The Immunologie Status of Patients withNonlymphomatous Cancer1
Chester M. SouthernSloan-Kettering Institute, New York, New York
SUMMARY
It is firmly established that many cancer patients have impaired immune responses. latrogenic immune-suppression contributes to this impairment in many patients, but the data indicate that there is also an impairment which is related to theneoplastic disease per se. The defect appears to be in responsesrequiring the mediation of cells, rather than in those whichare dependent upon the production and reaction of serum antibodies. The immunologie deficiency is most frequent and mostsevere in patients with widespread and debilitating cancer, butit is not a consequence of debility alone. There is no presentevidence that abnormal immune responsiveness precedes thedevelopment of the cancer, but since the methods for evaluatingimmune responses are crude and insensitive, this possibilitycannot be excluded.
INTRODUCTION
The detection of cancer-specific antigens in many tumors hasbeen achieved. The tumor-inhibiting effects of immune reactiondirected against such cancer-specific antigens is now well documented. The mechanisms by which host reactions influence thegrowth of autochthonous and syngeneic tumors are graduallybeing elucidated. Each of these areas has been considered byprevious participants in this symposium. But it is axiomaticthat if an immunologie response is to be effective against disease, the patient must be capable of producing an appropriateand adequate response. Therefore, it is appropriate that, in thissymposium on cancer antigens, we also consider the immunologie capability of the cancer patient.
There is no simple test of overall immune status. The approach to evaluation of a patient's immunologie status is, there
fore, the measurement of the quantity and quality of as manyas possible of the factors and the reactions which participatein or contribute to immune responses. The results of such testsprovide what can be called an immunologie profile of the patient. It is convenient to subdivide the immunologie profileinto four main categories: (a) serum antibodies, (b) serumfactors other than antibody which participate in antigen-antibody reactions or other host defenses, (c) specific immunereactions which are mediated directly by lymphocytes or other
1Support for this research was supplied in part by researchgrants from the American Cancer Society (T 229) and from theNational Cancer Institute, USPHS (CY 3215 and CA 08748).
"immunocytes," and (d) nonspecific cellular mechanisms which
may be involved directly in host defenses or which may participate in the process of antibody formation or cell-mediated
specific immunity.In a group of cancer patients there may be great variation
in types of cancer, general clinical status, extent and locationand rate of tumor growth, and type and intensity of treatment.Each of these variables may have an influence on the patient's
immune status. Radiotherapy and most chemotherapeuticagents are known to have immunosuppressive effects, and it isnecessary to distinguish these iatrogenic effects from results ofthe neoplastic disease itself. In this paper only the nonlym-phomatous types of cancer will be considered because primaryneoplastic diseases of the reticuloendothelial system presentimmunologie problems which set them apart, and because thisgroup is considered elsewhere in this symposium by Miller(30).
and late phase sera by the use of diethylaminoethyl cellulosecolumn fractionation. Antibody levels were just as high in thecancer patients as in the healthy controls. The mercaptoethanolstudies demonstrated that the pattern of 7 S and 19 S responsevaried greatly within the control group and also among thecancer patients, but there was no consistent difference in thisrespect between the two groups. Mean titers in patients andcontrols are shown in Chart 1.
The production of isoantibody to hoinografts of human tissue-cultured cell lines by cancer patients and healthy personshas also been studied in my laboratory using several serologietechnics (2, 18, 19, 58, 59). More than 20 cancer patients and30 healthy persons were studied after a primary homotrans-plant. About half of both groups had detectable antibody attiters of 1:10 to 1:80. In spite of the fact that rejection of thehomotransplants was markedly delayed in many of the cancerpatients (see below), there was no difference between patientsand controls in the frequency of detectable antibody, time ofappearance of antibody, or maximum antibody titers. Therewas a striking difference in the persistence of antibody, butit was in those patients whose ability to reject the homotrans-
plant was impaired that the isoantibody persisted longest (19,59). In both cancer patients and normals, the antibody activityin sera collected 2 to 4 weeks after homotransplantation wasusually destroyed by mercaptoethanol, indicating that theisoantibody was predominantly 19 S globulin (and this presumption was checked by G200 Sephadex fractionation). The
9 HEALTHY CONTROLSO Total Antibody
D MCE Resistant (7SÃŒA MCE Sensitive (195)
246Weeks After Yellow Fever Vaccine
Chart 1. Mean titers of hemagglutination-inhibiting antibodiesto yellow fever virus in healthy persons and patients with carcinoma following live virus vaccine. Serum dilution titers of totalantibody and mercaptoethanol-resistant (7 S) antibody are shownby scale at left. Mercaptoethanol-sensitive (19 S) antibody isshown by scale at right. All titers are just as great in the patientsas in the controls.
