728 BRITISH MEDICAL JOURNAL voLumE 293 20 SEPTEMBER 1986

cannot be taken by itself as a justification for such trials. Each trialmust be justified on its merits, which involve far broader issues thancan be discussed here. Indeed, in the light of more than 35 studiesconfirming the benefits ofmaintenance treatment for schizophreniathe original trial would be unjustifiable if proposed today. Trials ofnew drugs for patients who have already relapsed would be judgedon a different basis.

The follow up study was conducted at the academic unit for clinicalpsychopharmacology at Guy's Hospital Medical School, London. We aregrateful to Dr R Gaind and Dr D Bennett; the community psychiatric nursesand medical records staff of St Olave's and St Francis hospitals; Dr A ASchiff ofE R Squibb and Sons; and Mrs Carol Neale and Mrs Phyl Maskell,who typed all the drafts of the manuscript.

References1 Hirsch SR, Gaind R, Rohde PD, Stevens BC, Wing JK. Outpatient maintenance of chronic

schizophrenic patients with long-acting fluphenazine: double-blind placebo trial. Br Med J1973;i:633-7.

2 Wing JK, Cooper JE, Sartorius N. The measwemen and clasion of psychiatric smptms.Cambridge: Cambridge University Press, 1974.

3 Mindham RH, Gaind R, Anstee BH. Comparison of orphenadrine and placebo on the control ofphenoehiazine-inducedparkinsonism. PsycholMed 1972;2:406-13.

4 Knights A, Okasha MS, Salih MA, Hirsch SR. A trial of fluphenazine versus flupenthixol inmaintenance of schizophrenic outpatients. Brj Psychiary 1979;135:515-23.

5 Platt S, Weyman A, Hirsch SR, Hewett S. The social behaviour asessment schedule (SBAS):ratonale, contents, scoring and reliability of a new interview schedule. Soc Psychiay1980;15:43-55.

6 Platt S, Hirsch S, Weyman A. Social behaviow assessment schedul (SBAS). 3rd ed. Windsor:NFER-Nelson, 1983.

7 Stevens BC. Role of fluphenazine decanoate in lessening the burden of chronic schizophrenics inthe community. P ycholMed 1973;3:141-58.

8 Brockington IF, Kendell RE, Leff JP. Definitions of schizophrenia: concordance and predictionof outcome. Psychol Med 1978;8:387-98.

9 Curson DA, Barnes TRE, Bamber RW, Platt SD, Hirsch SR, Duffy JC. Long-term depotmaintenance of chronic schizophrenic outpatients: the seven year follow-up of the MedicalResearch Council fluphenazine/placebo trial. 1. Course of illness, stability of diagnosis, and therole of a special maintenance clinic. BrJ Psychiaty 1985;146:464-9.

10 Curson DA, Barnes TRE, Bamber RW, Platt SD, Hirsch SR, Duffy JC. Long-term depotmaintenance of chronic schizophrenic outpatients: the seven year follow-up of the MedicalResearch Council fluphenazine/placebo trial. II. The incidence of compliance problems, side-effects, neurotic symptoms and depression. BrJ Psychiaty 1985;146:474-80.

11 Curson DA, Barnes TRE, Bamber RW, Platt SD, HIrsch SR, Duffy JC. Long-term depotnaintenance of chronic schizophrenic outpatients: the seven year follow-up of the MedicalResearch Council fluphenazine/placebo trial. III. Relapse postpoement or relapse prevention?The implications for long-term outcome. BrJ Psychiary 1985;146:474-80.

12 Barnes TRE, Milavich G, Curson DA, Platt SD. Use of the social behaviour asessment schedule(SBAS) in a trial of maintenance antipsychotic therapy in schizophrenic outpatients: pimozideversus fluphenazine. Soc Psychiany 1983;18:193-9.

