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Saturday 8 August 1964

IMMUNOLOGICAL ASPECTS OF CANCER *

M. F. A. WOODRUFFM.D., M.S., D.Sc. Melb., F.R.C.S., F.R.C.S.E., F.R.A.C.S.

PROFESSOR OF SURGICAL SCIENCE IN THE

UNIVERSITY OF EDINBURGH

* Almroth Wright lecture delivered on May 25, 1964, at the Wright-Fleming Institute of Microbiology, St. Mary’s HospitalLondon, W.2.

IT used to be taught that tumours are autonomous; irother words that they are independent of all the con-straints which maintain normal tissue equilibriums. Thisidea is fortunately dead; it is high time that it was buried,The discovery by Charles Huggins and others oj

hormone-dependent tumours established an importantclass of exceptions to the rule. The observation thatsome tumours, including ones which show no evidence 01hormone dependence, undergo spontaneous regression(Everson and Cole 1956, Brunschwig 1963, Fullerton andHill 1963, and others) or, conversely, suddenly reappearafter a period of complete quiescence (Woodruff 1961),suggests that non-endocrine factors are also important,and confirmatory evidence of this is provided by thefrequent failure of both naturally circulating tumour cellsand experimental autotransplants of human tumours toestablish metastases.Let me cite some examples. First a case of spontaneous

regression reported by Fullerton and Hill (1963):A woman underwent subtotal gastrectomy in 1947 at the age

of 58 for carcinoma of the stomach. The tumour was found toinvolve the transverse mesocolon, but in view of the poorcondition of the patient complete resection, which could haveinvolved removing a segment of colon, was not attempted. Thediagnosis of diffuse gastric carcinoma was confirmed histo-logically. A second laparotomy was performed later in thesame year to see whether the residual mass was resectable; but,as it did not appear to be so, nothing was done beyond remov-ing a small piece of tissue for section. Histological examinationconfirmed the diagnosis of carcinoma. The patient lived untilOctober, 1963, when she died as a result of hypertension andrespiratory-tract infection. At necropsy there was no evidenceof carcinoma in the abdomen or elsewhere in the body.Next, an example of the sudden reappearance of a

tumour after a long latent interval:The patient was a woman age 50 in whom a former colleague

of mine, Mr. V. T. Pearse, performed a radical mastectomy fora scirrhous carcinoma of the left breast. Three years previouslythe patient had had a melanoma removed from her right foot,but there had been no evidence of either local recurrence ormetastasis of this tumour. After the mastectomy she wasgiven deep X-ray therapy, and within a few weeks there weretens of thousands of subcutaneous melanomatous nodules inthe irradiated area. Metastases then developed in the liver andother organs, and within a few months the patient was dead.It seems clear that either the trauma of the operation or theradiotherapy, or both together, had disturbed the tumour-hostequilibrium, and there is no knowing how much longer themelanoma cells might have remained quiescent in the absenceof this stimulus.

Thirdly, let us consider the frequent failure ofdisseminated tumour cells to form metastases.

Early reports by Warren Cole and others (Cole et al.1954, Engell 1955, Cole 1959) of the presence of largenumbers of neoplastic cells in the venous blood leavingtumours have recently been criticised on the ground thatmany of the cells which were diagnosed as neoplasticwere in fact normal cells. Even when full allowance ismade for errors of this kind, however, it is difficult to

escape the conclusion that only a small proportion ofneoplastic cells which gain entry to the blood-stream

actually form metastases. Moreover, experimental auto-transplants of human tumours in patients with advancedcancer, in the form of either intravenous injections of cellsuspensions or small solid transplants, often either fail totake or are destroyed within a few weeks (Southam et al.1957, Grace and Kondo 1958, Grace and Lehoczky 1959,Southam and Brunschwig 1961, Howard 1963).Much work will be required to elucidate fully the nature

of the non-endocrine factors which may limit tumour

growth, but there are grounds for believing that some atleast of them are immunological. Let us examine the dataon which this hypothesis is based.