antibody titers detected by the immune adherence technic,which requires the first four components of complement (C'l,
4, 2, 3c), and those obtained by the passive hemagglutinationtechnic, which does not require complement, were parallel inthe two groups (58). Thus, we have found no evidence ofabnormality in quantity or quality of the primary antibodyresponse to virus infections or to allogeneic tissue antigens incancer patients. Several other studies of serum antibody production by nonlymphomatous cancer patients have been published; most agree that there is no abnormality attributableto the cancer per se.
Leskowitz et al. (22) studied production of serum antibodiesin patients with cancer (types not specified but presumablygynecologic carcinomas) using pneumococcal polysaccharides,diphtheria toxoid, and tetanus toxoid as antigens. The cancerpatients developed higher antibody titers to the polysaccharideantigens than did the controls. The antitoxin responses werenot significantly different in the two groups.
Lytton and Hughes (28) also studied antibody response totetanus toxoid in patients with carcinoma and, contrary to theabove results, found that their cancer patients had a significantly poorer antitoxin response than did the controls. Thecancer patients had not received radiotherapy or anticancerchemotherapy, and surgery was excluded as the cause of diminished antibody response. In both of these studies the controls were patients with non-neoplastic diseases.
Levin et al. (26) studied antibody responses to tularemiaand E. coli antigens in 10 patients with metastatic cancer. Sixwho were being treated with 6-mercaptopurine had little or noantibody response, but the four patients who were not receivingchemotherapy all produced antibody at apparently normallevels.
Balch (4) studied the anamnestic responses to diphtheriavaccine in a group of cachectic cancer patients and found it nodifferent from the response of healthy controls and well-nourished noncancer patients.
Electrophoresis and immunoelectrophoresis have been usedextensively in recent years to study serum proteins in all typesof disease. In nonlymphomatous cancer the only deviationsfrom normal that occur with any frequency are increased concentrations of beta globulins, which are not known to haveimmunologie functions, and the decreased albumen concentration which occurs in any condition in which there is chronicnutritional depletion. Gamma globulins are apparently normalin patients with nonlymphomatous cancer (39, 60).
NONSPECIFIC SERUM FACTORS
Serum complement levels of cancer patients as judged bytotal hemolytic activity or immune adherence were withinthe normal range or higher (29, 50, 61). Levels of the secondcomponent of complement were normal (57), and in a recentstudy of all nine serum complement components by Me Kenzieet al. (29), C'8 and C'9 were usually higher in cancer patients
than in healthy controls.About 12 years ago, Pillemer (40) described a complement-
fixing reaction between serum globulins and zymosan (a preparation of yeast consisting largely of polysaccharides) which
he attributed to "a primordal type of antibody" which he
named properdin. Serum reactions which caused lysis of variousGram-negative bacteria and which neutralized T-2 bacterio-
phage were thought to be due to the same material. He postulated that this serum protein represented a nonspecific hostdefense mechanism which could react with a wide variety ofpotentially pathogenic microorganisms. During a period ofacrimonious debate concerning the nature of this "properdinreaction," it gradually became evident (34, 36) that muchproperdin activity is due to classic antibodies ("natural antibodies") which react specifically with antigenic materials such
as polysaccharides, which are widely distributed in nature andhave an identical or cross-reacting antigenic structure. Thusit appeared that the properdin reaction was not due to a nonspecific "primordal antibody" but rather, to paraphrase Pille-mer's words, was the reaction of classic antibody with a pri
mordal antigen. Recent data support the concept that thereis indeed a unique serum protein that participates in theproperdin reaction which is neither classic antibody nor complement (37), that is, true properdin. Nevertheless it seemsquite certain that most tests of properdin activity have measured antibody rather than this unique protein.
