ABSTRACTS OF MEMBERS PAPERS 401

PART I: ABSTRACTS OF MEMBERS' PROFFERED PAPERS ENHANCEMENT OF CHEMICALLY- Pregnant rats were injected with ethyl- INDUCED NEOPLASIA BY PROXI- nitrosourea to induce cerebral gliomas in a MAL ENTERECTOMY. R. C. N. WIL- high proportion of the offspring. With a dose LIAMSON, F. L. R. BAUER and R. A. MALT, of 40-50 mg/kg body weight the average United Bristol Hospitals and MlVassachusetts latent period for these brain tumours is 246 General Hospital, Boston, U.S.A. (Introduced days. Cultures have been prepared at by M. 0. Symes). different times after transplacental exposure Azoxymethane-induced tumours in the but before a tumour is visible. It has been rat, morphologically indistinguishable from reported previously (Roscoe and Claisse those arising in man, affect the duodenum, (1976) Nature, Lond., 262, 314) that there is jejunum and colon, but usually spare the a marked difference in the behaviour of ileum (Ward, J. M. (1975) Vet. Pathol., 12, cultures prepared 138-145 days p.i. and 2 165). Proximal small-bowel resection (PSBR) days p.i. These experiments have been causes prompt ileal hyperplasia, with a lesser extended and are reported here. Cultures response in the colon. To test the possible prepared 111-112 days p.i. were similar to adjuvant effect of postresectional hyper- those derived 138-145 days p.i. They con- plasia on intestinal carcinogenesis, rats tained cells like those found in tumour (n= 76) were submitted to 5000 PSBR 10 cultures which can predominate in culture days after the last of 16 weekly injections of and are tumourigenic. Similar cells observed azoxymethane (10 mg/kg s.c.) or vehicle. in cultures prepared 90-91 days p.i. appear Controls were unoperated. Nucleic acid to be lost on sub-culturing. These cells were contents of upper ileal mucosa in rats receiv- not seen when cultures were initiated at still ing vehicle alone showed increments of 76% earlier times (60, 34-35, 5, 3, 2 days p.i.). (RNA) and 680, (DNA) 3 nionths after However it has been shown that cultures PSBR (P < 0-001). The number and distri- prepared 2 days p.i. yield cells which are bution of intestinal tumours were noted in tumourigenic on prolonged culturing. These rats sacrificed at 30 weeks, or dying of cancer results provide a framework for further in the preceding 4 weeks. Intestinal tumours analysis of the latent period and more detailed occurred in all but 1 of the rats receiving studies are in progress.

No. tumours par rat (after azoxymothane) Duodenojejunum Ileum Colon Ear canal Control 18 1 3 0.1 1-6 0-8 PSBR 16 0 8 nil 2 * 9 1 2 NS NS P<0-02 NS

azoxymethane, but in none of those injected FIBRINOLYTIC ACTIVITY ASSOCI- with vehicle. Proximal enterectomy raised ATED WITH RAT BRAIN CELLS the incidence of colonic tumours but failed to EXPOSED TRANSPLACENTALLY TO induce ileal carcinogenesis, despite promoting THE CARCINOGEN ETHYLNITRO- marked mucosal hyperplasia. We conclude SOUREA. T. A. HINCE and J. P. ROSCOE, that postresectional hyperplasia cannot over- Department of Cell Pathology, School of Patho- come the relative insusceptibility of the ileum logy, Middlesex Hospital Medical School. to neoplasia, but that it enhances the induc- tion of tumours in colon previously exposed An increased fibrinolytic activity of to chemical carcinogens. tumour and transformed cells, as a result of increased amounts of plasminogen activator, AN INVESTIGATION OF ETHYL- has been proposed as another marker for NITROSOUREA-INDUCED CARCINO- transformed cells (Jones et al. (1976) Cancer GENESIS IN THE RAT BRAIN BY AN Res., 36, 2863). In this study we have investi- IN VIVO-IN VITRO METHOD. J. P. gated the fibrinolytic activity of cell lines RoSCOE and P. J. CLAISSE, Department of derived from cerebral gliomas of the rat brain Cell Pathology, School of Pathology, Middlesex and cultures derived from the brains of rats Hospital Medical School. after their transplacental exposure to the 402 B.A.C.R. 18TH ANNUAL GENERAL MEETING

carcinogen ethylnitrosourea (ENU) using inflammatory response with consequenit leak- an in vivo/in vitro system (Roscoe and Claisse, age of serum; the plasminogen would then be 1976, Nature, Lond., 262, 314). Using a fibrin- locally activated by the tumour cells which overlay method (Jones et al., 1975, Cell, 5, *Would then divide at a faster rate. 323) we have demonstrated that cell lines derived from a cerebral glioma and from rat brains 111-112 days after their exposure to the carcinogen show a very high level of fibrinolytic activity. In contrast, control cells CONTROL OF HAEMOPOIETIC and cultures derived from the brains of STEM-CELL POPULATIONS. E. G. animals exposed to buffer alone show low WRIGHT, Paterson Laboratories, Christie Hos- levels of activity. A detailed study of the pital and Holt Radium Institute, Manchester. relationship between fibrinolytic activity, (Introduced by R. Schofield). measured as the percentage of total colonies giving lysis, and cell colony size indicated Haemopoietic cells are derived from com- that there was a marked difference between mon pluripotential spleen-colony-forming transformed and control cells. Transformed stem cells (CFU-S). The majority of these cells showed a rapid rise in fibrinolytic stem cells, in the normal steady state, are activity with increasing colony size and not proliferating, but can be stimulated into reached a plateau level >70%0 at colony cell cycle by a decrease in their population sizes of below 70 cells/colony; wihereas con- size. The nature of CFU-S proliferation con- trol cells showed a linear increase with trol is not known. There is, however, evidence colony size and much reduced levels of that it is "local" rather than humoral. The activity: <30%0 at larger colony sizes, presence of proliferating and non-proliferat- >130 cells-colony. Thus in this system an ing stem cells in, respectively, the bone mar- increased amount of fibrinolytic activity is row and spleens of phenylhydrazine (PHZ)- associated with malignant transformation. treated mice (Rencrieca et al. (1970) Blood, 36, 764) has afforded the opportunity to investigate the presence and role of local factors responsible for the control of CFU-S SV40-3T3 CELL PLASMINOGEN ACTI- proliferation. This has been done by measur- VATOR-MEDIATED INITIATION OF ing the proliferative activity of femoral and MITOSIS IN QUIESCENT 3T3 CELLS. splenic CFU-S resulting from the addition of P. WHUR, M. GORDON, D. C. WILLIAMS, C. radiation-killed splenic or bone-marrow cell URQUHART and E. WRIGHT, Marie Curie populations. When bone-marrow cells from Foundation, Oxted, Surrey and Imperial PHZ-treated mice are incubated with irradi- College, London and Royal Free Hospital, ated spleen cells taken from the same mice, Medical School, Londont. there is a marked fall in the proportion of femoral CFU-S in DNA synthesis. In the The number of dividing cells ill a popula- converse experiments, rapid triggering of tion of quiescent 3T3 cells in low serum splenic CFU-S is achieved. Changes in increases, significantly in the presence of CFU-S proliferation have also been demon- SV40-3T3 cells and added plasminogen. This strated in other situations, where cell popula- effect is attributable to plasminogen activa- tions containing proliferating and non- tion (Whur et al. (1976) Nature, Lond., 260, proliferating CFU-S are mixed. It is not, 709). The effect of plasminogen activation on therefore, a phenomenon specifically related mitosis decreases as serum stimulation to the PHZ-treated mouse. The effects on becomes optimal, suggesting that the former the proliferative activity of CFU-S resulting may potentiate the latter. Scanning electron from the incubation of haemopoietic cells micrographs show that plasminogen activa- with irradiated cell populations suggest that tion causes fissures to open between previously some part or parts of these populations con- confluent 3T3 cells; thus the diffusion boun- tain material capable of altering the rate of dary layer may become disrupted, leading to stem-cell proliferation. It seems probable a reinitiation of mitosis by serum growth that these findings represent some aspect factors. This mechanism may also operate of the local physiological CFU-S proliferation- in vivo. Tumour cells would initiate a local control process. ABSTRACTS OF MEMBERS PAPERS 40.3

THE SPECIFIC STAINING OF staining. Normal monocytes showed weak, SUGARS IN THE HISTOCHEMICAL uniform staining for sialic acid and little ANALYSIS OF BONE MARROW staining for other sugars. Malignant mono- AND THE MYELOID LEUKAEMIAS. cytes stained weakly and irregularly for R. W. STODDART, W. JACOBSON and R. D. surface sialic acid and showed very little stain COLLINS, Strangeways Research Laboratory, for galactose or mannose; some appeared to Cambridge. and Department of Pathology, show caps. In acute lymphoblastic (human Vanderbilt University, Nashville, Tennessee, and murine) and chronic lymphocytic leuk- U.S.A. aemias the malignant cells showed much less staining for sialic acid than their normal coun- In many glycoproteins of cellular surfaces, terparts, both at the plasmalemma and nu- mannosyl residues lie in the "cores" of the clear membranes. Staining for mannose was oligosaccharides, while sialic acid is always at unaltered. There were no differences between the non-reducing (exterior) terminals. Galac- B and T lymphocytes. The malignant cells of tosyl groups are usually either terminal or nodular and thymic lymphomas gave a sub-terminal to sialic acid. Fluorescent- similar result. In all types of Hodgkin's labelled concanavalin A (FL-ConA) can be disease, the neoplastic lymphocytes were used to stain for a-mannosyl (or a-glucosyl) characterized as showing a reduction in groups; the similar derivatives of Ricinus sialic acid and in sub-terminal galactosyl communis haemagglutin (FL-RCA) and of groups; Reed-Sternberg cells were weakly aprotinin (FLA) stain for /-galactosyl groups stained for all sugars. Related, but rather and sialyl groups respectively. Fresh smears, more complex abnormalities were seen in or paraffin sections of methanol-fixed bone myeloma, Waldenstrdm's macroglobulin- marrow were used. Myeloblasts, myelocytes aemia and leukaemic reticuloendotheliosis. and leucocytes showed staining of their surfaces, cytoplasm and nuclear membranes with all three stains. Granules in eosinophils and basophils stained in each case, but those of neutrophils bound only FL-ConA. In AN ABNORMAL SURFACE PROTEIN megakaryocytes, there was intense cyto- OF TUMOUR CELLS. R. W. STODDART and plasmic staining with FL-RCA and FL- M. R. PRICE, Strangeways Research Labora- ConA; the platelets stained strongly with tory, Cambridge, and Cancer Research Cam- FLA, FL-RCA and FL-ConA. Staining of paign Laboratories, University of Nottingham. chromatin was seen in several cell types, but Investigations of a membrane-bound pro- was most intense (with each stain) in late tein of pI 4 00, which is present in the plasma erythroblasts. In all malignant cells of the membranes of hepatomas and mammary myeloid series there was a general reduction of carcinomas of the rat, have been extended staining with FLA and an increased binding to determine its subcellular location, its of FL-ConA at the plasmalemma. relation to the pathology of the tumours, its chemistry and its occurrence in other species. ABNORMAL SACCHARIDES OF lodination has shown that the protein is HUMAN LYMPHOID LEUKAEMIAS accessible at the surface of hepatoma cells. AND ALLIED LYMPHOMAS. W. Traces of a similar protein are present in the JACOBSON, R. W. STODDART and R. D. COL- nuclear membranes of normal hepatocytes LINS, Strangeways Research Laboratory, Cam- and are greatly elevated in malignancy. In bridge, and Department of Pathology, Vander- regenerating liver it is maximally elevated U.S.A. at Day 3, but it is far below the level in bilt University, Nashville, Tennessee, tumour cells, and does not occur at the plas- Fluorescent-labelled lectins and aprotinin malemma. In human, canine, feline, murine have been used to study the defects in the and porcine tumours, similar proteins have cell surface and intracellular saccharides of a been found. Foetal tissues (rat and human) range of reticuloses. Materials were fixed in contain related proteins of lower pI. The anhydrous methanol and used for paraffin levels of the protein are not related to the sections, or were freshly prepared as spreads. histological class of tumour, its invasiveness Autofluorescence was eliminated by a short- or antigenicity, its degree of vascularity or treatment with osmium tetroxide, before the extent of lymphocytic infiltration. There 404 B.A.C.R. 18TH ANNUAL GENERAL MEETING is evidence for its being a glycoprotein. Its J. natn. Cancer Inst., 51, 1119), casein appearance during carcinogenesis has been (Hendrick and Franchimont (1974) Europ. studied. J. Cancer, 10, 725), and various less specific biochemical markers (Coombes et al. (1977) THE SYNTHESIS OF co-LACTAL- Lancet, i, 132) have been observed in meta- BUMIN BY HUMAN MAMMARY static breast cancer, but it is necessary to CARCINOMAS. K. L. WOODS, D. H. COVE, establish wA-hether these markers have a useful A. HOWELL and D. A. HEATH, Department of role in clinical practice. The serial assay of Medicine, University of Birmingham. serum carcinoembyronic antigen in patients undergoing treatment suggests that liver a-Lactalbumin is the major whey protein and bone metastases will cause an elevation of human milk. Using a sensitive radio- more readily than does local recurrence. immunoassay we have sought evidence for Serum casein is elevated in a proportion of the synthesis of this protein by human mam- patients with primary breast cancer (240') mary carcinomas. The cytosol fraction 14/38 of varying histological grade. In a small carcinomas contained measurable o-lactal- series of patients wN-ho have undergone bumin. The presence of a-lactalbumin was bilateral mastectomy there is a lower incidence closely associated wNith the presence of of casein positivity than in a larger series oestrogen receptor, and the concentrations after bilateral mastectomy. Preliminary of ox-lactalbumin and of oestrogen receptor studies indicate that neither marker is showed a linear correlation. Serum levels of elevated in direct response to certain cyto- o-lactalbumin were studied in 50 patients toxic drug regimens. wvith breast cancer and compared with those of healthy control subjects. In normal women, the proportion having detectable circulating THE COMPETITIVE NATURE OF A-lactalbumin varied from about 80O/ in 06-METHYLGUANINE MISCODING young adults to about 200/' in post-meno- DURING DNA SYNTHESIS. P. J. Abbott pausal subjects. At all ages the level was and R. Saffhill, Paterson Laboratories, Christie generally below 10 ng/ml. The breast-cancer Hospital and Holt Radium Institute, Man- patients showed the same proportion and chester. range of detectable serum levels as age- matched controls. Serum ox-lactalbumin wNas The synthetic DNA-like polynucleotide measured prospectively in 100 patients poly(dC-dG) has been methylated in vitro with a variety of breast conditions. Although with either dimethyl sulphate (DMS) or the marked differences were found between potent carcinogen N-methyl-N-nitrosourea patients with benign and malignant breast (MNU) and the levels of the various methyla- diseases, these were entirely due to the tion products determined. Treatment with differeing age structures of the two groups. either DMS or MNU resulted in the formation It is concluded that although synthesis of of 3-methylguanine, 7-methylguanine and o-lactalbumin occurs in about a third of 3-methylcytosine whilst MNU-methylation human breast carcinomas, assay of this also produced 06-methylguanine and phos- protein in blood is unlikely to help in the photriesters. The methylated polymers were diagnosis or management of breast cancer. then used as templates for E. coli DNA However, the presence of o-lactalbumin in polymerase I in an in vitro assay and the tumour cytosol is related to the oestrogen- amounts of complementary and non-com- receptor content and may indicate the plementary base incorporation measured in tumour's hormone responsiveness. concurrent assays. The DMS-methylated polymer did not produce any mis-incorpora- A COMPARATIVE STUDY OF TWO tion, indicating that the products of DMS- TUMOUR MARKERS IN BREAST methylation do not miscode. The MNU- CANCER. F. SEARLE, K. D. BAGSHAWE and methylated polymer directed the incorpora- G. GOKA, Department of Medical Oncology, tion of thymine but not of adenine. Presum- Charing Cross Hospital, London. ably this was due to the presence of o6- methylguanine (a promutagenic base) in the Marked serum elevations of carcino- template. The thymine incorporation, how- embryonic antigen (Chu and Nemoto (1973) ever, varied with the ratio of the 5'-triphos- ABSTRACTS OF MEMBERS PAPERS 405 phates of deoxythymidine and deoxycytidine COMPETITIVE BINDING OF CYCLO- in the assay, and wtas less than the o6- PHOSPHAMIDE AND ITS META- methylguanine content of the template. BOLITES WITH CYCLIC-AMP-BIND- These results indicate that 06-methylguanine ING PROTEINS M. J. TISDALE, Depart- is capable of miscoding during DNA synthesis ment of Biochemistry, St. Thomass Hospital but the miscoding competes with the normal Medical School, London. incorporation of cytidine. 