Immunogenetics of Tumour TransplantationMuch important information has come from the study

of transplanted tumours, but great caution is required ininterpreting the experimental findings.As Jacob Furth (1963) has pointed out, the history of

research in tumour immunology can be divided into threeeras.

The first began with Ehrlich’s (1906) discovery thattumour transplants often failed to take in mice which hadpreviously received a transplant of the same or anothertumour. This observation was confirmed and extended bymany workers, including Bashford and his colleagues inthe laboratories of the Imperial Cancer Research Fund.Unfortunately, owing largely to the lack of geneticallyuniform strains of laboratory animals and the failure tomake or to take into consideration comparable observa-tions with transplants of normal tissues, many investiga-tors thought that they were discovering something abouttumours when in fact they were studying the laws oftransplantation, and many erroneous conclusions weredrawn. This period dragged on for many years and has noprecise end-point, but its death-knell was sounded byWoglom in a pungently critical article published in 1929.In retrospect perhaps the most interesting observationsduring this period were those of J. B. Murphy of theRockefeller Institute on the role of the lymphocyte inresistance to tumours, and his monograph on this andrelated subjects, which was published in 1926, is wellworth re-reading.The second era saw the development of precise immuno-

genetic analysis of the phenomena of transplantation ofneoplastic and normal tissues by Snell, Gorer, Medawar,Mitchison, and a host of other investigators (for reviewsee Woodruff 1960). It was made possible by the develop-ment of inbred strains of animals, which was initiated byLittle at Bar Harbor (see Little 1941).

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The main conclusions which emerge from this work

may be summarised very briefly: -

1. Homotransplants, whether of normal or neoplastic tissue,as a general rule, evoke a state of immunity in the recipient, as aresult of which they are sooner or later destroyed.

2. The antigens concerned are genetically determined, andthe corresponding genes, which are termed histocompatibilitygenes, are inherited as mendelian dominants.

3. Immunisation does not occur if the recipient possesses allthe antigens present in the donor.

4. Even when the donor does possess antigens lacking in therecipient, the transplant may escape destruction (a) if theseantigens are " weak ", i.e., capable of evoking only a weakreaction; (b) if the mechanism of immunological response isweakened or abolished in one of the ways to be described later;or (c) if, as often happens with tumour transplants, the tissue ofthe transplant has the capacity to survive in the face of apowerful immunological reaction.

5. It is sometimes possible to demonstrate antibody in theserum of homograft recipients by red-cell agglutination orhxmolysis, leucocyte agglutination, complement-fixation,in-vitro cytotoxic tests, immunofluorescent techniques, andvarious other procedures. One of the other procedures, whichwould assuredly have interested Almroth Wright, is the

opsonisation test of Bennett, Old, and Boyse (1963), in whichspecific immune serum stimulates phagocytosis of tumour cellsby peritoneal macrophages from normal animals.Such antibody may play a role, and sometimes a

dominant role, in the process of destruction of a graft.Often, however, cellular attack is of decisive importance,and homografts may be destroyed by this means in theabsence of demonstrable antibody. Moreover, the

presence of antibody may under certain conditions actuallyfacilitate the growth of a transplant. This phenomenon,which in the case of tumour transplants may be quitespectacular, was discovered by Kaliss (Kaliss and Molomut1952, Kaliss 1955, 1957, 1962), and is known as

enhancement.The third era, which overlaps with the second, dates

from the discovery that, with some tumours, animals iso-geneic with the one in which the tumour originated maybe made resistant to transplants of the tumour or maydevelop antibodies active in vitro against it.The first observation of this kind was made by Gross