Although it may now be inappropriate to discuss studies ofserum properdin reaction under the heading of nonspecificserum factors, the data are clearly pertinent to the presentdiscussion to the immune status of cancer patients because theserum of many cancer patients fails to give this reaction. Intests performed by Pillemer using his zymosan technic (41)on coded sera from healthy persons and cancer patients, manyof the latter had no detectable properdin activity, and theabsence of this reaction was correlated with an impaired abilityto reject homotransplants (56). Subsequent studies in my laboratory using Pillemer's technic showed that nearly 40% of
patients with advanced cancer had no detectable properdinactivity (less than 1 unit per ml) and 54% had less than 4units per ml. Only 5% of our healthy controls gave negative results and only 23% had titers of less than 4 units perml (47). These data are summarized by diagnostic categoriesin Table 1. Sera of cancer patients also showed a "zone" re
action more frequently than the controls. This term is usedfor sera which had properdin activity when diluted but notwhen undiluted. Such a result suggests the presence of sometype of inhibitor which is effective only in undiluted serum.It is possible that negative properdin tests were due to greaterconcentration of such inhibitors rather than actual absenceof properdin. This interpretation is also consistent with theobservation (47) that, when large amounts of serum globulin with extremely high properdin activity were administeredto patients with properdin titers of zero, the properdin activitydisappeared very rapidly, although when the same preparationswere administered to patients with detectable levels of serumproperdin, their titers were increased and remained elevatedfor a time consistent with a normal metabolic breakdown ofhuman globulins.
The European literature contains numerous papers reportinglow properdin activity in cancer patients, usually based onbacteriolytic or bacteriophage neutralization tests. It now seemscertain that these various tests of "properdin" activity did
not measure a single serum factor, but regardless of the identityor significance of these reactions, it is clear that many cancerpatients are deficient in certain serum factors which reactwith microorganisms and microbial products.
SPECIFIC CELL-MEDIATED IMMUNE MECHANISMS
Laboratory technics for the study of cell-mediated specificimmune reactions are currently under investigation in manylaboratories, as described earlier in this symposium (5, 7).They have been successfully applied to several discrete problems but are not yet adequate for the evaluation of immunologiecompetence of individuals. Instead, a clinical evaluation ofcell-mediated immunity requires direct participation of thepatients in tests of delayed hypersensitivity or homograft rejection and often requires parallel tests on healthy controlsas well.
As yet the only exception to this generalization is the blastictransformation of lymphocytes on exposure to phytohemag-glutinin or antigens to which the lymphocyte donor is sensitized. This laboratory test has been used to study the responsiveness of lymphocytes from patients with leukemia andlymphoma and often reveals poor responses (30), but patientswith other neoplastic diseases have not yet been studied, tomy knowledge.
