ~ and Genetics 3, St. James's Hospital and Trinity College Dublin, Northern Region

Genetic Services 2, Newcastle, U.K.

Bone marrow transplantation (BMT) is the most effective treatment, for chronic myeloid leukaemia (CML). As 95% of CML patients show the characteristic Philadelphia chromosome (Phi), reemergence of the recipient cloneis detectable. However, low numbers of Ph ~ positive cells may give rise to leukaemic relapse. The Philadelphia rearrangement involves a reciprocal

t rans loca t ion be tween chromosomes 9 and 22 with the production of a bcr-abl fusion protein with altered tyrosine kinase activity. The leukaemia specific transcript can be detected using reverse transcriptase PCR (RT-PCR) allowing molecular monitoring of minimal residual disease. In an effort to see if BMT is successful in curing CML, we assessed disease free survival (DFS) in 18 patients fol lowing BMT for chronic myeloid leukaemia (CML) using three methods for detecting residual disease - karyotyping analysis, RT-PCR of the bcr/abl rearrangement for PCR of short tandem repeat sequences (STRs). Fifteen patients received total body irradiation (TBI) and cyclophosphamide (CTX) as conditioning therapy while three patients were conditioned with Busulphan and CTX. Follow-up time ranged from 6 months to 9 years. Thirty-three of 55 karyotype analyses performed were informative for sex chromosomes. Ph ~ positivity was detected transiently on one occasion early post BMT. Sex chromosome analysis indicated donor haematopoiesis in all cases. Bcr/abl posit ivity was measured in 11 patients and STR polymorphisms were assessed in 12 patients on 21 occasions. Bcr/abl transcripts were absent in 9 patients from 6 months to 7 years and these patients were complete donor chimaeras at all times tested. Bcr-abl transcript was found in 2 patients, one of whom showed transient early positivity while the second patient exhibited recipient cells at the same timepoints by STR-PCR. Thus, in patients who are in haematological remiss ion post BMT, a combinat ion of karyotyping, sex chromosome marker analysis, PCR of bcr/abl and assessment of ehimaeric status correlates well with disease free survival.

SELECTIVE EXCRETION OF TUMOUR DERIVED FACTORS IN THE URINES OF CANCER PATIENTS

E O'Brien 1, A. Duggan 2, S. O'Herlihy 2, G. O'Sullivan 3, E Shanahan ~, J. K. Collins 1,2.

Departments of Medicine ~, Microbiology 2 and Surgery 3, University College and Mercy Hospital, Cork.

Tumours produce a variety of biologically active factors including transforming growth factors (TGF's) and immune suppressive factors (ISF's) . Also tumours induce immune responses with resulting circulating immune complexes. All these factors have access to the renal filtration system and therefore can potentially be excreted. In addition, there is a possibility that such factors can influence the dynamics of renal function modifying the profile excreted. In this study, tumour influences on urinary protein excretion was examined in patients with a variety of urinary tract cancers. It was found that tumour bearing patients excrete abundant quantities of putative tumour derived bio logica l ly active factors, such as TGF's , when compared to age and sex matched controls. The urinary protein excretion was measured quantitatively and qualitatively using high pe r fo rmance l iquid c h r o m a t o g r a p h y (HPLC) and po lyac ry lamide gel e lec t rophores is (PAGE). Signif icant differences were found for both of these parameters between tumour bearing patients and controls, which had relatively simple protein profiles. However, between patients with different malignancies, qualitative and quantitative differences were also seen. The initial conclusion from these studies indicated that tumour presence may influence the renal handling of proteins possibly through the release of biologically active cytokines by the turnout. To ascertain if the urinary excretion of tumour

Voi. 164 241 No. 3

derived factors could be directly related to tumour burden, a mouse human tumour xenograft model was developed. A s igni f icant cor re la t ion was found between tumour size/ progression and the release of TGF's in the urine. Urine has potential as a non-invasive sampling source for tumour derived factors including biolOgically active factors and antigens. This excretion appears to be related to tumour burden and hence an indicator of tumour presence/recurrence/response to therapy.