13 Davis JM. Overview: maintenance therapy in psychiatry. I. Schizophrenia. Am I Psychiany1975;132: 1237-45.

14 Johnson DAW. Haloperidol decanoate and the treatment of chronic schizophrenia. In: JohnsonDAW, ed. Therapeutics today. Vol 2. New York: Adis Press, 1982.

15 Freiman JA, Chalmers TC, Smith H, Kuebler RR. The importance of beta and the type enrrorand sample size in the design and interpretation of the rndomied control trial. NExglJ Med1978;299:6904.

16 Fleiss JL. Statistical methodsfor rates and proportios. New York: Wiley, 1973:1976-94.

(Accepted 28 July 1986)

Plasminogen activators in human colorectal neoplasia

J S K GELISTER, M MAHMOUD, M R LEWIN, P J GAFFNEY, P B BOULOS

AbstractA crucial step in the transition from adenomatous polyp toinvasive colorectal cancer is the degradation of the epithelialbasement membrane. Plasminogen activators may play a part inregulating the extracellular protease environment necessary forthis to occur. Both functional and antigenic activity of the twoprincipal activators ofplasminogen, tissue plasminogen activatorand urokinase, were measured in 30 colorectal cancers, matchedsamples of mucosa, and eight adenomatous polyps. Both polyps(p<0-01) and carcinomas (p<0c001) had raised urokinaseactivities compared with normal mucosa, the activity beinghighest in the carcinomas. Activity of tissue plasminogenactivator, however, was diminished in both polyps (p<0-01) andcarcinomas (p<0001) compared with normalmucosa, the valuesbeing lowest in carcinomas. Plasmin generation by urokinase-incontrast with tissue plasminogen activator-is fibrin independentand thus less subject to physiological control.

Department of Surgey, University College London, The Rayne Institute,London WC1E 6JJ

J S K GELISTER, FRCS, surgical research fellowMR LEWIN, PHD, senior research fellowP B BOULOS, MS, FRcS, senior lecturer in surgery

National Institute of Biological Standards and Control, London NW3 6RBM MAHMOUD, Msc, senior scientific officerP J GAFFNEY, DSc, FRCPATH, senior scientist

Correspondence to: Mr Gelister.

This capacity to produce urokinase may be an important factorcontributing to the malignant potential of adenomatous polypsand to the invasive quality of established carcinomas.

IntroductionThe generation of proteolytic enzymes, including those of thefibrinolytic system, has been implicated in the development andspread of cancer. Fibrinolytic activity results from the conversion ofthe proenzyme plasminogen to the active proteolytic enzymeplasmin, induced by plasminogen activators. Such activators existin two well characterised forms-tissue plasminogen activator andurokinase--which may be distinguished immunologically and bydifferences in molecular weight. The principal physiological role oftissue plasminogen activator is to maintain the vascular tree free offibrin; but a corresponding role for urokinase is less clear.

In malignant tissue plasminogen activators have been ascribedvarious functions by the activation of plasmiinogen. These includedestruction oftumour associated fibrin' and the basement membraneglycoprotein components fibronectin and lanminin,. as well as activa-tion oflatent collagenase.2 Aberrations in production ofplasminogenactivator, both qualitative and quantitative, have been associatedwith experimental' and human cancer.4 MoSt studies of fibrinolysisin human cancer have focused on overall tissue plasnilnogenactivator activity.516 The results, however, have been contradictoryand it was -not possible to distinguish urokinase from tissueplsiogen activator, as these activators have been characterised

only comparatively recently. By employing indirect immunologicalmethods, however, it has been reported that urokinase is producedby maigant colorectal tissue but not by normal mucosa,7 and thisfinding was supported by an immunoperoxidase study whichlocalised urokinase in colorectal tumours but not in normal

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mucosa.8 Urokinase has therefore been suggested as a tumourmarker.9 To date there have been no specific quantitative studies oftissue plasminogen activator and urokinase in either normal ormalignant colorectal tissue. We decided to analyse and quantify byspecific and sensitive techniques, both immunological and func-tional, the activities of urokinase and tissue plasminogen activator inmalignant, premalignant, and normal colorectal mucosa.