3-Methylcytidine, which has been shown to lead to mis-incor- There is a similarity in the biochemical poration wsith RNA polymerase (Ludlum effects of cyclophosphamide and cyclic AMP. (1971) Biochirn. biophys. Acta, 247, 412) Both produce hyperglycemia and cause an does not miscode in our DNA polymerase I increase in tyrosine transamminase, ornithine system. The competitive nature of the 06- decarboxylase and alkaline phosphatase acti- methylguanine miscoding is of interest: vity. This suggests that cyclophosphamide presumably it could be another of the many or its metabolites may interact with cyclic- factors determining the tissue specificity of AMP-specific proteins. A 4-hydroxyl substi- methylating carcinogens. tuent in the 1,3,2-oxazaphosphorine ring is required for inhibition of AMP binding to both AMP phosphodiesterase and to the regulatory subunit of the AMP-protein kinase CORRECTION OF CHANGES IN holoenzyme. Binding to the latter causes an LIVER METABOLITES OF MICE activation of the kinase and results in a dis- FOLLOWING CURATIVE TUMOUR sociation into regulatory and catalytic sub- RESECTION. K. C. CALMAN, R. A. Mc- units. The inhibitor constant, Ki, for the ALLISTER and M. SOUKOP, Department of inhibition of AMP binding to the protein Clinical Oncology, Gartnavel General Hospital, kinase holoenzyme (0-19 mM) correlates well Glasgow and Department of Surgery, We8tern with that for inhibition of the low Km form Infirmary, Glasgow. of the phosphodiesterase. In both cases inhibi- Earlier work (Calman and McAllister (1975) tion is of the competitive type. Although the Br. J. Surg., 62, 161; Br. J. Cancer, 32, 247, Ki value for inhibition of phosphodiesterase BACR presentation, Swansea, 31 March by 4-hydroxycyclophosphamide is much 1976), demonstrated in the non-involved higher than the ID50 value, it causes a time- liver of mice bearing a TLX-5 lymphoma, dependent inactivation of the enzyme prob- C3H mammary tumour, or Sarcoma 180, ably due to the release of N,N-di(2-chloro- significant alterations in metabolites, in ethyl)phosphorodiamidic acid. Thus the low particular coenzyme A and citrate. Extension affinity binding to phosphodiesterase could of this work has been conducted with the act as a highly efficient mechanism for C3H mammary tumour and TLX-5 lym- enzyme inhibition. Although 4-ketocyclo- phoma systems. With the C3H mammary phosphamide resembles 4-hydroxycyclophos- tumour, significant depressions (P < 0.001) phamide in electron-donating properties, it is of CoA content of liver occurred in the inactive with respect to binding to AMP- presence of a primary tumour (mean weight specific sites. This probably results from the 0.5 g). Curative resection of this small difference in conformation of the rings of tumour caused a return of CoA levels to the these two compounds. normal range. In a second experiment, inoculation of a cell-free supernatant of the TLX-5 lymphoma into normal mice mirrored HYDROXYUREA the metabolic alterations, i.e. fall in CoA "SUICIDE" STUDIES and a rise in citrate levels which had been seen ON CLONOGENIC CELLS OF THE in tumour-bearing animals. Interestingly, LEWIS LUNG CARCINOMA. A. E. similar increases in spleen weight and con- BATEMAN and G. G. STEEL, Division of comitant involution of thymus were seen in Biophysics, Institute of Cancer Research, both groups of mice. In conclusion, further Sutton, Surrey. support is given to the suggestion that these Studies on cells synchronized in vitro have changes in liver metabolites are directly demonstrated that the level of killing by related to the presence of a tumour product. cytotoxic agents varies with the position of However, the nature of this is as yet unknown. the cells in the cell cycle. We present results 406 B.A.C.R. 18TH ANNUAL GENERAL MEETING

on Lewis lung tumour clonogenic cells treated antineoplastic agent. AMPh was, however, in vivo, which show variations in survival more effective than AMOH against trans- between S-phase and non-S-phase cells after plantable tumours containing comparatively treatment with cytotoxic drugs. The hydroxy- high levels of alkaline phosphatase. A positive urea suicide technique is used in vitro to correlation was observed between sensitivity measure the proportion of clonogenic cells to aniline mustard (AM) and tumour ,B- in S-phase both for untreated cells and for glucuronidase levels, thus confirming previous cells treated in vivo with cyclophosphamide findings (Connors and Whisson (1966) Nature, (CY) 1-(2-chloroethyl)-3-cyclohexyl-1-nitro- Lond., 210, 866). Although sensitivity to sourea (CCNU) and irradiation. Forty-five AMG1 also correlated with tumour /-glucuro- per cent of untreated clonogenic cells are in nidase activities this agent was less effective S phase, as thus determined, whereas up to than AM. 70% of cells surviving CY and 85% of cells surviving CCNU are in S. We conclude that S-phase cells are more resistant than G1 or VIABLE TUMOUR REGIONS INAC- G2 cells to these agents. CESSIBLE TO CHEMOTHERAPEUTIC AGENTS AND A POSSIBLE NEW O-PHOSPHATE AND O-GLUCURO- STRATEGY FOR INACTIVATING NIDE DERIVATIVES OF p-HYDRO- THEM. R. J. GOLDACRE, Chester Beatty XYANILINE MUSTARD: POTENTIAL Research Institute, London. LATENT ANTINEOPLASTIC AGENTS. Studies with systemic dyes have shown that P. WORKMAN* and J. A. DOUBLE, Depart- advanced tumours have large ischaemic ment of Cancer Research, University of Leeds. zones frequently containing substantial quan- tities of living tumour cells. The question is: The 0-phosphate (AMPh) and 0-glucuro- are these cells responsible for tumour recur- nide (AMG1) esters of p-hydroxyaniline rence after chemotherapy? mustard (AMOH) were synthesised as poten- Transplantations were made from both the tial selective agents for tumours containing vascular and ischaemic zones (as marked out high levels of phosphatase and ,B-glucuroni- by systemic dyes) of advanced (9-day) dase, respectively. Specificity would be Walker tumours after the rats bearing the dependent upon their localized conversion tumours had been given chemotherapy by to the potently cytotoxic AMOH catalysed melaphalan at various doses. As the dose by tumour enzymes (Bukhari, Everett and increased, the percentage of takes from the Ross (1971) Biochem. Pharnwac, 21, 963). vascular zone fell from 100% to zero, whereas Partitioning studies showed that AMPh and the takes from the ischaemic zone remained AMG1 were more polar than AMOH, due to fairly constant at about 20%. the presence of the ionized phosphate and This shows clearly that chemotherapeutic glucuronate moieties. The chemical half- agents do not reach all stem cells in advanced life (t1/2) of the mustard group of AMG1 in tumours. Modifying the chemical structure aqueous solution (21 min) was longer than of drugs is unlikely to affect the lack of trans- that of AMOH (12 min); AMPh (tl/2== port in ischaemic regions, and a new strategy 13 min) was, however, as reactive as AMOH. is required for dealing with the inaccessible Enzyme kinetic studies have shown that cells. The following experiments suggest a AMPh was hydrolysed more rapidly by acid possible solution. and alkaline phosphatases of mouse bone Advanced Walker tumours after chemo- marrow and small intestinal mucosa than by therapy nearly all recurred when left in situ, the corresponding enzymes of transplantable but when transplanted whole to new hosts, mouse tumours. Km values for normal and no tumours grew. However, when the ischae- neoplastic mouse tissues were similar. In mic zone was transplanted after removing addition, AMPh was rapidly hydrolysed by the vascular (killed) shell, many tumours blood serum phosphatases. It was thus grew. Therefore, the stem cells in the ischae- unlikely that AMPh would be a selective mic zone are unable to penetrate the killed * Present address: MRC Clinical Oncology and (formerly vascular) shell which has no blood Radiotherapeutics Unit, The Medical School, Hills supply since its vessels were cut for the Road, Cambridge. transplantation. ABSTRACTS OF MEMBERS PAPERS 407

A comparable impenetrable shell was SERA. C. E. Newman, C. H. J. Ford and generated in situ by a combination of sero- H. J. STOKES, University Department of Sur- tonin, which selectively shuts down tumour gery, Queen Elizabeth Hospital, Birmingham; blood supply, followed after 4 h by melphalan. G. J. O'NEILL, G. D. Searle Research Labora- The serotonin doubled the cure rate, and tories, High Wycombe; and R. A. THOMSON, trebled the survival rate (half life after treat- Regional Immunology Laboratory, East Bir- ment) of rats bearing advanced Walker mingham Hospital, Birmingham. tumours. Thirty xenoantisera have been prepared in goats against lung cancers. Haemaglutinat- SPECIFICITY OF IgG ANTIBODIES ing, haemolytic and lymphocytotoxic anti- IN HODGKIN'S DISEASE. D. B. JONES, bodies were removed by sequential absorp- E. V. ELLIOTT,* S. V. PAYNE and D. H. tions with human spleens, and the immuno- WRIGHT, University Department of Pathology globulin (Ig) fractions precipitated. Each and *Tenorvus Laboratory, Southampton. absorbed Ig was examined for selective uptake by the tumour cells against which it Hodgkin's spleen tissue cultured for 72 h was prepared. The test system is an indirect in the presence of 14C-leucine shows increased immunofluoreseent (IF) test using cryostat- incorporation into secreted IgG measured cut sections from specimens of the original by a specific immunoprecipitation technique, tumour, snap frozen and stored in liquid N2, when compared with controls. IgG prepared and a rabbit anti-goat gammaglobulin fluores- by affinity chromatography from the cein-isothiocyanate conjugate. Every Ig culture supernatant of one patient with a showed selective localization by the original high synthesis rate was capable of binding tumour cells. Titres ranged from neat to 1/32. to human peripheral blood lymphocytes. In most cases, non-specific fluorescence was Angibody capable of binding to human lym- observed against connective tissue and phocytes was also present in the serum of endothelial cells. This was always similar to this patient and could be typed as IgG K;A. that seen with an Ig prepared in the same way In a further series of pretreatment Hodgkin's against a mycosis fungoides tumour. Signifi- sera screened by lodinated-protein-A, 20% of cant selective localization of this Ig by lung patients showed IgG binding to human tumour cells was not observed. Absorbed lymphocytes. However, when further exam- Igs showed positive tumour-cell fluorescence ined on peripheral lymphocyte subpopula- when examined for selective localisation by tions and lymphoid cell lines, the specificity lung tumours of the same and different of this antibody was not restricted to T cells histological types, suggesting a surprisingly as suggested by del Giaco et al. (1976) high cross-reactivity. A xenoantiserum pre- Biomedicine, 25, 79). Further, when tested pared against cultured oat-cell carcinoma in a 51Cr-release assay, none of these sera cells had high titres of haemolytic (1/96) were able to kill lymphocytes in the presence haemagglutinating (1/512) and lymphocyto- of complement; preliminary results suggest toxic (1/3072) antibodies which were removed this lack of cytotoxicity may be due to the by absorption. After absorption, the IF subclass of IgG present. Binding sera fre- titre against lung tumour cells was 1/1200. quently showed other tissue autoantibody The fractionated Ig has an IF titre of 1/80 specificities and this suggests that the anti- to 1/160. This reagent has been carefully lymphocyte-antibody present may be an assessed against a panel of 8 lung cancer additional disease-associated autoantibody sections comprising 2 of each of the histolo- rather than an aetiologic factor associated gical groups viz. oat-cell, anaplastic, adeno with a lymphocyte war. and squamous. In 7/8, the tumour-cell G. S. del Giaco et al. (1976). Anti-lympho- concentration of IF-detected antibody is cyte-antibodies in Systemic Lupus Erythe- apparent at titres of 1/80 to 1/160. The control matosus and in Hodgkin's Disease: A Com- Ig (mycosis fungoides) does not demonstrate parison by Immunofluorescence. this tumour-cell concentration. This evidence suggests the selective localization of tumour- cell antigens on human lung cancer cells. IMMUNOFLUORESCENT STUDIES These may be tumour-specific, tumour-associ- OF HUMAN-LUNG-CANCER ANTI- ated or even normal cellular antigens selec- 27 408 4B.A.C.R. 18TH ANNUAL GENERAL MEETING tively cOncentrated in the tumour-cell A TWO-STAGE ASSAY FOR membrane. TUMOUR-DIRECTED CELL-MEDIAT- ED IMMUNITY. A. J. COCHRAN, R. M. MACKIE, L. J. OGG, A. M. JACKSON, C. E. Ross and G. TODD, University Departments THE DEMONSTRATION OF of Pathology and Dermatology, The Western DEPRESSED LEVELS OF T LYMPHO- Infirmary, Glasgow. CYTES IN BREAST CANCER PATIENTS IS DEPENDENT ON THE In an attempt to overcome problems of the METHOD USED. R. H. WHITEHEAD, one-stage leucocyte migration inhibition assay G. P. ROBERTS, J. THATCHER and L. E. we are investigating a two-stage test. In HUGHES, University Department of Surgery, Stage I Ficoll-Hypaque (FH) separated Welsh National School of Mkedicine. mononuclear cells are incubated with formalin- fixed cells (FC) of the appropriate tumour There are a number of conflicting reports type, of other tumour types and with on the proportion of E-rosetting cells (T formalinized normal cells. After 24 h the lymphocytes) detectable in patients with migration-inhibiting activity of the various breast cancer. However, different rosetting supernatants is assessed relative to the super- techniques have been used in each study, and natant of FH cells incubated in the absence of it was felt that this might be the cause of the formalinized cells. The indicator-cell popula- differing results obtained. We have therefore tion is gravity-sedimented peripheral blood compared three standard rosetting tech- leucocytes from a normal individual. Active niques: supernatants resulted more often from co- cultre of melanoma leucocytes (ML) with (a) short incubation period of 1I h at melanoma cells (17/39-44%) than from 4 °C in PBS cultures of ML with other types of cells (b) overnight incubation at 4°C in PBS (6/41-150%). Active supernatants infrequently and resulted from culture of normal FH cells (c) 1-h incubation at 4°C in 5% FCS. with FC (control FH cells/melanoma FC, In addition, the effect of methodology on 1/30 (3%o) control FH cells/other FC, 4/22 cancer-serum-induced inhibition of E-rosette (180 ). Active supernatants were most fre- formation by normal lymphocytes has been quent with FH cells from Stage II melanoma studied. It was found that when the number patients (Stage I, 1/6 (17%), Stage II, of E-rosetting cells was determined using 14/25 (56%) and Stage III, 2/8 (25%). incubation at 4°C for 1 I h, levels in women In combinations of ML with melanoma FC with breast cancer and an age-matched the reaction frequency increased with the control group were both significantly lower number of FC preparations tested. This was than the levels obtained for a young control not seen with combinations of ML and other group. There was no difference between the FC or control leucocyte cultures with mela- 3 groups when rosetting was performed in noma or other FC. The direct and two-stage 5% FCS or by overnight incubation, as the assays were concordant in about 700% of proportion of E-rosetting cells was then concurrent tests. higher in the first two groups. Inhibition of E-rosette formation by incubating normal lymphocytes in breast cancer serum could A CONTROLLED TRIAL OF ACTIVE be demonstrated using a short incubation IMMUNOTHERAPY IN THE MAN- period (1 1 h) but not after overnight incuba- AGEMENT OF STAGE IIB MALIG- tion or after incubating the treated lympho- NANT MELANOMA. M. B. MCILLMURRAY, cytes in saline overnight at 4 °C before M. J. EMBLETON, W. G. REEVES, M. J. S. rosetting. These findings explain the previous LANGMAN and M. DEANE,* Department of conflicting results, and suggest the presence Therapeutics, Cancer Research Campaign of a factor(s) on the surface of T lymphocytes Laboratories, Department of Immunology, of cancer patients and in the sera of these University of Nottingham and *the Plastic patients which binds reversibly to the lympho- Unit, City Hospital, Nottingham. cyte surface and in some way masks the Within two years of operation about three- E-receptor site. quarters of all patients with malignant ABSTRACTS OF MEMBERS PAPERS 409