GENETIC ANALYSIS OF MUTATIONS ASSOCIATED WITH LEUKAEMIA, WITH PARTICULAR EMPHASIS ON MYELOID

MALIGNANCIES AND THEIR PROGNOSTIC VALUE

G. O'Keeffe, S. McCann t, K. Sweeney. Biology Department, D.I.T., Kevin Street, Dublin 8 and

~Haematology Department, St. James's Hospital, Dublin 8.

The acquisit ion of mutations in oncogenes and tumour suppressor genes is thought to be central to the development of malignant disease. The course of disease progression with myelodysplastic syndrome to acute myeloid leukaemia (AML) provides an adv~intageous system to study genetic change s in human malignancy. The pattern of genetic changes Can b e monitored throughout the development and progression of the disease. The initial mutation is unknown but its occurrence may bere la ted to the overall load of random mutations which are a consequence of both ihtrinsic DNA mutations and external mutagens. The mechanism of progression from a haematological aberration to overt leukaemia involves successive genetic changes resulting in abnormal control of ~ell proliferation and differentiation. Archival bone marrow slides were used in this study. Proteinase K:SDS digest ion fol lowed by phenol/ chloroform ext rac t ion and ethanol precipi ta t ion, y ie lded sufficient DNA for several PCR amplifications. Two oncogenes have been analysed for point mutations, N-Ras at codons 12, 13 and 61 and FMS at codons 301 and 969. These regions are 'hot spots' for mutations. The following table summarises the data from the main patient group. Percentage figures represent the proportion of point mutations found at that codon based on the information gathered to date.

Oncogene FMS FMS N-Ras. N.Ras Codon 301 969 12+13 61' Total (n=164) 44% 26% 66% 32% MDS (n=43) 20% 22% 43% 31% AML (n=34) 21% 29% 21% 22% Lymphoid Leukemias (n=36) 44% 25% 43% 22%

The highest inc idence , par t icular ly in MDS, of-point mutations is in N-Ras at codons 12+13. Point mutations are commonly found in N-Ras and FMS in acute leukemia and myelodysplasia. Serial studies, especially in MDS may provide useful information on leukaemic transformation.

PCR OF SHORT TANDEM REPEAT SEQUENCES ALLOWS MOLECULAR MONITORING OF TRANSPLANTATION OF

UV B IRRADIATED PRECURSORS IN RODENTS

A. O. Neill 1, M. Lawler 1, S. R. McCann t, D. Pamphilon 2. 1Department of Haematology, St. James's Hospital, Trinity College Dublin and 2Regional Transfusion Centre, Bristol, U.K.

Graft versus host disease (GvHD) and graft rejection (GR) remain serious problems following allogenic BMT and are

probably mediated by donor dendritic cells and donor T cells respectively. Ultraviolet light (UV) may have potential in the prevention of GvHD and GR as it can abolish allostimulation in vitro. We have addressed the efficacy of UV B in abolishing GvHD while preserving stem cell viability in an in vivo model. UV B treated marrow was given to mice in semi allogenic (n=48) or fully allogenic (n=24) BMTs. A sensitive PCR assay system, based on the amplification of short tandem repeat sequences (STRs) was developed which allowed serial assessment of the kinetics of engraftment o fUV B irradiated precursors. 12 STR markers were assessed for strain specific polymorphisms between three strains of mouse (CBA/J, Balb/c and C57/B 16). Recipients of UV B marrow were studied serially using tail ve in samples . Three lamps which d e l i v e r e d d i f fe ren t wavelengths of UV B light were assessed. The XX15B and TL12 lamps (Philips) deliver a broad spectrum UV B wavelength (250-375nm) while the TL01 (Philips) delivers a much narrower range (300-320nm). The broad range lamps were more efficient at abrogating alloreactivity while preserving stem cell function as judged by the CFU-GM assay. This was unexpected as the broad lamps do deliver some UV C (200-280nm) which we have found to be damaging to stem cells. Serial studies by STR- PCR indicated that UV B treated progenitors could rescue marrow function in semi allogenic BMT, though at higher UV B i r rad iance levels , the degree of engraf tment may be compromised. In the more immunoreactive allogeneic model, 75% of mice receiving 50Jm 2 from a broad spectrum UV B lamp were predominantly donor chimeras and showed sustained engraftment with survival up to 24 weeks post BMT. STR markers are useful in assessing chimerism post BMT in rodents in a serial fashion and initial results indicated that UV B treated marrow can reconstitute haemopoiesis in mismatched BMT. Establ ishment of an opt imal wavelength of UV B which preserves stem cell function while abrogating dendritic and alloreactive T cells may provide a window of opportunity for the prevention of GvHD and graft rejection..