Patients and methodsTissue was obtained fresh from 30 patients after a resection for colorectal

cancer. Samples were removed by an independent pathologist from thecentre and edge of the tumour and from macroscopically normal mucosasurrounding the tumour and at the edge of the resection. Samples were alsoobtained from eight patients having adenomatous colorectal polypsremoved. An independent histological diagnosis was made in all patients. Alltissue was washed in phosphate buffered saline, frozen in liquid nitrogen,

729

ResultsThe table summarises the results.Fibrin plates-No lysis was seen on plasminogen free plates that served as

negative controls. There was significantly less activity in carcinoma homo-genates than in normal mucosa (p<0001) but there was no differencebetween either the centre and edge of the tumours or between distant andsurrounding mucosa. Adenomatous polyps had activity intermediatebetween that of malignant tissue and normal mucosa but did not differsignificantly from either.

Tissue plasminogen activator-Normal mucosa had consistently raisedactivities of tissue plasminogen activator compared with both carcinomas(p<0 001) and adenomatous polyps (p<001), as measured by bothbioimmunoassay and enzyme linked immunosorbent assay (table). Therewas no difference between tissue from the tumour edge or centre. Mucosaadjacent to the tumour, however, showed less tissue plasminogen activatoractivity than distant mucosa (p<001) by both assays. Though polypsshowed tissue plasminogen activator activities just higher than carcinomason enzyme linked immunosorbent assay (p<005), activities found bybioimmunoassay were in the cancer range.

Median (range) plasminogen activator activity in colorectal neoplasia

Assay Distant mucosa Polyps Tumour centre

Fibrin plate lysis area (mm) 395 (255-644) 294 (223-352) 263 (58-544)Tissue plasminogen activator (IU ml) Bioinmnunoassay 4-1 (1-5-16-0) 1-3 (0-8-3-9) 0-8 (0-2-3-5)

Enzyme linkedummunosorbent assay 2-1 (1 1-6-0) 1-2 (0-5-5-0) 0-7 (0-3-1-5)

Urokinase (IU/ml) { Bioimmunoassay 0-2 (0-2-0-9) 0-4 (0-2-1-0) 0-7 (0-24-2)Enzyme linked immunosorbent assay 0-7 (0-3-43) 1-3 (0-9-1-8) 2-7 (04-96)

and stored at - 70'C. Weighed portions from each sample were disintegratedby a cryofragmentation technique'" utilising a Mikro Dismembrator II (BBraun) with suspension of the resultant powder in a fixed weight to volumeratio of phosphate buffered saline containing azide (1 g/40 ml). Activity ofthe homogenates was assessed by the following methods.

Fibrin plates were made by a standardised technique" using Kabi L gradefibrinogen at 1 g/l in imidazole buffered saline, pH 7-4. A sample offibrinogen was purified on a lysine-Sepharose column (Pharmacia) to removeplasminogen and used as a negative control. Thirty microlitres ofsample wasassayed in triplicate on each of two plates and the results expressed as lysisareas after incubation for 21 hours at 370C. The assay was performed on allsamples from the centre of the tumours, polyps, and distant mucosa and on15 samples from the edge of the tumours and surrounding mucosa.Bioimunoassay, employed to measure specific functional activity of tissue

plasminogen activator and urokinase in the homogenate supernatants,has been described in detail'2 and was performed for both activators in30 samples from the centre of the tumours and distant mucosa, in 15 samplesfrom the edge of the tumours and surrounding mucosa, and for tissueplasminogen activator in eight and urokinase in six polyps. The procedureentails coating multiwell plates with the IgG fraction from a rabbit antiserumto purified human melanoma tissue plasminogen activator or purified humanurokinase. Plasminogen and the chromogenic substrate S2251 (KabiVitrum)were added to each well and the resultant colour change read in a Titertekautomatic multiscan reader as optical density at 405 nm. Results (expressedin IU/ml homogenate) were determined from a standard dilution curve oftheWorld Health Organisation standard for urokinase or tissue plasminogenactivator.Immunoassay of plasminogen activators-A two site enzyme linked