Recurrence rate D)eath rate a- a Vaccinate(c Control Vaccinated Control 3 mouiths 3/8 0/7 2/8 0/7 6 months 4/8 2/7 3/8 0/7 12 months 4/8 4/6 4/8 0/6 melanoma who have regional nodes involved wNere monitored immunologically before treat- (Stage IIb) will have obvious recurrences ment, and at 3, 6 and 12 months after treat- and more than half of these will have died ment, in order to look for changes in immune (Lane, N., Lattes, R. and Malm, J. (1958) function wvhich might correlate with their Cancer, 11, 1025. Inmprovements in results clinical course, and thus provide a test with are therefore only likely to ensue if some prognostic significance. Immunocompetence other treatment can be added to excision. of patients was assessed by skin tests, using Animal studies indicate that host resistance the recall antigens PPD and Varidase, to transplantable tumours is enhanced when measurement of blood components, and by BCG and tumour-cell vaccines are given lymphocyte transformation ini vitro wvith together as contact immunotherapy, provided various agents. Attempts wvere made to that the tumour load is small, suggesting that evaluate tumour-directed immunity using such a combination mav be a more effective in vitro tests for both leucocyte-mediated immunostimulant than either given alone and antibody-mediated activity against mela- (Bast, R. C. et al. (1974) New Engl. J. Med. noma extracts or cultured cells. Th-ere wvas a 290, 1413) Fifteen patients with Stage IIb trend towAards earlier tumour recurrences malignant melanoma were randomly allocated in patients wvith poor skin reactivity to PPD to either a treatment group in which they and Varidase at the beginning of treatment. received a vaccine (3 x 107 live BCG and The in vitro measurements fluctuated through- 5 X 107 autologous irradiated tumour cells) out the time course for each patient, and or a control group who received no further none revealed any significant differences treatment after surgical eradication of their between patients with good or bad prognosis, disease. The recurrence and death rates in or between vaccinated and control patients. the two groups one year later are shown in It is concluded that in vitro monitoring using the table. present techniques is of no practical value in These results suggest that active immuno- prognosis of malignant melanoma. therapy may cause tumour enhancement in some patients with malignant melanoma, and RESULTS, FOR 27 MONTHS OF that claims of benefit from uncontrolled and FOLLOW-UP, OF A STRATIFIED unrandomized studies should not be readily RANDOMIZED TRIAL OF INTRA- accepted. DERMAL BCG IN ADDITION TO CON- VENTIONAL TREATMENT IN PATIENTS WITH LUNG CANCER. IMMUNOLOGICAL MONITORING H. M. ANTHONY, K. E. MADSEN, M. K. OF PATIENTS UNDERGOING ACTIVE MASON and G. H. TEMPLEMAN, University IMMUNOTHERAPY FOR STAGE IIB Department of Immunology, Leeds General MALIGNANT MELANOMA. M. J. Infirmary and Killingbeck Hospital, Leeds. EMBLETON, M. B. MCILLMURRAY, J. H. RANSOM and W. G. REEVES, Cancer Research Random allocation of 75 men with con- Campaign Laboratories and Departments of firmed bronchial carcinoma to BCG (0-1 ml Therapeutics and Immunology, University of Glaxo BCG i.d. monthly to 6 months) or Nottingham. control within a stratification system based on conventional therapy and other prognostic Fifteen patients with Stage IIB malignant factors, showed no significant prolongation of melanoma were allocated to a group receiving life by BCG, by sequential analysis of 23 a vaccine (3 x 107 live BCG and 5 x 107 pairs or by life table analysis (computer autologous irradiated tumour cells) following program kindly loaned by Dr P. G. Smith of tumour resection, and a control group who Oxford). The latter analysis shoNwed signifi- were given no further treatment. All patients cant prolongation of survival in "acceptable 410 B.A.C.R. 18TH ANNUAL GENERAL MEETING clinical" condition (P - 0.0049) or good mouse DNA and 10-12 viral genes per general condition (P -- 0-0112) for BCG- haploid genome in DNA from the FV- treated radical radiotherapy patients. For transformed Friend cell. A significant almost all groups, BCG had greater effect on minor proportion (20-300//) of the FV prolongation of "acceptable clinical" or good eDNA probe anneals only to virus general condition than on survival to death. related sequences in the transformed- BCG also reduced weight loss. These effects cell DNA, indicating that additional could be due to increase in T-lymphocyte FV-related sequences are integrated proportion by BCG (Anthony et al. (1975) in the transformed-cell DNA. The Clin. exp. Immunol., 20, 40) or to stimulation Friend virus consists of a helper of macrophages. In patients with squamous lymphatic leukaemia virus (LLV) and carcinoma, peripheral blood lymphocytes the erythroid-specific defective spleen- and monocytes directly correlated with focus-forming virus (SFFV). We have length of survival (P < 0-02, P < 0.04) in isolated by end-point dilution and keeping with partial immune control. For cloned a cell line producing only the oat cell carcinoma, lymphocyte numbers LLV component. The 70S RNA from inversely correlated with "Survival" (P < LLV can be used to remove the LLV 0.04) as did the trend with monocyte number sequences from FV eDNA, resulting (P < 0.03) suggesting resistance to immune in enrichment of the erythroid-specific cytolysis in oat cell carcinomas and a stimu- sequences and/or the sequences in- lating effect for immune attack. volved in transformation of the target cell.

C-TYPE RNA TUMOUR VIRUSES: ISOLATION AND CHARACTERIZA- CELL-MEDIATED RESPONSE TO TION OF A COMPLETE DNA COPY SIMIAN ONCORNAVIRUSES IN OF THE ERYTHROID-SPECIFIC WOMEN DURING PREGNANCY. L. FRIEND VIRUS GENOME. I. B. PRAG- THIRY, S. SPRECHER-GOLDBERGER, M. Bos- NELL, W. OSTERTAG* and J. PAUL, Beatson SENS and F. NEURAY, Institut Pasteur du Institute for Cancer Research, Wolfson Brabant and Free University of Brussels. Laboratory for Molecular Pathology, Glasgow (Introduced by F. J. Lejeune). and *Max Planck Institut fur Exp. Med., Baboon type-C virus and Mason-Pfizer G&ttingen, West Germany. virus (MPV) were added to short term human Friend virus (FV) is a C-type RNA leucocyte cultures and induced a high level tumour virus which induces an erythro- of thymidine incorporation, due to virus leukaemia in susceptible mice, and the trans- replication. Killed viruscs caused a limited formed erythroid cells can be maintained in but significant level of thymidine incorpora- tissue culture (Friend et al. (1966) Nat. Canc. tion in some leucocyte cultures, indicating Monogr., 22, 505). These cells can be stimu- that some individuals possess lymphocytes lated to differentiate along the erythroid sensitized to antigens carried by one of the pathway by addition of aprotonic solvents viruses. Cells chronically infected with each such asdimethylsulphoxide (Friend etal. (1971) virus, or not infected, were treated with Proc. Nat. Acad. Sci., U.S.A., 68, 378; Ostertag mitomyein C; one type of infected culture et al. (1972) Nature, New Biol., 243, 203). We specifically stimulated some leucocyte cul- have synthesized and characterized a viral tures, but responses to the infected cells 3H-labelled complementary DNA (eDNA) were not always associated with responses derived from the Friend virus genome. to the corresponding virus. Because oncor- Hybridization analysis has shown that: navirus particles have been described in placentas, lymphocyte responses to th-e (a) The FV eDNA is a full-lengthl copy of Baboon virus and to MPV were studied in the Friend virus genome. 30 women at the end of pregnancy and in 37 (b) The base-sequence complexity of the non pregant women. Lymphocyte responses viral genome is 4 x 106 daltons. to cells infected with either the Baboon (c) There are 4-7 viral genes homologous to virus or with MPV were found in 36% and the FV genome in normal (DBA/2) 2.60% of the pregnant and non-pregnant ABSTRACTS OF MEMBERS PAPERS 411 w8omen, respectively, and were most frequent as a tool to detect one (or more) neutraliza- in -women with many gestations. The number tion antigens to one (or more) "putative" of responses to one of the two virus particles human melanomaviruses. was not different in pregnant and non- pregnant women, but increased with the number of gestations, since they were found CROSS-REACTIVITY OF ANTISERA in 0% of gravida 0, in 16°/ of women with TO ONCOGENIC RNA VIRUS PRO- 1-4 gestations, and in 530o of women with TEINS WITH HUMAN LEUKAEMIA 5-7 gestations. Antigens similar to those of CELLS. A. PILLAI,*, N. HOGGt and R. T. D. Baboon virus or MPV may be expressed OLIVER, *Imperial Cancer Research Fund, during gestations. Department of Medical Oncology, St Bartholo- mew's Hospital, London, and tImperial Cancer Research Fund, Tumour Immunology Unit, University College, London. VSV PSEUDOTYPES PRODUCED IN HUMAN MELANOMA CELL LINES. Antisera raised in rabbits against PAGE- N. VAN TIEGHEM, D. LITEANU, A. F. VERCAM- separated proteins from disrupted Moloney MEN-GRANDJEAN, P. VANDENBUSSCHE, D. virus have been tested against a panel of DEKEGEL, L. BEAUMONT and F. J. LEJEUNE, leukaemia cells from 17 patients w%Nith AML, Universite Libre de Bruxelles, Institut Pasteur 6 patients with ALL, 5 patients with CLL, du Brabant and Institut Bordet, Bruxelles. 7 remission lymphocytes from 7 patients with acute leukaemia and lymphocytes from Three human melanoma sublines w-ere 8 normal laboratory controls. A standard investigated for viral particles. Electron microcytotoxicity assay writh absorbed wean- microscope studies showed a high production ling rabbit serum as a complement source was of melanosomes and viral particles budding used. Reactivity was greatest in serum into the cisternae ofthe endoplasmic reticulum against gp 79/80 (18/28 positive with from in cells derived from a subcutaneous meta- 12-80% cytotoxicity) and least with serum stasis (HM6B-A). This viral expression was against p30 (6/28 positive with from 10-50% related to melanin expression, and could be cytotoxicity). Intermediate reactivity was switched on or off by adding to, or subtract- observed with antisera to p15 (envelope), ing tyrosine from the culture media. When p15 and p12. Reactivity against remission infecting an amelanotic subline (HM6B-N), lymphocytes was considerably less than provided by the same patient, with a VSV against leukaemia cells, although 3/15 cells thermolabile mutant (tl) there was produc- tested did show slight reactivity (12-42%). tion of a VSV pseudotype (Zavada, J. (1972) Cross-absorption experiments with different Nature, New Biol., 240, 124). The coat of types of leukaemia cells suggest that the progeny VSV was modified: the thermolabile determinants detected on ALL and CLL virus had become thermostable. This cell cells are the same as on AML. Studies are in line did not show virus expression either progress, using the lysostrip technique, to after the tyrosine test or after treatment clarify the relationship of the determinants with halogenated pyrimidines (Lowy, D. R., detected by these sera to normal tissue Rowe, W. P., Teich, N. and Hartley, J. W. antigens, B2 microglobulin and HLA. (1971) Science, 174, 155). The amount of VSV pseudotype particles was increased 2500 x in the presence of (5-JUdR). Reverse transcriptase activity in the culture super- EFFECT OF HYPERTHERMIA ON natant was barely detectable. The VSV THE IMMUNOCOMPETENCE OF pseudotypes could be neutralized by the NORMAL AND VX2 TUMOUR-BEAR- patient's serum. When treating another ING RABBITS. S. A. SHAH and J. A. amelanotic subline (HM6A) from the same DICKSON, Cancer Research Unit, University origin with DL-DOPA (8-0 x 10-5 M/ml) Department of Clinical Biochemistry, Royal it was possible to detect a few virus-like Victoria Infirmary, Newcastle-upon-Tyne. particles. Thermostable pseudotypes were FolloNwing effective heat treatment of a also obtained in the presence of this drug. primary cancer in man and in animals, Thus, VSV pseudotype particles could be used tumour mnetastases also disappear wvith cure 412 B.A.C.R. 18TH ANNUAL GENERAL MEETING of the host. With the rabbit VX2 carcinoma, responses to treatments such as i.v. BCG heat applied locally to the tumour is more immunotherapy, but dogs involved with effective than total-body heating, and it is metastatic disease showed lower responses. believed that, an altered response of the Responses of BCG-treated tumour bearers animal's immune system may be involved were lower than those seen in healthy BCG- in this difference (Dickson (1976) Int. Syymp. treated controls. The use of autochthonous Cancer Therapy by Hyperthermia and Radia- tumour target cells was unsatisfactory, as tion. Am. Coll. Radiology Press, Baltimore, short-term cell cultures were resistant to Md. p. 134). In the present study, 9/19 VX2 lysis in the 5-Cr-release assay when compared tumour (15-20 ml) bearing rabbits treated by with established allogeneic cell lines. The Local Radiofrequency Heating (LRFH, 47- non-specific effector cell rosetted with human 50°C/30 min), and 1/8 rabbits treated by erythrocytes, a canine T-cell marker, but LRFH followved 8 days later by Total-body carbonyl iron treatment also reduced non- Hyperthermia (TBH) at 42°C (1 h on each specific cytotoxicity in some cases. Certain of 3 successive days) wvere cured. Skin target cell lines, e.g. osteosarcoma, were response to challenge with Dinitrochloro- particularly sensitive to non-specific lysis. benzene (DNCB) or tumour extracts, and Lymphocytotoxicity detected in the allo- the anamnestic response to bovine serum geneic 51Cr release assay was therefore not albumin (BSA) in tumour bearing rabbits directed at tumour specific antigens and was increased following LRFH with untreated present also in healthy controls. Spontaneous or (LRFH + TBH) treated rabbits. In vitro, canine neoplasms appeared to lack histolo- lymphocytes plus serum from cured animals gical-type-specific antigens capable of elicit- caused greater inhibition (30-650O)) of tumour ing a cell-mediated cytotoxic response in the cells than lymphocytes plus serum from tumour-bearing host. untreated animals (20-300/o). In normal rabbits, LRFH or TBH did not affect the skin response to DNCB. The response to BSA was reduced by up to 1/25 normal level STUDIES ON THE MICROCYTO- following LRFH and by up to 1/80 normal TOXICITY TEST: THE UPTAKE OF level after TBH. The data support the AMINO ACIDS BUT NOT NUCLEO- previous postulate that TBH suppresses the SIDES PROVIDES A DIRECT AND immune system. QUANTITATIVE MEASURE OF TARGET CELL SURVIVAL. R. C. REES and C. G. BROOKS, Cancer Research Cam- paign Laboratories, University of Nottingham. CELL-MEDIATED CYTOTOXICITY IN TUMOUR-BEARING DOGS. G. R. BET- Optimal labelling of tumour cells with TON, Oncology Unit, Department of Clinical radionucleosides required that these precur- Veterinary Medicine, University of Cambridge. sors be present at high concentration, because many tumour-cell targets did not utilize Dogs bearing spontaneous neoplasms were exogenous nucleoside efficiently when present tested for peripheral blood lymphocyto- at low concentration. However, even using toxicity using 51Cr-labelled allogeneic tumour relatively high concentrations of radio- target cells. Where possible, sequential testing nucleoside, large discrepancies between radio- was performed during the course of therapy. nucleoside uptake and -cell survival assessed Analysis of results obtaiiied with melanoma-, by cell counting were often found. Analysis osteosarcoma- and mammary-carcinoma- revealed that two types of soluble factors bearer peripheral blood lymphocyte prepara- released by lymphoid cells were responsible tions tested against tumour target cells of the for the discrepancies. same and different histological types shoved no evidence for type specificity. Healthy (a) Competitive inhibitors of nucleoside control donors also exhibited non-specific uptake, removable by washing. cytotoxicity in a proportion of donors, that (b) Factors which caused an irreversible was not significantly different from that disruption of tumour cell nucleoside observed in tumour bearers. Sequential metabolism without any apparent testing oftumour bearers showed no consistent effect on cell survival. ABSTRACTS OF MEMBERS PAPERS 413 In contrast to the severe problems encoun- liquid N2 after programmed freezing, and tered with radionucleosides, radio-labelled almost full cytotoxic activity recovered after amino acids were taken up equally avidly by thawing. These experiments show that potent all tumour cells tested, and provided a direct cytotoxic activity can be generated in lym- and precise measure of target-cell survival, phocyte culture, and that some target-cell because neither competitive nor non-com- systems may require a microcytotoxicity test petitive interference with amino-acid uptake to detect cytotoxicity. The exact culture caused by lymphocytes or lymphocyte factors requirements for the development of cytoxi- was detectable. The use of the y-emitting city and the nature and mechanisms of its 75Se-methionine as precursor permitted a effector phase are at present under investi- simple and rapid method of quantitating gation. target-cell survival in the microcytotoxicity test.