DETECTION OF ALLELIC CHANGES AT THE APC TUMOUR SUPPRESSOR GENE ON CHROMOSOME 5 IN

DUKE'S B COLORECTAL CARCINOMA

B. Curran, M. A. Bennett, D. T. Croke, M. Leader. Departments of Biochemistry and Pathology, Royal College

of Surgeons in Ireland, Dublin 2.

Fami l ia l adenomatous polyposis is a rare, dominant ly inherited disorder, which, if left untreated, progresses to colorectal carcinoma in young adults. In benign adenomatous polyps, one copy of the adenomatous polyposis coil (APC) tumour suppressor gene, may be inactivated by germline or somatic mutation, followed by somatic deletion of the second copy. APC is also implicated in up to 40% of sporadic colorectai carcinoma cases, among whom Duke's B patients have the most variable outcome. In this study, two oligonucleotide primer pairs were used in the polymerase chain reaction (PCR) to amplify DNA closely linked to the APC gene. Both regions include simple sequence repeats (microsatellites), which are highly polymorphic among individuals, in terms of number of repeat units. Microsatellite polymorphisms are more informative than other loss of heterozygosity (LOH) assays because the expected rate of heterozygosity among patient cohorts is high. We have calculated allele frequencies and percentage heterozygosity from 60 Irish control subjects.

242 Irish Associat ion f o r Cancer Research

Results were comparable with similar data from Dutch and American populations. Two PCR reactions were combined in a multiplex format to increase the informativity of the assay. A frequency of heterozygosity of 83% for at least one of these two genet ic po lymorph i sms in the Irish popula t ion was estimated. This constitutes a highly informative LOH assay, compared with restriction fragment length polymorphisms (RFLP's) which have a maximum informativeness of 50% of patients. This "multiplexed" PCR was used in the assessment of LOH and allelic changes (shifts) in Duke's B tumours. Tumour from 6 of 26 patients who died within 3 years of resection had detectable DNA changes, compared with 2 of 27 patients alive at least 5 years post-resection. The assay will be applied to a larger pat ient populat ion to invest igate its potential as a prognostic indicator.

DEVELOPMENT OF OPTIMISED IN VITRO SYSTEM FOR MEASUREMENT OF INDIVIDUAL PATIENT

RESPONSE TO CYTOTOXIC THERAPY, PRIOR TO SURGERY

M. Sheridan ~, I. Reid ~, C. Mothersill, C. B. Seymour, T. Walshe 1, T. P. Hennessy I.

Dublin Institute of Technology, Kevin Street, Dublin 8 and 1Department of Surgery, St. James's Hospital, Dublin 8.

Evaluation of the response of tumour tissue to combined chemotherapy (chemoDXT) is generally retrospective and is based on average results obtained in clinical trials. The purpose of this study is to develop an in vitro predictive testing system whereby individual patient response to chemoDXT can be evaluated prior to commencement of therapy. Prediction of patient response to therapy is estimated by means of an explant culture system using pre-treatment endoscopic biopsies taken from patients with oesophageal carcinoma. The effectiveness of therapy is determined by its ability to reduce the growth of explanted fragments of biopsy relative to untreated cultures (controls) after 2 weeks in culture.

Results for the first 14 patients show that 12 of the biopsies cultured grew successfully. Of these, 7 showed a reduction of up to 60% of the mean growth area relative to control. 5 were reduced by more than 60% relative to control, 12 responded to c h e m o t h e r a p y a lone and 12 to a combina t ion of chemoradiotherapy. Table I shows a sample of the individual responses for the first 4 pat ients . These results c lear ly demonstrate that biopsies from individual patients vary in their response to therapy, particularly radiotherapy, and suggests that there may be merit in predictive evaluation as an aid to treatment planning. The ability to culture biopsy specimens enhances the clinical revelance of the system as an individual predictor of patient response to treatment.