immunosorbent assay using goat and rabbit antibodies to tissue plasminogenactivator and urokinase respectively was employed. The assay was per-formed for both activators in 16 matched samples of tumour centre, edge,and distant mucosa; for eight polyps; and for tissue plasminogen activatoralone in 16 samples of surrounding mucosa. The procedure was similar tothat described by Rijken et al.'" Results were determined from a standarddilution curve of the appropriate activator and expressed in IU/ml.Zymography-Sodium dodecyl sulphate polyacrylamide gel electro-

phoresis of selected supernatants was performed according to the method ofLaemmli using a 10% separating gel."' Standards of urokinase and tissueplasminogen activator at appropriate dilutions were run as markers with testsamples. Gels were subsequently washed in 2% Triton X-100 and overlaidon to plasminogen enriched fibrin agarose plates and incubated at 370C.'9Gels were photographed before and after staining with Coomassie blue whenclear lysis bands were visible.

Statistics-Results were expressed as medians and ranges and analysed bythe Mann-Whitney U test.

Urokinase-Malignant tissue had significantly higher activities ofurokinase detected by both assays as compared with the low values found innormal mucosa (p<0001). There were no differences between centre andedge of the tumours. Surrounding mucosal samples, examined only bybioimmunoassay, were not different from distant mucosa. Eight adeno-matous polyps, however, had significantly higher immunological urokinaseactivities than normal mucosa (p<001) and three of six polyps assessed bybioimmunoassay showed values in the cancer range.Zymograms of normal mucosal supernatants from 14 patients showed a

dominant tissue plasminogen activator band; nevertheless, a high molecularweight urokinase band (figure) was always evident, and in four adenomatouspolyps both bands were seen. Twelve carcinoma supernatants examined bythis technique also showed both a tissue plasminogen activator and a highmolecular weight urokinase band, often with the urokinase band appearingmore rapidly. No bands corresponding to low molecular weight urokinase orto complexes of either urokinase or tissue plasminogen activator with theirfast acting inhibitor were seen.

Discussion

These results show that the development of malignancy in thecolorectal epithelium was almost invariably accompanied by in-creased production of urokinase. This enzyme was, however,present in very low concentrations in normal colorectal epithelium,as shown by bioimmunoassay and enzyme linked immunosorbentassay and confirmed by zymography. This finding is in contrastwith a single previous zymographic study which failed to detecturokinase in normal colorectal mucosa,7 prompting claims that thisenzyme is tumour specific. In adenomatous polyps the activities ofurokinase and tissue plasminogen activator were also significantlydifferent from those in normal mucosa and mirrored the changesseen in carcinomas. The findings in polyps accord with observationsin experimental carcinogenesis, where aberrations in production ofplasminogen activators often precede the morphological features ofmalignancy. 16

It was not possible to define the cellular origin of plasminogenactivators or to account for the possible variability in cellularcomposition among tissue samples. Probably, however, trans-formed epithelial cells are the source of plasminogen activators(particularly urokinase) within tumours, as other studies haveshown the production of these enzymes by epithelial cells derivedfrom both colorectal carcinomas' 18 and adenomatous polyps'8

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in culture. Immunocytochemical studies have also shown urokinaseto be associated with neoplastic epithelial cells in colonic cancer,819and it has been postulated that urokinase may be released by tumourepithelial cells into the stroma, where it has also been detected.8Though infiltrating white cells are a further potential source ofprotease activity in tumours, lymphocytes have been shown toproduce negligible amounts of plasminogen activators unlessthemselves transformed-for example, by human T cell lympho-tropic virus type IIF20-when appreciable amounts of plasminogenactivator, always including urokinase, were apparent. Moreover,the urokinase content of colonic mucosa from inflammatory boweldisease has been shown not to differ from normal,2' furthersupporting the view that the production of this enzyme is aparticular feature of transformed cells.