(1943, 1945), who demonstrated active immunisation ofinbred C3H mice to transplants of a tumour of recentorigin induced by methylcholanthrene in a mouse of thesame strain. This suggested that the tumour in questionpossessed one or more special antigens lacking in normalC3H tissues; but there remained the alternative possi-bility that the mouse in which the tumour originatedhappened to possess a mutant histocompatibility gene.Further experiments by Foley (1953b), and later byPrehn and his colleagues (Prehn and Main 1957, Prehn1960, 1962), incorporating a series of important controls,showed that many other chemically induced mousetumours were also capable of immunising animals of thesame inbred strain as the one in which the tumour

originated, and that this could be explained only bypostulating the existence of tumour-specific antigens.Such antigens have also been shown by similar methods

to occur in certain other types of mouse tumour, includingsarcomas induced by’ Cellophane films (Klein et al. 1963),polyoma virus tumours (Habel 1961, 1962a b, Sjogrenet al. 1961a b, Sjogren 1961, Sachs 1961), and variousleukaemias (Gorer and Amos 1956) including some knownto be of viral origin (Klein et al. 1962, Slettermark andKlein 1962), and in a few tumours in various otherspecies (for review see Old and Boyse 1964).

The methods used to induce immunity to a tumour inanimals isogeneic with the one in which the tumour

originated include (a) excision of a tumour transplant orligation of its blood-vessels (Foley 1953a, Prehn andMain 1957), (b) injection of tumour cells in numbers toosmall to produce lethal tumours (Gross 1943, Old et al.1962, Klein et al. 1962), (c) injection of heavily irradiatedtumour cells which are viable but incapable of continueddivision (Revesz 1960), and, in the special case of tumoursof viral origin, (d) infection with tumour- virus (Habel1961, Sjogren et al. 1961b, Sachs 1961), and (e) trans-plantation of a histo-incompatible tumour produced bythe same virus (Sjogren 1961, Habel 1962b).

Active immunity induced by some of these methods hasbeen transferred adoptively by means of immunised cellsto other animals of the same strain (Koldovsky 1961a, b,Klein et al. 1963, and others). It has also proved possiblein some instances to make an animal resistant to its ownautochthonous tumour (Prehn, 1960, 1962, Klein et al.

1960, Old et al. 1962).The chemically induced tumours vary greatly in their

immunising capacity; and different tumours, even ifinduced by the same chemical, appear to have individuallydistinct antigens. On the other hand, as a general rule,different tumours induced by the same virus possess thesame specific antigen, even when, as in the case of thepolyoma tumours, all demonstrable virus and virus-

antigen has disappeared from the tumour.Attempts to demonstrate tumour-specific antigens in

mouse mammary tumours (many, and possibly all, ofwhich are of viral origin) by the methods described abovehave for the most part yielded negative or equivocal results(Gross 1947, Foley 1953a, Axelrad and van der Gaag1959, Revesz 1960). Symes and I observed, however,that when mice of our A strain developed a spontaneousmammary carcinoma or received a transplant of a mam-mary carcinoma originating in another member of thisstrain, the spleen and regional lymph-nodes becameenlarged and on histological examination showed a plasma-cell reaction. After excluding various other possiblecauses we concluded that these changes could be accountedfor only on the basis of an immunological reaction evokedby specific antigen liberated from the tumour (Woodruffand Symes 1962a).

Immunological Properties of AutochthonousTumours

As already mentioned, it is sometimes possible to

immunise an animal against its own tumour; and this

suggests that some tumours may evoke a state of immunitywithout experimental intervention of any kind. Thetumours most likely to do this are chemically inducedtumours and tumours of viral origin. In view of the factthat chemical factors are now known to play an astiologicalrole in certain types of human cancer (notably cancer ofthe lung and the bladder), and the likelihood that virusesare concerned in human leukaemia and possibly also incancer of the breast and various other organs, it seems

possible that some tumours may be antigenic in the

person in whom they originate, and are in consequencesubject to at least some degree of immunological control.