Table I: Individual response of patients to therapy measured as a change in the mean growth area of the treated cultures relative to

the control (untreated cultures) for each patient.

Response to Therapy Patient Radiotherapy Chemotherapy Combination

1 ++ ++ 2 -- ++ ++ 3 ++ ++ ++ 4 ++ ++ Reduction of mean growth area by more than 60% relative to control No reduction of mean growth area relative to control

-- Increase of mean growth area relative to control

LJ.M.S. July, August, September, 1995

TUMOUR DERIVED IMMUNE SUPPRESSOR FACTORS AND APOPTOSIS

A. O'Mahony I, G. O'Sullivan 2, J. O'ConnelP, C. Lawlor 3, F. Shanahan 3, S. Nolan ~, J. K. Collins 1,3.

Departments "of Microbiology ~, Surgery 2 and Medicine 3, University College and Mercy Hospital, Cork.

The mechan i sms through which turnouts evade immunosurveillance and present in immunocompetent hosts remain to be fully elucidated. A putative mechanism is through the p roduc t ion by tumours of so lub le se lec t ive immunosupp re s s ive factors . We p resen t in vitro and s ign i f i can t ly in vivo evidence for this phenomenon in oesophageal squamous carcinoma. We have found that gastric and colorectal adenocarcinomas also produce soluble mediators of immune suppression. The precise mechanism of lymphoid suppression with these factors and the fate of such suppressed or anergic cells remains to be elucidated. Here we report on the involvement of apoptosis or programmed cell death as a consequence of tumour derived immune suppression. Apoptotic cell death was determined by light microscopy, flow cytometry and by DNA fragmentation analysis. We have found that tumour derived immune suppressor factors induce apoptotic cell death in normal and mal ignant ly t ransformed cel ls of myelo id lymphoid lineage. In the case of the squamous carcinoma derived immunosuppressor factor we have found that the factor induces a rapid rise in intracel lular cycl ic AMP pools in activating cells. Significantly, we also report some evidence for tumour derived induction of apoptosis in vivo. We conclude that tumour derived immunosuppressor factors selectively induce anergy which progresses to apoptosis in activated cells of lymphoid myeloid lineage. This phenomenon we propose accounts for the i n vivo immune s u p p r e s s i o n c l ea r ly demonstrable local to the tumour site in squamous carcinoma of the oesophagus.

THE POTENTIAL ROLE OF UROKINASE-TYPE PLASMINOGEN ACTIVATOR (UPA), THE UPA

RECEPTOR AND PAl 1 IN THE INVASION AND METASTASIS OF TUMOUR CELL LINES

D. Morrisey, D. Lynch, J. K. Collins, G. O'Sullivan. Departments of Microbiology and Surgery, University

College and Mercy Hospital, Cork.

Urokinase-type plasminogen activator (uPA) is proposed to play a significant role in the mechanism by which tumour cells spread from a primary lesion and develop into secondary tumours (metastases) at distant tissue loci. We have established in vivo and in vitro models in which to study the dissemination of oesophageal carcinoma cell lines. One cell line (OC2), despite being highly tumourigenic, never gives rise to metastases when injected subcutaneously in the athymic nude mouse. Two other cell lines (OCI, OC3) are less tumourigenic but have been known to generate metastatic lesions from small primary tumours. In the in vitro chick embryo heart invasion model system the OC2 cells exhibited less infiltrate capacity than either OC1 or OC3 cells which strongly invade the normal tissue. Significantly, OC2 cells were found to express less extracellular, intracellular and cell surface associated urokinase than either OC1 or OC3 ceils. Each cell line expresses the uPA receptor (uPAR) and its i nh ib i to r both on the ce l l sur face and extracellularly. To demonstrate conc lus ive ly . the role of

vol. 164 243 No. 3

urokinase we have transfected OC2 cells with the human uPA gene harboured on an expression vector. We have thus generated OC2 clones which harbour the uPA gene, express active urokinase and significantly express increased UPAR and P l l levels. These uPA-posi t ive OC2 clones display increased invasiveness in the chick heart model. However, t hey still do not form spontaneous metastases in the ~ithymic nude mouse. Thus it may be concluded that balanced interactions between uPA, uPAR and PAI l facilitate the early phase of tumour cell spread without predisposing the homogenous turnout cell lines to metastasis development.