Tissue plasminogenactivator

Urokinase *

Urokinase

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BRITISH MEDICAL JOURNAL VOLUME 293 20 SEPTEMBER 1986

of vascularity and is in keeping with current concepts of the widerdistribution and physiological role of tissue plasminogen activator.27Previous discrepancies regarding the fibrinolytic activity of normalas compared with malignant colorectal tissue are explained by thefibrin dependence of plasminogen activation by tissue plasminogenactivator and whether the methods used were fibrin dependent orindependent.28 Thus the enhanced fibrin plate activity of normalmucosa in this investigation was consistent with the raised activitiesof tissue plasminogen activator found in this tissue and withprevious fibrin plate studies.2

It has been postulated that the invasive edge of a tumour isassociated with enhanced protease activity and that malignant cellsmay induce host tissues to release proteolytic enzymes.' Tissuefrom the tumour edge and surrounding mucosa was therefore

F- G H-

Zymogram of normal mucosa and neoplastic colorectal tissue. A= International standard of urokinase (*54 kilodaltons, t31 kilodaltons) 5 IU/ml.B=International standard of tissue plasminogen activator (68 kilodaltons) 10 IU/ml. C-E=Normal mucosa. F-H=Carcinomas. I=Villous adenoma.

There was no relation between activity of plasminogen activatorand tumour grade or Dukes's stage in this study, though thenumbers were small. This may also relate to the comparatively wideranges of activity found, which we considered were a consequenceof biological variability, tumour heterogeneity, and methodology.In two of three patients with disseminated disease, however,urokinase activity (shown by bioimmunoassay) ranked first andthird highest.The generation of plasmin from plasminogen by urokinase,

unlike tissue plasminogen activator, is not fibrin dependent andmay therefore be regarded as less specific.22 Thus a tumourproducing urokinase may have an enhanced capacity to generateplasmin, a broad spectrum proteolytic enzyme with trypsin-likeactivity.23 Plasmin has been shown to stimulate cell turnover invitro24 and is thought to play a crucial part in the destruction ofbasement membrane,9 23 25 a key event in the transition frombenign polyp to carcinoma. Furthermore, the malignant be-haviour in vitro of cells derived from colorectal carcinomas andadenomatous polyps when stimulated by tumour promoterscorrelated with their capacity to secrete urokinase.'8 Moreover,antibodies to urokinase have been shown to inhibit experimentalmetastasis,26 further suggesting that this enzyme may have afunctional role in human colorectal cancer.

Tissue plasminogen activator has not previously been assayed innormal colorectal mucosa. This study clearly showed that this tissuewas rich in functional tissue plasminogen activator as comparedwith carcinomas and polyps. Probably this is not simply a reflection

examined and, while there were no differences between tumourcentre or edge, surrounding mucosa had less tissue plasminogenactivator activity compared with distant mucosa. Possibly methodo-logical problems related to the small size ofthe surrounding mucosalbiopsy sample may have contributed to the tissue plasminogenactivator findings. It has been suggested that colorectal and breastcancers produce the recently described fast acting inhibitor of tissueplasminogen activator,7 which has been associated with reducedfibrinolytic activity and thrombotic phenomena.3' Our studydoes not support this finding, immunological activities of tissueplasminogen activator not being higher than functional values andno bands corresponding to activator inhibitor complexes beingdetected by zymography.Thus the sequential changes in urokinase and tissue plasminogen

activator production that we found were in keeping with the conceptof an adenoma-carcinoma sequence in colorectal neoplasia. Whilethe progression ofcolorectal neoplasia is certain to be multifactorial,other proteolytic enzymes have recently been implicated in theinvasive capacity of malignant tumours.32 The physiology ofurokinase and the pattern of its production, supported by previousevidence implicating this enzyme in cancer, suggest that it may wellplay a part in the progression of both premalignant and malignantcolorectal tumours.