THE USE OF A 5lCr-RELEASE TEST FOR THE DETECTION OF COMPLE- SPONTANEOUS DEVELOPMENT OF MENT-DEPENDENT CYTOTOXICITY CYTOTOXIC ACTIVITY IN CUL- OF RAT HEPATOMA-BEARER TURES OF LYMPH NODE CELLS SERUM. M. R. PRICE, Cancer Research FROM TUMOUR-BEARING RATS. Campaign Laboratories, University of Not- R. A. ROBINS, Cancer Research Campaign tingham. Laboratories, University of Nottingham. A short-term 5ICr-release test was devel- During in vitro experiments attempting to oped for the detection of complement- induce cellular cytotoxicity by syngeneic dependent cytolytic activity of sera from lymphocytes to chemically induced rat donors bearing an aminoazo-dye-induced rat tumours, and to boost the cytotoxicity of hepatoma for transplanted tumour cells. lymph node cells from rats exposed to tumour Cytotoxicity was evident in the serum of in vivo, it was observed that lymph node donors bearing i.p. implants of hepatoma, cells from rats bearing a transplanted methyl- but not in sera from animals bearing s.c. cholanthrene-induced sarcoma became highly transplants or immunized with y-irradiated cytotoxic when cultured without addition tumour tissue. Sera from syngeneic multi- of tumour antigen. This cytotoxicity could parous donors sensitized to tumour-associated be very strong: significant reduction in embryonic antigens, also failed to exhibit a target-cell survival wvas observed with 100 cytotoxic response against tumour cells. effector cells per wvell in a 125IUdR post- Cytotoxic tumour-bearer sera displayed indi- label microcytotoxicity test (effector: target vidually distinct, tumour-specific reactivity ratio of 1:5); at E:T ratios of 10:1, over against hepatoma target cells which, with 90% cytotoxicity was obtained. In contrast, selected sera, was still detectable at final cultured lymph node cells from normal rats dilutions of 1/100. Although these sera con- only showed low levels of cytotoxicity at tained tumour-specific IgG antibody demon- relatively high E:T ratios. Cultured lymph strable using the indirect membrane-immuno- node cells from tumour-bearing and normal fluorescence test, cytolytic activity fraction- rats were also tested in a 5ICr-release assay. ated in the 19s region of Sephadex G200 gel- Cytotoxicity w%as not detected in this assay, filtration column eluates. This reactivity was even with a 17-h incubation period and high removed by absorption w-ith cells of the same E: T ratios using lymphocyte preparations hepatoma as that borne by serum donors, shown to be highly cytotoxic in a 24-48-h whereas absorption with cells of other post-label microcytotoxicity test. At least 4 hepatomas was without effect. The test days of culture were necessary for augmented developed is both objective and reproducible cytotoxicity to be detected, but cytotoxicity and, used in conjunction with the charac- did not increase further between 4 and 7 days terized syngeneic serological reagents avail- of culture. Yields of lymphocytes were norm- able, it should prove of value for the quanti- ally between 40 and 5000 after 7 days. tation of tumour antigens associated with Cultured lymphocytes could be stored in chemically induced rat hepatomas. 414 B.A.C.R. 18TH ANNUAL GENERAL MEETING ANTIGENIC HETEROGENEITY geneic hamsters with polyoma-virus-trans- WITHIN PRIMARY 3-METHYL- formed BHK 21/C13 fibroblasts, grew rapidly CHOLANTHRENE-INDUCED RAT and invaded adjacent host muscle and con- SARCOMAS. M. V. PIMM, Cancer Research nective tissue. Host inflammatory cells mere Campaign Laboratories, University of Not- observed, dispersed singly and in groups tingham. within tumour tissue, and these cells appeared to mediate focal necrosis of tumour cells Studies by Prehn (1970) J. natn. Cancer -which could not be accounted for simply Inst. 45, 1039, with primary 3-methylcho- by lack of tumour vascularization or coagula- lanthrene (Mc)-induced mouse sarcomas have tion necrosis. Ultrastructural studies revealed demonstrated the possibility of antigenic the presence of plasma cells and aggregates heterogeneity within individual established of lymphocytes and phagocytes among the tumours, so that a transplant line initiated tumour cells, which are indicative of a host with tissue from one part of a primary sar- immune response against the tumour (Moore, coma was occasionally antigenically distinct Nisbet and Haigh (1973) Br. J. Cancer, 28, from a line established from another part of Suppl. 1). Ultrastructural examination of the same tumour. In the present study the contacts between lymphocytes and tumour immunogenicities of in vivo lines established cells did not reveal the presence of any from primary Me-induced rat sarcomas have specialized intercellular junctions although been compared with those of lines initiated protrusion of lymphocyte pseudopodia into from tumour recurrences at the site of the tumour cells and tumour-cell lysis were primaries' surgical excisions. Lines from 2/4 observed. Endocytosis and destruction of primary sarcomas showed little or no immu- tumour cells by polymorphonuclear leuco- nogenicity, as assessed by protection to cytes (PMNs) and mononuclear phagocytes challenge afforded by graft excision or were observed; whereas PMNs engaged in implantation of irradiated tissue. In contrast, microphagocytosis (i.e. phagocytosis of small lines from all 4 recurrences were immuno- portions of tumour cells) the mononuclear genic, giving protection against up to phagocytes attempted to entire 5 x 106 tumour cells. Most importantly, with engulf cells. all 4 tumours, lines established from recur- The mononuclear phagocytes had the ultra- rences were antigenically distinct from lines structural appearance of "activated macro- from their original primary sarcomas, so that phages" (Carr (1973) The Macrophage: a immunization with regrowth lines gave no review of ultrastructure and function, Acad. protection to the lines from the primaries, Press) and appeared to mediate much of the and vice versa. These studies support the tumour fibroblast destruction as observed concept that primary Mc-induced tumours in other tumour systems (Evans (1973) Br. may be antigenically heterogeneous, and J. Cancer, 28, Suppl. 1, 19). Accumulation demonstrate that outgrowth of a second, of intercellular material around many of the antigenically distinct, tumour follows surgical tumour fibroblasts appeared to afford these removal of the primary. These findings also cells protection from direct contact and have implications for the design of immuno- attack by host inflammatory cells. therapy protocols for recurrences or meta- stases from experimental or even human tumours. ALVEOLAR MACROPHAGE CYTO- TOXICITY IN THE DOG. N. T. GORMAN, Oncology Unit, School of Veterinary Clinical ULTRASTRUCTURAL STUDIES OF Studies, Cambridge University. INTERACTIONS BETWEEN HOST INFLAMMATORY CELLS AND The cytotoxicity of alveolar inacrophages TUMOUR CELLS WITHIN TRANS- from a total of 33 dogs has been examined PLANTABLE HAMSTER FIBROSAR- using the 51Cr-release assay with allogeneic COMAS. R. G. P. PUGH-HUMPHREYS, long-term tissue-culture cells as targets. It Experimental Pathology Unit, Department of has been found that alveolar macrophages Zoology, Aberdeen University. from unstimulated dogs (8) do not exhibit any cytotoxicity. However, in the case of Transplantable malignant fibrosarcomas, those animals which received i.v. BCG (10) produced initially by s.c. injection of syn- and developed diffuse granulomatous lesions ABSTRACTS OF MEMBERS PAPERS 415 of the lung, the cytotoxicity was marked; this A high dose of CA similar to that used by was not found in 3 dogs which had received Fisher et al. (1976) J. natn. Cancer Inst., 56, intrathoracic BCG. The observed cytotoxicity 571, had little effect on primary tumour appeared to be non-specific, with a lack of growth but significantly enhanced metastases. selectivity between cells of neoplastic origin Iv. C. parvum given to these mice before or or normal canine kidney at the ratios exam- after CA treatment caused no further ined (10:1, 20:1, 40:1). An attempt has tumour inhibition, but significantly reduced been made to specifically immunize 9 dogs metastases. However, the number of meta- against allogeneic tumour cells in one of the stases in mice which were given combined following ways: C. parvum and CA was not significantly different from that found in control mice, (a) 3 i.v. injections of 5 x 108 cells and significantly greater than that found in (b) 3 i.v.injections of 5 x 108 cells plus mice which received only C. parvum. Thus 3 mg BCG (Glaxo Laboratories) CA effectively counteracted the beneficial (c) 3 i.v. injections of 5 x 108 cells antimetastatic action of C. parvum. A low sonicated with Freund's adjuvant plus dose of CA (human equivalent) or 0 5 mg sparine heat-killed BCG. did not alter primary tumour growth or the In these experiments it was found that only antimetastatic action of C. parvum. dogs which received i.v. BCG demonstrated We conclude that all drugs being given to any cytotoxicity. This however, lacked patients to counteract the side effects of specificity for the immunizing cell. In a immunotherapy should be examined experi- further series of 3 dogs which received more mentally to determine their effect on both immunizations with Freund's adjuvant, cells primary and secondary tumours. and heat-killed BCG, non-specific cyto- toxicity has been observed. Examination of the supernatants of cultures of alveolar macrophage from both normal dogs and those which had received i.v. BCG failed to reveal THE MECHANISM OF THE ANTI- any soluble factor which could produce the TUMOUR EFFECT OF GLUCANS AND observed cytotoxicity. FRUCTOSANS. A COMPARISON WITH C. PAR VUM. R. BOMFORD & C. MORENO, Department of Experimental JImmu- nobiology, Wellcome Research Laboratories, Beckenham, Kent. EFFECTS OF CORTISONE ACETATE AND SPARINE ON THE PRIMARY The antitumour activity induced by glu- LEWIS LUNG CARCINOMA AND ITS cans (lentinan, yeast cell walls, pseudonigeran, PULMONARY METASTASES AND ON dextran, DEAE-dextran and dextran sul- THE ACTION OF C. PARVUM. T. E. phate) and fructosans (levan and carboxy- SADLER, P. D. E. JONES, H. D. MITCHESON methyl-levan) was compared with the activitv and J. E. CASTRO, Urology and Transplan- of C. parvum. The following effects on tumour tation Unit, Royal Postgraduate Medical systems in CBA mice were assayed: (a) adju- School, Harnmersmith Hospital, London. vant activity on the immune response against tumour-specific transplantation antigens Corynebacterium parvum inhibits the (TSTA) with a methylcholanthrene-induced growth of a variety of animal tumours and fibrosarcoma; (b) cytostatic activity of peri- it is now undergoing clinical trials. In man, toneal macrophages against radiation-induced this vaccine causes undesirable side-effects, leukaemia cells; and (c) inhibition of nodule including nausea and pyrexia. Hydrocor- formation in the lungs following i.v. injection tisone and sparine have been used to relieve of fibrosarcoma cells. these symptoms. In this study, the effects of All the polysaccharides induced cytostatic cortisone acetate (CA) or sparine on the macrophages, but the dextrans and levans did growth of the primary Lewis lung carcinoma so only after i.p. and not i.v. injection. Only and its pulmonary metastases and the action lentinan, yeast cell walls and pseudonigeran of C. parvum were investigated in C57BL were active in the lung-nodule-inhibition mice. test; and only lentinan and dextran sulphate 416 B.A.C.R. 18TH ANNUAL GENERAL MEETING showed slight adjuvant activity for TSTA. Inst., 58, 91), the deficiency being established It is concluded that the antitumour activity earlier in the gut than in bone marrow. We induced by these polysaccharides is pre- have currently been investigating the possi- dominantly non-specific macrophage-media- bility that normal proliferating tissues of the ted and much wNreaker than that found wxAith mouse can be protected from MTX toxicity by C. parvum. the administration of purine and pyrimidine nucleosides or bases. It was demonstrated that a pyrimidine alone could not protect from the toxic effects of MTX. Howrever, SYNERGISTIC COMBINATION OF combinations of thymidine (TdR) and hypox- CHEMO- AND IMMUNO-THERAPY anthine (Hx) (together with allopurinol (Ap)) IN A MOUSE TUMOUR SYSTEM. M. T. do protect the mouse from MTX toxicity, SCOTT, Department of Experimental Immuno- with an efficacy comparable to folinic acid. biology, Wellcome Research Laboratories, Furthermore, rescue of L1210-tumour-bear- Beckenham, Kent. ing animals with TdR/Hx/Ap combinations, Treatment of a chemically induced mouse following MTX treatment, can produce a solid fibrosarcoma, using either non-specific median survival in excess of that achieved (C. parvum 350 jug i.v.) or specific active with folinic-acid rescue. It would seem that (s.c. C. parvum mixed with 5 x 105 irradi- purine/pyrimidine rescue techniques may ated tumour cells) immunotherapy, 4 days exploit selective differences in salvage- after a single dose of cyclophosphamide pathw,ay utilization between tumour and (200 mg/kg) was synergistically more effec- normal tissues, and may have clinical applica- tive than either C. parvum or drug treatment tion. alone. A contributory factor may be that cyclophosphamide pretreatment has been shown to potentiate the specific antitumour immunity that arises from C. parvuminter- DIHYDROFOLATE-REDUCTASE AC- action with tumour antigen. Systemic C. TIVITY IN MOUSE GUT AFTER parvum before cyclophosphamide will poten- TREATMENT WITH METHOTREX- tiate the antitumour effects of the drug; ATE, AND RESCUE WITH VARIOUS previously ineffective low doses becoming AGENTS. A. H. CALVERT and K. R. effective. No similar potentiation of the HARRAP, Department of Biochemical Pharma- effects of another alkylating agent, Melpha- cology, Institute of Cancer Research, Suttonz, lan, was evident. Surrey. Recently, considerable attention has been paid to the selective protection of normal ENHANCEMENT OF THE ANTI- tissues from the effects of methotrexate TUMOUR EFFECTIVENESS OF (MTX) by the use of purines and pyrimidines, METHOTREXATE THROUGH SELEC- w%ith the object of increasing the.therapeutic TIVE PROTECTION OF NORMAL index of this drug (Straw et al. (1977) J. natn. TISSUES. G. A. TAYLOR, G. P. BROWMAN Cancer Inst., 58, 91). The toxicity of MTX and K. R. HARRAP, Department of Biochemical is dependent on the time for which a 950/ Pharmacology, Institute of Cancer Research, inhibition of dihydrofolate reductase (DHFR) Sutton, Surrey. is maintained (Goldie et al. (1972) Eur. J. Cancer, 8, 409; Jackson- and Harrap The use of folinic acid (citrovorum factor) (1973) Arch. Biochem. Biophys., 158, 2). to limit the toxicity of methotrexate (MTX) Therefore it is important to assess this para- to normal proliferating tissues, is an meter in addition to a study of the total established clinical procedure during intensive plasma and tissue levels of MTX in those MTX therapy ((1975) Cancer Chemother. Rep., animal models used for testing rescue proto- 6). In previous reports we have shown that cols. A technique has been developed for MTX induces a purineless state in bone mar- measuring the in vivo inhibition of DHFR, row and small intestine when administered making allowance for the extracellular fluid to mice (Talbot et al. (1976) Br. J. Cancer, contribution of MTX to the total, and the 34, 321; Straw et al. (1977) J. natn. Cancer kinetic constants of the enzyme concerned. ABSTRACTS OF MEMBERS' PAPERS 417