This hypothesis would explain the pattern of histo-logical reaction sometimes found in the lymph-nodes inthe vicinity of tumours-commonly described in patho-logists’ reports as reactive hyperplasia-and also the

phenomena of spontaneous regression and frequentfailure of circulating tumour cells to form metastases, to

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which reference has already been made. On the other

hand, as the British Medicaliournal (1964) remarks, " the

most immediately obvious feature of malignant tumoursis that they are accepted by the tissues as part of the body,and do not excite the sort of vigorous reaction provokedby bacterial and other foreign invaders. Thus the tumourflourishes until it kills its host ".

This apparent contradiction can be resolved if we postu-late that tumours which evoke an immunological reactionare either destroyed at an early stage-possibly beforetheir presence is even suspected-or succeed in escapingfrom control. Possible escape mechanisms may be:

1. Loss of specific antigen.2. Inhibition of immunological response: (a) non-specific,

(b) specific. -

3. Non-immunological escape (development of capacityto grow progressively in the face of an immunological reaction).Loss of tumour-specific antigen would clearly deprive

the postulated immunological defences of their pointd’appui. We have obtained evidence (Woodruff andSymes 1962b) which strongly suggests that this occurswith the mouse mammary tumours we have studied,when they are serially transplanted in the strain of origin.It seems reasonable to postulate that similar antigenicdeletion may sometimes occur also in tumours which arenot transplanted but remain sufficiently long in theindividual in which they arise. The particular tumourswe have used are not very suitable, however, for

investigating this, because they kill too quickly.Some degree of non-specific depression of the capacity

for immunological response is seen in patients with

neoplastic diseases of lymphoreticular tissue (Fairley andMatthias 1960, Barr and Fairley 1961, Fairley and Akers1962), and possibly also to some extent in patients withother types of advanced cancer (see Southam et al. 1957).This probably accounts for the prolonged survival ofskin homografts which has been observed in such patients(Snyderman 1959), and partly also perhaps for theirunusual susceptibility to infection.

Specific inhibition of an immunological reaction, as

Billingham, Brent, and Medawar (1956) have pointedout, may occur at three levels-afferent, central, andefferent. According to their definition afferent inhibitiontakes effect by direct inactivation of antigen, or bypreventing its release (for example from a homograft)or its access to a seat of response; central inhibition affectsthe machinery of antibody formation or some comparableimmunological process; and efferent inhibition preventsthe effectors of the immune reaction from exercising theiraction. Specific immunological tolerance is an example ofcentral inhibition; enhancement (vide supra), in the

light of recent work by Moller (1963a, b, c), seems to bedue partly to afferent and partly to efferent inhibition.There is a considerable body of evidence that specific

inhibition can occur with tumours passaged in animalsisogeneic with the primary host (Koldovsky 1963,Koldovsky and Svoboda 1962); and if this is true it wouldbe surprising if it could not occur also, on occasion, in theprimary host itself. The criteria for distinguishingbetween the various types of specific inhibition have beendiscussed by Medawar and others (see Woodruff 1964),and some attempts have been made to apply these to thestudy of the tumour-bearing animal. The time seems tobe ripe for a full-scale investigation.

Finally, there is the possibility that mechanisms of anon-immunological kind may enable a tumour to survive

in the face of a powerful immunological reaction directedagainst it. It may be difficult, however, to distinguishbetween such a phenomenon (if it occurs) and specificefferent inhibition of the immune response in the form,for example, of neutralisation of antibody by largeamounts of antigen liberated by the tumour cells (seeFeldman and Sachs 1957, 1958); and so far there does notseem to be any convincing evidence which bears on thispoint.

Immunotherapy of CancerIn all forms of anti-cancer therapy the objective is to

destroy the tumour without seriously damaging the host.When the tumour is localised this can often be achieved

by surgical excision or, if the tumour is sufficiently radio-sensitive, by radiotherapy; but it is now clear that manypatients with apparently localised tumours are harbouringundetected but widespread metastases. Thus, for example,about a quarter of the patients with stage-i cancer of the--breast who are treated by radical mastectomy die as theresult of distant metastases, some of which were alreadypresent, presumably, when the primary tumour wastreated.