JSKG and PBB acknowledge support from Smith Kline and French andfrom the special trustees of University College Hospital.

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References1 Peterson HI, Kjartansson I, Kersan-Bengsten KB, Rudenstam. CM, Zettergren L. Fibrinolysis in

huma"n malignant tumosurs. Acta CkirScand 1973;139:219-23.2 Liotta LA, Goldfarb RH, Brnmdage R, Siega GP, Terranova V, Garbisa S. Effect ofpasioe

activator (urokinase), plamoin, and tdrombin on glycoprotein and collagenous components ofbasement membrane. CancerRes 1981;41:4629-36.

3 Quigley JP. Proteolytic enzymes of normal and malignant cells. In: HyDes RO, ed. Swrfaces ofxormdnland malgnant cedls. Chichester: John Wiley and Sons, 1979:247485.

4 Corsanti JG, Celik C, Camiolo SM, et al. Plasninogen activator content of human colon tumorsand normal mucosae: separaton of enzymets and partial purification.jNCI 1980;65:345-5l1.

5Clifton EiE, Grossi MD. Fibrinolytic activity of humarn tumors as measured by the fibrin platemethod. Cancer 1955;8:1146-54.

6 Malone JM, Wangensteen MD, Moore WS, Keown K. The fibrinolytic system. A key to tumormetastasis?Ams Surg 1979;10:342-9.

7 Tissot JD, Hauert J, Bachmann F. Characterisation of plasminogen activators from normalhuma-n breast and colon and from breast and colon carcinomas. Intl Cancer 1984;34:295-302.

8 Kohpg S, Harvey SR, Weaver RM, Markus G. Localization of pamognactivators in humancolon cancer by immunoperoxidase staining. CaxcffrRes 1985;4S: 1787-96.

9 Dano K, Andreassen PA, Grondahl-Hansen J, KristensenP, Nielsen LS, ShriverL. Plasminogenactivators, tissue degradation and cancer. Adv CaxcerRes 1985;4: 138-265.

10 de Coasart L, Marcusson RW. A simple quantitative assay of tissue pamognactivator.J GlinPaduol 1982;3S:980-3.

11I Marsh NA. Fibrinolsis. Chichester: John Wiley and Sons, 19811:213-4.12 Mahmoud M, Gaffiney PJ. Blomuoasy(BIA) of tissue plasminogen activator (tPA) and its

specific inhibitor (tPAlInh). Tkromub Haensos 1985;53:356-9.13 Rijken DC, Van Hinsbergh VWM, Sens EHC. Quantitation of tissue type pamoenactivator

in hulman endothelia cell cultures by use of an enzyme immutnoassay. Tkromb Res 1984;33:145-53.

14 Laemmli UK. Cleavage of structural proteins during assembly of the head of bacteriophage T4.Natur 1970,22:6805.

15 Granelli-Piperno A, Reich E. A study of protease and protease-inhibitor complexes in biologicalflukids.J ExpMed 1978;148:223-34.

16 Gree Uj, Rumsby PS, Roscoe JP. An increasein plsioeactivatormRNA occurs at anearlystage in ethylnirroourea induced transformation of rat brain cells. CarmognVesit 1986;7:477480.

17 Carretero F, Fabra A, Adan J, et al. Fibrinolytic activity ofhuman invasive tumours and derivedprimary cultures. In: Davidson JF, ed. Progress infibrinolsis. London: Churchill Livingsone,1985:276-9.