Delayed hypersensitivity reactions of patients with nonlym-phomatous cancer have been studied by several investigatorsusing tuberculin and other microbial'antigens. Positive reac
tions to these tests require not only the immunologie capacityto respond but prior exposure to the antigen, and this exposure is usually left to chance. When the occurrence, intensity,and time of exposure are unknowns, the absence of positivereaction to any one antigen cannot be interpreted as an immunologie defect for any individual, but failure to react toany of a group of frequently encountered antigens is indicativeof anergy, and even in studies with a single test antigen, response rates in different populations can be compared. Studiesof this type permit the general conclusion that there is adeficiency of delayed hypersensitivity reactions among patientswho have advanced nonlymphomatous cancer, although thisdeficiency is not as marked as among lymphoma patients. Inour study (24) only 30% of patients with nonlymphomatouscancer and 12% of lymphoma patients had positive tuberculintests to middle strength PPD, whereas 72% of patients withnonneoplastic nontubercular diseases had positive reactions.In two studies (9, 27) tuberculin response rates were not significantly different in carcinoma patients, but Hughes andMackay (17) found a distinct relationship to the extent of thedisease. In their group of patients with generalized or regionalspread of cancer, only 35% and 47% respectively had positivetests, while those with only local cancer reacted like the non-cancer control group (75% and 81% respectively). Soloweyand Rapaport (46) used a battery of 5 microbial antigens ofwhich streptokinase and streptodornase gave the most positivereactions; they found that 73% of the cancer patients wereanergic to this antigen preparation in contrast to only 24%of the controls. They also found that the degree of reactionwas generally less in the cancer patients. Lamb et al. (21)
other diseases.Assayedleukemia;CML, chronic myeloeytic leukemia;>82
(5%)116
(6%)31216
(11%)61111624
(8%)4
(12%)316
(20%)Zone5
(14%)11318
(18%)822212122
(15%)63212512449
(16%)6
(7%)by
the zymosan technicofLSA,lymphosarcoma; RSA,reticulum cell
used a similar battery of microbial antigens and also foundthat, among 32 patients "in poor condition" from carcinoma,
38% were anergic to all of the tested antigens whereas noneof the 27 carcinoma patients "in good condition" were anergic.
None of the patients in this study had received chemotherapyor hormonal therapy during the 4 weeks preceding the test.Hauler and Amos (14) had very similar results. Advancedcancer patients had a lower incidence of positive skin tests toall five test antigens than did their healthy relatives, and 5 ofthe 18 patients, but none of the controls, had complete anergy.Logan (27) found only 29% positive skin tests with mumpsantigen in patients with advanced carcinoma but 82% in patients with early cancer and about the same in healthy controls.
A more significant measure of immune responsiveness is obtained if the initial exposure to the test antigen is controlledas part of the investigative procedure, for a preexisting immunity might persist in a patient who is no longer capable ofinitiating an immune response. This requires use of a testantigen with which patients would not normally have anycontact. We (24) conducted a study of this type by sensitizingpatients and normal subjects with a primary dose of dinitro-
fluorobenzene (DNFB) and testing 14 days later for a delayed-type hypersensitivity. Seventy-five percent of the healthy per
sons and the same proportion of patients with nonneoplasticdiseases had positive reactions, but only 23% of the patientswith cancer reacted. (Only 11% of lymphoma patients responded in this test.)
Several methods have been used to study the homograft rejection reaction in man. The most extensive studies in cancerpatients were done with subcutaneous homotransplants of cellsuspensions of tissue-cultured lines of human cells. This technic
gives reproducibility of quantity, quality, and antigenic composition of the graft which cannot be achieved with solid tissuegrafts or with direct homografts from donor to recipient. Theprocedure is technically simple, is easy to explain to both patients and healthy volunteers, and was readily accepted bythem. Unfortunately, cell lines derived from normal tissues(amnion or fibroblast cells) proved unsatisfactory for thispurpose because they did not produce a detectable growth orhost reaction in any recipient. Certain of the cancer cell lines,notably the epidcrmoid carcinoma lines HEp3 and HEp2 (31)and the osteogenic sarcoma line RPMI-41 (32), were ideal
Rejection patterns of primary subcutaneous homotransplants of human cancer cell linesHEp2, HEp3, or RPMI-41 in cancer patients, patients with nonneoplastic diseases, and
healthy persons.o Many cancer patients were unevaluatable because they died within 2 weeks of receiving
the homotransplant. Prompt rejection began during the third week and was complete by 8(usually 6) weeks. Slight delay is defined as maximum nodule size during the third or fourthweek but complete rejection by 6 to 8 weeks. Definite delay is continued increase in nodulediameter for at least 4 weeks but eventual complete rejection. Marked delay is continuednodule growth for at least 6 weeks and rejection not observed because of death or excisionof nodule. Patients who died with nonregressing homotransplants between 3 and 6 weeks areclassified as if regression would have started the following week.
because, in healthy recipients, limited propagation of the transplanted cells together with the rejection reaction of the recipient produced a palpable subcutaneous nodule which increasedto a maximum of 2 to 5 cm by 12 to 14 days and regressedrapidly thereafter. This behavior in healthy recipients provides a base line against which deviations from the normal rejection pattern can be evaluated.