If the tumour has already metastasised, or has spreadlocally to involve vital structures, complete excision is

impossible. Conceivably, if the bulk of the tumour isremoved, natural defence mechanisms may sometimessuffice to deal with the remainder, but this certainly doesnot seem to happen commonly, and many would doubtwhether it happens at all. Hence additional methods oftreatment are urgently needed. Great efforts are beingmade, as we all know, to develop safe and effective

chemotherapeutic agents for treating cancer; and,although progress has been slow, these may provide theeventual solution to the problem. But it would be foolishto neglect the alternative approach suggested by the studyof tumour immunology.The following procedures are being investigated or

seem to merit investigation:1. Attempts to maintain, or if possible intensify, the host

reaction to the tumour.

2. Treatment with immune serum from isogeneic, allo-

geneic, or heterospecific donors.3. Treatment with immunologically competent cells from

normal isogeneic, allogeneic, or heterospecific donors.4. Treatment with immunologically competent cells from

preimmunised isogeneic, allogeneic, or heterospecific donors.

If the tumour possesses specific antigens but has escapedfrom control as the result of some form of inhibition of the

immunological response or by a non-immunologicalescape mechanism, it might be directly attacked bymeasures designed to intensify the host reaction, bypassive immunisation with antiserum, or by adoptiveimmunisation with cells from normal isogeneic donors orimmunised donors of any strain or species. The effective-ness of these various procedures will clearly depend on themechanism by which the tumour has escaped from control.If, for example, an anti-tumour effect were obtained withimmunologically competent cells from both normal andpreimmunised donors, this would suggest that the host,through a central failure of the response mechanism, hadbecome specifically tolerant of the tumour antigens. If,on the other hand, an effect were obtained with cells frompreimmunised donors only, this would suggest some formof afferent inhibition-without, however, excluding thepossibility of central inhibition, since the transferredcells might not have survived long enough in the host to

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damage the tumour, or might themselves have becomespecifically tolerant of the tumour antigens.

If the tumour lacks specific antigens, the attack cannotbe directed in the same way, though we might still beable to direct it in non-immunological ways-e.g., byconcentrating the agent (antibody or cells) in the tumour,or by preliminary subsidiary procedures designed to makethe tumour more susceptible than normal tissues to

immunological attack. Alternatively, the immunologicalprocedure might be non-directed (i.e., might damagetumour and host indiscriminately) but might be followedby some other type of directed attack.The procedures under discussion can of course be

combined with each other or with other forms of treat-ment such as surgical excision, radiotherapy, and chemo-therapy. It might also be possible to use immunologicalprocedures to direct other forms of attack, for exampleby coupling tumour-specific antibody with a cytotoxicagent as suggested by Nairn et al. (1963).

Let us consider now some examples of the applicationof these procedures to the treatment of animal and humantumours.

Attempts to Maintain and Augment the Host ReactionCohen and Cohen, in a series of experiments extending

from 1953 to 1960 (see Cohen 1964), showed that theradiosensitivity of mammary adenocarcinomas in C3Hmice could be increased by inoculating the animal withtissue of its own tumour which had been excised andirradiated in vitro, and somewhat similar results haverecently been obtained by Haddow and Alexander (1964)with chemically induced sarcomas.. Koldovsky (1962)found that the survival of CBA mice with spontaneousmammary tumours was almost doubled by subtotalremoval of the tumour followed two to five days later bythe injection of alcohol-killed tumour cells.