18 Friedman E, Urmacher C, Winawer S. A model for human colon carcinoma evolution based onthe differential response of cultured preneoplastic, premalinant, and maligant cells to12-0-tenaddcanoyl-phorbol-13-acetate. CancerRes 1984;44:1568-78.

19 Burtin P, Chavanel G, Andre J. The plsmin systen in human colonic tumors: an immuno-fluorescence study. Intl Cancer 1985;35:307-14.

20 Hinuma S, Honda S, Tsukamoto K, Sugamara K, Hinuma Y. Production of plainoenactivators by human T-ell eukaemia virus-transformed human T cell lines. Br J .Cancr1985;51:753-9.

21 Verspaget HW, De Bruin PAF, Neterman IT, et al. Changes in plsminonactivator activity ininflammatory bowel disease (IBD). Gut 1986;27:A630.

22 Matsuo 0, Rifken DC, Colen D. Comparison of the relative fibrinolytic and thrombelyticproperties of tissue plasminogen activator and urotinase in vitro. Tkromb Hamost 1981;45:225-9.

23 Duffy MJ, O'Grady P. PLasminogen activator and cancer. Eur J Cancer Cln Oncol 1984,20:577-82.

24 Whur P, Sik-ox JJ, Boston JA, WilliamsfDC. Plasminogen activator transforms the morphology ofquiescent 3T3 monolayers and iniiatesgrowth. BrJ Cancer 197939:718-30.

25 O'Grady RL, Upfold LI, Stephens RW. Rat mammary carcinoma cells secrete active collagenaseand activate latent enzyme in- the stroma via plasminogen activator. Int J Cancer 1981;28:509415.

26 Ossowski L, Reich E. Antibodies to pl n activator inhibit human tumor metastasis. Cell1983;33:616-9.

27 Granelli-Piperno A, Reich E. Plasminogen activators of the pituitary gland: enzyme characterisa-tion and horonl modulation.I CellBiol 1983;97:1029-37.

28 Gelister JSK, Mahmoud M, Gaffney PJ, Boulos PB. Proteinase-like peptidase activities inmalignant and non-malignant gastric tissue. Brj Surg 1986;73:84-5.

29 Szczepanski M, Lucer C, Zawadzki J, Toloczko T. Procoagulant and fibrinolytic activities ofgastric and colorectal cancer. Intl Cancer 1982;30-.329-33.

30 Wooley DE. Collagenase immunolocalisation studies of human tumours. In: Liotta LA, Hart IR,eds. Ttowr vaio and meastas. The Hague: Martiaus Nijhoff, 1982:391-404.

31 Julian-Vague I, Moerman B, De Cock F, Ailland MF, Collen D. Plasma levels of a specificinhibitor of tissue type plasminogen activator (and urokinase) in normal and pathologicalconditions. ThrombRes 1984;33:523-30.

32 Durdey P, CooperJC, Switala S, KingRFGJ, Williams NS. The role ofpeptidases incanceroftherectum and sigmoid colon. BrJ Surg 1985;f2:378-81.

(Accepted 26Jue 1986)

SHORT REPORTS

Clearance of psoriasis with low dosecyclosponPrevious studies have shown that T helper lymphocytes play an integral partin the pathogenesis of psoriasis.' 3 Cyclosporin A is a selective immuno-suppressive drug that specifically inhibits T helper cell function byimpairing the production and effects ofinterleukin-2.4 We therefore studiedthe efficacy of cyclosporin A in the treatment of psoriasis, using low dosesbecause of its dose related nephrotoxic effects.