This has been applied to the small intestine SOME KINETIC PARAMETERS OF from C57BI mice treated with 400 mg/kg of PURINE SALVAGE ENZYMES. D. C. MTX followed by rescue with either saline, TALBOT and K. R. HARRAP, Department of thymidine, hypoxanthine + allopurinol, Biochemical Pharmacology, Institute of Cancer hypoxanthine + allopurinol + thymidine or Research, Sutton, Surrey. folinic acid. In all the rescued groups, methotrexate levels in plasma, bone marrow In view of the use of hypoxanthine (Hx) and gut were higher than those in the saline in selective methotrexate (MTX) rescue control group. In all groups, inhibition of schedules (Strawr et al. (1977) J. natn. Cancer DHFR was greater than 9500 for the duration Inst., 58 91; Talbot et al. (1976) Br. J. Cancer, of the rescue period (5 days). These results 34, 321), and the toxic effects of adenosine suggest that the survival of the rescued (AR) to normal and malignant lymplhoid animals must depend upon an endogenous cells (Harrap et al. (1976) Br. J. Cancer, 34, supply of reduced folates, or purines and 321) it became important to understand the pyrimidines, rather than recovery of DHFR differences in purine salvage activity in a activity in the gut. number of cell types. We have compared kinetic parameters of hypoxanthine guanine phosphoribosyl transferase (HGPRT) (EC 2.4.2.8) from L5178Y cells growing in purine-free (Pu-) and purine-supplemented (Pu+) medium. A 5-fold increase in Vmax of EFFECTOR REGULATION: A POTEN- HGPRT from L5178Y (Pu+) was observed TIAL NEW CHEMOTHERAPEUTIC 8 weeks after transferring cells into culture. STRATEGY. R. M. PAINE and K. R. In the L5178Y (Pu-) line a 4-fold decrease in HARRAP, Department of Biochemical Pharma- Vmax was found. The L5178Y (Pu-) line had cology, Institute of Cancer Research, Sutton, a greater rate of purine synthesis de novo Surrey. than L5178Y (Pu+). Little change in the Kml At the last AGM we described how it was for Hx was observed in HGPRT from either possible to enhance the toxicity of adenosine line. Selective potentiation of the toxic effects (AR) to lymphoid cells with coformycin of AR by inhibitors of adenosine deaminase (Cf), a tight binding inhibitor of adenosine (ADA) (EC 3.5.4.4) depends on the relative deaminase (EC 3.5.4.4) (Harrap et al. (1976) rates of phosphorylation and deamination of Br. J. Cancer, 34, 309). In the case of mito- AR. Adenosine kinase (AK) (EC 2.7.1.20) from genically-stimulated lymphocytes it was mouse liver, spleen, marrow and L1210 cells apparent that the biochemical mechanism had a greater affinity for AR than ADA, but underlying cytotoxicity was a build up of the Vmax of ADA was -100-fold greater deoxyadenosine triphosphate (dATP) and than AK in these tissues. In L1210, the an associated inhibition of DNA synthesis deamination/phosphorylation ratio increased (see also Harrap and Paine (1977) Adv. Enz. markedly with AR concentration. This was Regln, 15). In the present investigation, we not observed in spleen, liveror marrow. Studies have found that deoxyadenosine (AdR) is have also indicated that AK from L1210 is toxic to cultured L1210 cells in presence of substrate-inhibited at high concentration of deoxycoformycin (dCf). Giant-cell formation AR. These studies show the importance of is detected by 24 h, due to inhibition of DNA the kinetic parameters of purine salvage synthesis in the face of continued RNA and enzymes in relation to Hx rescue and AR protein synthesis (imbalanced growth). The toxicity. primary metabolic lesion would appear to be inhibition of ribonucleotide reductase (EC 1.17.4.1) via a build up of the negative AN ASSESSMENT OF ORAL METHO- effector, dATP. Furthermore, treatment of TREXATE SYRUP. J. G. MCVIE, J. L1210-tumour-bearing animals with binary PAXTON, B. W. WHITING, M. SOUKoP and combinations of AdR and dCf produces K. C. CALMAN, Department of Clinical extension of survival time. We propose that Oncology, We.stern Infirmary, Glasgow. selective enhancement of the regulatory A new formulation of methotrexate (MTX) properties of effector molecules provides a was sought, to cope with the high doses in realistic means of inhibiting tumour growth. current practice. The standard tablet size 418 B.A.C.R. 18TH ANNUAL GENERAL MEETING is 2-5 mg, and so a syrup was prepared which Treosulfan is well tolerated, has a low level had a final concentration of 2 mg MTX per of gastrointestinal toxicity, is a predictable ml. It was tested in the clinic and was and transient marrow depressant, and gives accepted well by our patients. Samples of the response rates similar to other alkylating syrup were assayed 8 times throughout 32 agents. days of storage at room temperature or 4°C by a radioimmunoassay for MTX. There COLCHICINE ULTRASENSITIVITY was no alteration at all in the concentration OIF PERIPHERAL BLOOD LYMPHO- of MTX throughout the experiment. Six CYTES IN LYMPHOID MALIGNAN- patients had serial blood and urine samples CIES. J. H. SCARFFE, J. PRUDHOE and D. taken after ingestion of the oral syrup at a CROWTHER, CRC Department of Mledical dose of 50 mg/M2 and then a week later after Oncology, Christie Hospital and Holt Radium injection of an identical dose i.v. The relative Institute, Manchester. availability of the drug varied from 15 to 62%. This indicated that absorption of the The ultrasensitivity of chronic lymphatic oral drug had taken place in all the patients, leukaemia cells cultured for 20 h with but the bio-available levels were consider- colchicine, compared with normal lympho- ably less in all cases than when the drug was cytes, has been described by Thompson et al. given i.v. in the same dose. The mean (1972, Scand. J. Haemat., 9, 231). We have yt1/2 in hours was 4-14 h for the i.v. route and used this technique to study peripheral 3412 h by the oral route. There was marked blood lymphocytes in other lymphoid malig- individual variation in the handling of MTX nancies. Peripheral blood lymphocytes were in the 6 patients irrespective of the route of incubated at 370C in 5% C02 for 20 h at administration. We conclude that oral MTX concentrations of 0, 10-2, 10-3, 10-4, 10-5, may be given in a higher dose and possibly 10-6, 10-7 M colchicine in TC199. The cells more frequently than i.v. MTX. Further, were wet fixed on slides, stained and the MTX syrup is stable over 32 days, and has percentage of cells with pyknotic nuclei proved extremely palatable to large numbers counted. Twenty normal controls all showed of patients. less than 20% pyknosis at all concentrations less than 10-2 M colchicine. Twenty-six ill TREOSULFAN (DIHYDROXYBUSUL- controls, with diseases other than lymphoid PHAN) IN THE MANAGEMENT OF malignancies, showed a slightly higher range OVARIAN CARCINOMA. J. J. FENNELLY, of pyknosis up to 20% at concentrations less St Vincent's Hospital, Dublin. than 10-2 M. We were able to confirm the ultrasensitivity of chronic lymphatic leukae- Treosulfan (dihydroxybusulphan) is an mia cells in 17/18 cases studied. Forty and alkylating agent which was synthesized by 99% pyknosis at the lower concentrations of Feit in 1964. It has been found effective in 10-7 and 10-6 were observed, compared with Dunning Leukaemia and Lymphoma 8. the concentration of 10-2 M required for Because of reports of Lundvall, Sorenson pyknosis in normal lymphocytes. The one and Larsen (1973, Acta obstet. Gynaec. resistant case was initially sensitive but later Scand. Suppl. 22) of benefit in ovarian developed resistance, although the absolute carcinoma the author has evaluated Treosul- number of sensitive cells remained approxi- fan in 40 patients with ovarian carcinoma. mately the same. Peripheral blood lympho- Treosulfan was given as 250-mg capsules cytes in 17 cases of multiple myeloma were q.i.d. daily for 4 weeks on alternate months. not found to be ultrasensitive. However 20/51 Depression of white cell count and platelet patients studied with non-Hodgkin lymphoma count occurred in similar pattern to that of showed an abnormally high percentage of other alkylating agents, but recovery was pyknotic cells. It was expected that ultra- rapid. In addition a significant depression of sensitivity would be found more commonly haemoglobin occurred. in the well differentiated pathology groups,but Of 40 patients treated, 12 (30%) showed a results were similar for both well and poorly complete response for a mean duration of differentiated groups. None of the patients 15/12. Eleven (27%) showed a partial studied was frankly leukaemic, all the abnor- response for a mean of 7 months. 57% showed mal group had lymphocyte counts less than a total response. 7000/,ul. ABSTRACTS OF MEMBERS PAPERS 419

GROWTH KINETICS OF HUMAN have been related to growth inhibition induced TUMOUR XENOGRAFTS UPON by the same treatment. The FI measures the SERIAL PASSAGE IN IMMUNE- proportion of the total radiolabelled precursor DEPRIVED MICE. J. A. HOUGHToN and in the tissue sample which is incorporated D. M. Taylor, Radiopharmacology Department, into DNA within 1 h of administration. Institute of Cancer Research, Sutton, Surrey. The FI is not affected by variation in pre- cursor concentration achieved over a 10-fold Using the tumour systems previously range, which may occur in irregularly per- described (Houghton and Taylor (1976) Br. fused tumours. Significant growNth inhibition J. Cancer, 34, 313), preliminary studies have has been observed only when the cytotoxic shown increased growth rates -within the agent produced a considerable and prolonged first 10 serial passages in 5/6 lhuman colorec- depression in 3H-thymidine Fl. Tumour tal tumours maintained in immune-deprived growth rate returns to its pretreatment value CBA/LAC mice. Using bilateral implants, at a time wAhen Fl returns to the pretreatment the percentage of single tumour takes level (FI recovery time). Following adminis- (lecreases significantly with serial passaging, tration of cyclophosphamide, 5-fluouracil, or with hosts producing either two or no actinomycin D, the growtlh delay and FT tuniours. These values deviate frorn tlhose recovery time are always similar. The initial expected from a binomnial distribution after depression of 3H-thymidine FT into DNA the first one to two passages, more single is a poor indicator of the actual growth delay, takes beingr predicted than occur. Grow%th as different xenograft lines exhibiting the rates of individual tumours, calculated both same depression 1 to 2 days after treatment at 0 4 cm3 volume and during exponential may show considerably different FT recovery growth, can differ widely on very early times, w-hich are similar to the measured passages, and subsequently becomie more growth delay. How-ever, wNithin a tumour uniform after the first 2-4 passages. V'olume- line there is a relationship between the initial doubling times of tumours growing within depression of 3H-thymidine FI and both Fl the same animal are similar, and growth-rate recovery time and growth delay, which variation within a passage is such that the appears to be independent of the mechanism variance of growAth rates of tumours estab- by which the agent induces cell lished in different mice is greater than those kill. of tumours grow-ing in the sarne animal. Hence fast and slow-growing turmours occur in separate hosts. Tumours wThich occur as single takes also have very varied grow-th CYCLOPHOSPHAMIDE AND CIS- rates witlhin a passage. Results thus indicate DICHLORODIAMMINE PLATINUM a trend toward increased and more uniform (II): A PERSPECTIVE IN SCHEDUL- growth rates. However, actual growth rates ING. K. D. TEW and D. M. TAYLOR, Radio- and takes per mouse Avithin a passage may pharmacology Departnt/ent, Institute of Cancer l)e dictated by the host. This could depend Research, Suttonl. on the extent of individual host immune- Fractional incorporation (FI) of 3H-thymi- deprivation which may influence the rate of dine (proportion of total tissue 3H incor- cell loss wNNithin a tumour. porated into DNA) has been used success- fully as a parameter for judging temporal scheduling of cyclophosphamide (CY) and CHANGES IN 3H-THYMIDINE cis-dichlorodiammine platinum (DDP). A UTILIZATION AS A PREDICTOR OF difference in recovery time of FT following a GROWTH DELAY IN FOUR HUMAN dose of 100 mg/kg CY between tumour COLONIC TUMOUR XENOGRAFTS. (>12 days), gut (2-3 days) and bone marrow P. J. HoUGHTON, Institute of Cancer Research, (4 days) suggested a basis for a normal tissue- Sutton, Surrey. (Introduced by D. Ml. Taylor.) sparing drug regimen wNhen administering double-dose CY-DDP therapy. There were Changes in the fractional incorporation 0/10 sur-vivors when 100 mg/kg CY and (FI) of radiolabelled precursors into DNA 8 nmg/kg DDP wrere administered together have been examined in 4 xenograft lines 1/10 survivors when the doses were separated maintained in immune-deprived mice, and by 1 day and 10/10 survivors when separated 420 B.A.C.R. 18TH ANNUAL GENERAL MEETING by 4 days. This 4-day interval w-as con- for them. The differences in uptake are sidered to allow gut and bone marrow explicable by low aqueous solubility of CHP recovery, factors crucial to the survival of and MCHP, which persist in the peritoneum the animal, before the second insult. These for several days. This slowr uptake and sus- three combinations were similar in their anti- tained concentration in the tissues shown by tumour effect in being slightly more than CHP may explain its good antitumour additive. CY was more myelotoxic than activity. However, MCHP has similar DDP; DDP was more gut-toxic. The recovery pharmacokinetic behaviour but very low of bone-marrow cellularity was 2 days later activity. than Fl recovery. Peripheral white blood counts were reduced for a still longer period of time. The possibility of multiple drug administration based upon these findings THE MECHANISM OF INTER- remains to be elucidated. ACTION OF TWO PLATINUM CO- ORDINATION COMPLEXES WITH RADIATION IN CHO CELLS IN VITRO. A. H. W. NIAs and IRENA I. SZUMIEL,* PHARMACOKINETICS OF PLATI- Glasgow Institute of Radiotherapeutics and NUM ANTITUMOUR AGENTS IN Oncology, Belvidere Hospital, Glasgow. MOUSE ORGANS. B. W. MALERBI and The effects of two platinum coordination G. ABEL, Johnson -Matthey Research Centre, complexes have been compared on CHO cells Sonning Common, Oxon and Chester Beatty in vitro. While cis-dichlorobisisopropylamine Research Institute, London. (Introduced by trans-dihydroxy platinum IV (CHIP) is T. A. Connors.) very soluble in