THE CHANGING PA'I~ERN OF MALIGNANT MELANOMA IN NORTHERN IRELAND

P. J. Pedlow, M. Walsh, S. W. Lowry, R. J. Atkinson. Departments of Ontology and Pathlogy, The Queen's

University of Belfast.

The incidence of CMM is increasing worldwide. Metastatic disease remains resistant to treatment but early detection is known to improve survival. Preliminary results of a longterm study in Northern Ireland indicate that incidence has doubled over ten years. Earlier studies found a female to male ratio of 3 to 1, the highest reported in the literature. Now, however, the inc idence is r i s ing faster for men.~t-han for women. Consequently, the gap between the sexes is harrowing. There is evidence of earlier detection with more thin lesions than previously and also fewer ulcerated lesions. The most common site for men continues to be the head and neck, although the proportion of lesions on the trunk is increasing. There is a striking female predominance in the under 30 year age group. The findings have implications for specific targetting of the health education message.

ANTI-ERYTHROPOIETIC ACTIVITY OF A MURINE TUMOUR

J. J. A. McAleer ~, S. R. McKeown 2, M. AfrasiabP, T. R. J. Lappin 3, B. JoineP, K. V. HirsP, D. G. HirsP.

Departments of ~Oncology and 3Haematology, Queen's University of Belfast, Department of Biological &

Biomedical Sciences, University of Ulster, Jordanstown and CRC Gray Laboratory, Mount Vernon Hospital, Middlesex.

The carcinoma NT (CANT) is known to cause haematological changes in the host CBA mouse inc lud ing anaemia , splenomegaly and leucocytosis. We have previously shown that admin is t ra t ion of e ry th ropo ie t in (Epo) can reverse this anaemia ~J. Further studies have shown that the depression in haematocrit is dependent on tumour load. This suggests that a factor is produced by the tumour cells which interacts with the haematopoietic system. We have therefore tested tumour conditioned medium for interaction with Epo in vitro using the mouse spleen cell assay (MSCA), which is a method for measuring the biological activity Epo. Confluent CaNT cells were maintained for two or seven days in Dulbecco's medium with no additives except NaHCOy The harvested medium was

tested in the MSCA to assess its affect on the normal stimulation of spleen cells with Epo. Medium was also concentrated through 10 kDa and 30kDa molecular weight sieves. Both retentate and diffusate were tested. Tumour conditioned medium showed inhibi t ion of erythropoiet ic activity. The most effect ive inhibition was obtained with the retentate of the 10kDa sieve for media conditioned for seven days. The inhibitory factor was not retained by the 30kDa sieve. The results support the hypothesis that this tumour produces a factor between 10-30kDa which inhibits the biological action of Epo. The evidence suggests that the factor acts in a similar manner to interleukin 1. Reference 1. Joiner, B., Hirst, K. V., McKeown, S. R., McAleer, J. J. A., Hirst, D.G. Br. J. Cancer 1993; 68: 720.

ACTIVATION OF A CALCIUM, MAGNESIUM INDEPENDENT ENDONUCLEASE IN HUMAN

LEUKEMIC CELL APOPTOSIS

R. S. Fernandes, T. G. Cotter. Biology Department, St. Patrick's College, Maynooth, Co.

Kildare.

Cytotoxic drugs induce apoptosis in human tumour cell lines and this is characterised by fragmentation of the cell's DNA into nucleosome size units or multiples thereof. In the present study we demonstrate the nuclei isolated from three human haematopoietic cell lines, HL-6 U937 and K562, contain an endonuclease that is independent of Ca ++, and Mg § and Na § ions for its activity. This contrasts with what has previously been shown for a number of rodent cell types in which apoptosis has been studied. The lack of ion sensitivity ig also found in the nuclei of peripheral blood granulocytes, indicating that data is not peculiar to cell lines. The previously demonstrated lack of calcium flux in HL-60 cells undergoing apoptosis t~, and the current demonstration of a lack of an endonuclease dependent on this ion for its activity, suggests that the mechanism for apoptosis in human cells may be different from that in rodent ce i ls . I so l a t ed nuc lear DNA was ana lysed us ing gel electrophoresis and DNA fragmentation was quantified using a tritiated thymidine assay

Reference 1. Lennon, S. V., Kilfeather, S. J., Hallet, M. B., Campbell, T. G., Cotter, T. G. Elevations in cytosolic calcium are not required for apoptosis in human leukemic cells�9 Clin. Exp. Immunol. 1991; 87: 465- 471.