The cancer cell homograft studies (6, 48, 54, 55, 56) revealed that many cancer patients have an impaired immunecapacity which is evidenced as a lack of acute inflammatoryreaction to the foreign cells and a delay in rejection of theresulting subcutaneous nodule. Prolonged growth of the homo-transplants never occurred among 164 healthy persons whoreceived one or more of the human cancer cell lines, in 19 patients with debilitating non-neoplastic diseases, or in patientswith cured cancer or early disseminated cancer. However, persistence and growth of homotransplant nodules beyond thesix to eight weeks required for complete rejection by the healthyrecipients occurred frequently in patients with advanced cancer(Table 2). Of the 195 evaluatable studies in cancer patients,rejection was strikingly delayed in 31%. That is, regressionof the nodule did not start until 6 weeks or longer after transplantation. Only 42% of the cancer patients rejected theirhomotransplants as promptly as did the controls. Many ofthese patients, of course, received immunosuppressive types ofanticancer therapy which might have caused or contributedto the impairment of rejection mechanisms, but many of themwere not receiving any. chemotherapy or radiotherapy at thetime of, or for many weeks preceding, the transplants (23).Thus, the immunologie inadequacy of some cancer patientsmust be attributed to the cancer per se.
Homografts of cancer tissue directly from patient to recipient have also been studied in a few cancer patients, but theevaluation of graft rejection was usually not the primary objective. The composition and growth potential of the inoculumis variable and is not reproducible. In our studies (49) suchhomografts were usually rejected promptly, and the protracted
growth that occurred in two patients was probably attributableto the concurrent use of immunosuppressive drugs rather thancancer per se. Grace and Kondo (13) studied direct homograftsof cancer in 20 patients. A few had a somewhat longer persistence of the nodule than might have been expected, butall were rejected as soon or sooner than simultaneous homo-grafts of normal skin.
Isotopie skin homografts have also been used to study homo-graft reactions in man. The rejection reaction can be followedby macroscopic examination and by histologie examination ofsmall biopsies. Use of this method for clinical research requiresa surgical procedure for both donor and recipient, and repro-ducibility is limited by the number of studies that the skindonor will tolerate.
Gardner and Preston (10) found that in some patients withadvanced debilitating carcinoma rejection of skin homograftswas impaired. Mean rejection time among 23 carcinoma patients was 24 days, and in 5, rejection took 30 to 50 days. Incontrast, rejection time averaged 17 days in patients withcirrhosis and 12 days in other patients, and maximum rejectiontimes in these control groups were 38 and 12 days respectively.They also noted that there was less inflammatory response tothe homografts in the cancer patients.
Robinson et al. (43) studied skin homograft rejection in 25cancer patients (one Hodgkin's disease, but all others were non-
lymphomatous) and found that 19 of the 25 rejected skin ofhealthy donors (mostly children) in 6 to 18 days, but in theother six, rejection required 28 to 35 days. Rejection of simultaneous skin grafts from donors who had cancer wasusually slower—12 to 28 days in the 19 prompt rejectors and26 to more than 55 days in the slow rejectors. No healthy recipients were included in the study, but it seems clear that 6of these patients did have impaired homograft rejection.
In two other studies (12, 26), skin homografts were used todemonstrate the immunosuppressive effect of cancer chemotherapy, but the untreated cancer patients rejected their graftspromptly.
The lymphocyte transfer test is a special case of the homo-graft reaction in that the transplant is composed of immuno-logically reactive cells. Therefore, there may be a reaction ofthe graft against the recipient's tissues in addition to the re
action of the recipient against the graft. However, the totalreaction is small and persists only a few days.