In patients with advanced tumours Woodruff andNolan (1961) sought to improve the results of chemo-therapy by removing the spleen before administering thedrug and replacing it afterwards in the form of a cell

suspension. We hoped in this way to avoid, or at leastminimise, the immunological crippling which normallyresults from administration of the cytotoxic drugs at

present used in cancer chemotherapy, and which appearsto be due largely to the damage they inflict on lymphoidtissue. The results were inconclusive, but the safety of theprocedure was established, and the way is open for furtherinvestigations of the same kind.

Other investigators, notably Graham and Graham(1959,1962), have attempted to increase the host resistanceto human tumours by injecting tumour tissue, or tissueextracts emulsified with Freund’s adjuvant; but so far theresults have been disappointing. There appears to be arisk of inducing autoimmune disease by procedures ofthis kind, although so far this does not seem to have beenreported.

Passive Immunisation with Serum

The clinical results obtained with passive immunisationhave been disappointing (see Southam 1961). This may bedue partly to the fact that most of the patients treated havehad advanced tumours. In addition, however, it seems

likely, from what we know of homograft rejection, that

t The statement refers to autochthonous tumours, but it holds goodalso for tumours transplated to animals isogeneic with theanimals in which they originated. It does not hold (as will beseen later), when a tumour is able to grow in a foreign strain, andis transplanted to a member of this strain.

humoral antibodies alone may not be very damaging toforeign tissue-whether in the form of a homograft or anautochthonous tumour-except perhaps in the specialcase of the leukoses.

This form of treatment may also be highly dangerous,especially if heterospecific sera are used. The danger islikely to be reduced if the serum is first absorbed withnormal tissue of the recipient species, or alternatively ifthe serum donor has been made tolerant of such tissue inearly life, as in the experiments of Levi and Schechtman(1963); but the anti-tumour effect may then be greatlyimpaired or even lost.

Perhaps the most encouraging results with experi-mental tumours are those of Koldovsky and his colleaguesin Prague, who combined injection of immune serum withsurgical excision of the tumour (Koldovsky 1962) or

radiotherapy (Koldovsky and Lengerova 1960).

Transplantation of Immunologically Competent CellsKoldovsky’s group showed that the survival of A-strain

mice which received transplants of sarcoma i and othertumours could be prolonged by injecting them intra-venously with spleen cells from another A-strain mousewhich had been immunised by giving it a transplant ofthe same tumour and then destroying this in situ byrepeatedly ligating its vascular connections. The treat-ment was effective, however, only if it coincided with orpreceded transplantation of the tumour (Koldovsky andLengerova 1960, Koldovsky 1961a) or, if given later, wascombined with irradiation (Koldovsky and Lengerova1960) or surgical excision of the tumour (Koldovsky1962).

Loutit and Mathe, and their colleagues, showed thattransplanted leukaemia in mice could sometimes beeradicated by treatment in the form of potentially lethalor supralethal whole-body irradiation followed byinjection of allogeneic bone-marrow or spleen cells, andsuggested that this was due in part to an immunologicalreaction on the part of the transplanted cells (Barnes et al.1956, Barnes et al. 1959, Barnes and Loutit 1957, Matheand Bernard 1959a, b, Mathe 1960). All the mice whichwere freed of leukaemia in this way died of graft-versus-host disease; but, despite this, Mathe and his colleagueshave attempted to treat human leukaemia by the samemethod. By using multiple donors they have succeededin one case in inducing a state of stable chimaerism inrespect of the tissue of one of the donors, and in producinga remission of the leukaemia which has now persisted foralmost a year (Mathe et al. 1963, Dr. G. Mathe, personalcommunication).

In Edinburgh we have been studying the effect of

injection of allogeneic or heterospecific lymphoid cells,usually after whole-body irradiation or administration ofcytotoxic drugs in non-lethal dosage, on a variety ofanimal and human tumours.