DESIGN CONSTRUCTION, CLONING EXPRESSION AND BIOACTIVITY OF AN IL7 RECEPTOR TARGETED

FUSION TOXIN, DAB3ag-IL7

E. Sweeney ~.2, J. VanderSpek ~, J. Murphy 2, F. Foss ~. ~Sections of Medical Oncology and 2Biomolecular Medicine,

BUMC, Boston, MA 02118, USA.

Growth factor receptors are attractive entry points to the cytosol for the delivery of toxins liganded to defined growth factors: The IL2/I.2R growth factor/receptor complex has been used successfully to deliver diphtheria toxin, fused to IL2, to


IL2R+ cells. Leukaemic cells from ALL, AML, CLL and CML express the IL7R. To take therapeutic advantage of this we have designed, constructed, cloned and expressed an IL7 diphtheria toxin fusion protein (DAB~89-IL7) to target these cells. A synthetic gene encoding human IL7 was designed using E. coli codon usage bias. Four unique cloning restriction sites were introduced into the IL7 sequence through the "wobble" in the third position of the genetic code. The 465 base pair gene was const ructed by success ive PCR ampl i f ica t ions of 4 oligonucleotides spanning the length of the gene. A plasmid encoding our previously constructed DAB3ag-IL2 gene was digested with Sphl and Hind III to drop out the IL2 sequence. The IL7 gene was cloned in place of IL2 downstream of the bacterial DT gene, and the new plasmid encoding DAB389-tL7, was designated pES4. For expression, the DAB3sg-IL7 gene was placed under the control of a T7 promoter and expression induced in E. coli after infection with a lambda phage encoding T7 polymerase. SDS gel analysis of crude E. coli inclusion body extracts confirmed the expression of a protein at Mr 60kDa which is in agreement with the deduced combined Mr of DT and IL7. On Western blot analysis this protein immunoreacted with antibodies to both DT and ILT. Native gel electrophoresis and Western blot analyses indicated the presence of monomeric and multimeric forms of DAB389-IL7. The crude inclusion body extract inhibited protein synthesis in murine lymphoid IL7- dependen t 2E8 cel ls at an IC50 of 5x10-gM. DAB389-IL cytotoxici ty on other IL-7+ cell lines and fresh leukaemic samples is being evaluated. A study of the efficacy of DAB3s 9- IL7 in an IL7R+ leukaemia model in SCID mice and in a murine IL7 transgenic model is in progress.


AUTHENTIC PNA: PEPTIDE-BASED ANALOGUES OF DNA

N. M. Howarth*, M. Searcey, C. M. Topham, L. P .G. Wakelin.

St. Luke's Institute for Cancer Research, Department of Chemistry, University College, Dublin.

Reagents that bind specifically to complementary nucleic acids are of major interest. Considerable attention has been focused on developing oligonucleotide surrogates that maintain Watson-Crick or Hoogsteen base pairing to RNA or DNA targets but which do not incorporate the usual phosphodiester linkages, due to their susceptibility to nucleases and incompatibility with passive membrane transport. One recent advance in this area is the polyamide nucleic acids (PNA) developed by Nielsen.

Our approach to this quest involves the use of a peptide backbone to connect the base-containing subunits. The main advantages of using a peptide backbone are that these analogues will be resistant to nucleases, can be made resistant to peptidases and as the backbone charge will be controllable, they may be made to exhibit improved transport properties. We have designed a peptide nucleic acid in which the amino acid moiety is derived from homoserine (the DNA base replacing the original hydroxyl group). These base amino acids are interspersed with glycine and the bases are connected to one another via a six atom linker as in DNA. A model of a 12-mer PNA:DNA duplex has been built using a combined energy minimisation and restrained molecular dynamics (MD) protocol. Unrestrained MD studies are in progress and the synthesis of these compounds is well advanced. Progress in synthesis and computation studies will be presented.

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