water, cis-dichlorobiscyclo- In antitumour screening tests in BALB/C pentylamine platinum II (PAD) is insoluble mice, cis-diamminedichloroplatinum (II) and was dissolved in DMSO. Dose-response (DDP), cis-dichlorobis(cyclohexylamine) curves after a 15 min exposure to CHIP platinum (II) (CHP), and cis-dichlorobis anid PAD were similar in shape, wvith final (4-methylcyclohexylamine)platinum (II) exponential slopes of 16 and 14 jug/ml (MCHP) gave LD50 values of 16, 3200, and respectively. The shoulder of the CHIP 1180 mg/kg. Corresponding ID90 doses were curve was much larger (N-300) than that 2-4, 12 and 990 mg/kg respectively. To for PAD (N=7). elucidate these differences, these compounds Drug-radiation combination experiments w,ere injected i.p. into healthy BALB/C mice. showed a synergistic effect only when radia- Animals were sacrificed at intervals spanning tion follow%ed a drug dose level high enough 15 min to 14 days after injection, and plati- to reduce cell survival from the shoulder num analyses were performed on the liver, towards the exponential part of these dose- spleen, kidneys, heart, lungs, small intestine, response curves. After PAD, the highest large intestine, brain, skeletal muscle, bone enhancement ratio was 1 59, whilst a ratio of and skin. Although all these compounds were 1P73 was found after a comparatively lower stored in the liver, the effect was more marked dose of CHIP. No cycle-phase specificity for CHP and MCHP. In the kidneys, DDP was found following PAD alone, but com- produced an initial peak that declined binations with radiation showed more syner- rapidly, whereas CHP and MCHP produced gism in the G1 and late-S position of the cell lower steady levels. The large intestine cycle than in mid-S. showed a late rise in CHP and MCHP which These survival data, together with the was not observed wvith DDP. None of the results of other studies with PAD, including compounds showed superior ability to cross an absence of split-dose sparing and a pattern the blood-brain barrier. The pharmacokinetic of chromatid aberrations, are compatible behaviour of CHP and MCHP in the kidney with the "molecular theory of cell survival" may explain their low toxicity compared (Chadwick, Leenhouts, Szumiel and Nias with DDP. DDP is known to be excreted (1976) Int. J. Radiat. Biol., 30, 511) which mainly via the kidneys, but the high levels * IAEA Fellow, Department of Radiobiology of CHP and MCHP found in the intestines and Health Protection, Institute of Nuclear suggest that biliary excretion predominates Research, 03-195, Warsaw, Poland. ABSTRACTS OF MEMBERS' PAPERS 421 provides an explanation of the cytotoxic explained by the drug preventing or inhibiting action of the platinum complex and its repair of sub-lethal damage. It would appear synergistic interaction with radiation. therefore, that prolonged pretreatment with ICRF 159 reduces the cells ability to accumu- late sub-lethal damage. THE EFFECT OF ICRF 159 ON ACCUMULATION AND REPAIR OF RADIATION DAMAGE. I. W. TAYLOR STUDIES OF RESISTANCE TO and N. M. BLEEHEN, MRC Unit of Clinical ICRF 159 IN CELL LINE BS/159-1. K. Oncology and Radiotherapeutics, Cambridge WHITE and A. M. CREIGHTON, Imperial University Medical School. Cancer Research Fund, London. ICRF 159 has been shown to increase the The isolation of a cell line (BS/159-1), sensitivity to X-irradiation of exponentially derived from BHK 21S cells and showing growing EMT6 mouse tumour cells in vitro resistance to the antitumour drug ICRF 159, (Taylor and Bleehen (1977) Br. J. Canc(r, 36). has been previously reported (White and This is found only with ICRF 159 exposure Creighton (1976) Br. J. Cancer, 34, 323). times greater than 14 h, and only w%hen the Protein synthesis inhibitors (e.g. puromycin drug is given prior to irradiation. A 24-h expo- and cycloheximide) normally allow cells to sure to 200 ,ug ICRF 159 before irradiation progress into mitosis for 1 h only, after leads to a reduction in the radiation survival- which time cells will no longer cross the G2/M curve shoulder (Dq= 129 rad) compared with border. BS/159-1 cells, however, are not non-drug-treated controls (Dq=509 rad). This inhibited in this way, mitotic cells continue would suggest a loss of ability to accumulate to accumulate. This suggests that either the or repair sub-lethal damage. The split-dose protein requirement for mitosis is already radiation response was examined using cells met, or the protein-synthesis mechanisms in which had either a 6-h or a 24-h exposure BS/159-1 cells are resistant to these inhibitors. to 200 jig ICRF 159 before the two doses of The latter seems unlikely, since BHK 21S radiation and compared to control cells and BS/159-1 cells are equally sensitive to irradiated under similar conditions. In the puromycin inhibition of protein synthesis. ICRF 159-treated cells the drug was present ICRF 159 has no direct effect on protein- during the interval between radiation doses. synthesis mechanisms per se. In all three cases, the cells, whether drug- It is possible that ICRF 159 inhibits the treated or not, were found to have recovered function (either directly or indirectly) of a 75-85% of their previously measured Dq. protein required for mitosis. The availability The reduction in Dq found for cells treated and/or nature of this protein may be modified with ICRF 159 for 24 h, therefore, cannot be in BS/159-1 cells. 422 B.A.C.R. 18TH ANNUAL GENERAL MEETING PART II: POSTER EXHIBITS ABSENCE OF NUCLEOSIDE EFFECT Dilla (1972) Acta Cytol., 16, 26). The first IN CELLS IRRADIATED BY FAST peak of a characteristic histogram corresponds NEUTRONS. A. FERLE-VIDOVIC, D. to the fluorescence emitted by the DNA- PETROV16, J. SORIC, D. RENDIC and I. fluorochrome complex of cells in G1. The SLAUS, Institute Ruder Boskoric, Zagreb, emission of cells in G2 + M is double that of Yugoslavia. cells in G1 resulting in a second peak at, double the abscissa scale reading, channel Breakdown products of DNA can increase number. Two computer models are presented the survival of irradiated cells. This had been which can analyse the experimental data. studied extensively by employing deoxyri- The first employs age distribution theory bonucleosides in L cells (Petrovi6, Ferle- (Steel (1968) Cell & Tissue Kinet., 1, 193) to Vidovic, Habazin, Vukovic (1970) Int. J. give estimates of not only the proportions of Radiat. Biol., 18, 243) after X irradiation. cells in each phase, but also of the relative In the present work, L 929 cells were irradi- phase durations. This model can be used for ated by neutrons of different energies: populations containing a mixture of cycling 4-5 MeV mean energy and 14-5 MeV mono- and non-cycling cells, but it is concluded energetic neutrons. For comparison, cells that reliable estimates of the grow%th fraction were also irradiated by 60Co gamma rays. can only be obtained if the relative phase Following irradiation cells were treated by durations are known. Good agreement an equimolar solution of deoxyribonucleo- between the computed proportion in S phase sides (50 ,ug/ml), and effect on their survival and the 3H-TdR labelling index was found measured. Results showr that nucleoside in the five cell lines analysed. A second model treatment was efficient after the low LET based upon the theory presented by Hart- irradiation: gamma rays survival curves mann and Pederson ((1970) Cell & Tissue were altered by nucleosides in terms of Kinet., 3, 1), has been produced to analyse significantly increased extrapolation numbers the desynchronization of EMT6/M/CC cells only, but without Do change. Cells irradiated following mitotic selection synchronization. by neutrons from either of the two sources did Good agreement was obtained between the not respond to nucleoside treatment, and cytofluorimetric data and results from parallel consequently their survival curves remained 3H-TdR studies. unaltered. These results show that the nucleoside effect does occur after lowr LET irradiation, but apparently not following high LET irradiation. Since nucleosides as well as other cell breakdown products are released in irradiated tumours due to mass STUDIES ON A DNA CONTAINING cell destruction, such nucleoside effect could MATERIAL FROM P388 CELL possibly enhance the cell survival and thus LYSATES HIGHLY SENSITIVE TO affect the result of radiotherapy. Absence of IONIZING RADIATION. D. G. POPPITT the nucleoside effect in case of high LET and B. W. Fox, Paterson Laboratories, Christie irradiation may therefore be an additional Hospital and Holt Radium Institute, potential gain from neutrons in radiotherapy. Manchester. The sedimentation behaviour of a DNA complex material resulting from lysis of P388 lymphoma cells follow%iing X- and CELL CYCLE ANALYSIS IN VITRO gamma-irradiation has been studied on USING FLOW CYTOFLUORIMETRIC isokinetic sucrose gradients with an initial TECHNIQUES. J. V. WATSON, MRC sucrose concentration of 2000. A decreased Clinical Oncology and Radiotherapeutics Unit, sedimentation rate followNing irradiation Cambridge University Medical School. throughout the range from 5 rads to 10 krads has been observed. Post-irradiation incuba- Flow cytofluorimetric technology enables tion has suggested that a partial reconstitu- the DNA content of cells in a single cell tion of this material may occur within suspension to be estimated (Trujillo and van approximately 6 h. POSTER EXHIBITS 423

DEGRADATION OF ERROR PRO- tant) Wistar rats, producing a greater thera- TEINS IN HeLa CELLS. D. N. WHEATLEY, peutic index than can be achieved with M. R. GIDDINGS, M. S. INGLIS and J. H. chlorambucil alone. Similar results can be STEVENSON, Department of Pathology, Aber- obtained with prednimustine (Leo 1031) a deen University Medical School. prednisolone ester of chlorambucil. Previous work in this laboratory has shown that The possibility that changes in cell beha- chlorambucil induces morphological and viour seen in phenomena such as ageing, chemical changes in the structure of nuclear malignant transformation, differentiation and proteins of drug-sensitive cells, though not of mutation may be the result of error (or resistant ceBs (Riches and Harrap (1973) accumulation of errors) in protein biosyn- Cancer Res., 33, 389; Riches and Harrap thesis (e.g. Orgel (1973) Nature, 243, 441; (1975) Chem.-Biol. Interactions, 11, 291). Talmud and Lewis (1974) Nature, 249, 563; However, it will be shown that in the presence Bradley and Schimke (1973) in Intracellular of prednisolone, similar changes can be Protein Turnover, Academic Press, p. 311) produced in the chromatin of drug-resistant is currently receiving much attention. The cells. We have also found that the time hypothesis raises the question of whether sequence of steroid administration, in rela- a special surveillance mechanism exists tion to that of the alkylating agent, modifies through which error proteins are detected the pattern of DNA cross-linking in the and preferentially removed by cells, and the tumour, possibly by disruption of repair problems of what happens if it breaks down processes. or is overloaded. In HeLa cells allowed to incorporate amino acid analogues instead MYELOTOXICITY OF METHOTREX- of natural amino acids, we found no evidence ATE IN ANIMALS WITH PYOGENIC of preferential degradation of anomalous INFECTION. B. HARDING and I. C. M. proteins. In some cases, the consequences of MACLENNAN, NuJfield Department of Clinical analogue incorporation resulted in cells Medicine, Radcliffe Infirmary, Oxford. becoming degenerate with a subsequently elevated breakdown of all cellular proteins. This poster reports an investigation of the The results favour the hypothesis that hypothesis that increasedproliferative activity degradation follows first order kinetics for by neutrophil precursors induced by pyogenic both normal and abnormal proteins and is infection will result in an increase in the due to a common intracellular proteolytic susceptibility of these cells to damage by system operating in a stochastic manner. methotrexate (MTX). Rats were stimulated into prolonged increased neutrophil produc- tion by the induction of a unilateral pyo- ACQUIRED DRUG RESISTANCE: hydronephrosis. Consistent profound neutro- ENHANCEMENT OF THE INTRA- penia was seen when MTX was given during NUCLEAR REACTIVITY OF ALKYL- the first 2 days of infection, but thereafter ATING DRUGS. R. WILKINSON and K. R. greater neutropenia than that observed HARRAP, Department of Biochemical Pharma- in non-infected rats was only observed in cology, Institute of Cancer Research, Sutton, occasional animals. There was a significant Surrey. correlation between the degree of neutropenia induced by MTX and the day after MTX Alkylating agents find wide usefulness in upon which this occurred. It is argued that the treatment of malignant diseases, though the main target for myelotoxicity by MTX is their effectiveness is impaired frequently by the the development of acquired resistance. It myelocyte and that its precursors are becomes important therefore to devise relatively insensitive. schedules which, ideally, are antagonistic in terms of host toxicity and synergistic in MECHANISMS OF IN VITRO AND IN relation to their antitumour effects. Alkylat- VIVO RAZOXANE RADIOSENSITIZA- ing agents are frequently administered in TION. M. BARKER-GRIMSHAW, Chemo- combination with steroids: we have found therapy Department, Imperial Cancer Research that binary combinations of chlorambucil Fund, London. (Introduced by Q. Hellman.) and prednisolone can be administered to Razoxane (ICRF 159) produces radio- tumour-bearing (Yoshida sensitive and resis- sensitization in vitro and in vivo. Various 28 424 B.A.C.R. 18TH ANNUAL GENERAL MEETING mechanisms have been suggested to account (3H-deoxyuridine) and "salvage" (3H-thymi- for this effect, viz: the angiomorphic effect; dine) pathways after 5-fluorouracil treatment blockage of cell cycle progression at G2/M; has been used as a measure of the ability inhibition of repair of radiation-induced of that human tumour ]ine to utilise the DNA damage and general synergism with "salvage" pathway for thymidine triphos- other antitumour agents, but which, if any phate synthesis in the presence of thymidylate of these mechanisms is involved is not yet synthetase inhibition. There appears to be no clear. Experiments to test whether tumours correlation between the degree or duration treated with razoxane lhad an increased blood of thymidylate synthetase inhibition and flow as a result of the angiometamorphic gro-wth delay, following 5-fluorouracil admini- effect have been essentially negative. On the strationi, in these xenografts. The ability of other hand, a higher oxygen concentration the tumour to use the "salvage" pathway was found in such tumours even when treat- for thymidine triphosphate synthesis appears ment w%as delayed until 1 h before measure- to determine the response of these tumours ments were made. Compared with the inhibi- in the presence of de novo