In mice we have used the Landschutz ascites tumour,and a number of A-strain mammary carcinomas trans-

planted within the strain of origin.The Landschutz tumour is a relatively easy target

because it originated in a mouse of very different geneticconstitution to those used in our experiments. We havesucceeded firstly in prolonging the life of animals inocu-lated with the tumour three days before the start of treat-ment (Woodruff, Symes, and Stuart 1963), and morerecently in eradicating the tumour without killing thehost (Woodruff, Symes and Anderson 1963, and unpub-lished data). In these experiments rat cells were used, and

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of the various methods of treatment tested the mosteffective consisted of 400r whole-body irradiationfollowed by intraperitoneal injection of 30 millionthoracic-duct lymphocytes from rats immunised againstthe tumour.

The mammary carcinomas, on the other hand, weretransplanted in the strain of origin, and under thesecircumstances, as we have seen, the possibility of animmunologically directed attack is contingent on theirpossessing tumour-specific antigens. Whether or notthese tumours do possess such antigens is still a contro-versial question. Many investigators have concludedthat they do not; our own experiments (Woodruff andSymes 1962a, b), however, suggest that they do, at anyrate at an early stage in their life-history, but that theymay lose them later. It was expected therefore that thesetumours would be much more difficult to treat than theLandschutz tumours, and this has proved to be the case.

In our first experiments the tumour was inoculatedsubcutaneously; four days later the mice were irradiated(400r), and on the following day allogeneic (Woodruffand Symes 1962c) or heterospecific (rat) cells (Woodruff,Symes, and Stuart 1963) from either normal or pre-immunised donors were injected intravenously. Whencells from preimmunised donors were used, growth of thetumours was retarded and some tumours were completelydestroyed; but the mean survival of the mice was actuallyless, because those which were not killed by their tumourdied as the result of severe graft-versus-host disease.More recently, however, the survival of mice which wereinoculated intraperitoneally with the tumour has beensignificantly prolonged by irradiation and intraperitonealinjection of immune allogeneic spleen cells or thoracic-duct lymphocytes (Woodruff, Symes, and Boak, un-

published), though no complete cures have been obtainedas yet. Further experiments are being undertaken todetermine whether the results can be further improvedby the addition of one of the procedures such as splenec-tomy which have been shown to reduce the severity ofexperimental graft-versus-host disease (Biozzi et al.

1964).In dogs we have attempted to treat various spontaneous

tumours in a similar way (Woodruff, Symes, Boak,Nolan, Watt, and Head, unpublished). No animals havebeen cured, but some interesting observations have beenmade. One dog with a malignant melanoma, for example,which received whole-body irradiation followed by anintravenous infusion of spleen cells from two rabbitsimmunised against the tumour, appeared to developsudden severe pain at the site of the tumour withinminutes of the start of the infusion, and a few hours laterthe affected limb in the vicinity of the tumour had increasedin temperature. Other dogs, with osteogenic sarcomas,have shown temporary regression associated with exten-sive necrosis following whole-body irradiation andinfusion of either allogeneic or heterospecific (sheep)spleen cells.

Finally, we have treated ten patients with advancedcancer by intravenous or intraperitoneal injection of

allogeneic spleen cells or peripheral-blood leucocytes afteradministration of immuno-suppressive drugs of variouskinds. Eight of these cases have already been reported(Woodruff and Nolan 1963). Safety has, of course, been thefirst consideration, and no attempt has been made to

produce the maximal anti-tumour effect. In view of this,and the fact that the tumours were very advanced and had

resisted all other forms of treatment which were thoughtto be worth trying, spectacular remissions were not

expected. In every case, however, the treatment has beenfollowed by some change in either the patient’s symptoms,the physical signs, or the findings on serial biopsy, and itseemed reasonable to attribute at least some of these

changes to a direct action by the transplanted cells on thetumour.