Patients, methods, and results

We studied 10 patients with severe psoriasis that was unresponsive toconventional treatment (including methotrexate and psoralens and ultraviolet A)(table). Informed consent was gained from each patient before entry to the study.Cyclosporin A was taken once daily in the evening for 12 weeks. In eight patientsthe initial daily dose was 1-66 pmol/kg (2 mg/kg); the other two, who had verysevere active disease, were given 2-49 unmo1/kg (3 mg/kg). The dose was increasedby 0-83 &molI/kg (1 mg/kg) if there was no improvement after two weeks'

treatment; the agreed Maximum dose was, 3-32 ILmoUkg/day (4 mg/kg/day).Patients were assessed before treatment and at three days and one, two, three,four, six, eight, and 12 weeks. Before treatment and at each subsequent visit thefollowing investigations were performed: blood -pressure, urine analysis, ureaand electrolyte concentrations, serum creatinme concentration, liver function,and fulfl blood count. Blood cyclosporin concentrations were measured byradioinmmunoassay as whole blood trough concentration about 12 hours after thelast dose. The area of skin affected was determined at each visit and severity ratedon a scale of 0-10.

Psoriasis cleared completely in five of the 10 patients and there was anappreciable improvement in the other five. Of the five in whom psoriasis clearedcompletely, clearance was achieved by six weeks in case 1 and by eight weeks incases 2, 3, 4, and 5 (table). The therapeutic dos of cyclosporin A seeme to be2-49 pmol/kg/day (3 mg/kg/day); increasing the dose to 3.32 ltmoL'kg/day (4 mg/kg/day) did not induce clearance in those in whom the disease had not cleared atthe lower dose. There was no correlation between climcal response and wholeblood cyclosporin concentrations, and in all but one patient concentrations wereless than 0-62 tsmo1/l (750 ng/mI) on all occasions. Two patients experienced anincrease in blood pressure, and -in four serum creatinine concentration increasedby more than 10%/ during the study, thouQi the values were still within. thenormal range. Three of the six women developed hypertrichosis, cosstn Offine lanugo hairs on the face and. coarse terminal hairs on the legs. All threesaid that they were prepared to tolerate this side effect if their psoriasis- cleared

Clmnial data on patients with psoniasts partucpattng in study

Body area Blood pressure- Creatinine concentrationaffcted* (mm Hg) ("001l/) Mean (range)

Previous Cyclosporin A cyclosporin A bloodsystemiic- Start End Start End Start End dose concentrations

Case No Age/sex treatment of study of study of study of stsdy of study of study (pmo1/kg/day) (RsmoJA)

1 381F Methotrexate 5 () 100/80 12M8 .68 85 1-66-2-49 0-23(0-11-0 32)2 32/F PUVA 5 0 105/80 125/80 87 89 1-66-2-49 0-17 (011-0 25,)3 51/F PUTVA 5 0 140/80 120/75 85 75 2-49 0--31 (0-25-0-34)4 40/M PIJVA 2 0 125/85 130/85 91 112 1-66-2-49 0-30(0-16-0-39)S 55/F PUVA, Methotrexate 3 0 150/9 -130/90 80 73 1-66-2-49 0-51 (0-31-0 60)6 33/M PUVA, Methotrexate 7 1 135/90 140/80 94 76 1-66-3-32 0-47 (0-26-061)7 64/F Mcthotrexate,PUVA 5 1 125/85 130/80 65 65 1-66-3-32 0-26(Q16-0-30)8 42/F PUVA 2 1. 140/90 160/105 85 98 1-66-3-32 0-34(0 16-059)9 62/M PUVA, Methotrexat 5 2 120/85 140/100 84 94 2-49-3-32 0-63 (0544)-08)10 38/M Methotrexate,Razoxane 3 2 140/95 140/90 94 95 1-66-3-32 0-28(0-184034)

*0=<I%, 1=1-10%, 2=11-20%, 3=21-30%, 4=31-40%, 5=41-50%, 6=51-60%, 7=61-70%, 8=71-80%, 9=81-90%, 10=91-100%. PUVA=Paoraiens and ultravioletA beatimai.Couuesiw,:Slto madisiouualims-Creatiaine:1 MmoI10-01 mg/100 ml.Cyckosporin A: lMmol=1-2 mg.

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