thymidylate syn- tory effect of razoxane or radiation alone, the thesis inhlibition induced bv 5-fluorouracil. combination of the two in the treatment of sarcoma S180 was much more effective even when razoxane was given 3 h after the THE PIG AS A MODEL FOR TOXICITY radiation. Of the mechanisms of radiosensi- AND THERAPY TESTING OF CYTO- tization suggested for razoxane therefore that TOXIC DRUGS. S. E. BROWNLIE, J. G. of inhibition of repair of radiation-induced CAMPBELL, K. W. HEAD, P. IMLAH, H. S. DNA damage seems to be the most, likely. McTAGGART and J. G. MCVIE, Departmient of Clinical Oncology, Wrestern Infirmary, Glasgow. FACTORS DETERMINING THE A hereditary form of lymphoma associated RESPONSE TO 5-FLUOROURACIL with an autosomal recessive gene in Large IN HUMAN COLONIC TUMOUR White pigs is diagnosable before 3-4 months XENOGRAFTS. P. J. HOUGHTON, J. A. of age and fatal by about 15 months. The HOUGHTON and D. M. TAYLOR, Division of suitability of this condition for therapy test- Radiopharnkacology, Institute of Cancer ing of cytotoxic drugs has been investigated Research, Sutton, Surrey. in toxicity tests using normal pigs. Predniso- lone, dexamethasone, doxorubicin, cyclo- The relationship between the inhibition phosphamide and vincristine have been of 3H-deoxyuridine incorporation into DNA tested as single agents in normal and lympho- and growth delay followxving 5-fluorouracil matous pigs. The results of treatment mimic administration has been examined in 4 those expected in humans suggesting that humain colonic tumour xenografts growing in this is a good animal model for testing newN immune-deprived mice. The dose of 5- drugs and novel schedules of established fluorouracil producing 5000 inhibition of drugs. Prednisolone and dexamethasone used 3H-deoxyuridine incorporation in vivo (ID50) separately produced an increase in serum in 2 tumour lines was less than that found for albumin arid marked involution of the thymus normal (mouse) "limitinlg" tissues, but greater with "overshoot" on writhdrawal of drugs in in the other tumour lines. After 5-fluorouracil both normal and lymphoma pigs. A similar (100 mg/Kg ID90 in all tumour lines) only effect has been reported in human infants tumours of one line showved a depression in (Caffey and Silbey (1960) Paediatrics, 26, 3H-thymidine incorporation into DNA and 762). A striking reduction in circulating growth delay, whereas tumours of the other lymphocytes and in the size of lymph nodes lines showed an increase in uptake and occurred particularly in the lymphoma cases. incorporation of this nucleoside into DNA Beneficial effects were observed in red cell during the first, 4 days, and no growAth picture, neutrophil and platelet counts and delay. Recovery of 3H-deoxyuridine incor- on the general vigour and well-being of poration to the pretreatment level varied lymphoma cases. These effects were all more from 150 to over 600 h between tumour lines marked Mwith prednisolone than with dexa- after 100 mg/Kg 5-fluorouracil. The differ- methasone. As in man, doxorubicin was ence betwreen the recovery times for de novo cumulativ ely toxic at high doses in both POSTER EXHIBITS 425 normal and lymphoma pigs, producing gradient separation technique described by stunting of growth, buccal ulceration, alope- Cercek and Cercek. Two layers were obtained cia, diarrhoea, liver damage, leucopenia, with 6/10 normal bloods and 4/10 samples thrombo-eytopenia and cardiotoxicity. from cancer patients. The two layers have Remission has been achieved and maintained been compared. In both normal and cancer for over a year in one case given cyclophos- samples the upper layers contained from phamide and vincristine in combination with 92-9700 lymphocytes; the lower layers steroids. were more contaminated with non-lympho- cytic white cells. T (thymus-derived) cells were more predominant in the upper than CANINE OSTEOSARCOMA: COM- lower in both groups. The SCM test in 8/10 BINATION CHEMOTHERAPY AND normal subjects and 7/10 cancer patients CLINICAL STAGING. A. M. HENNESS, confirmed results originally reported by Clinical Oncology Service, University of Cali- Cereek et al. ((1974) Br. J. Cancer, 29, 345), fornia, U.S.A. (Introduced by L. N. Owen.) as regards the upper layers, i.e. more response There are few% reports on the use of anti- to phytohaemagglutinin (PHA) than to neoplastic drugs in tlherapy for canine myelin basic protein (MBP) in normals and osteosarcoma (OS); results w^ith these agents the reverse in cancer patients. In 2/10 normal have not been encouraging, either because subjects and 3/10 cancer patients the lympho- of their lack of effectiveness against the cytes were non-responsive to either substance. disease or due to their significant toxicity The lower layers, in both groups, were to the host. In this initial study of use of studied where yield permitted, but produced combination chemotherapy for canine OS, inconclusive SCM results. In 2 normal 11 dogs were given cytotoxic drugs following subjects, where the yield permitted study of amputation. Drugs in the standardized 6 PHA blastogenesis, the upper layers showed month protocol were: Adriamycin (30 mg/ more transformation. It is concluded that the M2), cyclophosphamide (50 mg/M2), and upper layers in all subjects studied contain a methotrexate (5 mg/in2) with citrovorum greater proportion of T lymphocytes, are less factor "rescue". A method of clinical staging contaminated by other white cells, are more of canine OS was devised, based on clinical responsive to PHA stimulation in culture and and radiographic findings at time of diag- give more definitive responses in the SCM nosis, to assist retroactively in the evaluating test. of therapy and survival data of these 11 animals. Their distribution by clinical stage was: 1, Ila; 4 Ila; 5, IlIb; and 1, IVb. SEPARATION OF HUMAN LYMPHO- At 8 months from time of diagnosis, 7 (64o0) CYTES FORMING MOUSE RED CELL were alive, and 5 (450 ) were clinically free ROSETTES. M. R. POTTER, Paterson of metastases. One (initially stage IIlb) Laboratories, Christie Hospital and Holt survived 23 months and then died suddenly Radium Institute, Manchester. (Introduced by without evidence of OS. A second (stage Ila) M. Moore.) is alive at greater than 41 months from time of diagnosis. Drugs in the protocol appeared Subpopulations of lymphocytes with to be well tolerated. receptors for heterologous erythrocytes can be identified by rosetting tests as exemplified by the formation, by human T lymphocytes, ANALYSIS OF TWO LYMPHOCYTIC of rosettes with sheep red blood cells (SRBC). LAYERS ACHIEVED BY FICOLL- Rosette formation between human lympho- TRIOSIL GRADIENT SEPARATION. cytes and mouse red blood cells (MRBC) C. R. PENTYCROSS, Department of Medical has been described more recently as a marker Oncology, Charing Cross HJospital, London. for B lymphocytes, or a subpopulation of B (Introduced by K. D. Bagshawe.) lymphocytes (Stathopoulos and Elliot (1974) Lancet, i, 600). MRBC rosette formation with During studies on the structuredness of human blood lymphocytes and the separation cytoplasmic matrix (SCM) of lymphocytes, of rosette forming cells as a method of B 2 separate interface layers wrere sometimes lymphocyte enrichment has been examined. obtained with a modified Ficoll-Triosil Blood lymphocytes were prepared by Ficoll- 426 B.A.C.R. 18TH ANNUAL GENERAL MEETING

Triosil gradient centrifugation and a small tests was seen for E rosettes in the SP group percentage of these cells (mean value 6%) (P < 0.002) and for EAC' rosettes in the BT formed spontaneous rosettes with MRBC group (P < 0-02). In the CP group the under conditions similar to those used for combination EAC' result was also significantly SRBC rosette formation. The proportion of reduced (P < 0-01). The possibility that the MRBC rosettes was increased (mean value difference found between the CP and control 16%) by treating the lymphocytes with groups is due to an effect of longer storage neuraminidase before rosetting. Neuramini- of blood from cancer patients prior to testing dase treatment of the. MRBC also increased is being investigated. the number of rosettes formed, but to a lesser extent (mean 11 %). Double marker tests demonstrated that lymphocytes forming CLINICAL CORRELATES OF IN MRBC rosettes were immunoglobulin (Ig) VITRO LYMPHOID FUNCTION. N. bearing cells, with a high proportion of IgM THATCHER, N. GASIUNAS and D. CROWTHER, bearing cells, but not all Ig bearing cells CRC Department of Medical Oncology, Christie formed rosettes. Depletion of the proportion Hospital and Holt Radium Institute, Man- of B lymphocytes in the population by nylon chester. fibre column filtration produced a correspond- ing fall in the number of MRBC rosette Lymphoid function was investigated using forming cells. Separation of rosette forming the peripheral lymphocyte count, E, EAC cells by Ficoll-Triosil gradient centrifugation rosettes and also direct, antibody dependent gave a pellet population enriched for B and PHA induced lymphocytotoxicity against lymphocytes and an interface population 51Cr labelled Chang cells. enriched for T lymphocytes. Tests on the (a) Influence of Pathology and Stage in degree of enrichment by re-rosetting with non-Hodgkin lymphoma. Thirty untreated MRBC produced variable results whereas patients were examined. Peripheral lympho- testing by SRBC rosette formation showed cytes, antibody dependent cytotoxicity and a consistent pattern of enrichment. E rosettes were reduced (P < 0-5) compared with controls. The reduction was statistically T AND B CELL POPULATIONS IN significant for patients with diffuse pathology. CANCER PATIENTS AND CONTROLS Patients with nodular pathology, early and USING FRESH AND FROZEN LYM- late stage disease, showed some reduction in PHOCYTES. C. H. J. FORD, C. E. NEWMAN test values but were not statistically signifi- and A. B. CARTER, University Department cant. of Surgery, Queen Elizabeth Hospital, Bir- (b) Influence of Surgical Removal of Primary Hypernephroma. Eighteen patients mingham. with untreated hypernephroma had signifi- Individual E and EAC' rosette tests and cantly reduced antibody dependent (P < a combination assay for measuring E, EAC' 0-01) and PHA induced cytotoxicity (P < and mixed rosettes have been used to measure 0.02) compared with the same patients 14 the numbers of T and B cells in the peripheral days post-nephrectomy or with normal blood of 96 blood transfusion donors (BT), 15 controls. Seven patients had demonstrable laboratory staff (LS), 36 pre-operative distant metastases pre-operatively, these patients with non-malignant surgical con- patients demonstrated a smaller post-opera- ditions (SP) and 40 cancer patients (CP). tive rise in cytotoxicity than the 11 non- Statistically significant differences were metastatic patients rendered clinically tumour obtained when comparing the three control free. groups (BT, LS, SP) with the CP group in (c) Influence of Chemotherapy and Reduc- the individual E test, P < 0-002 (74-7+ 9-7, tion in Metastatic Burden. Nineteen patients 79-4 + 8.7, 72-9 : 10 vs 66-2 + 15.5), com- with metastatic head and neck, gastric and bination E test, <0-02 to <0.002 (71-8 ± bladder carcinoma were treated with pulsed 8-9, 73-1 + 6, 71-9 + 8-8 vs 66-1 ± 10.7), courses of Adriamycin and 5-Fluorouracil. and when comparing the BT and CP groups The patients were immuno-suppressed (P < in the individual EAC' test, P < p-01 0-02) as indicated by peripheral lymphocyte (11.35 ± 4-3 vs 8-9 + 4-8). A significant count, E, EAC rosettes, direct and antibody effect of freezing on rosetting ability in both dependent lymphocytotoxicity. Therapy POSTER EXHIBITS 427 which induced an objective reduction in sonnel wvore surgical gowns, hats and masks, metastases reduced this pretreatment immu- and the animals wN-ere handled using sterilized nosuppression, but non-responding patients gloves. The room temperature wvas held at showed further immunosuppression. Lym- 25-27°C and the humidity (uncontrolled) phoid function as measured by these methods wvas about 4500. The breeding colony con- is therefore related to clinical parameters sisted of permanently caged pairs and trios. of tumour type and tumour burden. Productivity w-as similar to previous reports (Festing and King, 1974) and although some perinatal mortality was in evidence a mean HISTOCHEMICAL DETECTION OF number of 3-5 nu/nu per litter at wveaning ABNORMAL SACCHARIDES IN A was obtained. In most cases post-partum CHONDROSARCOMA. R. W. STODDART, matings occurred and the young were D. D. DZIEWIATKOWSKY and S. FITTON- delivered at about the time of weaning the JACKSON, Strangeways Research Laboratory, previous litter. Mortality rates for both the Cambridge. breeding colony and experimental mice were The Schwrarm chondrosarcoma of the rat, lowA. Some cases of the common wasting- which originated as a spontaneous osteochon- disease syndrome and skin abscesses were drosarcoma, produces a matrix wi-hich is found together wvith occasional instances abnormally soluble in chaotropic agents and of rectal prolapse. The colony has been lacks the repeating sequence of keratan maintained for more than 3 years and the sulphate. It was maintained by subcutaneous average life-span of homozygotes approached and intraperitoneal transplantation in hooded 9-12 months under these conditions. Neoplas- rats and large tumours and occasional tic tissues from canine melanoma, osteo- metastases were obtained. Samples were sarcoma, mammary carcinoma and lympho- fixed in anhydrous methanol or Zenker- sarcoma were made into cell suspensions and acetic acid and paraffin sections were made cultured in RPMI 1640 containing 10% FCS. by conventional procedures. These were Cells from 10 cultures were injected subcu- specifically stained for various sugars by taneously in duplicate (5 x 107 cells) into fluorescent-labelled lectins. Comparisons were 6-8 week old mice. Transplantation was made ith several cartilages of foetal, successful in 7 cases (14 mice) and growth juvenile and adult rats. The malignant was assessed by serial measurement of the chondrocytes showed peculiarities of their palpable tumours. Tumour samples were nuclear and surface sugars. The matrix was examined histologically and by electron highly abnormal and disordered. Soy-bean microscopy and were re-cultured in vitro. agglutinin stained 'cable-like" structures run- ning through it, which were not seen in any normal cartilage and wNrhich may have THE AETIOLOGY OF BREAST represented the unsubstituted residues of CANCER AND THE OESTROGENIC N-acetylgalactosamine bv which keratan METABOLITES OF FUSARIA. R. sulphate is normally linked to polypeptide. SCHOENTAL, Department of Pathology, Royal Concanavalin A stained glycogen intensely Veterinary College, University of London. and detected fine fluffy fibrils in the matrix Reviewing that may be disordered collagen. this subject and the many factors suspected as the causative agents, MacMahon, Cole and Brown ((1973) J. natn. ATHYMIC NUDE MICE: HUSBANDRY Cancer Inst., 50, 21), concluded that the AND TRANSPLANTATION STUDIES. 