Not very long ago to have suggested that immunologywould contribute to our understanding of the behaviourof tumours, and to the development of new methods oftreating them, would have seemed as absurd as AlmrothWright’s proposal to prevent typhoid by prophylaticvaccination with killed bacilli seemed to the War Officeat the time of the Boer War. Today, although the thera-peutic dividends from the immunological approach to thecancer problem are as yet very meagre, the suggestion nolonger seems absurd. How will it appear, I wonder, inanother sixty years’ time ?

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THE HISTAMINE-INFUSION TEST

J. H. LAWRIEM.B. Glasg., F.R.C.S., F.R.C.S.E., D.C.H.

RESEARCH ASSISTANT

G. M. R. SMITHB.M., B.Sc. Oxon.

SENIOR HOUSE-OFFICER

A. P. M. FORRESTM.D., Ch.M., B.Sc. St. And., F.R.C.S., F.R.C.S.E., F.R.C.S. Glasg.

PROFESSOR OF SURGERY

From the Department of Surgery, Welsh National School ofMedicine and United Cardiff Hospitals, Cardiff

THE most widely used and cited test of stimulatedgastric acid secretion is the augmented-histamine responsedescribed by Kay (1953). In this test the output of hydro-chloric acid is measured by nasogastric aspiration after thesubcutaneous injection of a large dose of histamine, the

side-effects ofwhich are covered

by an injection ofmepyramine.The response is

transient, rising toa peak in about 30minutes and re-

turning to basallevels in about 1hour. Precise esti-mation of acid out-

put thus dependson accurate gastricaspiration overthis short period.In expert handsand in large series,this test has beenshown to be repro-ducible ; but innormal ward prac-tice faults in

aspiration mayoccur and the

critical peak 15-minute collection may be lost. A furtherdisadvantage is the lack of agreement on the best methodof expressing the results of such a response, and variouscomputations of the four 15-minute collections followinghistamine have been used to express the output in

milliequivalents per hour (table i). These may be simplyadded together, or the second and third periods may

Fig. I-Dose-response curves from five

patients.

TABLE I-VARIOUS METHODS OF ESTIMATING " MAXIMAL " OUTPUTFROM AN AUGMENTED-HISTAMINE RESPONSE

be taken as a 30-minute output, or the " maximal"output may be calculated as some function of the peakperiod or of the two highest successive periods, as

suggested by Baron (1963).Most of these difficulties can be resolved if the response

is made to reach a " steady state " by administering thehistamine in a continuous intravenous infusion-a methodwhich has been standard practice in the experimentallaboratory and for research purposes in man (Teorell 1947,Obrink 1948, Adam et al. 1954, Johnston and Duthie1963). The studies reported here were undertaken todetermine whether the response to a continuous infusion

Fig. 2-Palmer pump, close-fitting disposable syringes, fine steril-ised paediatric scalp-vein needles, and connecting tubes used toset up histamine-infusion test.

of histamine could be adapted as a routine test of gastricfunction in man.

MethodAfter an overnight fast the nose and throat of the patient are

sprayed with 4% lignocaine. With the patient sitting up, a16 French gastric tube is passed slowly by the nose or mouth,largely by means of the patient’s own swallowing movements,into the stomach, until the tip of the tube is in the dependentpart of the body of the stomach, as indicated by the aspirationof resting gastric juice. In the earlier tests the tube was passedunder radiological control, but it became apparent that accuratepositioning of the tube could be verified during the test by any

variation in thevolume of the

aspirations.With the pa-tient flat on hisleft side, the

resting juice isaspirated and

discarded, anda basal hourcollectionmade.

A constant

intravenous in-fusion of hist-amine acid

phosphate e(0-04 mg. perkg. body-weight perhour) is thenstarted. Thisdose, whichgives a "maxi-

Fig. 3-Three successive histamine-infusion testsin a patient given 50 mg. mepyramine beforetest B.

Outputs: A (no mepyramine) 42-8 mEq. per hr.;B (mepyramine) 44-1 mEq. per hr.; C (nomepyramine) 46-0 mEq. per hr.