We performed lymphocyte transfer tests, using lymphocytesfrom the blood of healthy donors, in a series of 22 Hodgkin's
disease patients and 6 patients with nonlymphomatous neoplasms. We found no significant difference in response betweenhealthy controls and cancer patients, even in those with lym-phomas (25). Rattier and Amos (14) did find that their cancerpatients had significantly smaller reactions to lymphocytesfrom healthy donors than did healthy persons, but their patients and controls were not studied simultaneously. Threegroups of investigators (1, 14, 44) made the interesting observation that when the lymphocyte transfer test was performedwith lymphocytes from blood of patients who had carcinomaor lymphoma there was less reaction than was produced bylymphocytes from healthy donors tested simultaneously. If,as these investigators believe, the reaction to allogeneic lymphocytes is in part a graft-versus-host reaction, such a diminished response suggests that the lymphocytes of patientswith neoplastic diseases are functionally defective. If, on theother hand, the lymphocyte transfer test is purely a host-versus-graft reaction, then these results would imply that thelymphocytes of these patients are less able to stimulate immune responses in an allogeneic recipient than are cells fromnormal persons. These results resemble the observation thatskin grafts from carcinoma patients are rejected more slowlythan skin from healthy donors (3, 43).
NONSPECIFIC CELLULAR REACTIONS
The term nonspecific cellular reactions is used here to includethe participation of cells in those steps of specific immune reactions which are not restricted to specific antigens and thedirect participation of cells in host defense reactions whichoccur without relation to antigenic determinants. The first ofthese categories would include the uptake and transport of apreviously unencountered antigen by macrophages, as discussedby Nossal in this symposium (35), and various reactions thatoccur as a consequence of the reaction of antigen and antibody,such as degranulation of basophiles and contraction of smoothmuscle cells. The second category includes encapsulation offoreign matter or necrotic tissue by fibroblastic cells and theengulfing and digestion of microbes by polymorphonuclearneutrophiles and macrophages. It is often impossible to distinguish between these two roles because much is still unknownabout nonspecific defense mechanisms, about the steps in theinitiation and expression of specific immune reactions, andabout the interconversion of various wandering cells such asfibroblasts and macrophages. It is not surprising then thatthis aspect of immunology has received little attention inclinical research and that methods for such investigations arenot well developed.
Studies of the transfer of delayed hypersensitivity suggestthe existence of intermediate steps in the expression of cell-
mediated immune reactions and the need for investigation ofsuch steps. Injection of lymphocytes from blood of donorswho were highly sensitive to tuberculin resulted in the transferof sensitivity to healthy recipients, as evidenced by a delayedresponse to tuberculin when injected into the same area wherethe lymphocytes had been deposited, but the transfer of thesesame lymphocytes into patients with Hodgkin's disease failed
to transfer the tuberculin sensitivity in almost all cases (20,33). These results suggest that the Hodgkin's disease patients
lacked some mechanism which must participate in the interaction of tuberculin and immune lymphocytes to bring aboutthe inflammatory reaction of delayed hypersensitivity. However, similar studies in patients with carcinoma (14, 33) indicate that this defect does not commonly occur in nonlymphomatous neoplasia.
The cellular response to a nonspecific stimulus has beenstudied in cancer patients by Rebuck "skin window" technics,
in which the skin is abraded sufficiently to cause a slightexúdate which is collected on glass coverslips in the standardtechnic (42) or collected in isotonic salt solution in quantitativemodifications of this technic (38, 52). By both of these technics we (8, 11) found that patients with advanced canceroften display an unabated polymorphonuclear exudation for 24hours or more, in contrast to the conversion to a predominantlymononuclear cell response which is characteristic of normalpersons. Hersch et al. ( 15) had similar results in acute leukemiapatients and in carcinoma patients who were receiving chemotherapy, but the three untreated cancer patients in his serieshad normal responses.
The nonspecific phagocytic activity of the reticuloendothelialsystem was determined by Salky et al. (45) by measuring therate of clearance of a lipid emulsion from plasma after intravenous injection. Carcinoma patients showed higher-than-normal phagocytic activity.
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