'nature of the familial factors, genetic or D. R. MORGAN, Department of Clinical Veter- environmental, is unknown". I suggest that inary Medicine, University of Cambridge. possible aetiological factors of breast cancer, which not yet have been taken into considera- A breeding colony of nude mice was tion. are the non-steroidal oestrogenic secon- established using homozygous (nu/nu) males dary metabolites of microorganisms (mainly and heterozygous (nu/+) females, maintained of Fusarium spp.) such as zearalenone and under simple barrier conditions in isolation its congeners, which can be found in some from other animals. Cages, sawdust, food and batches of stored grains (compare Stoloff water were sterilized and maintenance per- (1976) in Mycotoxins and other Fungi related 428 B.A.C.R. 18TH ANNUAL GENERAL MEETING Food Pr-oblems, Adv. Chem. Series, 149, 23; 74.500/ and tumour cells wAere the predominant Hacking, Rosser and Dervish ((1976) Ann. cell type. Viable cells aggregated while on Appl. Biol., 84, 7). the Ficoll, and these balls of tumour cells The presence in human foodstuffs of oestro- readily adhered to and spread over the culture genic substances, active by the oral route surface to form epithelial islands and sheets could explain the occurrence of familial with virtually no fibroblast contamination. breast cancer. Members of a family usually partake from the same food, hlence wNould be THE ANTI-TUMOUR ACTION OF similarly exposed to its contaminants. It is SENSITIZED PIG LYMPH NODE worth noting, that the presence of oestrogenic CELLS, MEASURED BY REDUCTION substances is not likely to be detected by OF PULMONARY METASTASES IN taste, or by toxic effects following soon after MICE: OBSERVATIONS ON THE ingestion. They act insidiouslv. NECESSARY SPECIFICITY OF Specimens will be shown of Fusarium- SENSITIZATION. S. PRICHARD-THOMAS infected maize containing zearalenone, as well and M. 0. SYMES, Department of Surgery, as cultures of Fusarium graminearum on Br istol. various media kindly supplied by P. K. C. University of Austwick, Nuffield Institute of Comparative Pulmonary tumour metastases were Medicine, The Zoological Society of London, induced in A-strain mice by i.v. injection of and ofzearalenone-containing barley obtained 106 A-strain mammary carcinoma cells. In from A. Hacking, Ministry of Agriculture, some mice, a splenectomy alone wNas per- Fisheries and Food, Shardlow Hall, Shardlow, formed on Day 6. Other mice received, in Derby. addition, on Day 7 an i.v. injection of 2 x 107 unsensitized or sensitized mono- ISOLATION OF EPITHELIAL SHEETS nuclear cells, separated from the mesenteric OF HUMAN MAMMARY TUMOUR lymph node chain of a pig. The mice were CELLS. A. HOWELL, G. K. PANDA and N. killed on Day 14, their lungs fixed in Bouin's AHKTAR, Departments of Medicine and Cancer fluid, and the number of metastases counited. Studies, Birmingham University. The effect of splenectomy alone, and of splenectomy plus pig cells, in reducing the The mean cell yield from 37 human number of metastases, was assessed in com- mammary tumours using the collagenase parison wvith the numbers in untreated mice. di-aggregation technique of Lasfargues (J. The results, obtained by an analysis of Fogh (ed), Human Tumour Cells in vitro, variance using pooled data from a number of Plenum Press, 1975) was 3 90 x 107 eells/g experiments, were as follows: wet weight with a mean viability of 55.600. As judged by morphology lymphocytes Series I comprised of the cell Significance in 17-8% population on No. of reductionl of average. The presence of lymphocytes w%vas obser- pulmonary meta- confirmed by rosetting: all tumours tested Treatmetit vations stasis number contained cells with Fe and C3 receptors and Nil 27 also cells which formed E rosettes. Latex Splenectomy 18 <0 05 ingesting cells formed approximately Splenectomy + pig 10% cells (unsensitized) 10 NS of the total population. Adequate tumour Splenectomy + pig cell culture was not obtained possibly cells (sensitized to because of the presence of lymphocytes mouse tumour) 23 0-01. initially and certainly because of fibroblast overgrowth later. Cells spilled at the time of Series II of Nil 42 cutting tumours gave a mean yield of Splenectomy 27 NS 2.42 x 107/g original tumour wet weight Splenectomy + pig and a mean viability of 17-30. The high cells sensitized to proportion of dead cells interfered with mouse tumour :34 <0*01 tumour Sensitize(i to cell aggregation and adhesion to the mouse skin 17 (26)* NS culture surface. When dead cells were Sensitized to removed by centrifugation through Ficoll- human tumour 20 (28)* <0*05 Triosil the viability increased to a mean of * No. of "nil" treatments for this comparison. POSTER EXHIBITS 429 Thus only pig cells sensitized against the study. Additional evidence for the existence mouse tumour to be treated had a significant of macrophage mitogenic activity in inflam- anti-tumour effect. Cells sensitized against a matory exudate comes from the work of human tumour (Series II) were only margin- Adolphe's group in Paris (Adolphe et al. ally effective, producing the same degree of (1975) Nature, 253, 637), and other studies reduction in nodule number as splenectomly have implicated both fibroblasts (Cifone et alone (Series I). Furthermore, when these al. (1975) Expl Cell Res., 96, 96) and lympho- experiments were repeated using pig mono- cytes (Hadden et al. (1975) Nature, 257, 483) nuclear cells stimulated in vitro by culture for as potential sources of such factors; thereby 2 days in the presence of PHA, no anti- serving to emphasize the probable complexity tumour effect was obtained. The requirement of the cellular interrelationships and control for a population of pig cells sensitized against mechanisms which exist in chronic inflam- the tumour to be treated to obtain an anti- mation. tumour effect, suggests that the mechanism thereof is an adoptive transfer of immunity from pig to mouse. TARGET CELLS OF THE LEUKAE- MOGENS BUTYL AND METHYL NITROSOUREA. P. BAINES, Paterson Laboratories, Christie Hospital and Holt MONONUCLEAR PHAGOCYTE PRO- Radium Institute, Manchester. (Introduced by LIFERATION IN INFLAMMATION M. Moore.) AND IN VITRO. K. M. WYNNE and W. G. SPECTOR, Department of Pathology, St Bartho- Lymphoblastic leukaemia developing in lomew's Hospital Medical College, London. intact mice treated with a single i.v. dose of by M. Moore.) MNU or a chronic oral administration of (Introduced BNU usually present with thymoma. The Mononuclear phagocyte, or macrophage, spleen, lymph nodes and liver may also be proliferation is a well-established feature of involved. Occasionally the leukaemia may the chronic inflammatory response, although develop in the spleen or lymph nodes in the its influence upon macrophage activation, absence of thymic enlargement. Thymectomy and hence its exact contribution to lesion before MNU treatment greatly increases the progression and/or resolution, remain to be induction time and decreases the incidence elucidated. Cultured macrophages, in marked of leukaemias. The incidence of leukaemias in contrast to their inflammatory counterparts thymectomized BNU-treated mice depends in vivo, exhibit only minimal levels of DNA upon the dosing regime. The incidence synthesis and proliferation, a fact which can decreases and induction time increases if the be exploited in the design of a model system dose duration is reduced from continuous to investigate the possible existence of local feed to 4 weeks. A similar dose reduction has humoral mitogenic factors in inflammatory little effect on the characteristics of leukae- lesions. Exposure of in vitro macrophage mias developing in intact mice. A study of monolayers to cell-free inflammatory exudate surface markers on terminal leukaemic cells harvested from 4-day-old chronic lesions, shows that in mice with thymoma involve- resulted in a stimulation of DNA synthesis, ment 6+ve, Ig-ve leukaemias result. If no as evidenced by 3[H]TdR incorporation and thymus enlargement is observed the leukae- subsequent cell division. No response was mic cells are 9+ve, Ig-ve, or 0-ve Ig-ve. In observed prior to the 4th day, but by 7 days thymectomized mice 0-ve, Ig-ve or Ig+ve mean 3[H]TdR incorporation by exudate- leukaemias may develop. The leukaemias aris- treated cells had risen to 60% (range 46- ing in intact and thymectomized mice may be 830) as compared to 10% (range 0-2%) in derived from the same or different target the case of control cells, and direct counting cells. In order to investigate these possibili- techniques indicated that exudate-treated, but ties, neonatal thymuses were grafted into not control, cell populations doubled in thymectomized MNU- or BNU-treated mice. number between the 7th and 10th day of In most cases the grafted thymus did not culture. Further prolongation of the prolifera- bring about expression of 0 antigen on termi- tive response was not observed, under the nal leukaemic cells. The thymus may be culture conditions employed in the present either the site of a target cell population or 430 B.A.C.R. 18TH ANNUAL GENERAL MEETING the site required by the target cell population EXAMINATION OF THE MEMBRANE for expression of 0 and rapid proliferation. PROTEINS OF HUMAN MELANOMA Thus thymectomy may remove either the CELL LINES. G. P. ROBERTS, R. H. target cell or essential factors required for WHITEHEAD and L. E. HUGHES, University maturation of the target cell. Leukaemias Department of Surgery, Welsh National School develop in thymectomized hosts, grafted of Medicine, Cardiff. with neonatal thymus, only after a prolonged induction time and only rarely express 8 Cell-surface components play an important antigen. These results are consistent with role in regulation of cell growth, antigenicity there being a target cell population within and cellular recognition. There is increasing the evidence that the plasma membranes of thymus. tumour cells differ from those of normal cells, but these studies have been largely confined IMMUNOADSORBENT PURIFICA- to laboratory animal cells. Therefore, a study TION OF A RAT SARCOMA SPECIFIC has been made of the cell surface proteins of ANTIGEN. V. E. PRESTON and M. R. PRICE, human melanoma cell lines. The cell-surface Cancer Research Campaign Laboratories, proteins were labelled with 125I or 1311 in the University of Nottingham. presence of lactoperoxidase, and the labelled Soluble fractions retaining tumour specific proteins examined by SDS electrophoresis antigenic activity were prepared from a on a 5-22.5% acrylamide gradient followed 3-methylcholanthrene-induced rat sarcoma by autoradiography. As many as 24 cell- by 3 M KCI treatment of tumour tissue. surface proteins were detected in the indivi- These extracts, being initially highly hetero- dual melanoma cell lines; the molecular genous, were fractionated by immunoadsor- weights of the mercaptoethanol-reduced sub- bent procedures involving: (a) the binding of units ranged from about 10,000 to 240,000. tumour-specific antigen to syngeneic rat There were considerable differences in the anti-sarcoma antibodies immobilized upon protein profiles of the different melanoma Sepharose 4B (Pharmacia, Uppsala), (b) cell lines, only 10 of the proteins being elution of bound material with 3 M NaSCN common to all 4 melanoma cell lines exam- followed by rapid desalting of the antigenic ined, and 7 of these proteins were also protein upon Sephadex G25, and (c) passage detected on the cell surface of fibroblast cell of the antigenic protein fraction over an lines. Attempts were made to detect mela- immunoadsorbent containing normal rat noma-specific antigens by affinity chromato- serum IgG to remove contaminants which graphy of extracts of the labelled cells on non-specifically bind to substituted Sepharose immunoadsorbent columns prepared with an matrices. The material so obtained was antisera against melanoma cells. Electro- characterized by its capacity to neutralize phoretic examination of the proteins bound syngenic tumour specific antibody and to by the immunoadsorbent columns did not induce the formation of specific antibody in reveal any proteins common to all melanoma immunized rats. This antigenic fraction was cell lines but absent from other cell lines. further employed in the preparation of rabbit antisera which, following absorption with HISTOLOGICAL AND IMMUNO- unrelated rat sarcoma cells, were rendered LOGICAL monospecific for the immunizing sarcoma. RESPONSES IN THE Although the material isolated showed limited DRAINING LYMPH NODE DURING heterogeneity as judged by polyacrylamide TUMOUR GROWTH IN RATS. G. gel electrophoresis and gel filtration chromato- ROBINSON.* J. A. JONES and R. C. REES, graphy, the preparative procedure does allow *Department of Pathology and Cancer Research the' rapid recovery of fractions 'which are Campaign Laboratories, University of Notting- suitable for further separation and charac- ham. terization. Also, the availability of these Little detailed information exists regarding semi-purified antigen preparations as well the response of the draining lymph node to a as monospecific heteroantisera mav aid the developing tumour. Using a transplantable development of a quantitative radioimmuno- rat hepatoma (D192A) as a model, the assay for tumour specific antigens associated histological and immunological changes of with chemically induced rat tumours. lymph nodes regional and distal to the POSTER EXHIBITS 431 tumour site were studied at various stages morphological evidence of cell- and humoral- following tumour implantation into the mediated responses to tumour growth was hind limb. Histologically, an early cell- observed in distal nodes, but these were out mediated response was detected in the of phase with those shown by the draining lumbar node draining the tumour site. The node. The response of the draining and distal T-dependent paracortex showed a marked lymph nodes was monitored using the in vitro proliferation of cells, along with increased microcytotoxicity test. Cells from the lumbar numbers and prominence of post-capillary nodes displayed an early cytotoxicity against venules. This response was maintained for D192A and 15-day-old-embryo cell targets, the first 3 weeks of tumour growth, and then which decreased during tumour growth. the paracortex became depleted of lympho- Cells from the cervical lymph nodes showed cytes. Stimulation of the cortical lymph an increasing cytotoxic response towards follicles, with development of active germinal these cell targets. In addition, the presence centres and migration of plasma cells to the of serum antibody, specific for the developing medullary cords, was evident 11 days after tumour, was detected during the latter stages inoculation, and this humoral response of tumour growth by indirect membrane showed no signs of later inactivation. Similar immunofluorescence.